p73 in colorectal cancer

Linköping University Medical Dissertations No. 1108
p73 in colorectal cancer
Daniella Pfeifer
Linköping University, Faculty of Health Sciences
Department of Clinical and Experimental Medicine, Division of Oncology
SE-58185 Linköping, Sweden
Linköping 2009
Cover: Colonic crypts with neoplastic cells. Illustration by the author.
© 2009 Daniella Pfeifer
ISBN 978-91-7393-677-4
ISSN 0345-0082
Published articles have been reprinted with the permission of the copyright
holders.
Paper I © 2004 Oxford University Press, Carcinogenesis.
Paper III © 2006 Elsevier, International Journal of Radiation
Oncology*Biology*Physics
Illustrations made by the author, unless otherwise specified.
Printed by LiU-Tryck, Linköping, Sweden 2009
Målet är ingenting. Vägen är allt.
-Robert Broberg
ABSTRACT
Colorectal cancer (CRC) is the third most common cancer in the world, with
about 5000 new cases in Sweden every year. CRC is caused by mutation (inherited
or acquired) in genes, by gene variants and changed expression of proteins. The
primary way to achieve a curative result for CRC is to remove the tumor by
surgery. To reduce risk of recurrence chemo- or radiotherapy are given as a
complement to surgery. p73 is a structural and functional homologue of tumor
suppressor p53. However, p73 is rarely mutated in tumors, but rather overexpressed as compared to normal tissue. There are two main isoforms of p73, the
transactivation capable TAp73 and the truncated ΔNp73, which are involved in an
autoregulatory loop with TAp73 and p53.
The aim of this study was to investigate the role of p73 and related proteins in the
development and treatment of CRC. A G4C14-to-A4T14 polymorphism of p73
was studied in CRC patients and healthy controls (Paper I), and rectal cancer
patients who were randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper II). The AT/AT genotype of the p73
polymorphism may increase risk of CRC development and CRC patients with the
AT allele had a better prognosis. When dividing the cases into colon and rectal
cancer it was seen that in colon cancer the AT allele tended to be more favorable
for overall survival, while in rectal cancer the GC allele seemed to be more favorable. Rectal cancer patients, with a combination of GC/GC genotype, wild type
p53 and weak survivin expression survived longer after preoperative radiotherapy.
This was not observed in the patients only receiving surgery. The protein expression of p73 was further studied in the rectal cancer patients randomized to treatment with either surgery alone or preoperative radiotherapy and surgery (Paper
III). p73 was expressed higher in tumor tissue than in normal mucosa. Patients
with p73 negative tumors had a lower risk of local recurrence after radiotherapy, as
opposed to patients that had p73 positive tumors or patients with p73 negative
tumors that did not receive radiotherapy. Effects of γ-radiation was further studied
in colon cancer cell lines KM12C, KM12SM and KM12L4a regarding cell cycle,
survival fraction (clonogenicity), apoptosis and protein expression patterns of
mutated p53, TAp73, ΔNp73, survivin and PRL-3 (Paper IV). KM12C displayed
low survival fraction, low apoptosis, no cell cycle arrest and an upregulation of the
anti-apoptotic ΔNp73 after irradiation. KM12L4a showed a high survival fraction,
but high apoptosis, arresting of the cell cycle and upregulation of the radioresistance factor survivin. The effects of overexpression and knockdown of survivin
on TAp73, ΔNp73 and p53 expression in colon cancer cell lines HCT-116p53+/+
and HCT-116p53-/- with and without γ-radiation were studied (Paper V). Overexpression of survivin decreased wild type p53, whilst downregulation of survivin
lead to a simultaneous downregulation of TAp73 and ΔNp73, mRNA and protein,
both with and without γ-radiation. Knockdown of survivin also demonstrated an
increase in apoptosis.
In conclusion, we showed that the G4C14-to-A4T14 polymorphism of p73 and
p73 protein expression may be involved in CRC development, radiotherapy
response and survival. We further showed that TAp73, ΔNp73 and p53 were
regulated by survivin in colon cancer cells.
TABLE OF CONTENTS
SAMMANFATTNING ........................................................................................................... 11
ABBREVIATIONS .................................................................................................................. 13
LIST OF PUBLICATIONS ..................................................................................................... 15
INTRODUCTION ................................................................................................................. 17
Colorectal cancer ........................................................................................................... 18
Colon and rectum .................................................................................................................... 18
Risk factors................................................................................................................................. 19
Carcinogenesis .......................................................................................................................... 20
Sporadic, familial and hereditary CRC ................................................................................ 20
The adenoma-carcinoma sequence...................................................................................... 22
Prognosis .................................................................................................................................... 23
Treatment ....................................................................................................................... 25
Radiotherapy ............................................................................................................................. 26
Cell cycle ......................................................................................................................... 27
Cell death ........................................................................................................................ 28
Apoptosis ................................................................................................................................... 28
Other types of cell death ......................................................................................................... 29
p53 .................................................................................................................................... 31
p73 .................................................................................................................................... 32
Regulation of p73 ..................................................................................................................... 33
p73 in tumors ............................................................................................................................ 34
A G4C14-to-A4T14 polymorphism in p73 ........................................................................ 35
p73 and radiosensitivity .......................................................................................................... 36
Survivin ............................................................................................................................ 36
PRL-3 ............................................................................................................................... 37
Cox-2................................................................................................................................ 38
AIMS ...................................................................................................................................... 39
Specific aims ................................................................................................................... 39
MATERIALS AND METHODS .............................................................................................. 41
Patients (Papers I-III) .................................................................................................. 41
Cell lines .......................................................................................................................... 44
KM12C, KM12SM and KM12L4a (Paper IV) ................................................................. 44
HCT-116 (Paper V) ................................................................................................................ 44
Irradiation of colon cancer cell lines .......................................................................... 45
Cell transfection............................................................................................................. 46
cDNA .......................................................................................................................................... 46
siRNA ......................................................................................................................................... 47
Polymorphism genotyping .......................................................................................... 48
DNA extraction ........................................................................................................................ 48
Polymerase chain reaction...................................................................................................... 48
Restriction fragment length polymorphism ....................................................................... 49
Loss of heterozygosity ............................................................................................................. 50
Immunological protein detection............................................................................... 51
Immunohistochemistry .......................................................................................................... 51
Western blot .............................................................................................................................. 52
Cell cycle analysis .......................................................................................................... 54
Survival fraction analysis .............................................................................................. 54
Apoptosis detection ...................................................................................................... 55
M30 ............................................................................................................................................. 55
DAPI ........................................................................................................................................... 55
Real time polymerase chain reaction ......................................................................... 56
Statistical analysis .......................................................................................................... 58
RESULTS ............................................................................................................................... 59
Paper I.............................................................................................................................. 59
Paper II ............................................................................................................................ 60
Paper III .......................................................................................................................... 61
Paper IV........................................................................................................................... 62
Paper V ............................................................................................................................ 63
DISCUSSION ......................................................................................................................... 65
The p73 G4C14-to-A4T14 polymorphism in CRC risk and survival ................. 65
Expression of p73........................................................................................................... 67
Expression of p73 in colorectal cancer patients ................................................................. 67
Expression of p73 in colon cancer cell lines........................................................................ 68
p73 in relation to radiotherapy response................................................................... 69
p73 in relation to radiotherapy response in rectal cancer patients ................................ 69
Cellular radiosensitivity .......................................................................................................... 70
p73 in relation to radiotherapy response in colon cancer cells ....................................... 71
p73 and other proteins.................................................................................................. 73
p73 and p53 ............................................................................................................................... 73
p73 and survivin........................................................................................................................ 74
p73 and Cox-2........................................................................................................................... 75
CONCLUSIONS .................................................................................................................... 77
ACKNOWLEDGMENTS ....................................................................................................... 79
REFERENCES ........................................................................................................................ 81
SAMMANFATTNING
Cancer i tjocktarmen (kolon) och ändtarmen (rectum) är den tredje vanligaste
cancerformen i världen och i Sverige drabbas varje år ca 5000 personer av tjockoch ändtarmscancer. Antalet drabbade individer är högre i de industrialiserade
länderna än övriga världen, vilket tyder på att vissa omgivnings- och livsstilsfaktorer påverkar risken för tjock- och ändtarmscancer. Även vissa genetiska faktorer
påverkar risken för insjuknande i dessa cancerformer.
En gen är mallen för ett eller flera proteiner och en förändring (mutation) i en gen
kan innebära att motsvarande protein förändrar eller förlorar sin normala funktion.
En del gener har också nedärvda naturliga variationer, så kallade polymorfier, vilka
i sin tur kan leda till variation i funktionen hos motsvarande protein. Proteiner som
förändrat eller förlorat sin normala funktion bidrar till de speciella egenskaper som
finns hos cancerceller. Cancerceller till skillnad från normala celler delar sig
okontrollerat, är motståndskraftiga mot den programmerade celldöd (apoptos)
som normalt förstör skadade celler och kan sprida sig via blodbanan till andra
organ (metastasera).
Tjock- och ändtarmscancer behandlas främst med kirurgi, cellgifter och strålning
och tack vare förbättrade behandlingar har dödligheten minskat de senaste årtiondena. Genom att studera olika genetiska varianter, proteiner och förhållandet
mellan proteiner i cancerceller får vi större insikt i vilka faktorer som ökar risken för
tjock- och ändtarmscancer, men också vilka faktorer som påverkar hur effektiv en
behandling är för varje individuell patient.
Målet med denna avhandling var att studera proteinet p73 och hur den påverkar
risken för tjock- och ändtarmscancer och behandlingseffekten av sjukdomen. p73
tillhör samma proteinfamilj som “genomets väktare” p53 och båda har förmågan
att skydda cellen genom att stoppa celldelning och inducera apoptos i skadade
celler. Dock är p53 muterat i ungefär 50 % av alla tjock- och ändtarmscancrar.
SAMMANFATTNING 11
Genom att studera en polymorfi i p73 hos tjock- och ändtarmscancerpatienter och
friska blodgivare såg vi att de individer som har dubbel uppsättning av variantgenen har ökad risk att utveckla tjock- och ändtarmscancer och att de patienter som
är bärare av variantgenen hade en längre överlevnad (Paper I). Ändtarmscancerpatienter som fått strålbehandling överlevde längre om de hade dubbel uppsättning
av den ursprungliga p73-genen, icke-muterat p53 och lågt uttryck (mängd) av
survivin. Survivin är ett protein som normalt hindrar cancerceller från att dö
(Paper II). De ändtarmscancerpatienter som behandlats med strålning och som
hade p73 negativa tumörer fick mycket färre återfall än de patienter som hade p73
negativa tumörer men inte fått strålbehandling, eller som hade p73 positiva
tumörer (Paper III). För att närmare studera tjocktarmscancercellers motståndsmekanismer mot strålbehandling mättes bland annat olika typer av celldöd och
uttrycket av två olika varianter av p73, kallade TAp73 och ΔNp73, survivin och
PRL-3. PRL-3 är ett protein som uttrycks starkast i metastatiska tumörer och leder
till ökat motstånd mot strålbehandling. Den cellinje som inte dog genom apoptos
hade högt uttryck av ΔNp73, vilket är en variant av p73 som hindrar celler från att
dö, medan den som trots strålning i hög grad fortsatte dela sig hade ett högt uttryck
av survivin (Paper IV). Efter indikationer om att uttrycket av p73 proteinerna
TAp73 och ΔNp73 är kopplade till survivin undersöktes ett eventuellt förhållande
mellan dessa proteiner i tjocktarmscancerceller. I de fall där uttrycket av survivin
minskades i cellerna minskade också uttrycket av TAp73 och ΔNp73. Detta
fenomen sågs även i celler som behandlats med strålning. En minskning av survivin
i cellerna ledde även till att fler celler dog genom apoptos (Paper V).
Sammanfattningsvis pekar våra resultat på att p73 spelar en viss roll i utvecklingen
av tjock- och ändtarmscancer, men också avseende effekterna på strålbehandling
av ändtarmscancer. Vi har också visat att TAp73, ΔNp73 och p53 regleras av
survivin.
12 SAMMANFATTNING
ABBREVIATIONS
5-FU
APC
ATM
Caspase
cDNA
CMV
Cox-2
CRC
DAPI
DBD
DIABLO
DNA
dsRNA
FAP
FBS
Gy
HNPCC
HRP
IAP
IHC
K-RAS
LOH
Mdm2
MEM
MMR
mRNA
NSAID
OD
PCR
5-fluorouracil
Adenomatous polyposis coli
Ataxia telangiectasia mutated
Cysteine-aspartic acid proteases
Complementary DNA
Cytomegalovirus
Cyclooxygenase-2
Colorectal cancer
Diamidino-2-phenylindole
DNA binding domain
Direct inhibitor of apoptosis-binding protein with low pI
Deoxyribonucleic acid
Double stranded RNA
Familial adenomatous polyposis
Fetal bovine serum
Gray
Hereditary non-polyposis colorectal cancer
Horse radish peroxidase
Inhibitor of apoptosis
Immunohistochemistry
Kirsten-ras
Loss of heterozygosity
Mouse double minute 2
Minimal essential medium
Mismatch repair
Messenger RNA
Non-steroidal anti-inflammatory drugs
Oligomerization domain
Polymerase chain reaction
ABBREVIATIONS 13
PEST
PRL
RFLP
RISC
RNA
RNAi
RT-PCR
SAM
SF5
siRNA
SMAC
SNP
TA
TME
TNF
TNM
Penicillin-Streptomycin
Phosphatase of regenerating liver
Restriction fragment length polymorphism
RNA-induced silencing complex
Ribonucleic acid
RNA interference
Real time polymerase chain reaction
Sterile alpha motif
Survival fraction at 5Gy
Small interfering RNA
Second mitochondria-derived activator of caspase
Single nucleotide polymorphism
Transactivation
Total mesorectal excision
Tumor necrosis factor
Tumor node metastasis
14 ABBREVIATIONS
LIST OF PUBLICATIONS
This thesis is based on the following Papers, which will be referred to in the text by
their roman numerals (I-V):
I.
Pfeifer D, Arbman G, Sun XF
Polymorphism of the p73 gene in relation to colorectal cancer risk and
survival
Carcinogenesis (2005) 26:103-7
II.
Lööf J*, Pfeifer D*, Adell G, Sun X-F
Significance of an exon 2 G4C14-to-A4T14 polymorphism in the p73
gene on survival in rectal cancer patients with or without preoperative radiotherapy
Resubmitted to Radiotherapy & Oncology
*Authors contributed equally to this work
III.
Pfeifer D, Gao J, Adell G, Sun XF
Expression of the p73 protein in rectal cancers with or without preoperative radiotherapy
Int J Radiat Oncol Biol Phys. (2006) 65:1143-8
IV.
Pfeifer D, Wallin Å, Holmlund B, Sun X-F
Protein expression following γ-radiation relevant to growth arrest and
apoptosis in colon cancer cells with mutant p53
Submitted
V.
Pfeifer D, Sun X-F
Survivin regulates expression of p73 isoforms TAp73 and ΔNp73 in colon
cancer cell lines with and without γ-radiation
Submitted
LIST OF PUBLICATIONS 15
16 LIST OF PUBLICATIONS
INTRODUCTION
Cancer touches many people in one way or the other and most people know
someone who has or has had cancer. It is estimated that every third person in
Sweden will be diagnosed with cancer during their life time. Cancer is not a new
disease, in fact, the oldest descriptions of cancer date back to ancient Egypt
papyrus scrolls from around 1600 B.C. The origin of the word cancer is credited to
the ‘Father of Medicine’, Hippocrates, who used the terms carcinos and carcinoma
to describe tumors. In Greek these words refer to a crab, probably because the
finger-like spreading projections from a tumor called to mind the shape of a crab.
Cancer is a complex group of over 100 different types and can affect almost every
organ or tissue in the body of both humans and animals. Although all cancers are
different they have acquired the molecular, chemical and cellular traits needed for
an abnormal proliferation of cells (neoplasm). These changes include selfsufficiency in growth signals, insensitivity to growth-inhibitory signals, escape of
programmed cell death (apoptosis), limitless replicative potential, the ability to
grow new blood vessels to the tumor (angiogenesis) and the ability to invade other
tissues (invasion) and spread through the body (metastasis) [1].
During 2007 there were 50 100 cases of cancer diagnosed and reported to the
Swedish cancer registry. The most common cancers are breast cancer in women
and prostate cancer in men. Colorectal cancer (CRC) is the second most common
cancer in women and third most common in men [2]. In Sweden the prevalence of
cancer has increased, however thanks to better diagnostic methods and more
efficient treatment the survival rates have increased [3].
INTRODUCTION 17
Colorectal cancer
CRC is the third most common cancer in the world with 1.2 million people
diagnosed all around the world during 2007. In Sweden there were 5000 new CRC
cases, corresponding to about 11.5% of the total number of Swedish cancer cases.
Among the CRC cases most (65%) are located in the colon and the rest (35%) are
located in the rectum [3].
Colon and rectum
The large intestine extends from the final section of the small intestine (ileocecal
valve) to the anus. It is divided anatomically and functionally into three parts, the
colon, rectum and anus. Histologically the colon and rectum wall comprises four
distinct layers: mucosa, submucosa, muscle (muscularis propria) and serosa
(Figure 1).
Figure 1. Normal colon and rectum.
The colon is the site where water and salt from solid wastes are extracted before
they are eliminated from the body. The colon, which is approximately 1-1.5 m
long, is divided into four parts, the ascending colon, the transverse colon, the
descending colon, and the sigmoid colon. The rectum, which is the temporary
storage place for the feces, is approximately 12-15 cm in length. The anal canal
18 INTRODUCTION
measures 2 to 4 cm and flow of substance through the anus is controlled by the
anal sphincter muscle, an important feature for continence and control of defecation [4].
The michroarchitecture of the colon and rectum is characterized by crypts,
approximately 50 cells deep. The epithelial cells of the colon divide (proliferate)
and mature into specialized cells (differentiate) in the lower part of the crypt and
then migrate towards the lumen of the bowel. Mature cells in the upper part of the
crypt lose their capacity to divide and finally die by apoptosis [5]. The life span of a
mature colonic cell is about 4-8 days and there is a constant renewal of the luminal
epithelium which is subjected to a constant abrasion [6]. In some cases cells
divide, but do not differentiate, and cells start to fill the crypt (aberrant crypt
focus). This causes normal crypts to bud, develop further cellular changes and
grow above the surrounding mucosa, forming a small adenoma [7, 8]. An adenoma
is a benign neoplasm, and although it is known that colon cancers arise from
adenomas, the majority of adenomas never develop into cancer [5].
Risk factors
Multiple factors contribute to the development of CRC, dietary and life style
factors on one hand and genetic factors on the other [9]. CRC is more common in
North America, parts of Europe, Australia, New Zeeland and Japan than in eastern
Asia and Africa [3]. This together with the fact that populations migrating from a
low-incidence to a high-incidence geographical area show a similar incidence as
those living in the high-incidence area, points towards life style and dietary habits
being causative [10]. The exact causes are still controversial but epidemiological
studies indicate that diets that include low fruit, vegetable or fiber intake, high red
meat or saturated fat consumption increase the risk of developing CRC. Exposure
to caffeine, cigarette smoke and alcohol has also been suggested to increase risk.
Diets high in calcium and folate and regular physical activity are associated with a
reduced risk of developing CRC [11-15].
INTRODUCTION 19
Carcinogenesis
Carcinogenesis, meaning creation of cancer, requires a series of mutations in
oncogenes, tumor suppressor genes and mismatch repair (MMR) genes for the
transformation of a normal cell into a tumor cell. When an oncogene is mutated it
causes constant activation of a gene, while a mutation in a tumor suppressor gene
reduces or removes the activity of the gene product. Oncogenes normally stimulate cell division and control the degree of differentiation and tumor suppressor
genes are normally involved in slowing down cell division and activating apoptosis
[16, 17]. Mutations in oncogenes and tumor suppressor genes lead to uncontrolled cell division and inhibition of the normal apoptotic death process. MMR
genes, a class of “caretaker” genes, are responsible for repairing subtle mistakes
made in normal DNA replication or induced by exposure to mutagens. When
these genes are inactivated genetic alterations increase, meaning mutations in
other genes occur at higher rates [18-20].
There are two major ways for a tumor to acquire the multiple genetic alterations
needed for a malignant progression, either subtle alteration in single nucleotides or
large chromosomal changes resulting from chromosomal instability (CIN) [16,
21]. Nearly all solid tumors have an abnormal number of chromosomes (aneuploidy) and at gene and molecular level chromosome losses are evident as loss of
heterozygosity (LOH). Such changes in the chromosomal content can be advantageous for cancer cells, since they efficiently eliminate one allele of a tumor
suppressor gene [22, 23]. In addition there are many other genes that have been
implicated in carcinogenesis without being mutated. The proteins of these genes
have been shown to be expressed in higher or lower levels as compared to normal
cells, thereby changing the normal function of the cell [16].
Sporadic, familial and hereditary CRC
There are three major forms of CRC; sporadic, familial and hereditary (Figure 2).
The vast majority of CRCs are sporadic, meaning cancer in individuals who do not
carry any germline mutations associated with the disease and who lacks a family
history of CRC [5].
A number of the sporadic cancers show a familial aggregation, without fitting into
the mendelian inherited cancer model [24]. Individuals with one or more first
20 INTRODUCTION
degree relatives with sporadic CRC have a two to three fold increased risk of
developing CRC, indicating a mild inherited genetic predisposition to develop
adenomas and carcinomas [5]. This mild genetic predisposition is thought to
involve low-penetrance genes or gene variants [9]. A polymorphism is a variation
within a gene, often in a single nucleotide (SNP), where two or more of the variant
alleles exist at a frequency of at least 1% in a general population [25]. Every 100300 base in the human genome is a SNP and they account for about 90% of the
human genetic variation. Many SNPs occur in non-coding regions and have no
effect on cell function. A SNP in a gene usually encodes a functional protein, but
the effectiveness of the protein function may depend on the specific variation [13].
Gene polymorphisms can predispose to an increased risk of CRC and may also be
a reason for poor response to treatment [8, 9]. It is possible that environmental
factors partly determine which of the genetically predisposed individuals will
develop tumors [5, 26]. Polymorphisms in the adenomatous polyposis coli (APC)
gene are the most extensively studied polymorphisms with regard to CRC association. The APC I1307K polymorphism occurs almost exclusively in people of
Ashkenazi Jewish descent and results in a twofold increased risk of colonic adenomas and adenocarcinomas compared with the general population [27].
Figure 2. Familial adenomatous polyposis (FAP), hereditary non-polyposis colorectal
cancer (HNPCC) and other hereditary conditions causing colorectal cancer (CRC)
have a strong hereditary component with little environmental influence. There are also
mutations and gene variants that contribute to a familial CRC susceptibility, involving
interactions between genes and environmental factors. Mutations, possibly caused by
environmental factors, contribute to sporadic CRC.
INTRODUCTION 21
Other polymorphic variant alleles have been identified in multiple genetic association studies. These polymorphisms may be of great importance for the prevention,
prediction, and treatment of many common diseases, but very few polymorphisms
have been characterized enough to currently support routine use in the clinic [28].
Hereditary cancers have a clear genetic component, but they are different from
other classic genetic diseases, since patients with germline mutations are predisposed to cancer, but will not necessarily be afflicted with disease [29]. Hereditary
syndromes that predispose to CRC include familial adenomatous polyposis (FAP)
with a germline mutation in the APC gene and hereditary non-polyposis colorectal
cancer (HNPCC) with germline mutations in the MMR genes [9, 29]. These
hereditary syndromes and their genetic basis, have given great insight into the
nature of CRC development.
The adenoma-carcinoma sequence
During the evolution of normal epithelial cells to benign adenomas and malignant
carcinomas, mutational activation of oncogenes and inactivation of tumor suppressors occur in a temporal sequence, each specific for defined CRC stages [10,
29, 30]. Mutation in the tumor suppressor gene APC, observed in 70-80% of
sporadic colorectal tumors, is an early event leading to transformation from normal
mucosa to early adenoma [31]. Mutational activation of the K-RAS oncogene,
found in up to 50% of all CRCs, is usually associated with adenoma growth and
progression. K-RAS activates a signaling pathway that modulates cell growth and
survival [32]. Late adenoma progression is commonly accompanied by alterations
in the SMAD and transforming growth factor β (TGF-β) genes connected to
angiogenesis, cell proliferation and differentiation [10]. Inactivation of both alleles
of the tumor suppressor p53 marks the malignant transformation from adenoma to
carcinoma in about 45% of all CRCs [33]. The p53 protein is a transcription factor
that normally inhibits cell growth, stimulates cell death induced by cellular stress
and is important for genomic stability [10, 34]. The adenoma-carcinoma sequence
is illustrated in Figure 3.
22 INTRODUCTION
Figure 3. The adenoma-carcinoma sequence, originally reviewed by Fearon and
Vogelstein (1990).
Prognosis
The term prognosis is widely defined as the likely future outcome or course of a
disease, or an indication of recovery or recurrence. In the oncology field prognosis
often refers to survival, either overall survival or disease free survival. Overall
survival defines the chances of staying alive for individuals suffering from a disorder and disease-free survival analyzes the results of the treatment for localized
disease, such as surgery and/or adjuvant treatment. In disease-free survival the
endpoint is relapse rather than death. In general the prognosis of a cancer patient is
affected by factors such as the type of cancer, the extent to which the cancer has
spread (metastasis), or how closely the cancer resembles normal tissue (grade of
differentiation). Other factors that may also affect a patient's prognosis include
age, general health and effectiveness of treatment [35].
For CRC patients´ pathologic stage represents one of the most important prognostic factors. The Dukes´ system was the classic staging method for CRC,
however the tumor, node, metastasis (TNM) staging system is more detailed and
is most commonly used today. On occasion, Roman numerals I through IV are
used in CRC staging (Table 1). These numerals correspond with Dukes´ classes A
INTRODUCTION 23
through D [36]. The stages of cancer are based on the depth of invasion of the
bowel wall, extent of regional lymph node involvement and the presence of
metastatic disease in other tissues or organs. The 5 year overall survival of patients
with stage I CRC is 90% as opposed to patients with stage IV, who have only a 10%
5 year overall survival [37].
Besides stage there are other clinical and pathological factors that have been
associated with a worse prognosis. Histological poor differentiation of tumor cells
or tissue, lymphovascular invasion, perineural invasion, clinical obstruction of
bowel or perforation of bowel and an elevated preoperative plasma level of
carcinoembryonic antigen (CEA) are considered poor prognostic factors [38].
There are also a number of molecular features that may provide prognostic
information. The 15-20% of sporadic cancers with microsatellite instability (MSI),
which is changes in short repetitive sequences of DNA caused by a loss of MMR
function, has a better prognosis than those that have microsatellite stable tumors
[39]. In up to 70% of all CRCs there is a loss of chromosome 18q, harboring the
tumor suppressor gene deleted in colon cancer (DCC), which has been connected
to a worse prognosis [38, 40].
Table 1. Dukes´ and TNM staging for CRC
Dukes´stage
TNM
Stage
Description
A
T1-T2, N0, M0
I
The tumor penetrates into the mucosa
of the bowel wall but no further
B
T3-T4, N0, M0
II
Tumor penetrates into and through
the muscularis propria of the bowel
wall
C
any T, N1-2, M0
III
Pathologic evidence of colorectal
cancer in the regional lymph nodes
D
any T, any N, M1
IV
Cancer has metastasized to distant
parts of the body
24 INTRODUCTION
Treatment
The primary way to achieve a curative result for CRC is to remove the primary
tumor by surgery. In most cases of surgery the colonic or rectal segment, with a
certain margin, containing the tumor is removed. Lymph nodes associated with the
tumor are also removed. The removal and identification of lymph nodes containing tumor is important both for reducing risk of recurrence and for making
decisions about adjuvant therapy [41].
To reduce risk of recurrence adjuvant chemo- or radiotherapy is given after
surgery. For palliative care of patients that are not candidates for curative surgery,
focus lies on alleviating suffering and promoting quality of life and this can be
achieved by giving chemo- or radiotherapy [42, 43]. Chemotherapy includes
cytotoxic agents targeting fast dividing cells with the intent to destroy cancerous
cells outside of the surgical area. The most widely used agent for CRC treatment is
5-Fluorouracil (5-FU), which inhibits thymidylate synthase, a rate limiting enzyme
in the synthesis of nucleotides. 5-FU is usually combined with leucovorin, which
stabilizes 5-FU. 5-FU is currently often used in combination with either oxaliplatin
(FOLFOX), a platinum compound that binds to DNA, or with irinotecan
(FOLFIRI), which inhibits topoisomerase I. Both oxaliplatin and irinotecan
disturb the process of DNA replication [37, 44]. Monoclonal antibodies are also
used for treatment of CRC and seem to work well and improve survival together
with other chemotherapies [45].
In the case of rectal cancer the constraint of the pelvis limits surgical access, leading
to a lower likelihood of achieving negative resection margins while preserving the
function of the anal sphincter. Therefore the management of rectal cancer varies
somewhat from that of colon cancer, regarding surgical technique and the use of
radiotherapy [37]. An important parameter in rectal cancer surgery is the distance
from the lower tumor limit to the anal verge. A low rectal cancer, close to the anus,
is commonly removed by abdominoperineal resection or rectal amputation, which
requires permanent colostomy. For mid-range and higher rectal tumors anterior
total mesorectal excision (TME) is the standard. The use of TME has reduced
local failure rates after 5 years of follow up from 28% to 10-15%. Preoperative
radiotherapy is recommended for most patients with rectal cancer since it further
decreases the risk of local recurrence and increases survival [46, 47].
INTRODUCTION 25
Radiotherapy
Radiotherapy can be administered before or after surgery and at higher doses over
a shorter time or lower doses over a longer time course [37]. The short-course
preoperative radiotherapy, currently used for rectal cancer treatment in Sweden,
was evaluated in the Swedish Rectal Cancer Trial, where patients received a total
dose of 25Gy over 5 days. They found that preoperative radiotherapy enhanced
local control and prolonged survival compared to patients who underwent surgery
alone [48].
The goal of radiotherapy is damaging DNA in tumor cells, more specifically
causing single and double strand breaks in the DNA. Double strand breaks are
lethal for the cells and radiation can cause cell death by one of two major mechanisms: apoptosis or necrosis [49]. The relation of radiosensitivity and apoptosis has
been debated, where some investigators have reported that apoptosis is an important mechanism by which radiotherapy kills cells [50, 51], while others have
argued that apoptosis is not the predominant form of cell death after exposure to
ionizing radiation [52]. One of the principles of radiobiology is that an essential
difference exists between cell death and loss of reproductive capability for the
outcome of curative radiotherapy. Loss of colony forming ability is another key
event in radiation treated tumors. Also the cell cycle is strongly affected by radiation and radiosensitivity depends on cell cycle position and cell cycle progression
[49].
There is significant variation in the response to radiotherapy among patients even
if they have the same tumor stage. Despite rapid advances in knowledge of cellular
functions affecting radiosensitivity, there is still a lack of predictive factors for local
recurrence and normal tissue damage. Nevertheless there are molecular candidates
which have been associated with altered radiosensitivity. Several studies have
shown that apoptosis induction by agents used as cancer treatment, such as
ionizing radiation, is highly dependent on a normal p53 function [49, 53, 54]. The
anti-apoptotic proteins survivin and Cyclooxygenase-2 (Cox-2) have been
implicated in the radioresistance of rectal cancer [55-57]. Another protein found
to be significant for the outcome of radiotherapy is phosphatase of regenerating
liver (PRL), which may predict resistance to radiotherapy when overexpressed at
the invasive margin of rectal cancer [58].
26 INTRODUCTION
Cell cycle
The life of a cell is cyclic with phases of rest and activity, but also of reproduction
by dividing into two new identical daughter cells (mitosis). The cell cycle is highly
regulated by signals affecting so called checkpoints before each phase, in which the
cellular system decides whether the cell will continue through the cycle, arrest or
die. There are four different phases of the cell cycle. G1 is the gap between the last
mitosis and the beginning of DNA synthesis, where cells grow and prepare for
DNA replication. The S phase is the DNA synthesis phase, where all the DNA of
the cell is duplicated during a process called DNA replication. The G2 phase is
another gap phase, where cells can duplicate all other cell components and finally
prepare for the mitotic (M) phase, where the cell divides into two identical G1
daughter cells. Each of these daughter cells may immediately re-enter the cell cycle
or pass into a non-proliferative phase, referred to as G0 (Figure 4) [59].
Cells commonly respond to DNA damaging agents by activation of the cell cycle
checkpoints and arresting at a specific phase of the cell cycle to allow repair of
possible defects in the DNA. Ionizing radiation causes arrest in the G1, S and G2
phases of the cell cycle. The G1 checkpoint prevents replication of damaged DNA
before cells enter the S phase and the G2 checkpoint prevents the division of faulty
chromosomes during M phase [49].
Figure 4. The cell cycle.
INTRODUCTION 27
Cell death
Following DNA damage cells arrest the cell cycle to try and repair the damage,
although if the damage is too severe to repair cells are programmed to die by
apoptosis. Defects in DNA damage-induced apoptosis is one of the proposed
alterations that lead to the resistance of cancer cells to a variety of anti-cancer
agents. Increasing attention is being directed to other types of cell death, such as
necrosis and mitotic catastrophe, as factors partly deciding treatment outcome in
cancer. Understanding the regulation of apoptotic and non-apoptotic death
signaling pathways will better help us to evaluate their impact in tumor development and treatment response [60-62].
Apoptosis
Apoptosis was first described by Kerr et al in 1972. The Greek term apoptosis
means “falling off” and is based on the morphological characteristics of the dying
cells [63]. The morphology of an apoptotic cell includes cellular shrinkage,
membrane blebbing, condensed chromosomes, DNA fragmentation and finally
fragmentation of the cell into apoptotic bodies (Figure 5). During apoptosis
certain molecular signals become exposed on the surface and function as “eat me”
signals for macrophages. With the membrane bound apoptotic bodies being
destroyed without cell contents being released into the surrounding tissue no
inflammatory reaction is triggered [60].
Apoptosis is a tightly regulated cellular process and can be initiated by two
different forms of signals, extracellular ligands or intracellular stress signals. When
extracellular ligands, Fas, tumor necrosis factor α (TNFα) or TNF-related apoptosis inducing ligand (TRAIL), bind to their receptors, adaptor proteins are recruited to the intracellular domains of these receptors. Together they form the
death inducing signaling complex (DISC) which activates initiator caspases 8 and
10 [64]. Caspases are a family of cysteine proteases, normally present in the cell as
proenzymes that require proteolysis for activation of their enzymatic activity [65].
The active initiator caspases in turn cleave and thereby activate another class of
caspases, called effector caspases. Effector caspases cleave a number of vital
proteins, thereby degrading the cell [64, 65]. Intracellular stress signals such as
28 INTRODUCTION
DNA damage, oxidative stress or oncogene activation initiates permeabilization of
the outer membrane of the mitochondria, leading to the release of among others
cytochrome C [60]. This process is regulated by the bcl-2 protein family, which
contains both pro- and anti-apoptotic proteins [66]. The released cytochrome C
forms an apoptosome together with apoptosis activating factor 1 (Apaf-1) and
initiator caspase 9. The apoptosome activates effector caspases [67, 68]. Both the
extrinsic and intrinsic apoptotic pathways lead to the activation of the effector
caspases 3, 6 and 7, which are the main proteases that degrade the cells [60]. The
activity of effector caspases is controlled, partly by the inhibitor of apoptosis
proteins (IAPs), in turn controlled and inhibited by the pro-apoptotic second
mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding
protein with low pI (SMAC/DIABLO) proteins [69].
Apoptosis is one of the major barriers that must be overcome by tumor cells to
allow them to survive and proliferate in stressful conditions. One strategy for
avoiding apoptosis includes loss of wild type p53, which normally is a central
protein for induction of apoptosis after DNA damage. Another strategy is to more
or less constantly upregulate anti-apoptotic factors, such as bcl-2 or survivin [70].
It has also been proposed that failure to undergo apoptosis can result in treatment
resistance. For solid tumors, such as colorectal tumors, several anticancer agents,
such as 5-FU and radiotherapy, have been shown to trigger apoptosis in tumor
tissues [71, 72] and a high apoptotic index was related to less local recurrence in
rectal cancer after radiotherapy [71].
Other types of cell death
In contrast to apoptosis, necrosis is considered an uncontrolled form of cell death
and it is characterized by loss of membrane integrity and cellular swelling. The cells
rupture, which results in a release of cellular components into the surrounding
microenvironment of the cell and a consequent inflammatory response (Figure 5).
Necrosis is usually an effect of pathological trauma [60], and it is known that
necrosis is induced by anti-cancer drugs and radiation [73].
Mitotic catastrophe is a type of cell death that was first described in yeast, where
cells died as a result of abnormal chromosome segregation during mitosis. In
tumor cells it is mainly associated with deficient cell cycle checkpoints. Morpho-
INTRODUCTION 29
logic features of mitotic catastrophe cells are enlarged and multinucleated cells
(Figure 5). The G2/M checkpoint is responsible for blocking mitosis in case of
DNA damage and p53, which maintains a G2 arrest via p21 upon DNA damage
[74], might play a role in preventing mitotic catastrophe. In most cell types,
radiation damaged cells do not die immediately, or soon after irradiation [60, 61].
Cells that will die are generally indistinguishable from cells that will survive for one
or several cell divisions after irradiation. Some studies show that mitotic catastrophe is followed by apoptosis and it is still debated if mitotic catastrophe is a specific
cell death mode or just a trigger for later apoptosis [75].
Figure 5. Cell death after DNA damage.
30 INTRODUCTION
p53
The p53 protein has received significant attention since its discovery in 1979,
originating from its dominant role in tumor suppression and being the “guardian of
the genome” [76]. The p53 gene, TP53, is the most mutated gene in human
cancer. About 50% of all CRCs have non-functional p53 proteins due to mutations
in TP53 [34, 77]. Inactivation of p53 and proteins associated with the functions of
p53 are strongly associated with tumor development in both laboratory animals
[78, 79] and humans [80]. The p53 protein contains distinct DNA binding
domains (DBDs) for binding specific DNA sequences and transactivation (TA)
domains for transcription of genes. The p53 protein is functional as a tetramer and
contains an oligomerization domain (OD) for these protein-protein interactions
(Figure 6).
Activation of p53 following DNA damage leads to phosphorylation and stabilization of p53 by the Ataxia telangiectasia mutated (ATM) kinase [81]. The subsequent accumulation of p53 and binding to specific DNA sequences results in
transcriptional activation or silencing of target genes of p53, which encode
proteins that push cells into apoptosis via Bax, Noxa and Puma, and promote cell
cycle arrest in the G1 and G2 phase via p21 and growth arrest and DNA damage
45 (GADD45) [61]. Normally, when no DNA damage is present, p53 is kept very
low in cells. The primary regulator of p53 is mouse double minute 2 (mdm2),
which induced by p53 itself marks p53 for degradation by the 26S proteosome.
Mutant variants of p53 are often detected at constant high levels possibly due to
their inability to upregulate mdm2 [82, 83].
TP53 mutations would be expected to correlate with a more aggressive tumor
phenotype and an increased risk of death has been associated with p53 mutation in
CRC patients, mainly for those with otherwise good prognostic tumor characteristics [84]. Being a central mediator of DNA damage response, p53 can be expected
to also play a role in the sensitivity to cancer treatment. In general a loss of p53 is
associated with a more radioresistant phenotype in rectal cancer [53, 85].
INTRODUCTION 31
Figure 6. Both p53 and p73 contain a transactivation (TA), DNA binding (DBD) and
oligomerization (OD) domain. The p73 proteins contain an additional sterile alpha
motif (SAM). TP73 also contains two different promoters giving rise to TAp73 and
ΔNp73.
p73
After a 20 year search for genes related to p53, a new member of the p53 family
named p73 was described in 1997. Many similarities were found between the
family members, but also striking differences. The three most conserved domains
of the p53 family members are the TA domain, the specific DBD and the OD
(Figure 6) [86]. The highest similarity between p53 and p73 is found in the DBD
and it has been shown that p73 can bind to and activate p53 target genes, such as
p21, Bax and mdm2 [87] and thereby induce cell cycle arrest [88] and apoptosis
[89]. Less similarity is found in the OD, which may explain why p73 and wild type
p53 cannot form oligomeres with each other [90]. The p73 protein contains an
additional sterile alpha motif (SAM) domain, extending beyond the p53 core.
SAM domains are involved in protein-protein interactions and developmental
regulation [91, 92] and the p73 protein has been shown to be essential for neuronal development in mice and the survival and long-term maintenance of adult
neurons [93, 94].
32 INTRODUCTION
The p73 gene, TP73, contains two promoters, producing two different classes of
proteins. The P1 promoter, located upstream of exon 1 gives rise to a protein
named TAp73, which contains a functional TA domain, making it possible for the
protein to activate p53 targets and mimic p53 function. The P2 promoter, located
in intron 3 creates a truncated protein called ΔNp73 [95, 96] which acts as a
dominant negative inhibitor towards TAp73 and p53 [97]. The p73 proteins are
also found in multiple C-terminal splicing variants. Although several C-terminal
exons are cleaved of in the different variants, all variants retain the DBD and OD
(Figure 6).
Regulation of p73
The activity of p73 is regulated by several of the same mechanisms as p53. In
addition a number of new pathways have been described. As a response to DNAdamaging agents such as γ-radiation, cisplatin and taxol, it has been seen that ATM
induces the tyrosine kinase c-abl, which phosphorylates and activates p73 [98, 99].
Checkpoint kinases Chk1 and Chk2 can also activate p73 by stabilizing the E2F1
transcription factor. E2F1 has a direct binding site in the P1 promoter thereby
inducing TAp73 [100].
Figure 7. p53 and TAp73 are upregulated by DNA damage. p53 and TAp73 can bind
to the P2 promoter and upregulate ΔNp73. ΔNp73 in turn can bind to and downregulate p53 and Tap73, creating an autoregulatory loop. Certain p53 mutants are
capable of binding and downregulating p53 and TAp73.
INTRODUCTION 33
p73, just like p53, can activate the transcription of mdm2. Mdm2 however does
not degrade p73, instead it stabilizes p73 and negatively regulates it by competing
for binding to p300, a co-activator of p73 [101, 102].
The ΔNp73 isoform has a very important regulatory role blocking the transactivation activities of TAp73 and p53 and hence their abilities to induce cell cycle arrest
and apoptosis [97]. The ΔNp73 promoter, P2, also contains a very efficient
TAp73/p53 responsive element, which means that TAp73 and p53 can upregulate
ΔNp73, creating a dominant negative feedback loop [103, 104]. Some tumorderived mutants of p53 have an ability to bind and interact with TAp73. This
interaction occurs through the core DBD rather than the OD. This inactivation of
p73 by p53 mutants may confer a selective advantage in promoting tumorigenesis
(Figure 7) [105].
p73 in tumors
The p73 gene was mapped to chromosome 1p36, which was found to frequently
undergo LOH in among others neuroblastoma and breast cancer [86]. This led to
the beliefs that p73 was a tumor suppressor like p53, meaning that it by definition
is targeted to undergo loss of expression or function during tumorigenesis [106].
However, the fact that mice lacking p73 do not develop spontaneous tumors at all
[96] and that a number of studies have genotyped TP73 in altogether 1500 tumors
without finding any mutations significant for p73 function [107, 108] have
excluded p73 as a classic tumor suppressor [106]. In fact most studies, involving a
number of tumor types, show that p73 is overexpressed in tumors as compared to
normal tissues. The p73 protein has been found to be correlated to overexpression
in tumors from breast [106], lung [109], esophagus [110], stomach [111], colon
and rectum [112, 113]. Importantly p73 overexpression has been correlated with
parameters relevant for prognosis, where hepatocellular carcinoma patients with
p73 positive tumors had lower mean survival time [114] and p73 in breast cancers
was associated with lymph node metastasis and a higher pathologic stage [115]. A
study done at our lab showed that p73 independently predicted poor prognosis in
patients with colorectal cancer [116].
There is an emerging sense that the ΔNp73 isoform, rather than TAp73, might be
the relevant component in tumor-associated overexpression of p73. A correlation
34 INTRODUCTION
between the TAp73/ΔNp73 balance and survival has been shown in several tumor
types [117, 118]. This balance between isoforms may have escaped notice since
many early studies determined total p73 levels rather than those of specific
isoforms, due to lack of antibodies that distinguish between the different isoforms
[119, 120].
A G4C14-to-A4T14 polymorphism in p73
Two SNPs have been found in the p73 gene. A G-or-A variant found at position 4
and a C-or-T variant at position 14 in the untranslated region (UTR) of exon 2
(Figure 8). These two SNPs are in complete linkage disequilibrium, meaning that
only two alleles exist, namely a GC and an AT allele. This polymorphism lies just
upstreams of the initiating AUG of exon 2, in a region that might theoretically form
a stem-loop structure. It was suggested that this could potentially affect gene
expression, perhaps by altering the efficiency of translation [86].
This polymorphism has been studied in several types of cancer and it has been
shown that the AT allele decreases the risk of esophageal cancer in an Irish
population [121] and lung cancer in a Chinese population [122], while it increases
risk of lung cancer in a young American Caucasian population [123] and cervical
cancer in a Japanese population [124].
Figure 8. The G4C14-to-A4T14 polymorphism is found in exon 2 just upstream of the
initiating codon.
INTRODUCTION 35
p73 and radiosensitivity
Accumulating evidence shows that p73 plays a significant role in curative anticancer therapy. Similar to p53, p73 mediates cellular responses to cancer therapies,
including radiotherapy [125]. In cervical cancer it was seen that p73 increased
after irradiation and that it increased the transcription of p53 responsive genes
such as p21 and Bax, leading to cell cycle arrest and apoptosis [126]. It was also
shown that p73 overexpression was associated with cellular radiosensitivity after
radiotherapy and that ΔNp73 was significantly associated with radioresistant
cervical carcinomas, while an increase of TAp73 was observed in cervical cancer
cases sensitive to radiation [127, 128]. Given the possible role of TAp73 and
ΔNp73 in cancer and cancer treatment it is of great interest to determine how they
are regulated and what factors affect their relative expression levels.
Survivin
Survivin belongs to the anti-apoptotic IAP family and it plays a key role in a
simultaneous regulation of apoptosis and cell division [129]. Survivin is highly
expressed in embryonic tissues and in tumors, but is almost absent in normal adult
tissue [130]. An exception is normal tissues and cells with high mitotic activity
[131]. Survivin is mainly expressed in the G2/M phase of the cell cycle during cell
division, where it assures proper segregation of chromosomes during the division
of fast dividing cells [132]. The dominant functions of survivin during development and tumorigenesis however seem to be largely cell cycle independent [133].
Survivin is simultaneously involved in apoptosis inhibition by interfering with
initiator caspase 9 [134] and possibly also effector caspases 3 and 7 [132]. Survivin
has also been seen to directly interact with SMAC/DIABLO, which promotes
apoptosis by eliminating the anti-apoptotic effect of survivin and other IAPs [112].
Survivin is highly expressed in tumors, but not in normal tissue, which makes it a
potential biomarker for cancer. Overexpression of survivin in colorectal tumors has
been connected to a shortened disease-free or overall survival [56, 135]. Radioresistance in cancer cells overexpressing survivin has been observed in tumor cells
from cervix [136], pancreas [137], colon and rectum [57]. Survivin has therefore
36 INTRODUCTION
been suggested as a suitable target for radiosensitization. In fact knockdown of
survivin has been shown to decrease cell viability and reduce proliferation after
irradiation [57].
One signature feature of survivin is the high number of different molecules and
transcriptional networks that are directly or indirectly involved in the functions of
survivin [138]. One of the protein networks connected to survivin is the tumor
suppressor p53. The wild type p53 protein has been seen to transcriptionally
repress survivin [133]. Survivin seems to be upregulated in cells where p53
function has been lost by mutation [139, 140]. It has been shown that survivin
regulates expression and degradation of p53 through a caspase 3/mdm2 axis in a
human breast cancer cell line. The same study showed that ectopic survivin
increases the mRNA levels of the TAp73 and ΔNp73 isoforms [141].
PRL-3
Interest in PRL-3 and its role in cancer and metastasis was generated by the finding
that PRL-3 was upregulated in metastatic CRCs, while it was lower in primary
colorectal tumors and nearly undetectable in normal colonic tissue [142, 143].
The PRL-3 protein is, together with the other PRL family members, a tyrosine
phosphatase located in the cytoplasmic membrane. Protein tyrosine phosphatases
are involved in regulating diverse proteins involved in essentially all cellular
physiological and pathological processes [144]. In experimental models, PRL-3
promoted migration, invasion and metastasis of tumor cells [144-146].
In terms of patient outcome, high PRL-3 expression in primary CRC was significantly associated with liver metastasis of CRC and shorter survival time [147]. The
same trend has been seen in several other cancer types, such as breast, gastric and
liver cancer. PRL-3 has been indicated as a promising biomarker, especially for
metastatic progression, with predictive significance [148]. Overexpression of the
PRL proteins at the invasive margin of rectal tumors predicted a resistance to
radiotherapy [58]. A study on lung cancer cell line showed that both TAp73 and
p53 have the ability to up-regulate PRL-3 [149]. Still there are big question marks
regarding the signaling pathways that involve PRL-3 in both normal and abnormal
contexts and it is of interest to further investigate PRL-3 as a biomarker and
potential anti-cancer target.
INTRODUCTION 37
Cox-2
Cox-2 is an enzyme that normally converts arachidonic acids from the lipid
membrane of the cell into prostaglandin H2 (PGH2) [150]. Cox-2 has been
implicated in tumorigenesis and is overexpressed in among others colorectal
cancer [151, 152]. Knock out of Cox-2 in mice with a hereditary polyposis condition, resembling the human FAP, significantly reduced the number of colonic
polyps [153]. One of the most well defined molecular targets of non-steroidal antiinflammatory drugs (NSAIDs) is Cox-2 and epidemiologic studies have shown a
40-50% reduction in CRC incidence among users of NSAIDs [154]. It has been
shown that Cox-2 may mediate tumor development and progression by inhibiting
apoptosis and enhancing invasiveness of the tumor [155, 156]. Inhibitors of Cox-2
has been shown to have anti-tumor effects and several studies have looked at
inhibition of Cox-2 as enhancers to radiotherapy treatment. Treatment of mice
with selective Cox-2 inhibitors has shown to significantly enhance the radiation
response in a number of tumor types [157, 158].
38 INTRODUCTION
AIMS
The general aim of this study was to investigate the role of p73 and related proteins
in the development and treatment of CRC.
Specific aims
•
Investigate a G4C14-to-A4T14 polymorphism of the p73 gene in relation to
risk of CRC, LOH and clinicopathological factors.
•
Study the effect of a G4C14-to-A4T14 polymorphism of the p73 gene on
survival of rectal cancer patients treated with surgery alone or surgery together
with preoperative radiotherapy.
•
Relate the expression of p73 with preoperative radiotherapy and clinicopathological factors in rectal cancer patients.
•
Show effects of γ-radiation on cell cycle arrest, clonogenic abilities, apoptosis
and protein expression profiles of mutant p53, TAp73, ΔNp73, survivin and
PRL-3 after γ-radiation in colon cancer cells in vitro.
•
Examine a possible connection between survivin and p73 isoforms TAp73 and
ΔNp73 in colon cancer cell lines, before and after γ-radiation treatment, by
forced overexpression or knockdown of survivin.
AIMS 39
40 AIMS
MATERIALS AND METHODS
Patients (Papers I-III)
In Papers I-III in this thesis information about patient data such as gender, age,
tumor location and stage was obtained from surgical and pathological records.
Survival data was obtained from the Cause of Death registry, provided by the
Swedish National Board of Health and Welfare (Socialstyrelsen). The data on
apoptosis determined by Terminal deoxynucleotidyl Transferase Biotin-dUTP
Nick End Labeling (TUNEL), as well as expression of p53, survivin and Cox-2
were taken from previous studies at our laboratory [53, 55, 56, 71].
Paper I included DNA extracted from fresh frozen tissue of 179 patients who
underwent surgical resection for primary colorectal adenocarcinoma at Linköping
University Hospital between 1975 and 1986. The mean age was 71 years old
(range 35-95) in this randomly selected CRC patient group. The patients were
followed up until the end of 2003, by which time 54 patients had died from CRC.
Paper I also included 260 blood samples from randomly selected healthy individuals recruited in the same geographical region as the CRC patients. The control
individuals had neither gastrointestinal disease nor a history of tumors and the
mean age was 59 years (range 22-77). In Paper I formalin-fixed paraffin-embedded
tissue sections from 69 of the CRC patients were also included for immunohistochemistry (IHC) staining.
Papers II and III included formalin-fixed paraffin-embedded tissue sections from
rectal cancer patients that participated in a randomized clinical trial of preoperative
radiotherapy between 1987 and 1990 [48]. The patients were randomized to
receive either surgical treatment alone or preoperative radiotherapy and surgical
treatment. In Paper II DNA was extracted from surgical specimens of 138 patients,
where 73 patients were treated with surgery alone and 65 patients received
preoperative radiotherapy before surgery. The mean follow-up time was 86
MATERIALS AND METHODS 41
months (range 0-193) and the mean age of the patients was 66 years (range
38-85). In Paper III, IHC staining was performed on tissue sections from 131
patients with primary rectal tumors, of which 89 had adjacent histologically normal
tissue on the same tissue section as the primary tumor. Seventy four patients
received surgery alone and 57 received preoperative radiotherapy before surgery.
The mean follow-up time was 85 months and the mean age of the patients was 67
years (range 36-85). The study also included samples from 102 pretreatment
biopsies, 82 distant normal mucosa samples and 32 lymph node metastasis
samples. The endoscopic biopsies were taken by surgeons for clinical diagnosis
and distant normal samples were taken from the distant margin of the resection
and were histologically free from pre-tumor and tumor. Patient data is reviewed in
Table 2.
The patients included in this thesis have given consent for the material to be used
in scientific research. The use of the material has been approved by the local
Human Research Ethical Committees.
42 MATERIALS AND METHODS
Table 2. Clinicopathological characteristics of patients from Papers I-III
Characteristics
Papers
I
II
III
Patient Number
179
138
131
Diagnosis (year)
1975-1986
1987-1990
1987-1990
2003
2004
2004
Gender (Male/Female)
96/83
80/58
76/55
Age (year)
35-95
38-85
36-85
Right colon
77
-
-
Left colon
32
-
-
Rectum
67
138
131
6
9
8
117
94
85
51
35
34
-
-
4
I
14
37
33
II
72
41
41
III
56
50
45
IV
25
10
12
Followed up until
Tumor site
Differentiation
Well
Moderately
Poorly
Unknown
Tumor stage
MATERIALS AND METHODS 43
Cell lines
KM12C, KM12SM and KM12L4a (Paper IV)
The KM12 cell lines were originally established in the laboratory of Professor I J
Fidler (M.D. Anderson Cancer Center, Houston, TX) and kindly given to us. The
KM12 cells were created when human primary colon cancer cells from a patient
with Dukes´ B stage were either directly established in primary culture (KM12C)
or injected into the cecum and spleen of nude mice (KM12SM and KM12L4a)
[159]. The KM12SM cell is a spontaneous liver metastasis derived from implantation of KM12C in nude mice. The KM12L is an experimental liver metastasis,
where a KM12C-derived liver metastasis cell line was grown in culture before
injected into the spleen of nude mice and harvested from the liver. This procedure
was repeated until a fourth generation of metastatic cell line, KM12L4a, was
generated [159]. A karyotyping of the cell lines, showed that KM12C was near
diploid, KM12SM near tetraploid and KM12L4a near diploid, with a minor
tetraploid subpopulation [160]. The KM12 cells have a His to Arg mutation in
codon 179, found in the DBD, of p53, leading to a non-functional p53 protein
[161, 162].
The KM12 cells were cultured in Eagle’s Minimal Essential Medium (MEM) with
Earle´s salts, L-glutamine and non-essential amino acids (Sigma-Aldrich, Stockholm, Sweden), supplemented with 1.5% NaHCO3, 1 mM Na-Pyruvate (GIBCO
Invitrogen, Carlsbad, CA), 1X MEM vitamin solution (GIBCO Invitrogen), 1%
Penicillin-Streptomycin (PEST) (GIBCO Invitrogen) and 10% fetal bovine serum
(FBS) (GIBCO Invitrogen). The cells were grown as a monolayer in tissue culture
flasks at 37°C with 5% CO2 and passaged every few days to maintain exponential
growth.
HCT-116 (Paper V)
The two human colon cancer cell lines HCT-116, with wild type p53 (HCT116p53+/+) and mutated p53 (HCT-116p53-/-), were a kind gift from Dr. B Vogelstein
(Johns Hopkins University, Baltimore, MD). The HCT-116, derived from colon
cancer epithelial cells, is near diploid and MMR-deficient [163, 164]. The two wild
type p53 alleles in HCT-116p53-/- have been targeted by homologous recombina-
44 MATERIALS AND METHODS
tion, resulting in a mutated p53 with a 40 amino acid truncation [74, 120]. The
HCT-116p53-/- cells do not express detectable wild type p53 [74].
The HCT-116 cells were cultured in McCoy´s 5A medium (Sigma-Aldrich)
supplemented with 10% FBS (GIBCO Invitrogen), 1% PEST (GIBCO Invitrogen) and 1.5 mM L-glutamine (GIBCO Invitrogen). The cells were grown as
monolayer in tissue culture flasks at 37°C with 5% CO2 and passaged every few
days to maintain exponential growth.
Irradiation of colon cancer cell lines
In Papers IV-V cell cultures were irradiated with photons from a 4MV or 6MV
linear accelerator Varian Clinac 600C or Varian Clinac 600C/D (Varian Medical
Systems, Palo Alto, CA). The field size was 40x40 cm and the distance between the
radiation source and cells was 85 cm. The cells were placed on a 10 cm thick acrylic
glass plate and a 3 cm thick acrylic glass plate was placed on top of the cells (Figure
9).
Figure 9. The set-up for irradiation of cell lines.
MATERIALS AND METHODS 45
For Paper IV, KM12C, KM12SM and KM12L4a were seeded at a density of
20 000 cells/cm2, 24 h before irradiation. The cells were irradiated with single
doses of 0Gy, 10Gy or 15Gy at room temperature. The dose was chosen based on
pilot studies, where the criterion was achieving adequate apoptosis induction,
whilst remaining within biologically interesting doses. For Paper V HCT-116 were
seeded at a density of 12 000 cells/cm2, 48 h prior to irradiation. The HCT-116
cells, which had been previously treated with either mock small interfering
(si)RNA or survivin siRNA, were irradiated with single doses of either 0Gy or
4Gy. The goal of the irradiation was to induce DNA-damage and apoptosis
pathway activation and achieve an induction of survivin, p53 and p73 pathways.
During the treatment cells were cultured in medium as described earlier and the
unirradiated controls were simultaneously placed in room temperature to obtain
comparable conditions.
Cell transfection
Cell transfection is a method of introducing foreign DNA or RNA into mammalian
cells that normally take up and express externally applied DNA with very low
efficiency. By transfecting DNA together with an inducible promoter to produce
protein of interest or by knocking down specific genes, thereby abolishing protein
expression, it is possible to accurately define the role of genes and the protein they
encode in various cellular processes.
cDNA
In this study the survivin mRNA and protein was overexpressed by transfection of
HCT-116 colon cancer cells with a pCMV6-XL4 vector containing the full length
survivin complementary (c)DNA insert (Origene, Rockville, MD). Expression in
the transfected eukaryotic cell is driven by the human cytomegalovirus (CMV)
promoter, incorporated in the vector, which promotes constitutive expression of
the survivin insert. An ampicillin resistance gene allows the selection of the plasmid
in competent Escherichia coli. A significant increase in survivin mRNA and protein
was seen 48 h and 72 h after transfection. An pCMV6-XL4 vector lacking a cDNA
insert was used as a negative control.
46 MATERIALS AND METHODS
siRNA
RNA interference (RNAi) is a normal system in euakryotic cells, which uses
siRNA to control gene activity. The discovery of this system gave Andrew Fire and
Craig Mello the Nobel prize in 2006 [165]. Double stranded (ds)RNA is recognized and cut by the endonuclease Dicer into a small single stranded siRNA, about
21 base pairs long, which is incorporated into a RNA-induced silencing complex
(RISC). RISC is then guided by the siRNA to a complementary mRNA, which is
then cleaved and degraded, preventing the production of the corresponding
protein (Figure 10). Today this system is used as a powerful tool to investigate the
function of specific genes in cells and the exogenous siRNA pathway is parallel to
the endogenous pathway [166]. In this study a HP Validated siRNA (Qiagen,
Minneapolis, MN) targeting all mRNA transcripts of survivin was used. An
AllStars Negative Control (Qiagen), which is siRNA that has no homology to any
known mammalian gene, was used as a negative control. A significant decrease in
mRNA and protein level was seen 28 h and 48 h after transfection with siRNA.
Specific silencing was confirmed by measuring mRNA by real-time PCR (RTPCR) and protein by Western blot.
Figure 10. The principle of gene silencing by siRNA.
MATERIALS AND METHODS 47
Polymorphism genotyping
DNA extraction
For Paper I genomic DNA was extracted from 20 mg normal colorectal mucosa
from the distant resection margin, colorectal tumor tissue or normal peripheral
leukocytes by the means of Wizard® SV Genomic DNA Purification System
(Promega, Madison, WI). The extraction was performed according to the manufacturer´s instructions. The concentration and purity of DNA was measured with a
spectrophotometer.
For Paper II genomic DNA was extracted from 50 µm paraffin-embedded tissue
sections from rectal cancer patients, from normal lymphnodes, normal mucosa
from the distant margin of distant resection, and tumor samples. The extraction
was performed with Gentra Puregene Tissue Kit (Qiagen) according to the
manufacturer´s instructions. The concentration and purity of DNA was measured
spectophotometrically.
Polymerase chain reaction
Kary Mullis was awarded the Nobel Prize in Chemistry in 1993 for inventing
polymerase chain reaction (PCR) in the 1980s. The name comes from the DNA
polymerase used to amplify a desired part of the genome. Two primers, matching
opposite strands of DNA on either side of the region of interest, are used to
amplify the intervening segment of DNA by more than a million fold.
The PCR reaction usually starts with an initiation step where the DNA polymerase
is activated at high temperatures around 94-96°C. The first part of the 30-40
repeated cycles is a denaturing of the DNA at about 94°C where the two DNA
strands or the DNA strand and primer are separated. During the second part of the
cycle the temperature is lowered to 50-65°C to allow the primers to anneal to the
now single stranded DNA templates. Finally the DNA polymerase adds nucleotides to elongate the DNA strand from the primer site, creating a strand that is
complementary to the template strand. The temperature is set to be optimal for
the polymerase. When the given numbers of cycles are completed there is a final
elongation step where any remaining single-stranded DNA can be fully extended.
The PCR product can be separated by size through electrophoresis on an agarose
48 MATERIALS AND METHODS
gel, where shorter DNA strands move faster than longer DNA strands. By adding
ethidiumbromide, which binds to DNA, the separated DNA bands can be visualized under ultraviolet (UV) light. In Papers I-II a PCR was used to detect a
G4C14-to-A4T14 polymorphism in the p73 gene, TP73.
Important variables that can influence the outcome of PCR include the MgCl2
concentration and the cycling temperatures. Additives that promote polymerase
stability and specific primer can greatly improve sensitivity, specificity, and yield.
The advantage of PCR is that very little DNA is required to initiate the process and
relatively large amounts of pure and specific DNA sequences are generated [167].
Restriction fragment length polymorphism
Restriction enzymes, also known as restriction endonucleases, recognize specific
nucleotide sequences and cut the double stranded DNA at that specific site. A
restriction fragment length polymorphism (RFLP) is a genetic variation where one
of the alleles, but not the other, contains a restriction site for an endonuclease. The
technique is used to evaluate polymorphisms and mutations in a PCR derived
DNA sample. As a result of restriction enzyme cleavage a PCR product may either
retain its original size or be cleaved into smaller sized DNA strands. The DNA
strands of different sizes can be detected by electrophoretic separation on an
ethidium bromide agarose gel.
Figure 11. Sty I recognizes a nucleotide sequence which is present in the AT variant,
but not in the GC variant, of the G4C14-to-A4T14 polymorphism of p73.
MATERIALS AND METHODS 49
In Paper I a restriction enzyme named StyI, which recognizes and cleaves the AT
allele of the G4C14-to-A4T14 polymorphism in TP73 was used (Figure 11). In
Paper II allele specific primers were used instead of restriction enzyme cleaving,
since it eliminates the risk of false negative results due to inactive enzyme during
cleaving. To reduce the number of false negative results when using Sty I, a
heterozygotic sample was added to each enzyme cleaving run.
Loss of heterozygosity
By using regular PCR-RFLP a possible loss of an allele (LOH) can be found by
comparing the genotype of a certain polymorphism in a DNA sample derived from
normal cells with the corresponding DNA sample from tumor cells. A seemingly
homozygotic genotype in a tumor from a patient with a heterozygotic genotype of
the same polymorphism in normal cells can be considered as having LOH. In the
case of the G4C14-to-A4T14 polymorphism a patient with a GC/AT genotype in
a normal DNA sample would show only a GC allele or an AT allele in the corresponding tumor DNA sample.
50 MATERIALS AND METHODS
Immunological protein detection
Immunohistochemistry
With IHC it is possible to localize a protein in tissue sections by the use of labeled
antibodies. The discovery of IHC is credited to Coons and Jones who established
an immunofluorescent technique for the detection of bacteria during the 1940s
and 1950s. In the late 1970s IHC became a standard tool in diagnostic pathology
[168].
IHC typically begins with antigen retrieval, by pressure cooking, microwaving, or
heating tissue sections in baths of appropriate buffers, with the goal of unmasking
antigens hidden by formalin cross-links. The protein is detected by a specific
primary antibody, typically produced by immunizing an animal with the protein of
interest. Species-specific secondary antibodies bind to each primary antibody. The
secondary antibody is typically linked to biotin or horse radish peroxidase (HRP),
which converts a substrate from colorless to a colored end product, allowing
visualization of the primary antibody location (Figure 12). Several secondary
antibodies bind to each primary antibody thereby enhancing the signal. A counterstaining with hematoxylin helps visualizing the histological structures of the
slide. In Papers I and III a goat ABC Staining System (Santa Cruz biotechnology,
Santa Cruz, CA) with a biotinylated secondary antibody was used in combination
with an enzyme reagent with avidin and biotinylated HRP. A peroxidase substrate
with 3,3-diaminobenzidine (DAB) and peroxidase substrate was added for color
development.
To lessen the subjectivity when judging expression levels of the protein, the
stained slides were graded by two independent investigators in a blinded fashion.
Problems that may arise with IHC is non-specificity in staining, both immunologic
(similar antigen) and non-immunologic (binding of endogenous substrates to
visualization enzymes). In this case specificity of the primary antibody was tested
by Western blot, which shows a size specific band of the antigen detected by the
antibody. Non-immunologic false positive staining was circumvented by blocking
of endogenous peroxidases with H2O2 in methanol and by staining a number of
slides with a negative control antibody, directed against a protein not found in
humans, instead of the antibody specific for the protein of interest.
MATERIALS AND METHODS 51
Figure 12. Immunological protein detection. A primary antibody binds to the protein
of interest. Enzyme-linked secondary antibodies then bind to the primary antibody.
The enzyme substrate is activated by the enzyme creating a detectable colored or
fluorescent product.
Western blot
Western blot is an immunological method to determine the presence and/or
relative expression of a given protein in a cell lysate. The proteins of a cell lysate are
separated by size through Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). After separation the proteins are transferred to a Polyvinylidene Fluoride (PVDF) or nitrocellulose membrane, by adding an electric current
at a 90 degree angle to the gel, causing the proteins to migrate out of the gel onto
the protein attracting membrane. Towbin et al. were the first group to describe the
use of electrophoresis to transfer proteins from a gel to a membrane [169].
52 MATERIALS AND METHODS
The detection of protein on the membrane, performed much as in IHC, is based
on the use of a primary antibody against the protein and an enzyme-linked secondary antibody amplifying the signal from the primary antibody. In Papers I, IV-V a
HRP-linked secondary antibody was used together with an enhanced chemiluminiscence (ECL) system, producing luminescence in proportion to the amount of
protein (Figure 12). The image of the antibodies bound to the blot was either
imaged on photographic film (Papers I and IV) or by a digital charge-coupled
device (CCD) camera (Paper V).
Protein size approximations are made by comparing stained bands on the membrane to a size marker loaded during electrophoresis. To assure that all samples are
comparable an equal amount of total protein (20 or 30 µg) was loaded onto the
electrophoresis gel. The membrane was also re-probed with a so called housekeeping gene, β-actin, which is expressed in cells at constant level regardless of treatment. A relative quantitative measurement of the protein expression in each
sample was achieved by comparing the intensity of the detected protein band with
the intensity of the corresponding β-actin band.
MATERIALS AND METHODS 53
Cell cycle analysis
Tumor cells are often aneuploid, which means that they have lost big parts of or
gained extra chromosomes compared to healthy diploid cells. The nuclear DNA
content of a cell can be quantitatively measured by flow cytometry, which measures fluorescence from one single cell at a time, analyzing both ploidy distributions
(diploidy or aneuploidy) and in which cell cycle phase the cells are in. A fluorescent dye, such as propidium iodide, is added to a suspension of permeabilized
single cell suspension. The measurement is based on the principle that the stained
material has incorporated an amount of dye proportional to the amount of DNA.
The stained material is then measured in the flow cytometer and the emitted
fluorescent signal yields a signal which is proportional to the total fluorescence
emission from the cell. Data obtained is not a direct measure of cellular DNA
content and are therefore compared to reference cells with known amounts of
DNA. In Paper IV a modified Vindelöv protocol [170] was used and red blood
cells from chicken and trout were used as reference.
Survival fraction analysis
When cells are seeded as single cells and left to grow they eventually form a colony
of cells, emerged from that single cell. A colony is defined as a number of cells, or
as in the case of the KM12 cells where individual cell nuclei cannot be counted, as
a certain size of the whole colony. When cells are treated with a DNA damaging
agent, such as γ-radiation, cells stop proliferating to a certain extent, depending on
the sensitivity of the cells to the treatment. By comparing the number of colonies
in a treated culture to the number of colonies in an untreated cell culture a survival
fraction can be calculated and used as a measurement for cell viability with and
without a certain anti-cancer treatment.
54 MATERIALS AND METHODS
Apoptosis detection
The apoptotic process can be visualized both at the molecular level and at the
morphological level. One of each method is often used complementary to each
other to ensure measurement of late and early, molecular and morphological
apoptosis.
M30
Cytokeratin 18 (CK18) is an epithelial cell specific marker and in early apoptosis
caspases cleave CK18 revealing a neo-epitope, called M30. The M30 epitope was
characterized by Leers et al (1999). They also characterized a M30 monoclonal
antibody which detected this epitope [171]. M30-Apoptosense™ (Peviva, Stockholm, Sweden) is an enzyme-linked immunosorbent assay (ELISA) based on the
M30 antibody, representing a specific quantitative test for apoptosis in epithelial
cells. In Papers IV-V apoptosis was measured in cell lysates by means of the M30Apoptosense™ according to the manufacturers´ instructions. Apoptotic cells
detach from the surface and true apoptosis levels are difficult to detect, however
with M30-Apoptosense™ CK18 is detected in both cell lysate and growth medium.
DAPI
A basic method for detection of apoptosis is studying cell morphology under a
microscope. The DNA-specific dye diamidino-2-phenylindole (DAPI) displays
the cell nuclei in a blue fluorescence visible by UV light (Figure 13). DAPI can pass
through living cell membranes, but apoptosis increases cell membrane permeability and uptake of DAPI, leaving a stronger blue stain. In addition, the nuclear
morphology of a normal cell is round, clear-edged, uniformly stained, while an
apoptotic cell show irregular edges around
the nucleus, chromosome condensation,
heavier coloring and an increased number
of nuclear body fragments. In Paper IV
apoptotic cells were counted in an area of
about 100 cells by two independent
Figure 13. DAPI stained colon
investigators.
cancer cells.
MATERIALS AND METHODS 55
Real time polymerase chain reaction
RT-PCR is based on conventional PCR, where DNA is copied and amplified. The
goal is to precisely distinguish and measure the amount of specific nucleic acid
sequences, mRNA for example, in a sample, by measuring how quickly an amplification-derived fluorescent signal reaches a threshold. Reaching the threshold early
is an indicator of a higher number of target sequences at the start of amplification.
A limitation of the PCR is that DNA polymerases can only use DNA as a template.
Therefore the mRNA has to be converted into cDNA using the enzyme reverse
transcriptase in reverse transcription PCR. In Paper V a High Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, Foster City, CA) was used
according to manufacturer’s instruction.
The real time monitoring of the amplification is accomplished by using fluorescent
probes and instrumentation for reading the fluorescence. These probes are
sequence-specific oligonucleotides labeled with two fluorescent molecules, one so
called reporter and one quencher. When the reporter and quencher sit close
together on the probe, the quencher absorbs the fluorescent signal from the
reporter. During the amplification process the oligonucleotide is broken apart by
the DNA polymerase, separating the reporter and quencher, liberating the fluorescent signal of the reporter (Figure 14).
In paper V TaqMan® Gene Expression Assays were used, containing a ready-touse mix of specific primers and probes, with a FAM reporter and minor groove
binder (MGB) quencher, which is non-fluorescent. The instrumentation used for
detection in this study was the 7900HT Fast Real-Time PCR System (Applied
Biosystems).
Complementary software was used to determine the cycle time at which fluorescence reached a threshold level (CT). For accuracy and precision, quantitative data
must be collected at a point where samples are in an exponential amplification
phase, where amplification is reproducible. In a 100% efficient PCR the amplified
product doubles every cycle, meaning that a difference of 1 CT between two
samples corresponds to a double amount of target sequence in the sample with
higher CT.
56 MATERIALS AND METHODS
In this study a standard curve was constructed from a mRNA sample of known
concentration and used for extrapolating absolute quantitative information for
mRNA targets of unknown concentrations. To be able to compare samples
between runs the housekeeping gene β-actin was run for each sample with a
concurrent standard curve.
Figure 14. The principle of mRNA quantification with a fluorescent probe by means of
RT-PCR. R:Reporter, Q: Quencher
MATERIALS AND METHODS 57
Statistical analysis
The χ2 method was used to compare frequencies of genotypes and alleles in CRC
patients with the control population (Paper I) and to evaluate the relation of the
genotypes with clinicopathological and biological variables (Papers I-II). The χ2
method was also used to to test difference in expression of p73 between normal
mucosa tissues, primary tumor and lymph node metastasis and relation to clinicopathologic and biologic factors, both in the overall rectal cancer patient group and
in the subgroups (Paper III).
McNemar’s method was used for testing differences of the genotype/allele
frequency in normal colorectal mucosa and tumor (Paper I). McNemar´s method
was also used to test difference in expression of p73 between normal mucosa
tissues, primary tumor and lymph node metastasis and relation to clinicopathologic and biologic factors, both in the overall rectal cancer patient group and in the
subgroups.
Logistic regression was used for calculating odds ratio (OR), 95% confidence
intervals (CI) and trend test (Paper I).
Relation between the p73 polymorphism/p73 expression and survival of patients
was tested using Cox´s Proportional Hazard Model and survival curves were
calculated according to the Kaplan-Meier method (Papers I-III).
Because of the low number of patients with the AT/AT genotype, the AT/AT
genotype was grouped together with the GC/AT genotype in the statistical
analysis (Papers I-II).
In the in vitro studies (Papers IV-V) all experiments were performed in at least
duplicates and repeated three times and results are given as means ± standard error
of mean. Statistical significance between subgroups was evaluated with Student´s
t-test (Paper V).
In all Papers p-values cited were two-sided and p-values below 0.05 were considered significant.
58 MATERIALS AND METHODS
RESULTS
Paper I
In this study we investigated, in a case-control study, if a p73 G4C14-to-A4T14
polymorphism was related to risk of developing CRC. The polymorphism was also
studied in relation to protein expression and clinicopathological variables in CRC
patients. In addition, we compared normal DNA heterozygotic for the p73
polymorphism with tumor DNA from the same patient to detect LOH connected
to the p73 gene.
The frequencies of the three genotypes, GC/GC, GC/AT and AT/AT were
significantly different between CRC patients and healthy controls (p = 0.02). The
patients had a higher frequency of AT/AT (7% versus 2%), but a lower frequency
of GC/AT (30% versus 37%) (Paper I, Table I).
The protein expression of p73, determined by IHC, was higher in primary tumor
than in normal mucosa (p = 0.02) (Paper I, Table IV). When comparing polymorphic genotypes with p73 staining intensity in normal mucosa and primary tumor,
tumors with the AT/AT genotype tended to be less stained for p73. No such trend
was seen in normal mucosa (Paper I, Table III).
A significant correlation was found in the overall patient group between genotypes
and survival, where the patients with the GC/GC phenotype had a worse prognosis than those carrying the AT allele (p = 0.01) (Paper I, Fig.1.). When dividing the
patients into those with colon or rectal cancer it was shown that colon cancer
patients with a GC/GC genotype seemed to have a worse prognosis, while rectal
cancer patients with a GC/GC genotype seemed to have a better prognosis.
DNA from the matched normal and tumor tissues were available for 50 of the
heterozygotic cases and all showed a heterozygotic genotype in both normal and
tumor DNA, indicating no LOH.
RESULTS 59
In summary, the AT/AT genotype increased the risk of developing CRC. Colon
cancer patients carrying the AT allele had a better prognosis, whilst rectal cancer
patients with the AT allele had worse prognosis. The p73 protein was more
strongly expressed in tumor compared to normal tissue.
Paper II
In this study we further investigated if the G4C14-to-A4T14 polymorphism of p73
was related to survival of rectal cancer patients who did or did not receive preoperative radiotherapy in combination with surgery. In addition we studied a possible
relationship of the polymorphism with clinicopathological variables and expression of other proteins within this patient group.
In the overall group of patients there was no relationship between the G4C14-toA4T14 polymorphism and disease free survival, although in the radiotherapy
group patients with the GC/GC genotype tended to survive longer (Paper II,
Fig. 2.). In the radiotherapy group the patients carrying the AT allele more
frequently had positive p53 staining by IHC (indicating mutated p53) than those
with the GC/GC genotype (p = 0.01). The same phenomenon was observed with
survivin, where patients with the AT allele more often had strong survivin expression as opposed to patients with the GC/GC genotype (p = 0.03). Neither of these
relationships were observed in the non-radiotherapy group (Paper II, Table 2) and
no other clinicopathological variables were related to the genotypes.
Considering the relationship between p73 genotypes, p53 and survivin, a comparison was made between patients with GC/GC, negative p53 and weak survivin
expression and the rest. Patients in the radiotherapy group with the GC/GC,
negative p53 and weak survivin combination had significantly longer disease free
survival (p = 0.03), even after adjusting for TNM stage and tumor differentiation
(p = 0.01). There was no survival significance for p73 polymorphism genotype,
p53 and survivin in the non-radiotherapy group (p = 0.37) (Paper II, Fig. 3.).
In summary there seems to be a relation between p73 genotypes, p53 and survivin
expression. Rectal cancer patients with the GC/GC genotype together with
negative p53 and low survivin expression survived longer than those with other
genotype/phenotype combinations.
60
RESULTS
Paper III
In this study we investigated the expression of p73 in distant and adjacent normal
mucosa, primary tumor and lymph node metastasis from rectal cancer patients
who did or did not receive preoperative radiotherapy in combination with surgery.
We further studied the relationship between p73 expression, radiotherapy response and clinicopathologic and biologic factors.
In the overall cases cytoplasmic expression of p73 was significantly increased in
primary tumors when compared to normal mucosa (p = 0.0001), although there
was no difference in nuclear expression between normal mucosa and primary
tumor.
When comparing p73 expression between non-radiotherapy groups and radiotherapy groups the number of cases with positive nuclear p73 expression was higher in
irradiated normal mucosa (33% versus 12%, p = 0.02) and primary tumor (32%
versus 18%, p = 0.06), than non-irradiated (Paper III, Fig. 2.). The frequency of
normal mucosa with strong cytoplasmic expression of p73 was significantly higher
in the radiotherapy group than the non-radiotherapy group (53% versus 21%,
p = 0.003) (Paper III, Fig. 3.).
In further analysis the rectal cancer patients were divided into subgroups with
either a positive or negative p73 staining (nuclear, cytoplasmic or both). Patients
who had p73 negative tumors tended to have a lower rate of recurrence in the
radiotherapy group than in the non-radiotherapy group (0 versus 26%, p = 0.06)
(Paper III, Fig. 4.). In the radiotherapy group p73 expression was positively related
to Cox-2 expression. In further analysis of p73 and Cox-2 combined in the radiotherapy group it was shown that among 3 patients with p73/Cox-2 negative
tumors none had local recurrence, while among 7 patients with p73/Cox-2
positive tumors, 4 had local recurrence.
In summary we demonstrated that p73 was expressed higher in tumor than normal
mucosa and that after radiotherapy normal mucosa upregulated p73 more than
tumor did. We also showed that patients with p73 negative tumors, compared to
those with p73 positive tumors, may have a lower risk of recurrence after preoperative radiotherapy.
RESULTS 61
Paper IV
In this study we looked at the effects of γ-radiation on colon cancer cell lines
KM12C, KM12SM and KM12L4a, regarding cell cycle, survival fraction (clonogenicity) and apoptosis. In addition the expression patterns of mutated p53,
TAp73, ΔNp73, survivin and PRL-3 were studied, by means of Western blot, after
irradiation.
After irradiation with 10Gy, the near diploid KM12C accumulated transiently in
the S phase and G2 phase of the cell cycle, however 48 h after irradiation KM12C
cells continued through the cell cycle. In irradiated samples the cell cycle analysis
showed a subpopulation of giant cells (7.2% at 24 h, 9.0% at 48 h, 7.1% at 72 h
post-radiation) with a DNA content of about 4 times the normal content (4N) of
KM12C. The near tetraploid KM12SM cell line arrested permanently in the G2
phase after irradiation and displayed a 4N giant cell subpopulation (4.5% at 24 h,
7.2% at 48 h, 44% at 72 h post-radiation) (Paper IV, Fig 1.). The untreated
KM12L4a consisted of two subpopulations, one diploid and one near tetraploid.
KM12L4a arrested permanently in G2 after irradiation and did not display a 4N
giant cell subpopulation.
A clonogenic assay was used as a measurement of internal radiosensitivity, by
comparing survival fraction after irradiation among the 3 cell lines. The KM12C
was most sensitive with a radiated/non-radiated sample quota of 0.32 ± 0.06,
followed by KM12SM with 0.48 ± 0.02 and KM12L4a with 0.60 ± 0.05 (Paper IV,
Table 1).
Apoptosis was quantified by means of the M30-Apoptosense™ Elisa kit and
verified by DAPI staining. The M30 radiated/non-radiated apoptosis quota was
about 1.8 at 48 h and 1.6 at 72 h for KM12C, 2.0 at 48 h and 2.5 at 72 h for
KM12SM and 3.0 at 48 h and 72 h for KM12L4a (Paper IV, Fig. 2.).
The expression patterns of mutated p53, TAp73, ΔNp73, survivin and PRL-3 were
examined before and after irradiation. The KM12C featured a slight increase in
TAp73, prominent increase of ΔNp73 and PRL-3 after irradiation. KM12SM
showed an initial low expression of TAp73 and high expression of ΔNp73, which
both decreased after irradiation. Mutated p53 and survivin increased while PRL-3
decreased after irradiation in KM12SM. After irradiation an initially low expres-
62
RESULTS
sion of TAp73 and ΔNp73 decreased further, while the expression of survivin
increased. The KM12L4a entirely lacked PRL-3 protein (Paper IV, Fig. 3.).
In summary, KM12C showed low survival fraction, but low apoptosis and continued cell cycle progression, while KM12L4a showed a high survival fraction, but
high apoptosis and cell cycle arrest. The three cell lines, although sharing common
origin, showed different response to γ-radiation regarding mode of cell death and
protein expression patterns.
Paper V
In this study we investigated a possible connection between survivin and the p73
proteins in colon cancer cell lines HCT-116p53+/+ and HCT-116p53-/- by manipulating survivin levels with ectopic cDNA and siRNA. The effect of survivin knockdown on p73 expression and apoptosis with and without γ-radiation was also
investigated.
The transfection of survivin cDNA was successful in HCT-116p53+/+ and HCT116p53-/- as seen by an increase in survivin mRNA and protein. After transfection
there was no significant change in mRNA of TAp73 and ΔNp73, while there was a
small decrease of TAp73 protein in both cell lines and a small decrease of ΔNp73
protein in HCT-116p53+/+. The HCT-116p53+/+ also displayed a decrease in wild
type p53 in response to survivin overexpression (Paper V, Fig. 2 and Fig. 3.).
The expression patterns of survivin, TAp73, ΔNp73 mRNA and protein and p53
protein were examined before and after irradiation. In HCT-116p53+/+ there was no
change in the levels of survivin mRNA or protein, while TAp73 mRNA decreased
and TAp73 protein increased, ΔNp73 mRNA decreased and ΔNp73 protein
increased and p53 protein increased prominently, after irradiation. In HCT116p53-/- there was no change in the levels of survivin mRNA or protein, TAp73
mRNA was unchanged and TAp73 protein decreased, ΔNp73 mRNA decreased
initially and increased later and ΔNp73 and p53 protein increased, after irradiation
(Paper V, Fig. 4. and Fig. 5.).
The knockdown of survivin was successful in HCT-116p53+/+ and HCT-116p53-/- as
seen by a prominent decrease in survivin mRNA and protein both before and after
irradiation. After knockdown of survivin with siRNA in HCT-116p53+/+ TAp73 and
RESULTS 63
ΔNp73 mRNA and protein decreased, while p53 remained unchanged. In HCT116p53-/- knockdown of survivin resulted in no change in TAp73 mRNA and
decreased TAp73 protein, ΔNp73 mRNA and protein. After knockdown of
survivin in irradiated HCT-116p53+/+ levels of TAp73 mRNA and protein, ΔNp73
mRNA and protein and p53 protein decreased, when compared to mock siRNA
irradiated controls. In HCT-116p53-/- treated with both survivin siRNA and
irradiation TAp73 mRNA decreased while protein increased slightly and ΔNp73
mRNA and protein decreased as compared to mock siRNA irradiated controls
(Paper V, Fig. 4. and Fig. 5.). Knockdown of survivin in HCT-116p53+/+ and HCT116p53-/- lead to increased apoptosis, however only in HCT-116p53-/- did irradiation
further increase apoptotic levels in cells transfected with survivin siRNA (Paper V,
Fig. 6.).
In summary we showed that an overexpression of survivin does not regulate levels
of TAp73 and ΔNp73 mRNA and protein significantly, but decreases wild type
p53 in HCT-116 cell lines with and without functional p53. We also demonstrated
that forced downregulation of survivin leads to a downregulation of TAp73 and
ΔNp73 mRNA and protein in HCT-116 cell lines with and without functional
p53, both with and without γ-radiation. We further demonstrated an increase in
apoptosis in HCT-116p53+/+ and HCT-116p53-/- after survivin knockdown and that
only in HCT-116p53-/- did irradiation further increase apoptosis.
64
RESULTS
DISCUSSION
When p73 was first discovered it was thought to be a tumor suppressor just like the
previously known family member p53. The p73 gene and protein indeed showed
many similarities to p53, but it also had characteristics different from p53 regarding isoforms, expression and function [107, 119]. By studying p73 in vivo and in
vitro in this thesis, we have demonstrated that p73 is expressed higher in primary
CRC than in the corresponding normal mucosa, that p73 is involved in CRC risk
and radiotherapy response and that the anti-apoptotic protein survivin regulates
the two p73 isoforms TAp73 and ΔNp73.
The p73 G4C14-to-A4T14 polymorphism in CRC
risk and survival
We studied the G4C14-to-A4T14 polymorphism of p73 (Papers I-II). This
polymorphism, located just upstream of the initiating AUG of exon 2, is not a
functional polymorphism, but it has been hypothesized that the AT allele may
cause a stem loop structure, thus influencing the efficiency of translational initiation [86]. The distribution of genotypes GC/GC, GC/AT and AT/AT was
compared between CRC patients and a healthy control group (Paper I). A
significantly higher percentage of AT/AT genotypes among CRC patients pointed
towards an increased risk of developing CRC for individuals carrying the AT/AT
genotype. In a number of studies, reviewed in Table 3, this polymorphism and the
risk of developing different types of cancer has been looked at. The results of these
studies are confusing rather than conclusive as some studies show that the AT
allele increases risk of developing cancer [123, 124, 172-175], while others show
that the AT allele decreases the risk [121, 122]. Yet other studies show no correlation between the p73 polymorphism and cancer risk [176-179]. The reason for the
discrepancy in the different studies is unclear, but it may be influenced by different
carcinogenic mechanisms of the different cancers or different treatments given to
DISCUSSION 65
the patients enrolled in the studies. It may also be due to underlying differences in
genetic, environmental or social factors relevant to the polymorphism, in the
populations studied, especially in the control populations [122, 176]. Many of the
studies, including the present study, have a relatively low number of patients which
may also influence the results.
Apart from our studies only two other studies up to date have covered the p73
polymorphism and prognosis. In breast cancer the GC/GC genotype was an
independent prognostic factor for both worse overall survival and worse disease
free survival [180], while in pancreatic cancer patients with the GC/GC genotype
had longer time to progression and had a better clinical response [181]. We found
that in the overall group of CRC patients, those carrying the AT allele had significantly better survival (Paper I). When divided into colon cancer and rectal cancer
there was a difference in genotype of p73 and survival between the two tumor sites.
We also showed that the GC/GC genotype tended to correlate with a longer
survival in rectal cancer patients treated with preoperative radiotherapy (Paper II).
Again there is no clear explanation for the discrepancy between the different
cancers, especially between colon and rectal cancer, which are very similar.
Table 3. Review of G4C14-to-A4T14 polymorphism and cancer risk
Cancer type
Lung cancer
Head and neck cancer
Oropharynx cancer
Oral cancer
Endometrial cancer
Cervical cancer
Lung cancer
Esophageal cancer
Esophageal cancer
Breast cancer
Digestive tract cancer
Lung cancer
66 DISCUSSION
Risk
Population
AT increased
risk
Caucasian
Caucasian
Caucasian
Indian
Japanese
Japanese
Li et al, 2004 [123]
Li et al, 2004 [172]
Chen et al, 2008 [175]
Misra et al, 2009 [173]
Niwa et al, 2005 [174]
Niwa et al, 2004 [124]
AT decreased
risk
Chinese
Irish
Hu et al, 2005 [122]
Ryan et al, 2001 [121]
No risk
Chinese
Japanese
Japanese
Korean
Ge et al, 2007 [177]
Huang et al, 2003 [179]
Hamajima et al, 2002 [178]
Choi et al, 2006 [176]
The p73 polymorphism seems to be associated with both risk of developing a
number of cancers and prognosis. It has been hypothesized that the AT allele
influences the efficiency of translation initiation [86]. Another theory is that p73
function is altered by other functional polymorphism that may be in linkage
disequilibrium with the p73 polymorphism [123]. In Paper I we showed that
tumors with the AT/AT genotype tended to have a lower p73 expression. It would
be of interest to further study a possible correlation between p73 polymorphism
genotypes and the expression of TAp73 and ΔNp73. The G4C14-to-A4T14
polymorphism is located just upstreams of the P1 promoter and it is possible that
certain genotypes favors one of the two p73 promoters, thereby favoring expression of one or the other isoform, possibly altering the balance between the two p73
isoforms [122].
Expression of p73
Several extensive mutational analyses failed to demonstrate a significant frequency
of mutations in TP73. Instead studies in multiple tumor types have revealed that
p73 is overexpressed rather than mutated or deleted in human cancer [107].
Expression of p73 in colorectal cancer patients
We investigated the expression of p73 in normal colon and rectal mucosa, primary
colorectal tumors and in lymph node metastasis with IHC (Papers I and III). We
found that normal mucosa had weaker p73 expression as compared to primary
tumor. We found no difference in p73 expression between primary tumor and
lymph node metastasis. This is in concordance with what most other studies found
when looking at p73 expression in a number of normal tissues, such as lung, breast,
prostate, colon and rectum, and the corresponding cancers [106, 113, 116, 182,
183].
Attempts have been made to correlate p73 expression status with patient prognosis
and in CRC it was previously shown that patients with p73-positive tumors had
shorter survival than those with p73 negative tumors [116]. We did not find a
correlation between p73 overexpression with survival (Papers I and III). In both
the study by Sun [116] and the present studies total p73 was measured, with the
DISCUSSION 67
same antibody, and the discrepancy between these two studies may be due to
different patient material. The low number of cases in Papers I and III may also be
a factor causing discrepancy between studies. In Paper I and III an antibody that
recognizes the C-terminal of the p73 proteins was used, which means that both
N-terminal p73 isoforms were detected. This problem has been seen in a number
of studies both regarding antibodies used in IHC and Western blot, but also in
RT-PCR where primers against a domain found in both TAp73 and ΔNp73 has
been used [107]. Later studies have shown that the two N-terminal isoforms are
co-upregulated in a number of tumors, including CRC, as compared to the
corresponding normal tissue. It has also been shown that a high expression of
ΔNp73 is correlated with a worse prognosis [184, 185]. There are also a number of
C-terminal isoforms of p73, which may also contribute to different properties of
p73 [86, 186, 187]. In this thesis we only distinguished between the N-terminal
variants when possible, since they have proven distinct functions.
Expression of p73 in colon cancer cell lines
The expression of individual isoforms TAp73 and ΔNp73 was examined in colon
cancer cell lines KM12C, KM12SM, KM12L4a by means of Western blot (Paper
IV), HCT-116p53+/+ and HCT-116p53-/- by means of RT-PCR and Western blot
(Paper V). The two isoforms where co-expressed in all cell lines. There is an
autoregulatory feedback loop between ΔNp73 and TAp73/p53 [97], still many
studies looking at the expression of both isoforms in cell lines show that cells
expressing high levels of TAp73 also express high levels of ΔNp73 [188]. The
ΔNp73 protein has been shown to be rapidly downregulated upon DNA damage
though, while TAp73 and p53 increase and remain high [189].
In the KM12 cell lines, with mutated p53, TAp73 seemed generally slightly lower
than ΔNp73, except in KM12SM which had a noticeable higher expression of
ΔNp73 than TAp73 in untreated cells. In the two cell lines HCT-116p53+/+ and
HCT-116p53-/-, which are identical except for p53 status, there seemed to be no
difference in expression of the two isoforms of p73.
68 DISCUSSION
p73 in relation to radiotherapy response
In Sweden preoperative radiotherapy is routinely used as adjuvant treatment for
rectal cancer patients and has been shown to increase local control and improve
survival [48, 190]. Response of cancers to ionizing radiation varies widely and may
partly be explained by differences in cancer cell death-inducing effectors. It is
important to find the factors that may predict the patient’s response to treatment
in order to reduce overtreatment, adverse effects and normal tissue damage [191].
Numerous studies have confirmed that p73 is an important determinant for
chemotherapeutic efficacy in human cancer and that p73 is induced by a number
of cytotoxic drugs. Little work has been done on the role of p73 in radiosensitivity
in human cancer [192]. It has been shown that p73 is induced by γ-radiation [98,
99] and in one study on cervical cancer it has been seen that overexpression of p73
in radioresistant cell lines increased their radiosensitivity [192].
p73 in relation to radiotherapy response in rectal cancer patients
The expression of p73 was studied in normal mucosa, primary tumors and lymph
node metastasis from rectal cancer patients who did or did not receive preoperative radiotherapy in combination with surgery (Paper III). The number of cases
with strong p73 expression was higher in the patient group that received radiotherapy than those who did not, the increase was especially noticeable in normal
mucosa. It is well known that p73 is induced by γ-radiation [98, 99] and in cervical
cancer radiosensitive cases had higher p73 than radioresistant cases [127]. We
nonetheless observed that patients with p73 negative tumors tended to have a
lower rate of recurrence after preoperative radiotherapy. In patients who did not
receive preoperative radiotherapy, p73 expression was not related to recurrence
rates (Paper III). The fact that rectal cancer patients with p73 negative tumors had
better prognosis and that p73 expression in rectal tumors was positively correlated
to the anti-apoptotic factor Cox-2, may indicate that the effect comes from an
overexpression of the anti-apoptotic ΔNp73 rather than TAp73. This is probably
the case at least in tumor. It is possible that the p73 isoforms are expressed differently in normal and tumor tissue and that TAp73 is upregulated in normal
mucosa in response to irradiation.
DISCUSSION 69
As mentioned previously we showed that rectal cancer patients with the GC/GC
genotype tended to have a longer disease free survival when treated with preoperative radiotherapy and it was hypothesized that the genotypes of the
G4C14-to-A4T14 polymorphism may affect the transcription balance of the two
p73 isoforms (Paper II). In cervical cancer patients treated with radiotherapy it
was demonstrated that overexpression of ΔNp73 was mainly detected in radioresistant cases, while TAp73 overexpression was mostly seen in radiosensitive cases.
The ΔNp73 expression was also associated with a worse clinical outcome, while
TAp73 was associated with better survival in cervical cancer patients [128]. The
p73 protein, most probably TAp73, is able to induce both cell cycle arrest and
apoptosis as response to γ-radiation and it has been suggested that this may be a
compensatory effect when p53 is lost [90]. Studies in cervical cancer have shown a
correlation between p73 expression after irradiation and apoptotic index, especially in a p53 non-responding group [126]. We did not see a correlation between p73
expression and apoptosis in rectal cancer patients (Paper III).
Cellular radiosensitivity
In order to study molecular mechanisms of apoptotic proteins, such as p73, a
significant frequency of apoptosis was needed and therefore a relatively high dose
of γ-radiation was used on the KM12 cells (Paper IV).
Traditionally there are two forms of radiation-induced cell death; one is apoptosis
before cells reach the first mitosis after irradiation, and the other is reproductive or
mitotic cell death, usually evaluated by clonogenic assays [193]. Cell cycle phase
analysis, SF5 (clonogenic assay) and apoptosis assays were used to measure the
intrinsic radiosensitivity of KM12C and its two derivatives KM12SM and
KM12L4a with the aim to study protein expression of mutated p53, TAp73,
ΔNp73, survivin and PRL-3 in correlation to radiation-induced cell death (Paper
IV). After irradiation the KM12C cell line had a transient cell cycle arrest with
some giant cells and very low apoptosis, but also a low clonogenic ability. The
KM12SM showed a permanent cell cycle arrest with a high number of giant cells,
moderate apoptosis and clonogenic ability after irradiation. The KM12L4a had the
highest level of apoptosis, but also the highest clonogenic ability. KM12L4a also
had a permanent cell cycle arrest, however without any detectable giant cells. The
giant cells seen in cell cycle analysis are cells that have four times the amount of
70 DISCUSSION
DNA as compared to cells in G1. Mitotic catastrophe is typically seen after
irradiation and is characterized by failure to undergo a complete mitosis, leading to
multinucleated cells with an excessive amount of DNA [194]. Cell cycle analysis
and also the morphology of cells studied by DAPI indicated possible mitotic
catastrophe in the KM12 cells after irradiation. Due to lack of morphological and
molecular examination of mitotic catastrophe this could not be confirmed in this
study.
Regarding apoptosis the KM12C cell line was the most radioresistant and
KM12L4a was most radiosensitive, yet when looking at SF5 the relationship is the
opposite. It is controversial whether or not apoptosis affects the radiosensitivity of
cells, still it is widely accepted that cells that evade apoptosis are resistant to cell
killing by DNA-damaging agents [195]. Not all cells die immediately after treatment and time of death is highly dependent on cell type and toxic agent. Therefore
a complementary assay such as the clonogenic assay is needed as a complement to
study long-term viability of cells [195]. There are studies that show that apoptosis
and cell killing assessed by clonogenic assay are well correlated. Even so, in the case
of solid tumor cells many times it is not [195, 196]. There are also an increasing
number of reports suggesting that apoptosis also plays a role in and after the
clonogenic cell death [193], even if it is not necessary for the lethal effect [75]. It
seemed that KM12C may be inactivated mainly by post-mitotic cell death after
irradiation, while KM12L4a may be inactivated mainly by a rapid pre-mitotic
response to irradiation. Considering the relatively high dose of irradiation it is
probable that a number of cells also died by necrosis (Paper IV).
p73 in relation to radiotherapy response in colon cancer cells
It has been implicated that p73 is induced by γ-radiation and that for induction of
apoptosis it is the TAp73 isoform that is upregulated, while ΔNp73 is rapidly
downregulated [189]. It has also been shown that in certain tumors this balance is
disrupted and that high expression of ΔNp73 after DNA damage is a bad prognostic factor [197]. Whether a specific isoforms is in fact expressed in the specimens
from the Swedish rectal cancer trial could not be identified within Paper III. In
Papers IV-V we investigated the expression of specific isoforms TAp73 and
ΔNp73, by means of RT- PCR and Western blot, in colon cancer cell lines before
and after irradiation.
DISCUSSION 71
The three cell lines KM12C, originating from a Dukes´ B colon cancer, KM12SM
and KM12L4a, originating from KM12C, where irradiated with γ-radiation and
protein expression of TAp73 and ΔNp73 was measured (Paper IV). Mutated p53,
survivin and PRL-3, previously implicated in radiotherapy response by our group
were also studied in the KM12 cells [53, 56, 58]. KM12C had a marked increase in
ΔNp73 expression after irradiation, while TAp73 remained relatively unchanged.
This cell line showed relatively low apoptosis as measured by M30 and it is
possible that the mutant p53 status together with the ΔNp73 increase after
irradiation is partly the cause for such low levels of apoptosis. In KM12SM and
KM12L4a the levels of TAp73 and ΔNp73 were simultaneously decreased after
irradiation. In KM12SM there was an increase in the mutated p53, which may be
one explanation for the downregulation of at least TAp73. The specific mutation
found in the KM12 cells has been found to downregulate TAp73, which may give
cells further oncogenic potential [105]. Of course radiation-response is a multipathway mechanism and other pathways not studied here are certainly involved in
the complete response of the cells to γ-radiation.
The effect of γ-radiation on the expression of TAp73 and ΔNp73 in HCT-116
with a functional and non-functional p53 was studied (Paper V). In HCT-116p53+/+
TAp73 increased in response to irradiation, while it decreased in HCT-116p53-/-.
The ΔNp73 protein increased in both HCT-116p53+/+ and HCT-116p53-/-, although
most prominently in HCT-116p53-/-. As could be expected the levels of p53 increased after irradiation and other studies have shown that after DNA damage in
HCT-116 wild type p53 is 20-30 fold higher than p73 [198]. It has been shown
that ΔNp73 is rapidly degraded in response to DNA-damage to allow for an
increase in TAp73 and p53 to elicit cell cycle arrest and apoptosis [189], which
agrees with the response seen in HCT-116p53+/+. In HCT-116p53-/- the ΔNp73 is
downregulated, although so is TAp73. Studies have shown that TAp73 is not
dependent on p53 to induce cell cycle arrest or apoptosis. A possible explanation
for the simultaneous downregulation of TAP73 and ΔNp73 is that the lack of
functional p53 somehow disturbs the autoregulatory loop between ΔNp73 and
TAp73. TAp73 has been shown to be downregulated by mutant p53, however the
truncated variant of p53 in HCT-116p53-/-, has not been studied in that context
[199]. Both HCT-116 cell lines showed increased apoptosis after irradiation,
suggesting that although both wild type p53 and TAp73 are lost or downregulated
there are other compensatory mechanisms inducing apoptosis in HCT-116p53-/-.
72 DISCUSSION
p73 and other proteins
In this thesis we have seen indications that p73 is connected to proteins such as
p53 (Papers II and IV), survivin (Papers II and IV) and Cox-2 (Paper III). We
showed that rectal cancer patients with the GC/GC genotype of the p73 polymorphism, wild type p53 and low expression of survivin had a better prognosis after
preoperative radiotherapy than patients with other genotype/phenotype combinations (Paper II), which points toward a possible connection between the proteins.
We also showed that p73 was positively related to Cox-2 expression in rectal
cancer patients treated with preoperative radiotherapy. It is not clear whether this
association is due to interactions between these proteins or only a synergistic
effect.
p73 and p53
Several studies have established that there is a relationship between the two family
members, p73 and p53. They are associated through an autoregulatory loop,
where TAp73 and p53 can bind to the ΔNp73 promoter and induce its transcription and ΔNp73 in turn inhibits the activity of TAp73 and p53 [97]. TAp73 and
p53 do not form complexes with each other and induce cell cycle arrest and
apoptosis independently of each other [200], although in some cellular systems it
was indicated that wild type p53 is dependent on p73 to induce apoptosis [201].
Considering the high mutational rate of TP53 and that mutation in TP73 is
virtually non-existent in tumors, it was proposed that p73 could compensate for
the loss of p53 in certain cell types [126]. Experimental and clinical evidence
suggest that certain p53 mutants have acquired the ability to bind to and downregulate TAp73 via the DBD, thereby gaining an oncogenic function [202]. The
KM12 cells in Paper IV harbor a mutation which has been shown to be able to
bind and downregulate TAp73. In KM12SM a major increase of mutated p53 and
a simultaneous downregulation of TAp73 were seen. It is possible that this
increase may interrupt the activities of TAp73. To further elucidate this, a coprecipitation analysis would be needed.
DISCUSSION 73
p73 and survivin
Our studies (Papers II and IV) indicated a possible relationship between p73 and
survivin and we further investigated this possible association (Paper V). Many
experimental studies show that wild type p53 normally represses survivin and that
survivin is re-expressed in cancer cells without functional p53, in vivo and in vitro,
[139, 140]. It has also been suggested that TAp73 is capable of downregulating
survivin [203]. To this date only one study has looked at the regulation of the p53
family members by survivin, in breast cancer, where ectopic survivin downregulated p53, but upregulated TAp73 and ΔNp73 [141]. We looked at a possible
effect of survivin on the expression of TAp73 and ΔNp73 mRNA and protein and
p53 protein in HCT-116p53+/+ and HCT-116p53-/- (Paper V). Overexpression of
survivin did not show a significant effect on TAp73 or ΔNp73 in either HCT-116
cell line, which is not in concordance with what was seen in breast cancer where
TAp73 and ΔNp73 were overexpressed in cells overexpressing survivin [141]. Our
present study and the study in breast cancer showed that ectopic survivin expression downregulates p53 in HCT-116p53+/+ and MCF-7. When survivin instead was
knocked down by siRNA the levels of TAp73 and ΔNp73 mRNA and protein
decreased in both HCT-116 cell lines. The downregulation of p73 by survivin was
observed both before irradiation and after, showing that the effect is not dependent on DNA damage initiation. Survivin affected both mRNA and protein of
TAp73 and ΔNp73, with some exceptions, suggesting a transactivational or
translational effect. In this study it is not clear if survivin downregulates both
isoforms or if the downregulation of ΔNp73 is a consequence of diminished
TAp73.
Levels of survivin, TAp73 and ΔNp73 were measured simultaneously in KM12
cells after irradiation (Paper IV). In KM12SM survivin is very low and also TAp73
and ΔNp73 at one time point after irradiation. It is possible that this is the same
effect seen in the HCT-116 cell lines, nevertheless other effects on p73, such as
that of mutant p53 binding to p73 cannot be excluded in these cell lines after
irradiation.
74 DISCUSSION
p73 and Cox-2
We found a correlation between overexpression of p73 and Cox-2 in rectal cancer
patients who received preoperative radiotherapy (Paper III). In a previous study
on the same material it was shown that patients with strong Cox-2 had higher local
recurrence rates [55]. Rectal cancer patients with overexpression of both proteins
more often had local recurrence than those negative for both p73 and Cox-2. To
this date there are no other studies connecting p73 and Cox-2, however a functional connection between p53 and Cox-2 has been demonstrated [204]. Considering the homology of p53 and p73 it is possible that there is a functional
interaction between p73 and Cox-2. It is also possible that the correlation between
the radioresistance factor Cox-2 and p73 in rectal cancer patients, suggests that the
p73 expression observed is mainly ΔNp73, which just as Cox-2 is a negative factor
in radiotherapy response.
DISCUSSION 75
76 DISCUSSION
CONCLUSIONS
Based on the findings in Papers I-V included in this thesis the following conclusions can be drawn:
•
Individuals carrying the AT/AT genotype of the G4C14-to-A4T14
polymorphism of p73 had an increased risk of developing CRC and CRC
patients carrying the AT allele had a better prognosis.
•
Rectal cancer patients with the GC/GC genotype tended to survive longer
after radiotherapy, especially in combination with wild type p53 and weak
survivin expression.
•
After preoperative radiotherapy in rectal cancer patients, normal epithelial
cells had a more marked increase in p73 expression than primary tumor cells.
Rectal cancer patients with p73 negative tumors may have lower risk of local
recurrence after radiotherapy than those with p73 positive tumors.
•
KM12C, KM12SM and KM12L4a seem to have evolved different responses
after γ-radiation, evasion of apoptosis for KM12C and continued proliferative
ability for KM12L4a. The low apoptotic response to γ-radiation in KM12C
may be explained by an induction of ΔNp73.
•
Knockdown of survivin decreased levels of TAp73 and ΔNp73 in both HCT116p53+/+ and HCT-116p53-/-. The knockdown also caused an increase in
apoptosis, especially noticeable in HCT-116p53-/- after irradiation. Ectopic
overexpression of survivin downregulated wild type p53 in HCT-116.
CONCLUSIONS 77
78 CONCLUSIONS
ACKNOWLEDGMENTS
During my graduate study years I have had the pleasure to meet and work with
people who make going to work easy and fun. I would like to express my sincere
gratitude to everybody who in some way has contributed to this thesis. I would
especially like to thank:
All the colorectal cancer patients who gave their consent to donate the surgical
material for scientific research. You are the foundation for this thesis.
Swedish Cancer Foundation, Swedish Research Council, the Health Research
Council in the South-East of Sweden, Lions forskningsfond, for financial support.
Xiao-Feng Sun, my supervisor, for taking me in and introducing me to the fascinating world of cancer research. From day one you have shown your support and
your strong belief in me and my abilities and it has made me grow as a person and
as a researcher.
My room mates. Bibbi, you always have an answer to all my problems, big or small.
Gizeh, who no matter how busy you are, always has time to chat.
All my other colleagues at the Oncology lab for filling my days with interesting
discussions on pretty much all possible topics. With you guys there is always
something fun on the agenda! Rock band and tea at my place on Wednesday?
My colleauges at KEF for creating a friendly and enjoyable work environment.
Dr Janine T Erler for your kindness and help regarding revision of my thesis.
All of you who read and commented my thesis under construction.
Chatarina Malm. For always having time to help in the administrative jungle.
Sara Olsson, Erik Angland and Dan Josefsson for helping me with the irradiation of
my cells and for always making time for me and my experiments.
Birgitta Holmlund, for your technical assistance.
ACKNOWLEDGMENTS 79
To all my co-authors, especially Åsa Wallin and Jasmine Lööf. For good discussions on tricky results and just beeing so fun to work with!
My friends who know how to pull me back to reality when I get lost in my own
head. Hanna, no matter how many kilometers there are between us, I always feel
close to you. Bushdave, for always knowing when I need to have a lunch break
downtown. Maria and Fredrik, for good diving and travelling company and
spontaneous Friday night dinners. Karin, Ola, Cecilia, Dan and the kids, for many
fun board gaming nights.
Syjuntan. Charlotte, Cissi, Josefine, Karin and Marie. I am glad to have found
others who share my passion for both cross stitch and “fika”.
My new family, the Ohlssons. To Kerstin and Gunnar for being so warm and kind
and for your encouragment in both my work and my everyday life.
The Smidelik family. For having the biggest hearts in the world, always opening
your home to anyone who needs it and for all the fun coco banana family dinners.
My aunt, Susanna Smidelik. You are a true role model for strong and independent
women. You are a never ceasing source of encouraging words and love.
My mother, Agnes Malmström, for your endless love and support. I wish I had at
least half of your energy and commitment.
My sisters. Ester, the most stubborn, crazy and intelligent girl I know. I am glad
you share my passion for medicine. Sara, for always having the time to check in on
me, and ask how everything is going. May all your dreams come true! Emma, few
have been granted a bonus big sister like you.
Those of you who left this life to early. My father, Ivan Pfeifer. This was your
dream as much as mine and I wish you could have been here to share it with me.
My aunt, Vera, who always had time for “marhaság” and a story about our family
history. Ingvar, I was lucky to have an extra father who always lent a helping hand. I
miss you all so very much.
Stina and Quick. I may be a crazy cat lady, but life is easier with a purring cat on
your lap.
And Johan, for being there for me always. Jag älskar dig i vått och
torrt!
80 ACKNOWLEDGMENTS
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