Pathway Determinants of 5-Fluorouracil Sensitivity

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PATHWAY DETERMINANTS OF 5-FLUOROURACIL SENSITIVITY

TAN WEN LEE B.Sc. (Pharm)(Hons.), NUS

A THESIS SUBMITTED

FOR THE DEGREE OF MASTER OF SCIENCE

DEPARTMENT OF PATHOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

2009

ACKNOWLEDGEMENTS

First of all, I wish to thank Prof. Teh Ming for giving me an opportunity to study in the Department of Pathology.

I would like to express my heartfelt gratitude to my supervisor – A/Prof Richie Soong for giving me this opportunity to undertake this project. I am indebted to him for his invaluable understanding, guidance and support throughout this course.

I would also like to thank Prof. Limsoon Wong and Mr. Difeng Dong from School of Computing, National University of Singapore, and Ms. Marie Loh from Oncology Research Institute (Singapore) for their invaluable advice and inputs for the statistical analyses; Dr. Ross Soo from Department of Haematology Oncology, National University Hospital (Singapore) for his expert opinions in clinical research; Dr. Iduna Fichtner from Max Delbrück Centre for Molecular Medicine, Berlin-Buch, (Germany) for the provision of tumor xenograft materials; Dr. Robert Hewitt, Dr. Rajeev Singh, Dr. Chon Boon Eng, Ms. Almeda and Ms Wentong Yang from NUH-NUS Tissue Repository (Singapore) for the provision of human tissue samples and clinical information and, Ms. Cecilia Lee from Institute of Molecular and Cell Biology (Singapore) for the provision of human cancer cell lines.

Lastly, I would like to thank my family members, my hubby, Eng Lee and all my lab colleagues from Translational Interface, Cancer Science Institute (Singapore) for their companionships and their help in one way or another.

CONTENTS

List of Tables ...... 3 List of Figures ...... 4 ABSTRACT ...... 5 CHAPTER 1: LITERATURE REVIEW ...... 6 1.1 Colorectal Cancer ...... 6 1.2 5-fluorouracil ...... 7 1.2.1 Transport ...... 9 1.2.2 Metabolism ...... 10 1.2.3 Mechanism of Action ...... 14 1.3 Fluorouracil-based therapies ...... 15 1. 4 Molecular determinants of 5FU sensitivity ...... 18 1. 4.1 Candidate gene approach ...... 18 1.4.2 Genome-wide approach ...... 24 1.5 Study approaches ...... 30 1.5.1 Quantification of mRNA expression using real-time PCR ...... 31 1.5.2 Regulation patterns of genes in response to 5FU treatment ...... 35 1.5.3 RNA interference (RNAi) ...... 35 1.6 Scope of Study ...... 38 CHAPTER 2: MATERIALS AND METHODS ...... 41 2.1 Cell lines ...... 41 2.2 Cell sensitivity analysis ...... 41 2.3 Xenografts ...... 42 2.4 Primary Tumors ...... 42 2.5 Gene expression analysis ...... 42 2.6 Small interfering RNA (siRNA) transfections ...... 49 2.7 Statistical analysis ...... 49 CHAPTER 3: RESULTS ...... 52 3.1 Correlation between gene expression and 5FU sensitivity ...... 52 3.2 Identification of baseline gene expression associated with 5FU sensitivity ..... 60 3.3 Identification of post-treatment gene expression changes associated with 5FU sensitivity ...... 65 3.4 Modulation of 5FU sensitivity by CTPS2 siRNA ...... 69 CHAPTER 4: DISCUSSION ...... 73 CONCLUSION ...... 79 REFERENCES ...... 80 APPENDIX ...... 94 PUBLICATIONS ...... 95

List of Tables

Table 1: Overview of studies on gene/protein expression levels in relation to 5FU

chemosensitivity.

Table 2: Summary of genome-wide and proteomic studies to identify determinants for

5FU-based chemosensitivities.

Table 3: List of 96 genes selected for LDA analysis.

Table 4: Characterization of 5FU sensitivity: (A) IC50 values of 21 colorectal cancer cell lines; (B) % of tumor shrinkage in 14 xenografts treated by 5FU; (C) Recurrence data of

25 stage II or III colorectal cancer patients treated with standard 5FU and leucovorin

therapy.

Table 5: Associations (χ2 test) between 5FU sensitivity and clinicopathological

parameters

Table 6: Candidate genes with differential gene expression (p<0.05) identified by

unsupervised clustering analysis for xenografts and primary tumors.

Table 7: Differential expression of genes in xenograft and patient series (p<0.05).

List of Figures

Figure 1: Chemical structure of uracil and 5-fluorouracil (5FU).

Figure 2: 5FU metabolism.

Figure 3: Pathways of fluoropyrimdine and folate metabolism and downstream events.

Figure 4: Activation of DNA repair mechanisms and stress response in relation to 5FU

effects.

Figure 5: Diagram illustrating the concepts for the TaqMan probe assay.

Figure 6: TaqMan® LDA.

Figure 7: The RNA Interference pathway.

Figure 8: Genes analyzed in this study for their roles in transport and metabolism of 5FU

and its downstream events.

Figure 9: Unsupervised hierarchical clustering of samples based on the expression of 96

genes for (A) cell lines, (B) xenografts and (C) primary tumors.

Figure 10: Difference in expression levels of CTPS2 and NME4 between clusters in (A) xenografts and (B) patients.

Figure 11: Patterns of gene expression at 0, 4, 8, 12, 24 and 48 hours after 10 μM 5FU treatment for (A) RKO and (B) SW837.

Figure 12: CTPS2 and GAPDH mRNA expression levels in SW620 cells transfected with

(A) CTPS2 siRNA and (B) GAPDH siRNA 48 and 120 hours after transfection. Growth curves of cells either treated or untreated with 10 μM 5FU after 48 hours of transfection of (C) CTPS2 siRNA, (D) GAPDH siRNA and (E) negative control siRNA.

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ABSTRACT

Purpose: Large inter-patient variability in patient response to the chemotherapeutic agent, 5-Fluorouracil (5FU), makes markers for predicting 5FU response highly desirable. We describe here a pathway-based gene expression analysis of the relative

roles of all known components of 5FU transport, metabolism, its downstream effects and

co-factor (folate) metabolism in determining 5FU sensitivity in cell lines, xenografts and

tumours.Experimental design: The expression of 96 rationally-selected genes was measured by low-density array real-time PCR in 21 colorectal cancer cell lines, 14 xenografts and 25 tumors. Samples were grouped by unsupervised hierarchical clustering and the groups tested for associations with respective sensitivity, response and patient survival data.

Patterns of expression changes of the 96 genes in 5FU sensitive and resistant cells following a time course of 5FU treatment were also clustered to identify the most defining expression changes, and their component genes. siRNA knockdown was performed to verify the influence of the top candidate on 5FU sensitivity in-vitro.

Results: 5FU sensitivity associated with clustered groups for xenografts and tumours, but not cell lines. CTPS2 and NME4 were the genes common to the significantly

differentially regulated genes in xenografts and tumors. CTPS2 was also in the group of

genes with the most distinct expression change following 5FU treatment in sensitive and

resistant cell lines. siRNA knockdown of CTPS2 reduced sensitivity to 5FU.

Conclusion: Pathway-based analysis in multiple cancer models identifies CTPS2

expression as a major determinant of 5FU sensitivity.

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CHAPTER 1: LITERATURE REVIEW

1.1 Colorectal Cancer

Colorectal cancer (CRC) is one of the leading causes of cancer deaths worldwide

(Greenlee et al., 2000). In Singapore, CRC is the most prevalent cancer in both men and

women combined and the incidence is increasing very rapidly with about 1000 new cases diagnosed annually (Singapore Cancer Registry Report No. 6). The probability of developing CRC increases with age, with 1 in 124 males and 1 in 149 females aged between 40 to 59 years old developing CRC, increases to 1 in 29 males and 1 in 33 females for age between 60 to 79 (Greenlee et al., 2000).

Surgical resection remains the primary treatment for patients with CRC. Resected samples are then sent for pathological evaluation which provides prognostic information based on tumor staging and facilitates treatment decision. Chemotherapy and/or radiotherapy is often given in adjuvant settings, and less commonly as neoadjuvant treatment. For patients whose surgery is deemed non-curative, for example, in metastatic colorectal cancer, chemotherapy or radiotherapy may be used as primary treatment

(palliative). In colon cancer, whereby recurrence to distant sites like liver, bone and lungs

is not uncommon, systemic chemotherapy given in adjuvant settings is more preferred to

radiotherapy (Midgley et al., 1999). In contrast, about 50% of recurrences of rectal

cancer localize in the pelvic region and hence, local radiotherapy is a preferred treatment

for relapse prevention.

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Forty to 60% of the CRC patient population is diagnosed with stage II or stage III disease

(Aranha et al., 2007). After curative surgery, stage III patients experience 50-60% chance

of developing disease recurrence. Published clinical trials have proved the beneficial

effects of adjuvant chemotherapy in improving curative rates in resected stage III cancer

(Wolmark et al., 1993; Moertel et al., 1995; Bleiberg et al., 2000). As such, the use of

adjuvant chemotherapy for stage III cancer has since become the standard care. In

contrast, patients with stage II colorectal cancer, the use of adjuvant therapy remains

controversial and is not routinely recommended (Benson et al., 2004). However, the

recurrence rate for stage II patients is about 20%, which is considerably high. This poses

a dilemma for clinicians in deciding the appropriateness of adjuvant chemotherapy in

stage II disease, so not to expose patients to unnecessary side effects while ensuring

clinical benefits.

1.2 5-fluorouracil

In 1957, fluoropyrimidines were rationally designed based on the observation that rat hepatomas utilize radiolabelled uracil more rapidly than normal tissues, indicating that uracil metabolism was a potential target for chemotherapy (Heidelberger et al., 1957). 5- fluorouracil (5FU), then synthesized with the presence of a fluorine atom on the fifth carbon of naturally occurring uracil is readily taken up and metabolized by cells, thus interfering with uracil metabolism (Figure 1). The more rapid uracil metabolism in tumor cells provides 5FU the advantage of being tumor selective as the rate of 5FU uptake goes hand-in-hand with uracil. With well demonstrated cytotoxicity, 5FU has since been widely used in the treatment of a number of cancer types, including colorectal, breast,

7 LITERATURE REVIEW

head and neck cancer (Longley et al., 2003). Though being tumor selective, 5FU also causes normal tissues toxicities like nausea, vomiting, diarrhea, myelosuppression,

mucositis, hand-foot disease, and, more rarely cardiac and neurotoxicities (Grem et al.,

2000).

Figure 1: Chemical structure of uracil and 5-fluorouracil (5FU). The hydrogen atom at the carbon-5 position of the pyrimidine ring is replaced by a fluorine atom in 5FU.

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1.2.1 Transport

As a synthetic nucleoside, uptake of 5FU into cells has been shown to occur via the same

non-concentrative, saturable mechanism as uracil (Wohlhueter et al., 1980), via an active,

concentrative mechanism (Yamamoto et al., 1981), or via nonfacilitated diffusion (Kessel et al., 1967). It was later reported that 5FU enters human erythrocytes via same carriers that transport purine nucleobases and nonfacilitated diffusion (Domin et al., 1993).

It is now recognized that uptake transporters responsible for translocation of nucleoside and nucleobases are members of the equilibrative nucleoside transporters (ENT,

SLC29A) family and concentrative nucleoside transporter (CNT, SLC28A) family

(Pastor-Aglada et al., 1998). The ENTs (comprises 4 isoforms, ENT1-4) transport hydrophilic nucleosides and nucleoside analogs by bi-directional facilitated diffusion

(Baldwin et al., 2004). They accept both purine and pyrimidine nucleosides but differ in their sensitivity to inhibition by nitrobenzylthioinosine (NBMPR). Human ENT1

(hENT1, an equilibrative sensitive, es-type transporter) is inhibited by nanomolar concentration of NBMPR, whereas hENT2 (an equilibrative insensitive, ei-type transporter) is unaffected by low concentrations of NBPMR. Besides purine and pyrimidine analogs, ei-type transporter also transports nucleobases (Ritzel et al., 2001).

On the other hand, CNT transporters are Na+-dependent and show different substrate specificity and pharmacological properties (Pastor-Aglada et al., 1998). A number of isoforms (CNT1-3) are known to exist: hCNT1 (a concentrative NBMPR-insensitive thymidine selective or cit-type transporter) is pyrimidine-preferring; hCNT2 (a

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concentrative insensitive and formycin B-selective or cif-type transporter) is purine-

preferring; hCNT3 (a concentrative NBMPR-insensitive or cib-type tranporter) is broadly

selective, transporting both purine and pyrimidine nucleosides and also nucleobases

(Pastor-Aglada et al., 1998, Ritzel et al., 2001). In another study investigating the role of

hCNT1 in cytotoxicity of 5’-deoxy-5-fluorouridine (5’-DFUR), a 5FU prodrug, it was

found that 5’-DFUR is an hCNT1 substrate, but 5FU is not (Mata et al., 2001). Efflux

transporters for 5FU cytotoxic intracellular metabolite has first been identified by Guo et

al. in an in-vitro transport assay (Guo et al., 2003). Like drug uptake mechanism, efflux

transport could have important implication in chemotherapeutic cytotoxicity as efflux

mechanism also determines drug availability in tissues.

1.2.2 Metabolism

Biotransformation of 5FU is important for its clinical effectiveness. The balance between

anabolism and catabolism determines the availability of 5FU in tissues (Heggie et al.,

1987). As a uracil analogue, 5FU undergoes the de novo anabolic and catabolic pathways used by uracil. The anabolism of 5FU to nucleotides occurs through several pathways

(Figure 2 and 3):

(1) a reversible conversion of 5FU to 5-fluoro-2’-deoxyuridine (FdUrd) by thymidine phosphorylase (TP), followed by phosphorylation to 5-fluoro-2’-deoxyuridine-5’- monophosphate (FdUMP) by thymidine kinase (TK) (Hartmann and Heidelberger et al.,

1961);

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(2) a transformation of 5FU to 5-fluorouridine (FUrd) by uridine phosphorylase (UP),

followed by phosphorylation to 5-fluorouridine monophosphate (FUMP) by uridine

kinase (UK) (Skold, 1958);

(3) a direct conversion of 5FU to FUMP by orotate phosphoribosyltransferase

(OPRT) which directly transfers a ribose phosphate from phosphoribosyl pyrophosphate

(PRPP) to 5FU (Reyes et al., 1969).

Another pathway for FdUMP formation is via dephosphorylation of fluorodeoxyuridine-

5’-diphosphate (FdUDP), which is converted from fluoridine-5-diphosphate (FUDP) by

ribonucleotide reductase. FdUMP and FdUDP are substrates for thymidine kinases,

resulting in the formation of fluorodeoxyuridine-5’-triphosphate (FdUTP). FUMP formed

from pathways described in (2) and (3) is sequentially phosphorylated to FUDP and

fluorouridine-5’-triphosphate (FUTP).

Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme of 5FU

degradation, catabolising approximately 80% of 5FU to dihydrofluorouracil (DHFU)

(Diasio and Harris, 1989). Dihydropyrimidinase (DHP) subsequently converts DHFU to

5-fluoroureido-propionic acid (FUPA), followed by conversion to fluoro-β-alanine

(FBAL) by β-alanine synthase.

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Figure 2: 5-fluorouracil (5FU) metabolism. 5FU is converted to three main active metabolites: fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). 5FU is directly converted to FUMP by orotate phosphoribosyltransferase (OPRT) phosphoribosyl pyrophosphate (PRPP) as the co-factor, or indirectly by fluorouridine (FUR), acting through uridine phosphorylase (UP) followed by uridine kinase (UK). FUMP is sequentially phosphorylated to fluorouridine diphosphate (FUDP) and fluorodeoxyuridine-5’- diphosphate (FdUDP), which can either be phosphorylated or dephosphorylated to FdUTP and FdUMP, respectively. Alternatively, 5FU is converted via thymidine phosphorylase (TP) to flurodeoxyuridine (FUDR), which is then phosphorylated by thymidine kinase (TK) to FdUMP. Dihydropyrimidine dehydrogenase (DPD) catabolizes 5FU to dihydrofluorouracil (DHFU) (Adapted from Longley et al., 2003).

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Figure 3: Pathways of fluoropyrimdine and folate metabolism and downstream events. The anti-tumor activity of 5FU is via inhibition of thymidylate synthase (TS), incorporation into RNA and DNA. The prominent candidate fluoropyrimidine predictive indicators are highlighted in large font and bold (Adapted from Soong and Diasio, 2005). 3UP: 3-ureidoproionoic acid; 3UP: 3-ureidopropionase; 5FU: 5-fluorouracil; BAL: b alanine; CDA: Cytidine deaminase; CE: Carboxylesterase; CK: Cytidylate kinase; CYP450: Cytochrome P450; DHF: Dihydrofolate; DHFR: Dihydrofolate reductase; DHP: Dihydropyrimidinase; DHU: Dihydrouracil; DNA: Deoxyribonucleic acid; DNAPol: DNA polymerase; DNMT: DNA methyltransferase; DPD: Dihydropyrimidine dehydrogenase; dUDP: Deoxyuridine diphosphate; dUMP: Deoxyuridine monophosphate; dUrd: Deoxyuridine; dUTP: Deoxyuridine triphosphate; dUTPPP: Deoxy-UTP- pyrophosphatase; F: fluoro; MS: Methionine synthase; NDK: Nucleotide diphosphate kinase; NDPase: Nucleoside dihposphatase; NMK: Nucleotide monophosphate kinase; NSase: Nucleosidase; NTase: 5’- nucleotidase; OPRT: Orotate phosphoribosyltransferase; RDR: Ribonucleotide diphosphate reductase; RNA Ribonucleic acid; RNAPol: RNA polymerase; SAH: S-adenosylhomocysteine; SAM: S- adenosylmethionine; THF: Tetrahydrofolate; TK: Thymidine kinase; TP: Thymidine phosphorylase; TS: Thymidylate synthase; UDP: Uridine diphosphate; UKL Uridine kinase; UMP: Uridine monophosphate; UNSases: Uridine nucleosidase; UP: Uridine phosphorylase; Urd: Uridine; UTP: Uridine triphosphate.

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1.2.3 Mechanism of Action

The antitumor activity of 5FU is exerted by its metabolites, mainly FdUMP, FdUTP and

FUTP. At least three mechanisms of action have been identified for 5FU toxicity:

inhibition of thymidylate synthase (TS), incorporation into RNA and also DNA (Figure

3).

FdUMP inhibits TS enzyme through the formation of a stable ternary complex with 5,10-

methylene-tetrahydrofolate (CH2THF), thereby blocking the binding of natural substrate

dUMP (Sommer et al., 1974). The failure of dUMP binding prevents deoxythymidine

monophosphate (dTMP) synthesis, which is important for deoxythymidine triphosphate

(dTTP) synthesis. Disruption of dTTP synthesis leads to deoxynucleotide pool

imbalances, thus interrupting DNA synthesis.

CH2THF, the reduced-folate cofactor essential for the tight binding of FdUMP to TS is

derived from folic acid in diet. Figure 3 (Soong et al., 2005) summarizes the key enzymes

involved in folate metabolism and illustrates the role of CH2THF in TS inhibition. Folate

deficiency has been shown to incur resistance to 5FU therapy (Branda et al., 1998).

Besides disrupting the dTMP and dTTP pools, TS inhibition also leads to accumulation

of dUMP which in turn results in increased levels of dUTP. Together with FdUTP, dUTP can be misincorporated into DNA by DNA polymerase (Longley et al., 2003). There are

two enzymes which prevent the misincoporation of FdUTP and dUTP into DNA: (1)

dUTP pyrophosphatase (dUTPase) which hydrolyses FdUTP to FdUMP and; (2) uracil-

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DNA-glycosylase, a DNA repair enzyme which hydrolyses the fluorouracil-deoxyribose

glycosyl bond of (F)dUMP on DNA, thereby leaving an apyrimidinic site in DNA

(Mauro et al., 1993). The lesion in DNA is recognized by an apurinic/apyrimidinic endonuclease, then resulting in single strand break, which is subsequently repaired. The efficiency of DNA repair mechanism is further weakened by the depletion of dTTP.

Furthermore, 5FU substituted DNA is a weaker substrate for uracil-DNA-glycosylase as compared to uracil.

The incorporation of FUTP into RNA by the action of RNA polymerase also contributes to 5FU cytotoxicity. Various effects that occur as a result of 5FU-RNA incorporation include decrease in net RNA synthesis, disruption of normal RNA processing and function, inhibition of mRNA polyadenylation and alteration of secondary structure of

RNA (Longley et al., 2003).

1.3 Fluorouracil-based therapies

For the past few decades, 5FU has been administered intravenously in various forms of

different schedules, and in both adjuvant and palliative settings. Firstly used as a single

agent, 5FU has an overall response rate (RR) of 10-15% (Advanced Colorectal Cancer

Meta-Analysis Project, 1992; Thirion et al., 2004). In view of the poor clinical outcome,

many strategies have been developed to improve survival benefits and overcome drug

resistance. Few of the early biochemical modulators used to increase 5FU cytotoxicity

includes leucovorin (LV), levamisole, methotrexate (MTX) and interferon. Levamisole

has been found to be effective in treating colon cancer when combined with 5FU

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(Moertel et al., 1990). However, the long term clinical outcome of this combination remains unclear (Cassidy 1994; Chlebowski et al., 1994). The use of MTX and interferon has not been translated into clinical practice today as the results from randomized trials failed to support their clinical benefit (Thirion et al., 2001; Punt et al., 2002). LV, a folinic acid, helps to increase the intracellular reduced folate pools, thus increasing the stability of folate-FdUMP-TS ternary complex. The maintenance of a stable ternary complex is essential for 5FU cytotoxicity. A meta-analysis of 9 randomized clinical trials demonstrated a response rate in favor of 5FU/ LV over 5FU alone (23% vs 11%) but did not improve overall survival (OS) (Advanced Colorectal Cancer Meta-Analysis Project,

1992).

As rapid degradation by DPD greatly affects 5FU bioavailability, the oral Uracil/Ftorafur

(UFT) formulation have been developed to reduce 5FU degradation by saturating DPD activity with uracil. It has been shown that UFT/LV offered similar response rates as

5FU/LV with fewer side effects (Douillard et al., 2002). In the late 1990s, capecitabine, a

5FU oral prodrug, was designed to by-pass DPD degradation in the liver. It has been shown that capecitabine produced higher response rate than 5FU/LV (25% vs 16%), but time to disease progression and survival were not significantly different for both treatment groups (Hoff et al., 2001). Fewer side effects were observed in patients treated with capecitabine, except hand-foot syndrome which develops in 25% of these patients

(van Cutsem et al., 2004).

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Novel combinations of 5FU and its analogs with agents that have different mechanisms

of actions like irinotecan and oxaliplatin further improves clinical outcome in colorectal

cancer. The MOSAIC trial showed that stage II or III colon cancer patients treated with

5FU, LV and oxaliplatin (FOLFOX) achieved better disease-free survival at 3 years as

compared to infusional 5FU/LV therapy (78.2% vs 72.9%), although no difference was

noted for OS (Andre et al., 2004). Randomized trials have shown that irinotecan given in combination with 5FU/LV (FOLFIRI) to be superior to 5FU/LV regimen (Saltz et al.,

2000; Douillard et al., 2000). Saltz and colleagues reported that metastatic colorectal cancer patients treated with irinotecan, 5FU and LV had a significantly higher RR, longer progression-free survival (PFS) and longer OS than those treated with 5FU and LV alone

(Saltz et al., 2000). Similar observations were also reported by Douillard et al. (2000).

Other newer FDA-approved chemotherapeutic agents including monoclonal antibodies like bevacizmab and cetuximab have been used in combination for the treatment of metastatic colorectal cancer. Bevacizumab, targeting the vascular endothelial growth factor, was the first targeted drug to show clinical benefit in metastatic CRC (Fakih

In a combined analysis of 3 randomized studies, metastatic colorectal cancer patients treated with 5FU/LV/bevacizumab had improved RR, longer PFS and OS (Fairooz 2005) compared to 5FU/LV alone. Cetuximab, which targets the epidermal growth factor receptor provide good clinical efficacy when used in combinations with 5-fluorouracil and/or irinotecan (Reynolds et al., 2004). In the CRYSTAL phase III trial, patients who received FOLFIRI and cetuximab had a significantly longer progression-free survival and higher response rate than patients who had FOLFIRI only (van Cutsem et al., 2007).

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1. 4 Molecular determinants of 5FU sensitivity

1. 4.1 Candidate gene approach

The ability to predict 5FU response from its metabolism and cytotoxic mechanisms was

first demonstrated using candidate gene approaches. Early studies have associated

resistance to 5FU with the loss in activity of uridine kinase (Reichard et al., 1962),

uridine phosphorylase (Goldberg et al., 1966) and pyrimidine phosphoribosyltransferase

(Kasbekar et al., 1963), leading to changes in 5FU metabolite levels. Soon after TS

inhibition by 5-FdUMP was identified as an important determinant for 5FU cytotoxicity,

leading the degree of TS inhibition and TS levels to be deemed as potential indicators for

response. Many studies have demonstrated the relationship between elevated TS

expression/activity and 5FU resistance (Berger et al., 1985; Clark et al., 1987; Johnston

et al., 1992; Johnston et al., 1995). The general consensus associated low TS

expression/activity with improved response to 5FU (Johnston et al., 1995; Leichman et

al., 1995; Bathe et al., 1999; Wong et al., 2001; Fernandez-Contreras et al., 2006).

However, some reports have shown no relationship between these parameters (Findlay et

al., 1997; Allegra et al., 2003; Johnston et al., 2003) or reverse association, i.e. high TS expression provides disease-free survival benefit (Berglund et al., 2002; Edler et al.,

2002). Polymorphism studies have revealed the presence of variable number tandemly

repeated polymorphic regulatory sequences (VNTR) in the TS enhancer region (TSER) correlates with TS expression, with triple repeat (3R) having higher TS expression than

double repeat (2R) (Horie et al., 1995; Pullarkat et al., 2001). Clinical studies later showed that homozygous 3R/3R patients had a lower clinical response as compared to

18 LITERATURE REVIEW others (Pullarkat et al., 2001; Etienne et al., 2002), or did not obtain a survival benefit from 5FU (Iacopetta et al., 2001). In the second repeat within the VNTR, the presence of a functional G>C single nucleotide polymorphism (SNP) at bp 58 was associated with reduced TS expression (Kawakami et al., 2003). Another TS polymorphism consisting of a 6 bp deletion at bp 1494 in the 3’-untraslated region is associated with reduced TS expression in colorectal tumors (Ulrich et al., 2000).

The catabolic enzyme, DPD, is another important determinant for responsiveness to 5FU since its level and activity greatly affects 5FU availability. DPD activity is inversely associated with 5FU sensitivity: 5FU resistance being observed in patients having high

DPD level/activity (Ishikawa et al., 1999) whilst patients with DPD deficiency suffering from severe toxicities (Tuchman et al., 1985; Milano et al., 1999). DPD deficiency has been linked with a DPD exon 14 deletion variant and thus identified as a potential indicator of 5FU response (Meinsma et al., 1995). Nevertheless, there are few studies which failed to observe such inverse relationship between DPD expression/activity and

5FU response (Jakob et al., 2005; Kinoshita et al., 2007; Yamada et al., 2008).

Other genes or proteins involved in the metabolic pathway identified as molecular markers for 5FU response includes TP, OPRT and TK. The role of TP in determining

5FU sensitivity is unclear. Metzger et al. reported that high TP level in colorectal tumors is associated with nonresponse to 5FU (Metzger et al., 1999) but opposite findings have also been reported (Schwartz et al., 1995; Yasuno et al., 2005). Colorectal cancer patients with high OPRT activity were reported to have better 5FU efficacy (Sakamoto et

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al., 2007) and better disease-free and overall survival when treated with 5FU based chemotherapy (Ochiai et al., 2006). However, no relationship between OPRT level and

5FU efficacy was observed by Ishida et al. (2005). Decreased TK activity has been shown to be associated with 5FU insensitivity (van der Wilt et al., 1998).

As CH2THF is crucial in maintaining a stable ternary complex with TS and 5-FdUMP,

folate metabolism becomes the focus for 5FU response prediction. Intracellular CH2THF concentration is mainly controlled by MTHFR. Several polymorphisms are commonly found in the MTHFR gene, with 677C>T polymorphism in exon 4 and 1298A>C polymorphism in exon 7 being most commonly linked with altered TS activity (Etienne et al., 2007). The 677C>T polymorphism is frequently associated with enhanced 5FU activity due to increased CH2THF levels (Cohen et al., 2003; Etienne et al., 2004).

Higher 5FU sensitivity was observed in mutated A1298C variants of MTHFR (Etienne et

al., 2004).

A promising molecular marker related to the downstream mechanisms of 5FU is p53.

Loss of p53 function may contribute to 5FU resistance as p53-deficient cells fail to undergo p53-mediated apoptosis (Longley et al., 2002). Mutations in p53 occur in more than 50% of colorectal tumors, often resulting in p53 overexpression (Brett et al., 1996).

Tumor overexpression of p53 was associated with lower response rate and higher rate of

deterioration radiologically and clinically. Others have also provided evidence for the correlation between mutations/deletions of p53 with 5FU resistance (Lowe et al., 1993;

Bunz et al., 1999; Liang et al., 2002).

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Some studies have investigated these determinants in minimal combinations, as summarized in Table 1.

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Table 1: Overview of studies on gene/protein expression levels in relation to 5FU chemosensitivity.

Gene/protein Sample Outcome References RNA UP, TP 8 colorectal UP and TP expression were Mader et al., cancer cell predictive for 5FU cytotoxicity in 1997 lines 5/7 cell lines DPD, TP, TS 33 colorectal Colorectal tumors responding to Salonga et al., tumors 5FU had low DPD, TS and TP 2000 DPD, TS 5 xenografts, Low TS and DPD was associated Fujiwara et al., 85 gastric with 5FU sensitivity 2002 tumors DPD, TS 37 colorectal Patients with low TS and DPD had Ichikawa et al., tumors better response rate and longer 2003 median survival time DPD, OPRT, 37 colorectal UFT-responding tumors had a Ichikawa et al., UP tumors statistically higher OPRT/DPD 2003 ratio survived longer than those with low ratio DPD, TS 23 colorectal Increase in TS in 5FU-sensitive Inoue et al., tumors colorectal cancers 2005 DPD, TP, TS 4 colorectal Higher sensitivity to 5FU of cell Amatori et al., cancer cell lines with the lowest TS 2006 lines expression DPD, OPRT, HCT116 DPD and TP levels were inversely Okumura et al., TP, TS, UP xenotransplant correlated with 5FU sensitivity 2006 DPD, OPRT, 49 colorectal DPD levels and OPRT/DPD ratio Kinoshita et al., TP, TS tumors is weakly correlated with 5FU 2007 sensitivity DPD, OPRT, 103 colorectal Patients with high OPRT had Yamada et al., TP, TS tumors longer disease-free and overall 2008 survival

Protein OPRT, UP NUGC- 5FU resistance was due to reduced Inaba et al., 3/5FU/L cell activities of 5FU anabolizing 1996 line enzymes DPD, TP, TS 41 colon Intratumoral TS protein expression Cascinu et al., tumors was inversely correlated with 1999 response to chemotherapy DPD, TP, TS 40 rectal High TS and low TP was Jakob et al., tumors associated with nonresponse to 2005 5FU

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DPD, TP, TS 62 colorectal Patients with low TS and DPD had Ciaparrone et tumors longer disease-free survival and al., 2006 overall survival DPD, TP, TS 967 colorectal Low TS and DPD were prognostic Soong et al., tumors for worse outcome in patients 2008 treated by surgery alone; low DPD, TP and TS were prognostics for better outcome in patients treated with 5FU p53, TS 122 colorectal No relationship between TS and Berglund et al., tumors p53 expression with 5FU response 2002 p53, TS 706 colorectal None of the markers could be used Allegra et al., tumors to predict benefit from 5FU 2003 treatment p53, TS 967 colorectal None of the markers was Popat et al., cancer significantly associated with 2006 survival

23 LITERATURE REVIEW

1.4.2 Genome-wide approach

As 5FU metabolism is a complex event and its biochemical action involves multiple

targets, multiple genes involved in pyrimidine metabolism and activity may be associated

with 5FU sensitivity. Approximately 50 genes identified from Kyoto Encyclopedia of

Genes and Genomes (KEGG) pathway database are found to be involved in the normal pyrimidine metabolism which is important for thymidine and cytidine synthesis

(http://www.genome.ad.jp/kegg/pathway.html). Insufficient folate supply due to impaired folate metabolism may also affect 5FU action as MTHF is crucial for stabilizing the tertiary complex formed with 5FU and thymidylate synthase. Also, there is growing body of evidence which suggests the involvement of many other genes/proteins in the transport

mechanism, stress response and DNA repair in influencing 5FU sensitivity.

The role of efflux transporters in determining FP response has been investigated by Guo

and coworkers in an in-vitro transport assay (Guo et al., 2003). Using MRP8-transfected

LLC-PK1 cells, the investigators demonstrated that MRP8 (ABCC11) functions as a

resistance factor for 5FU by mediating the efflux of 5-FdUMP, the intracellular cytotoxic

metabolite of 5FU. The role of MRP8 in 5FU resistance is also confirmed by Oguri and

colleagues (Oguri et al., 2007). The high degree of structural resemblance between

MRP8 and MRP9 (Tammur et al., 2001, Yabuuchi et al., 2001) suggests that MRP9 may

participate in 5FU efflux. MRP5-transfected cells were found to be resistant to 5FU (Pratt

et al., 2005). However, a review by Borst and coworkers on MRP4 and MRP5 found no

evidence that the transport of nucleotide analogs results in resistance in vivo (Borst et al.,

24 LITERATURE REVIEW

2007). Further investigation on the role of 5FU transport in clinical resistance to 5FU is

clearly needed.

Overexpression of DNA repair genes gives rise to tumor resistance to DNA-damaging agents (Yu et al., 2006). Deficiency in DNA mismatch repair (MMR) genes MLH1,

MSH2, MSH6 and PMS2 was shown to have a negative impact on overall survival in

CRC, suggesting that MMR status may contribute to decision making regarding treatment approaches (Gologan et al., 2005). Ide et al. has reported an association between low

MLH1 expression with better disease-free survival rate in 94 colorectal cancer patients

(Ide et al., 2008). Cell lines deficient of Smug-1, a DNA glycosylase in base excision

repair mechanism, were found to be highly sensitive to 5FU cytotoxicity. Figure 4 shows

the genes involved in the DNA repair mechanisms and stress response in relation to 5FU

effects. However to date, their influence has not been well characterized.

25 LITERATURE REVIEW

5FU DNA damage PARP1 & 2

p53 ATR/ATM DNA repair UNG, SMUG1, XRCC1, HMGB1, MLH1, MSH2, MSH6, PMS2, Exo1

CHK1 CHK2 Apoptosis Bcl-2

p53

Cell cycle inhibition

Figure 4: Activation of DNA repair mechanisms and stress response in relation to 5FU effects. 5FU-induced DNA damage leads to activation of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia related (ATR). ATM phosphorylates Chk2 while ATR phosphorylates Chk1. Both Chk2 and Chk1 phosphorylates p53, leading to cell cycle arrest, apoptosis and enhanced DNA repair. Poly(ADP-ribose) polymerase (PARP)-1 and PARP-2 proteins are also play an important role in DNA repair.

26 LITERATURE REVIEW

With the advent of microarray technology, gene expression profiling has been applied to identifying determinants of 5FU response at a genome/proteome-wide level (Table 2).

Several model systems have been investigated for 5FU-based therapies in the treatment of colorectal cancer, including the cancer cell lines, tumor xenografts and primary as reviewed by Mariadason et al. (Mariadason et al., 2004). These studies analyzed basal gene expression profiles and identified determinants for pre-treatment drug response.

Genome-wide expression analysis has also been applied to identifying determinants from after-drug treatment by identifying genes that were altered following drug exposure (De

Angelis et al., 2006; Boyer et al., 2006).

27 LITERATURE REVIEW

Table 2: Summary of genome-wide and proteomic studies to identify determinants for 5FU-based chemosensitivities.

Sample Compounds Outcome References RNA 60 human 1400 Negative correlation between DPYD Scherf et al., cancer cell compounds expression and 5FU potency 2000 lines including 5FU and analogues 39 human 55 anticancer Survivin was highly expressed in 5FU Dan et al., cancer cell agents resistant cells while members of the 2002 lines aldo-keto reductase and aldehyde dehydrogenase families and galectin 4 were highly expressed in 5FU responsive cells 85 human 5FU 20 genes of the highest correlation to Zembutsu et cancer 5FU chemosensitivity were not al., 2002 xenografts previously reported 30 colon 5FU and Expression of 50 genes not previously Mariadason cancer cell camptothecin reported were correlated with 5FU- et al., 2003 lines induced-apoptosis and significantly predicted response to 5FU 23 rectal 5FU and Expression levels of 54 genes not Ghadimi et cancer radiation previously reported were significantly al., 2005 patients different between 5FU responders and non-responders MIP101 5FU Low SPARC expression in resistant Tai et al., colorectal cell line 2005 cancer cells 3 colorectal 5FU 81 genes were identified to be related Shimizu et cancer and to 5FU sensitivity al., 2005 corresponding resistant sublines 22 colorectal 5FU and Expression levels of TNFRSF1B, Matsuyama cancer analogues SLC35F5 and OPRT were significantly et al., 2006 patients different in 5FU responders and non- responders 30 human 5FU Tumors with low DPYD expression Ooyama et cancer analogues had high sensitivity to 5FU based al., 2006 xenografts therapy, except S-1

Protein Colon cancer 5FU Metabotropic glutamate receptor 4 was Yoo et al., cell lines overexpressed in 5FU resistant cells 2004

28 LITERATURE REVIEW

Colon cancer 5FU Lower expression of the alpha subunit Shin et al., cell lines of mitochondrial F(1)F(0)-ATP 2005 synthase in 5FU-resistant cells SW480 cells 5FU After 5FU treatment, cyclophilin A, Wong et al., cytokeratin (CK) 19, CK8, ras-related 2008 nuclear protein, heat shock protein (hsp) 27 and peroxiredoxin 6 (Prx 6) were upregulated whereas hsp60, CK18, CK9, carbamoylphosphate synthetase I, alpha-enolase, hsp70, nm23 and beta-actin were down- regulated

29 LITERATURE REVIEW

1.5 Study approaches

In recognizing the value of identifying determinants for 5FU pre-treatment response prediction and the lack of promising markers for clinical implementation, a multiple-gene approach based on real-time quantitative RT-PCR was employed in this study, focusing on genes from fluoropyridimine- and closely related cellular pathways. This pathway based analysis of best characterized genes enables us to better investigate the combined effects of gene expression relevant to 5FU mechanism as well as limits the potential genes of interest and avoids too many irrelevant, false positive or negative markers.

Furthermore, genome-wide analyses require a reasonably large sample size to accurately determine the predictive value of a gene and this requirement is often unmet.

In order to identify factors robustly related to 5FU sensitivity, we have investigated cell lines, tumor xenografts and clinical samples. Cell lines and tumor xenografts allows assessment of tumor-directed effects, which is less achievable in humans. Furthermore, it was reported that antitumor effects in human tumor xenograft models correlate well with clinical effects (Tashiro et al., 1989).

Examination on regulation patterns of 5FU pathway genes in response to 5FU timepoint treatment in resistant and sensitive cell lines was used as another approach to identifying potential determinants for 5FU sensitivity. RNAi technology was used to validate the determinants identified from gene expression studies of pre- and post-drug treatment.

30 LITERATURE REVIEW

1.5.1 Quantification of mRNA expression using real-time PCR

Quantitative RT-PCR (Q-PCR) is sensible method for quantifying gene expression levels and could be realized with small amount of tissue. However, quantification of many genes laborious as primers and probes specific for each gene need to be individually designed. Although high-throughput gene expression profiling with microarray is a powerful alternative and allows more genes to be investigated, it requires more sample tissues and there is a higher probability of having false positive or negative markers that still require validation with Q-PCR.

The introduction of low-density array (LDA) based on the principle of TaqMan Q-PCR has made the quantification of multiple genes easier and faster than before, requiring much less template cDNA than conventional Q-PCR. The chemistry employed in the real-time Q-PCR is to use a Taq polymerase that has a 5′exonuclease activity. The principle of the process was first described by Holland et al. (Holland et al., 1991) and it was utilized with fluorescent and quencher dyes by Livak et al. (Livak et al., 1995). In the TaqMan probe-based approach, the probe contains one fluorophore (6-carboxy- fluoroscein) at its 5′ terminus and a quencher (6-carboxytetramethyl- rhodamine;

TAMRA) at its 3′ terminus. When in close proximity, the two dyes formed a quenched system and no fluorescence was observed. However, when the probe hybridized to its template, the 5′exonuclease activity of the Taq polymerase would hydrolyse the probe during PCR amplification. Once the fluorophores were separated from the quencher, the emissions of the fluorophore were no longer being quenched and fluorescence was detected (Figure 5). The cycle number at which the real-time fluorescence signals is

31 LITERATURE REVIEW detected represents the progression of the PCR reaction and is used as an indicator of successful target amplification (Wilhelm et al., 2001). This cycle number, also known as

the threshold cycle (CT) is defined as the PCR cycle in which the increase in fluorescence generated by the accumulating PCR amplicons exceeds the mean baseline fluorescence and is proportional to the number of target copies present in the sample (Gibson et al.,

1996; Jung et al., 2000). Thus, the development of real-time PCR also allows true quantitation of the target nucleic acids. Quantitation of real-time PCR can be performed in two ways - relative quantitation and absolute quantitation. Relative quantitation describes changes in the amount of a target sequence compared with its level in a related matrix.

Generally, relative quantitation provides sufficient information and is simpler to develop.

On the other hand, absolute quantitation states the exact number of nucleic acid targets present in the sample, based on the CT of the sample which can then be compared with similar data collected from a series of standards by the calculation of a standard curve

(Freeman et al., 1999).

LDA is a 384-well micro fluidic card that enables 384 simultaneous real-time PCR reactions, determining the expression of multiple, user-defined gene clusters (Figure 6).

This array allows for 1 to 8 samples to be run in parallel against 12 to 384 gene expression targets that are pre-loaded into each well on the card. The reproducibility of

LDA have also been demonstrated and results were compatible with the conventional single gene based Q-PCR (Abruzzo et al., 2005; Steg et al., 2006; Lu et al., 2007).

32 LITERATURE REVIEW

Figure 5: Diagram illustrating the concepts for the TaqMan probe assay. When in close proximity, the reporter dye (R) and the quencher (Q) formed a quenched system and no fluorescence is observed. Upon exonuclease digestion during the PCR reaction, the fluorigenic dye is released from the close proximity of the quencher dye and is hence able to fluoresce and be detected by the fluorimeter (Adapted from Applied Biosystems, USA).

33 LITERATURE REVIEW

Figure 6: TaqMan® LDA. (A) Plate layout; (B) LDA format of 96 genes (Format 96a).

34 LITERATURE REVIEW

1.5.2 Regulation patterns of genes in response to 5FU treatment

Tumor resistance to 5FU may manifest at the start of chemotherapy or develop during treatment. Alteration in gene regulation after exposure to 5FU could be the key to 5FU resistance. It has been shown that 5FU treatment for 24 hours in resistant cell lines

resulted in p53 accumulation, up-regulation of p53-target genes on DNA damage

response, cell cycle regulation and apoptotic pathway (De Angelis et al., 2006). Another

gene expression analysis identified genes like PDF, CCNG2, AVEN, SSAT and JAG1 to be consitutively dysregulated in 5FU resistant cells and transiently altered following 5FU exposure for 0, 6, 12 and 24 hours (Boyer et al., 2006).

Multi-timepoint temporal gene expression analysis following 5FU treatment could offer insights into understanding the 5FU effects on gene regulation and identifying sets of genes responsible for these changes. By comparing the regulation patterns of genes in

5FU resistant and sensitive samples, we could possibly identify genes that govern 5FU resistance if there is distinctive difference in their respective regulation patterns.

1.5.3 RNA interference (RNAi)

RNA interference (RNAi) is a defense strategy which was first described in

Caenorhabditis elegans (Fire et al., 1998). It is a sequence-specific, post-transcriptional gene silencing process initiated by double-stranded RNA (dsRNA) molecules (Fire et al.,

1998; Figure 7). These dsRNAs are first cleaved by Dicer to produce functional siRNAs

(19-23 nucleotides long). Subsequently, these siRNAs are loaded into the RNA-induced silencing complex (RISC), which contains helicase that unwinds the duplex (Nykanen et

35 LITERATURE REVIEW

al., 2001). The siRNA strands direct the RISC to the target mRNA and cleave the

complementary mRNA. The mRNA is rapidly degraded, resulting in reduction or

blocking of protein expression.

RNAi technology has been widely applied in cancer research and treatment (Martin et al.,

2007, Bruserud et al., 2007). There are many advantages of using siRNA over other

methods, such as chemical inhibitors and dominant negative mutants, to knockdown gene expression. First of all, virtually any gene can be silenced by siRNA which is highly specific. On the contrary, there are not many chemical inhibitors available for gene silencing and most of them are non-specific. Direct comparison of an optimized phosphorothioate-modified ASO with a siRNA directed against the same target mRNA site found that the siRNA was approximately 100 to 1000-fold more efficient (Bertrand et al., 2002). Second, the synthesis of siRNA is more straight-forward and less time consuming than constructing dominant negative mutants. Lastly, siRNA silencing is more specific and has longer sustained silencing than chemical inhibitors and dominant negative mutants (Bertrand et al., 2002). This could be due to the protection of the siRNAs from intracellular degradation by its incorporation into the RISC.

Although siRNA is highly specific, there are off-target effects which need to be minimized in order to have a meaningful siRNA application. Off-target effects can be divided into two types of responses: (a) the induction of nonsequence-specific silencing pathways via Type 1 Interferon pathway and (b) the silencing of targets that have partial complementarity to the siRNA.

36 LITERATURE REVIEW

Figure 7: The RNA Interference pathway. Long double-stranded RNA (dsRNA) is cleaved by the RNase III family member, Dicer, into small interfering RNAs (siRNAs) in an ATP-dependent reaction. These siRNAs are then incorporated into the RNA-induced silencing complex (RISC). The single-stranded antisense strand guides RISC to messenger RNA that has a complementary sequence, which results in the endonucleolytic cleavage of the target mRNA.

37 LITERATURE REVIEW

1.6 Scope of Study

The overall aim of this study is to identify and validate determinants of 5FU sensitivity based on a comprehensive examination of genes involved in its transport, metabolism and activity (Figure 8). The specific aims are:

1. To rank genes (or combinations thereof) involved in 5FU transport, metabolism and activity according to their association with 5FU sensitivity in cell lines, xenografts and human tissue samples

Hypothesis: Expression levels of genes involved in 5FU transport, metabolism and activity influence overall cellular 5FU sensitivity. By ranking the genes (or combinations thereof) according to strength and consistency of association with 5FU sensitivity in cell lines, xenografts and human tissue samples, the strongest determinants of 5FU sensitivity can be prioritized for further investigation.

2. To characterize how modulation of expression of prioritized genes affects 5FU sensitivity in vitro

Hypothesis: Modulation of the expression of genes that have a determining role in 5FU sensitivity will change 5FU sensitivity and reveal mechanisms for this change.

38 LITERATURE REVIEW

Figure 8: Genes analyzed in this study for their roles in transport and metabolism of 5FU and its downstream events.

39 LITERATURE REVIEW

5FU: 5-fluorouracil; 5-Fo-THF: 5-Formimino-THF; 5-Fm-THF: 5-formyl-THF; 10-Fm-THF: 10-formyl- THF; 5-met-THF: 5-methyl-THF; 5,10-met-THF: 5,10-methenyl-THF; 5,10-metn-THF: 5,10-methylene- THF; (F): Fluoro; ABCC4: ATP-binding cassette, sub-family C, member 4; ABCC5: ATP-binding cassette, sub-family C, member 5; ABCC11: ATP-binding cassette, sub-family C, member 11; ABCC12: ATP- binding cassette, sub-family C, member 12; AK3: Adenylate kinase 3; ALDH1L1: Aldehyde dehydrogenase I family, member L1; AMT: Aminomethyltransferase; ATIC: 5’-aminoimidazole-4- carboxamide ribonucleotide formyltransferase; ATR: Ataxia telangiectasia and Rad3 related; ATM: Ataxia telangiectasia mutated homolog; BAL: β-alanine; bcl-2: B-cell CLL/lymphoma 2; CANT1: Calcium activated nucleotidase I; CDA: Cytidine deaminase; CHEK1: CHK1 checkpoint homolog; CHEK2: CHK2 checkpoint homolog; CMP: Cytidine monophosphate CDP: Cytidine diphosphate CTP: Cytidine triphosphate; CTPS: CTP synthase; Cyd: Cytidine; DHF: Dihydrofolate; DHFR: DHF reductase; DPYS: Dihydropyrimidinase; DPYD: Dihydropyrimidine dehydrogenase; dUDP: Deoxyuridine diphosphate; dUMP: Deoxyuridine monophosphate; DUT: dUTP pyrophosphatase; dUTP: Deoxyuridine triphosphate; dUrd: Deoxyuridine; dTMP: Deoxyuridine triphosphate; DTYMK: Deoxythymidylate kinase; dUMP: Deoxyuridine monophosphate; ENTPD: Ectonucleoside triphosphate diphosphohydrolase; Exo1: Exonuclease 1; FTCD: Formiminotransferase cyclodeaminase; GART: Phosphoribosylglycinamide formyltransferase; HMGB1: High-mobility group box 1; MLH1: mutL homolog 1; MSH2: mutS homolog 2; MSH6: mutS homolog 6; MTFMT: Mitochondrial methionyl-tRNA formyltransferase; MTHFD: Methylenetetrahydrofolate dehydrogenase; MTHFD1L: Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) I-like; MTHFR: 5,10-methylenetetrahydrofolate reductase (NADPH); MTHFS: 5,10- methenyltetrahydrofolate synthetase; MTR: 5-methyltetrahydrofolate-homocysteine methyltransferase; NME: Non-metastatic cells; NT: Nucleotidase; NUDT2: Nudix (nucleoside diphosphate linked moiety X)- type motif 2; PARP-1: Poly (ADP-ribose) polymerase 1; PARP-2: Poly (ADP-ribose) polymerase 2; Postmeiotic segregation increased 2; RRM: Ribonucleotide reductase; SHMT: Serine hydroxymethyltransferase; SLC28A1: Solute carrier family 28, member 1; SLC28A2: Solute carrier family 28, member 2; SLC28A3: Solute carrier family 28, member 3; SLC29A1: Solute carrier family 29, member 1; SLC29A2: Solute carrier family 29, member 2; SLC29A3: Solute carrier family 29, member 3; SLC29A4: Solute carrier family 29, member 4; SMUG1: Single-strand-selective monofunctional uracil- DNA glycosylase 1; THF: Tetrahydrofolate; TK: Thymidine kinase; TP: Thymidine phosphorylase; TP53: Tumor protein p53; TS: Thymidylate synthase; TXNRD: Thioredoxin reductase; TYMP: Thymidine phosphorylase; UCK: Uridine-cytidine kinase; Urd: Uridine; UDP: Uridine diphosphate; UMP: Uridine monophosphate; UMPS: Uridine monophosphate synthetase; UNG: Uracil-DNA glycosylase; UTP: Uridine triphosphate; UPB1: β-ureidopropinase; UppppU: P1,P4-Bis(5'-uridyl) tetraphosphate; XRCC1: X- ray repair complementing defective repair in Chinese hamster cells 1

40 MATERIALS AND METHODS

CHAPTER 2: MATERIALS AND METHODS

2.1 Cell lines

Twenty one colorectal cancer cell lines of human origin were investigated. Caco-2,

Colo201, Colo205, Colo320DM, DLD-1, HCT116, HT29, LoVo, LS513, LS174T, RKO,

SW403, SW480, SW620, SW837 and WiDR were obtained from American Type Culture

Collection (ATCC, Rockville, MD). CCK-81, CoCM-1, HCC56, OUMS23 and RCM1

were obtained from Japanese Collection of Research Bioresources (Osaka, Japan). Cells

were routinely cultured in DMEM or RPMI1740 supplemented with 10% fetal bovine

serum (Invitrogen, Madison), 50,000 units penicillin and 50mg streptomycin (Sigma, St

Louis, MO) at 37°C in a humidified atmosphere containing 5% CO2.

2.2 Cell sensitivity analysis

Cells were seeded in 100 µl medium onto 96-well microtitration plates (Nunc, Rochester,

NY) with an initial cell density of 5000 per well. After 24 hours, 100 µl of medium

containing 5FU (Sigma) with graded concentrations ranging from 0.01 µM to 1000 µM

was added to the wells and cells were incubated for another 48 hours. Cytotoxicity was

assessed by methyl thiazolyltetrazolium (MTT)-based CellTiter 96® Non-Radioactive

Cell Proliferation assay (Promega, Madison, WI) according to manufacturer’s

instructions and analyzed at 570 nm. All investigations were performed in triplicates. IC50 values were determined from curves plotted according to the dose-response function of

GraphPad Prism software (GraphPad Software Inc., San Diego, CA).

41 MATERIALS AND METHODS

2.3 Xenografts

Fourteen colon carcinoma xenografts were kindly provided by Dr. Iduna Fichtner at the

Max-Delbrück-Center for Molecular Medicine Berlin-Buch (Berlin, Germany). The

antitumor activity of 5FU in the 14 xenografts has been previously characterized

(Fitchner et al., 2004). Non-response to 5FU was defined as tumor shrinkage of less than

50% after drug treatment.

2.4 Primary Tumors

Total RNA and clinicopathological data of 25 stage II/III colorectal frozen tumors of patients treated with 5FU-based chemotherapy was kindly provided by NUH-NUS Tissue

Repository, Singapore under protocols approved by the Institutional Review Board,

National University of Singapore. Tumor RNA provided was extracted from whole

human tissue of tumor content ≥50% using the TRI reagent (MRC, Cincinnati, OH, USA)

according to manufacturer’s instructions. Twelve patients were females and 13 were

males, with ages ranging from 45 to 82 year-old (median 62). Four cases were stage II

disease. Treatment consisted of standard 5FU and leucovorin administration for at least 1

cycle after tumor resection. Lack of response to 5FU treatment is defined as recurrence

within 5 years.

2.5 Gene expression analysis

A total of 92 genes involved in the pyrimidine and folate metabolism were identified

from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database

(http://www.genome.ad.jp/kegg/pathway.html), whereas those in the transport and

42 MATERIALS AND METHODS downstream mechanisms were obtained from literature review (Table 3). Four housekeeping genes (18s, ACTB, B2M and GAPDH) were also included into the assay.

43 MATERIALS AND METHODS

Table 3: List of 96 genes selected for LDA analysis.

Gene symbol Gene name Alias GenBank References Accession No.

Housekeeping genes 18S 18S ribosomal RNA - X03205.1 Banda et al., 2008 ACTB Actin, beta - NM_001101.3 Banda et al., 2008 B2M Beta-2-microglobulin - NM_004048.2 Banda et al., 2008 GAPDH Glyceraldehyde-3-phosphate - NM_002046.3 Banda et al., dehydrogenase 2008

Transporters ABCC4 ATP-binding cassette, sub-family C MRP4 NM_005845.3 Borst et al., (CFTR/MRP), member 4 2007 ABCC5 ATP-binding cassette, sub-family C MRP5 NM_005688.2 Pratt et al., (CFTR/MRP), member 5 2005 ABCC11 ATP-binding cassette, sub-family C MRP8 NM_032583.3 Guo et al., (CFTR/MRP), member 11 2003 ABCC12 ATP-binding cassette protein C12; ATP- MRP9 NM_033226.2 Yabuuchi et binding cassette transporter sub-family C al., 2001 member 12 SLC28A1 Solute carrier family 28 (sodium-coupled CNT1 NM_004213.3 Griffith and nucleoside transporter), member 1 Jarvis, 1996 SLC28A2 Solute carrier family 28 (sodium-coupled CNT2 NM_004212.2 Griffith and nucleoside transporter), member 2 Jarvis, 1996 SLC28A3 Solute carrier family 28 (sodium-coupled CNT3 NM_022127.1 Ritzel et al., nucleoside transporter), member 3 2001 SLC29A1 Solute carrier family 29 (nucleoside ENT1 NM_004955.2 Griffith and transporters), member 1 Jarvis, 1996 SLC29A2 Solute carrier family 29 (nucleoside ENT2 NM_001532.2 Griffith and transporters), member 2 Jarvis, 1996 SLC29A3 Solute carrier family 29 (nucleoside ENT3 NM_018344.4 Baldwin et transporters), member 3 al., 2005 SLC29A4 Solute carrier family 29 (nucleoside ENT4 NM_153247.2 transporters), member 4

Metabolism (pyrimidine) AK3 Adenylate kinase 3 AK6 NM_016282.2 Kegg (pyrimidine) CANT1 Calcium activated nucleotidase I ENTPD8 NM_138793.2 Kegg (pyrimidine) CDA Cytidine deaminase CDD NM_001785.2 Kegg (pyrimidine) CES1 Carboxylesterase 1 ACAT NM_001266.4 Kegg (pyrimidine) CES2 Carboxylesterase 2 CE-2 NM_003869.4 Kegg (pyrimidine) CTPS CTP synthase - NM_001905.2 Kegg (pyrimidine)

44 MATERIALS AND METHODS

CTPS2 CTP synthase II - NM_019857.3 Kegg (pyrimidine) DPYD Dihydropyrimidine dehydrogenase DPD NM_000110.3 Kegg (pyrimidine) DPYS Dihydropyrimidinase DHPase NM_001385.2 Kegg (pyrimidine) DTYMK Deoxythymidylate kinase TYMK NM_012145.2 Kegg (pyrimidine) DUT dUTP pyrophosphatase dUTPase NM_001948.3 Kegg (pyrimidine) ECGF1 Thymidine phosphorylase NTPDase- NM_012145.2 Kegg 1 (pyrimidine) ENTPD1 Ectonucleoside triphosphate NTPDase- NM_001776.4 Kegg diphosphohydrolase 1 3 (pyrimidine) ENTPD3 Ectonucleoside triphosphate NTPDase- NM_001248.2 Kegg diphosphohydrolase 3 4 (pyrimidine) ENTPD4 Ectonucleoside triphosphate NTPDase- NM_004901.2 Kegg diphosphohydrolase 4 5 (pyrimidine) ENTPD5 Ectonucleoside triphosphate NTPDase- NM_001249.1 Kegg diphosphohydrolase 5 6 (pyrimidine) ENTPD6 Ectonucleoside triphosphate ITPase NM_001247.2 Kegg diphosphohydrolase 6 (pyrimidine) ITPA Inosine triphosphatase NM23-H1 NM_181493.1 Kegg (pyrimidine) NME1 Non-metastatic cells 1 NM23-H2 NM_000269.2 Kegg (pyrimidine) NME2 Non-metastatic cells 2 NM23-H3 NM_002512.2 Kegg (pyrimidine) NME3 Non-metastatic cells 3 NM23-H4 NM_002513.2 Kegg (pyrimidine) NME4 Non-metastatic cells 4 NM23-H5 NM_005009.2 Kegg (pyrimidine) NME5 Non-metastatic cells 5 NM23-H6 NM_003551.2 Kegg (pyrimidine) NME6 Non-metastatic cells 6 NM23-H7 NM_005793.3 Kegg (pyrimidine) NME7 Non-metastatic cells 7 PNP NM_013330.3 Kegg (pyrimidine) NP Nucleoside phosphorylase DNT NM_000270.2 Kegg (pyrimidine) NT5C 5’,3’-nucleotidase, cytosolic CN1A NM_014595.1 Kegg (pyrimidine) NT5C1A 5’-nucleotidase, cytosolic IA CN1B NM_032526.1 Kegg (pyrimidine) NT5C1B 5’-nucleotidase, cytosolic IB GMP NM_033253.2 Kegg (pyrimidine) NT5C2 5’-nucleotidase, cytosolic II UMPH NM_012229.2 Kegg (pyrimidine) NT5C3 5’-nucleotidase, cytosolic III CD73 NM_016489.1 Kegg 1 (pyrimidine) NT5E 5’-nucleotidase, ecto (CD73) dNT-2 NM_002526.1 Kegg (pyrimidine) NT5M 5’,3’-nucleotidase, mitochondrial APAH1 NM_020201.3 Kegg (pyrimidine) NUDT2 Nudix (nucleoside diphosphate linked PNPASE NM_001161.3 Kegg moiety X)-type motif 2 (pyrimidine)

45 MATERIALS AND METHODS

PNPT1 Polyribonucleotide nucleotidyltransferase R1 NM_033109.2 Kegg 1 (pyrimidine) RRM1 Ribonucleotide reductase M1 polypeptide R2 NM_001033.3 Kegg (pyrimidine) RRM2 Ribonucleotide reductase M2 polypeptide p53R2 NM_001034.1 Kegg (pyrimidine) RRM2B Ribonucleotide reductase M2 B - NM_015713.3 Kegg (pyrimidine) TK1 Thymidine kinase 1 - NM_003258.4 Kegg (pyrimidine) TK2 Thymidine kinase 2 TR1 NM_004614.3 Kegg (pyrimidine) TXNRD1 Thioredoxin reductase 2 TR3 NM_003330.2 Kegg (pyrimidine) TXNRD2 Thioredoxin reductase 2 TP NM_006440.3 Kegg (pyrimidine) TYMS Thymidylate synthase TS NM_001071.1 Kegg (pyrimidine) UCK1 Uridine-cytidine kinase 1 URK1 NM_031432.1 Kegg (pyrimidine) UCK2 Uridine-cytidine kinase 2 UK NM_012474.3 Kegg (pyrimidine) UMPS Uridine monophosphate synthetase OPRT NM_000373.2 Kegg (pyrimidine) UPB1 Ureidopropinase, beta BUP1 NM_016327.2 Kegg (pyrimidine) UPP1 Uridine phosphorylase 1 UP NM_181597.1 Kegg (pyrimidine) UPP2 Uridine phosphorylase 2 UP2 NM_173355.2 Kegg (pyrimidine)

Metabolism (folate) ALDH1L1 Aldehyde dehydrogenase I family, FTHFD NM_012190.2 Kegg member L1 (folate) AMT Aminomethyltransferase GCE NM_000481.2 Kegg (folate) ATIC 5’-aminoimidazole-4-carboxamide AICAR NM_004044.4 Kegg ribonucleotide formyltransferase (folate) DHFR Dihydrofolate reductase - NM_000791.3 Kegg (folate) FTCD Formiminotransferase cyclodeaminase LCHC1 NM_006657.2 Kegg (folate) GART Phosphoribosylglycinamide PRGS NM_000819.3 Kegg formyltransferase (folate) MTFMT Mitochondrial methionyl-tRNA FMT1 NM_139242.3 Kegg formyltransferase (folate) MTHFD1 Methylenetetrahydrofolate dehydrogenase DCS NM_005956.2 Kegg (folate) MTHFD1L Methylenetetrahydrofolate dehydrogenase MTC1THF NM_015440.3 Kegg (NADP+ dependent) I-like S (folate) MTHFD2 Methylenetetrahydrofolate dehydrogenase NMDMC NM_006636.3 Kegg (NADP+ dependent) (folate) MTHFR 5,10-methylenetetrahydrofolate reductase - NM_005957.3 Kegg (NADPH) (folate)

46 MATERIALS AND METHODS

MTHFS 5,10-methenyltetrahydrofolate synthetase - NM_006441.2 Kegg (folate) MTR 5-methyltetrahydrofolate-homocysteine MS NM_000254.2 Kegg methyltransferase (folate) SHMT1 Serine hydroxymethyltransferase 1 CSHMT NM_004169.3 Kegg (soluble) (folate) SHMT2 Serine hydroxymethyltransferase 2 GYLA NM_005412.4 Kegg (folate)

DNA repair/apoptosis/ cell cycle regulation ATM Ataxia telangiectasia mutated homolog AT1 NM_000051.3 Yu et al., 2006 ATR Ataxia telangiectasia and Rad3 related FRP1 NM_001184.3 Yu et al., 2006 bcl2 B-cell CLL/lymphoma 2 Bcl-2 NM_000633.2 Christmann et al., 2003 CHEK1 CHK1 checkpoint homolog Chk1 NM_001274.4 Christmann et al., 2003 CHEK2 CHK2 checkpoint homolog Chk2 NM_007194.3 Christmann et al., 2003 Exo1 Exonuclease 1 HEX1 NM_003686.3 Christmann et al., 2003 HMGB1 High-mobility group box 1 HMG1 NM_002128.4 Yu et al., 2006 MLH1 mutL homolog 1, colon cancer, HNPCC2 NM_000249.2 Yu et al., nonpolyposis type 2 2006 MSH2 mutS homolog 2, colon cancer, HNPCC1 NM_000251.1 Yu et al., nonpolyposis type 1 2006 MSH6 mutS homolog 6 HNPCC5 NM_000179.2 Yu et al., 2006 PARP-1 Poly (ADP-ribose) polymerase 1 ADPRT NM_001618.3 Christmann et al., 2003 PARP-2 Poly (ADP-ribose) polymerase 2 ADPRT2 NM_005484.3 Christmann et al., 2003 PMS2 Postmeiotic segregation increased 2 HNPCC4 NM_000535.4 Christmann et al., 2003 SMUG1 Single-strand-selective monofunctional UNG3 NM_014311.1 Christmann uracil-DNA glycosylase 1 et al., 2003 TP53 Tumor protein p53 p53 NM_000546.3 Yu et al., 2006 UNG Uracil-DNA glycosylase UDG NM_003362.2 Christmann et al., 2003 XRCC1 X-ray repair complementing defective RCC NM_006297.1 Yu et al., repair in Chinese hamster cells 1 2006

47 MATERIALS AND METHODS

Total RNA was extracted from the respective cell lines and whole tumor xenograft

tissues using the TRI reagent (MRC) according to manufacturer’s instructions. Total

RNA yields and purity were determined spectrophotometrically at 260 and 280 nm. Real-

time RT-PCR was carried out using the Low Density Array (LDA) system (Applied

Biosystems, Foster City, CA), preloaded with inventoried TaqMan® Gene Expression

Assays into 384-well micro-fluidic cards, according to the manufacturer’s instructions. In brief, 1µg of total RNA was reverse transcribed using the High Capacity cDNA Reverse

Transcription Kit (Applied Biosystems). The reaction mixtures were incubated at 25°C for 10 min, followed by 37°C for 120 min and 95°C for 5 min. Nine µl of cDNA samples

(equivalent to 400 ng of total RNA), along with 41 µl of nuclease-free water and 50 µl of

2 × PCR master mix, were loaded into each sample port on the micro-fluidic card.

Thereafter, the card was centrifuged twice for 1 min at 331 x g at room temperature and then sealed. Real-time PCR and relative quantification were performed using an ABI

PRISM 7900 HT Sequence Detection System. The expression values of target gene were normalized to GAPDH levels using SDS 2.2.1 software (Applied Biosystems).

48 MATERIALS AND METHODS

2.6 Small interfering RNA (siRNA) transfections

Pre-designed Silencer Select siRNA (CTPS2, sense strand: 5’-

GGUUCGAGGUAAACCCUAATT-3’ and antisense strand: 5’-

UUAGGGUUUACCUCGAACCGA-3’; GAPDH, sense strand: 5'-

GGUCAUCCAUGACAACUUUTT-3' and antisense strand: 5'-

AAAGUUGUCAUGGAUGACCTT-3; Negative Controls, sense strand: 5’-

UAACGACGCGACGACGUAATT-3’ and antisense strand: 5’-

UUACGUCGUCGCGUCGUUATT-3’) were purchased from Applied Biosystems.

SW620 cells were transfected with CTPS2 siRNA (5 nM) or GAPDH siRNA (10 nM) or

negative controls using siPORT NeoFX transfection agent (Applied Biosystems) at 50%

cell confluence incubated in serum-free Opti-MEM I medium (Invitrogen) according to the manufacturer’s instructions. Cells were treated with 10 μM of 5FU 48 hours after

transfection. To measure the number of viable cells, cells were stained with Trypan blue

solution (Promega) at 24, 48 and 72 hours after 5FU treatment. Experiments were done in

duplicate.

2.7 Statistical analysis

Using statistical software R version 2.7.1 (R Foundation for Statistical Computing,

Vienna, Austria), unsupervised hierarchical clustering analysis was carried out to

examine gene expression relationships and to identify sample groupings in each series.

The function heatmap.2 in library gplots within R version 2.7.1 was used for the heatmap

and cluster generation. The default distance/dissimilarity matrix was used, which equates

49 MATERIALS AND METHODS to the Euclidean distance measure. Multiple testing correction was performed by

Bonferroni correction.

Two-sample t-test was used to identify differentially expressed gene between sample groupings with significance of p<0.05. Gene expression analysis over time was carried out for RKO and SW837 using Short Time-series Expression Miner (STEM) software

(Ernst et al., 2006) to identify significant temporal expression profiles and the genes associated with these profiles. A schema of analyses is shown in Figure 9.

50 RESULTS

Figure 9: A schema of analyses done in the 3 series (cell lines, xenografts and patients) in identifying determinants of 5FU sensitivity.

51 RESULTS

CHAPTER 3: RESULTS

3.1 Correlation between gene expression and 5FU sensitivity

To determine if 5FU sensitivity correlates with gene expression in the first instance,

unsupervised hierarchical clustering analysis was performed separately on cell line,

xenograft and primary tumor RNA levels. For cell lines, a wide range of 5FU IC50 values

(0.7-32.7 µM) were observed (Table 4a), however there was no correlation between 5FU sensitivity and gene expression (Figure 10A). This was the case either using a cut-off for sensitivity of 10 µM (Rossi et al. 2007), 7.91 µM (median) or 8.95 µM (mean).

Nevertheless, related cell lines such as HT29 and WiDR (ATCC), SW480 and SW620

(ATCC), as well as COLO201 and COLO205 (ATCC), grouped together, supporting the

accuracy of the clustering analysis.

The characterization of 5FU sensitivity in xenografts is reported in Table 4b. Xenograft

cases clustered into three major groups (Figure 10B), for which one group (I) of 4 cases

consisted entirely (100%) of responders, while only 2/9 (22%) cases in the other group

were responders (p=0.02). Xenograft sample 5854 clustered alone, indicating it to be an

outlier. As a result, this case was not included in subsequent analysis.

5FU sensitivity in primary tumors characterized as tumor recurrence within 5 years is shown in Table 4c. In primary tumors, gene expression patterns also displayed a trend for

correlation with 5FU response (Figure 10C). In one major group (I), all 4/4 (100%) cases

were responders, while only 17/21 (71%) cases were responders in the other major group

52 RESULTS

(II) (p>0.05). There was no significant difference in age, sex and tumor stage between the groups (Table 5).

53 RESULTS

Table 4: Characterization of 5FU sensitivity: (A) IC50 values of 21 colorectal cancer cell lines; (B) % of tumor shrinkage in 14 xenografts treated by 5FU; (C) Recurrence data of

25 stage II or III colorectal cancer patients treated with standard 5FU and leucovorin therapy. ‘R’ refers to responder and ‘NR’ refers to non-responder.

Cell Line IC50 ± S.D. (μM) Caco-2 5.6 ± 1.6 CCK81 5.0 ± 1.3 CoCMI 9.8 ± 1.5 Colo201 8.5 ± 1.5 Colo205 12.2 ± 1.5 Colo320 3.4 ± 1.4 DLD-1 2.2 ± 1.4 HCC56 17.6 ± 1.3 HCT116 8.0 ± 1.1 HT29 7.9 ± 1.3 LoVo 3.1 ± 1.4 LS174T 4.4 ± 1.4 LS513 13.1 ± 1.5 OUMS23 0.7 ± 2.7 RCM1 9.5 ± 1.6 RKO 19.5 ± 1.4 SW403 32.7 ± 1.3 SW480 7.2 ± 1.5 SW620 9.3 ± 1.3 SW837 0.7 ± 2.1 WiDR 7.4 ± 1.2

54 RESULTS

Xenograft Tumor shrinkage 5FU sensitivity 5676 + R 5677 – NR 5679 – NR 5682 + R 5734 + R 5735 + R 5771 + R 5776 – NR 5841 – NR 5854 – NR 5896 – NR 6044 – NR 6228 – NR 7271 + R a + indicates ≥50% tumor shrinkage – indicates <50% tumor shrinkage

55 RESULTS

Tumor Recurrence 5FU sensitivity NT00/0008 – R NT00/0025 + NR NT00/0031 – R NT00/0068 – R NT00/0076 – R NT00/0126 – R NT00/0137 – R NT00/0160 – R NT00/0189 – R NT01/0005 – R NT01/0007 – R NT01/0019 – R NT01/0035 – R NT01/0057 – R NT02/0012 – R NT02/0019 – R NT02/0028 – R NT02/0048 – R NT02/0049 – R NT02/0060 – R NT02/0062 – R NT02/0094 + NR NT03/0014 – R NT03/0048 + NR NT04/0099 + NR

a + indicates tumor recurrence within 5 years – indicates no tumor recurrence within 5 years

56 RESULTTS

57 RESULTTS

Figure 10: Unsupervised hierarchical clustering of samples based on the expression of 96 genes for (A) cell lines, (B) xenografts and (C) primary tumors. Red indicates decreased expression, and green indicates increased expression. Cases sensitive (red) and resistant (blue) to 5FU are indicated in the bar above the heatmaps.

58 RESULTS

Table 5: Associations (χ2 test) between 5FU sensitivity and clinicopathological parameters. ‘R’ refers to responder and ‘NR’ refers to non-responder.

Total (%) 5FU sensitivity (%) p R NR All cases 25 (100%) 21 (84%) 4 (16%)

Age, y 1.0 <50 3 (12%) 3 (100%) 0 (0%) ≥50 22 (68%) 18 (82%) 4 (18%)

Sex 1.0 Female 12 (48%) 10 (83%) 2 (17%) Male 13 (52%) 11 (85%) 2 (15%)

Stage 0.5 II 4 (16%) 3 (75%) 1 (25%) III 21 (84%) 18 (86%) 3 (14%)

59 RESULTS

3.2 Identification of baseline gene expression associated with 5FU sensitivity

Since the cell line groupings did not correspond with their sensitivity to 5FU, thus, no candidate gene was derived from this series. Using a two-sample t-test, 8 out of 96 genes showed significant difference in expression between the 2 main clusters in xenografts

(Table 6). For primary tumors, the expression of 41 genes was significantly different.

Two genes, cytidine triphosphate synthase type II (CTPS2) and non-metastatic cells 4

(NME4) were present in both lists and hence were considered the top priority candidate determinants of 5FU sensitivity. Indeed, CTPS2 and NME4 expression was higher in responders than non-responders in both xenografts and primary tumors and the difference in CTPS2 expression is significant (Figure 11).

60 RESULTS

Table 6: Candidate genes with differential gene expression (p<0.05) identified by two sample t-test for xenografts and primary tumors. Genes that occur in both lists of xenografts and primary tumors are highlighted in bold.

Fold change (Sensitive/ Gene Code Gene t statistic p-value resistant)

Xenografts ACTB Actin, beta 5.6 <0.01 1.98 18S 18S ribosomal RNA 3.2 0.01 4.57 CANT1 Calcium activated nucleotidase I 3.0 0.01 0.54 MSH6 mutS homolog 2, colon cancer, nonpolyposis type 1 2.7 0.02 1.29 NME4 Non-metastatic cells 4 2.6 0.02 1.69 CTPS2 CTP synthase II 2.4 0.03 2.25 NT5C 5’,3’-nucleotidase, cytosolic 2.3 0.04 2.19 PARP1 Poly (ADP-ribose) polymerase 1 2.4 0.04 1.98

Primary Tumours B2M Beta-2-microglobulin 11.5 <0.01 0.17 TXNRD1 Thioredoxin reductase 1 8.7 <0.01 0.31 NT5E 5’-nucleotidase, ecto (CD73) 6.3 <0.01 0.13 ECGF1 Thymidine phosphorylase 6.0 <0.01 0.25 UCK1 Uridine-cytidine kinase 1 5.5 <0.01 0.32 ABCC4 ATP-binding cassette, sub- family C (CFTR/MRP), member 4 4.7 <0.01 0.26 RRM1 Ribonucleotide reductase M1 polypeptide 4.6 <0.01 0.37 ENTPD1 Ectonucleoside triphosphate diphosphohydrolase 1 4.2 <0.01 0.13 MTFMT Mitochondrial methionyl-tRNA formyltransferase 4.2 <0.01 0.40 UPB1 Ureidopropinase, beta 4.1 <0.01 0.15 ATM Ataxia telangiectasia mutated homolog 4.0 <0.01 0.21 MTHFR 5,10-methylenetetrahydrofolate reductase (NADPH) 4.0 <0.01 0.29 CES1 Carboxylesterase 1 4.0 <0.01 0.16 DPYS Dihydropyrimidinase 3.8 <0.01 0.07

61 RESULTS

ENTPD3 Ectonucleoside triphosphate diphosphohydrolase 3 3.8 <0.01 0.16 BCL2 B-cell CLL/lymphoma 2 3.8 <0.01 0.26 PARP2 Poly (ADP-ribose) polymerase 2 3.7 <0.01 0.32 DPYD Dihydropyrimidine dehydrogenase 3.6 <0.01 0.16 NT5C3 5’-nucleotidase, cytosolic III 3.6 <0.01 0.23 RRM2B Ribonucleotide reductase M2 B 3.5 <0.01 0.44 NME3 Non-metastatic cells 3 3.4 <0.01 0.28 ALDH1L1 Aldehyde dehydrogenase I family, member L1 3.4 <0.01 0.34 NUDT2 Nudix (nucleoside diphosphate linked moiety X)-type motif 2 3.4 <0.01 0.30 MTHFD2 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 3.2 <0.01 0.38 CTPS2 CTP synthase II 3.1 <0.01 0.40 NME5 Non-metastatic cells 5 6.3 0.01 0.29 SLC28A3 Solute carrier family 28 (sodium-coupled nucleoside transporter), member 3 3.0 0.01 0.34 ENTPD4 Ectonucleoside triphosphate diphosphohydrolase 4 3.0 0.01 0.32 CHEK2 CHK2 checkpoint homolog 2.9 0.01 0.45 DHFR Dihydrofolate reductase 2.7 0.01 0.55 NME7 Non-metastatic cells 7 2.7 0.01 0.41 MSH2 mutS homolog 2, colon cancer, nonpolyposis type 1 2.7 0.02 0.50 NME4 Non-metastatic cells 4 2.6 0.02 0.34 NME6 Non-metastatic cells 6 2.5 0.02 0.47 TK2 Thymidine kinase 2 2.5 0.02 0.33 UPP2 Uridine phosphorylase 2 2.4 0.03 0.06 ATR Ataxia telangiectasia and Rad3 related 2.4 0.03 0.36 MLH1 mutL homolog 1, colon cancer, nonpolyposis type 2 2.3 0.03 0.47 CES2 Carboxylesterase 2 2.3 0.03 0.19

62 RESULTS

p<0.01

0.05

0.04

0.03

0.02 CTPS2 mRNA CTPS2 0.01

0.00 Non-response Response

p=0.02

0.15

0.10

0.05 NME4 mRNANME4

0.00 Non-response Response

63 RESULTS

B. p>0.05

0.20

0.15

0.10

CTPS2 mRNA CTPS2 0.05

0.00 Non-response Response

p>0.05

2.0

1.5

1.0

NME4 mRNANME4 0.5

0.0 Non-response Response

Figure 11: Difference in expression levels of CTPS2 and NME4 between clusters in (A) xenografts and (B) patients.

64 RESULTS

3.3 Identification of post-treatment gene expression changes associated with 5FU

sensitivity

To identify genes for which expression changes after 5FU treatment best discriminated

5FU sensitivity from resistance, SW837 (5FU sensitive) and RKO (5FU resistant) cells

were seeded at an initial density of 1 x 105/ml for 24 hours. Cells were treated with 10

μM of 5FU for 0, 4, 8, 12, 24 and 48 hours, after which cells were harvested for RNA and

analyzed for the expression of the selected 96 genes.

Genes with similar expression change patterns were grouped based on Pearson

correlation coefficient, and these groups ranked according to degree of similarity in

change patterns (Figure 12). The model profiles (patterns) are generated by the software in two steps. Firstly, the system exhaustedly listed all possible patterns according to the

user parameter c. “c” represents possible stages that a gene can be regulated between time

points. For example, if c=1, it means that a gene can be up-regulated, down-regulated, or

unchanged, and if c=2, it means that a gene can be up-regulated for 1 or 2 stages, down-

regulated for 1 or 2 stages or unchanged. Thus, there are in total (2*c+1)n-1, where n is the

number of time points. The first time point will always be regarded as unchanged. In our

experiment, we have set: c=2, n=6. Secondly, the software selected a subset of patterns

from the exhaustedly generated patterns, promising that the selected patterns are

representative and distinct from each other, that is, to maximize the minimal distance

between any of the two patterns. Since the optimal solution to the second step is Non-

deterministic Polynomial-time Hard (NP-Hard), a heuristic algorithm is proposed by

Ernst and colleagues (Ernst et al. 2005).

65 RESULTS

The top-ranked patterns were surmised to be the ones that defined sensitivity and resistance in the respective cell lines. Interestingly, CTPS2 was identified in the top groups of both RKO and SW837, making it the highest priority candidate gene of

66 RESULTTS

67 RESULTTS

Figure 12: Patterns of gene expression at 0, 4, 8, 12, 24 and 48 hours after 10 μM 5FU treatment according to reducing statistical significance from left to right, top to bottom for (A) RKO and (B) SW837. The component genes of the top three concurrently regulated groups are displayed on the left.

68 RESULTS

3.4 Modulation of 5FU sensitivity by CTPS2 siRNA

To verify the direct influence of CTPS2 on 5FU sensitivity, the effects of siRNA knockdown of CTPS2 expression were examined, with GAPDH siRNA treatment as a

control. 5FU-sensitive cell line (<10 µM) with relatively high CTPS2 levels would be an

ideal choice for CTPS2 knockdown and measuring the change in 5FU sensitivity. SW620

as an adherent cell line was chosen for this investigation as its CTPS2 level was fourth

highest among all 5FU sensitive cell lines. Using chemical transfection agents, siRNA

transfections of the top 2 cell lines in rank (CCK81, CoCM1) were unsuccessful while

transfection of Colo201 (third in rank for its CTPS2 level) was not attempted as these

cells are in suspension. Cell lines grown in suspension culture remain challenging

transfection targets.

More than 50% reduction of CTPS2 and GAPDH RNA levels was achievable in SW620

cells following 48 hours treatment with respective siRNA (Figure 13A and 13B),

although full CTPS2 expression were restored after 120 hours treatment. Cells transfected

for 48 hours with siRNA were then treated with 10 μM 5FU (IC50 of SW620 at 48 hours)

and cell viability was measured at 24, 48 and 72 hours. At 48 hours post-5FU treatment,

viability of CTPS2-knockdown cells was the same as that of transfected cells without

5FU treatment (Figure 13C), indicating a resistance to 5FU of CTPS2-knockdown cells.

This result contrasts with that of cells transfected with negative control or GAPDH

siRNA (Figure 13D and 13E). For these cells, 40-60% reduction in cell growth was observed at 48 hours post-5FU treatment, consistent with reduction expected from using an IC50 dosage. At 72 hours post 5-FU treatment (120 hours post-siRNA treatment), the

69 RESULTS number of viable CTPS2-knockdown cells became 53% of non-treated cells. This is likely due to the lower CTPS2 knockdown at 120 hours post-siRNA treatment (Figure

5A), and the known transient nature of siRNA knockdown.

70 RESULTS

1.2 1 0.8 0.6 0.4 CTPS2 mRNA 0.2 0 Negative control CTPS2 (48 hours) CTPS2 (120 hours) siRNA

1.2 1 0.8 0.6 0.4

GAPDH mRNA 0.2 0 Negative control GAPDH (48 hours) GAPDH (120 hours) siRNA

71 RESULTTS

Figure 13: CTPS22 and GAPDDH mRNA expression levels in SW620 cells transfected with (A) CTPS2 siRNA and (B) GAPDH siRNA 48 and 120 hours after transfection. Growth curves of cells either treated (unfilled circles) or untreated (filled circles) with 10 μM 5FU after 48 hours of transfection of (C) CTPS2 siRNA, (D) GAPDH siRNA and (E) negative control siRNA.

72 CONCLUSION

CHAPTER 4: DISCUSSION

Most investigations on determinants of 5FU sensitivity until now have been candidate

gene approaches. These have mostly been on genes/proteins involved in the metabolism and cytotoxic mechanisms of 5FU, with thymidylate synthase (TS) (Peters et al., 1995) and dihydropyrimidine dehydrogenase (DPD) (Diasio et al., 2001) being the most prominent. TS has been studied as a major target of 5FU activity, and numerous reports have associated elevated TS expression/activity with 5FU resistance (Johnston et al.,

1995, Berger et al., 1985, Clarke et al., 1987, Johnston et al., 1992). DPD is the rate- limiting step in 5FU clearance, and its deficiency has been correlated to 5FU toxicity and increased sensitivity (Diasio et al., 1988; Ishikawa et al., 1999; Milano et al., 1999).

However, not all studies on TS and DPD have been in agreement (Soong and Diasio

Findlay et al., 1997; Berglund et al., 2002; Allegra et al., 2003; Johnston et al., 2003;

Kinoshita et al., 2007; Yamada et al., 2008), indicating other factors are involved.

Gene expression profiling has also been applied to identifying determinants of 5FU response at a genome-wide level (Scherf et al., 2000; Zembutsu et al., 2002; Mariadason et al., 2003; Shimizu et al., 2005; Matsuyama et al., 2006). Testing response to multiple drugs on the NCI panel of 60 cell lines, Scherf et al. identified that DPD is highly negatively correlated with 5FU response (Scherf et al., 2000). Similar to the NCI panel of

60 cell lines, the cell lines used in our study were exposed to 5FU for 48 hours. The IC50 values for the overlapped cell lines from both experiments were similar except for SW620 which is shown to be more 5FU resistant by Scherf et al. Mariadason et al. studied a panel of 30 colon cancer cell lines and identified 420 genes, enriched in DNA replication

73 CONCLUSION and repair and protein processing/targeting, to be significantly associated with 5FU sensitivity (Mariadason et al., 2003). The 30 colon cancer cell lines described by

Mariadason et al. were exposed to 5FU for 72 hours. Most of the IC50 values of overlapping cell lines reported by Mariadason et al. were similar except for SW480 and

SW620 which seemed to be more resistant, whilst RKO and SW403 seemed to be more sensitive to 5FU. Moreover, based on the IC50 values, 2 of the four cell lines tested by

Scherf et al. (Colo205, HCT116) were found to be more sensitive to 5FU than that of

Mariadason et al. Zembutsu and colleagues examined 9 anticancer agents in 85 cancer xenografts and identified 20 novel genes related to 5FU sensitivity (Zembutsu et al.,

2002). Shimizu et al. analyzed 3 colorectal cancer cell lines and corresponding resistant sublines and identified 81 genes related to 5FU sensitivity (Shimizu et al., 2005). A derivative panel of these genes has since been validated with TNFRS1B, SLC35F5 and

OPRT identified as a “Response Index” (Matsuyama et al., 2006).

In this study, CTPS2 was identified as a determinant of 5FU sensitivity through an

integration of pathway-based real-time PCR analysis of three cancer models, an

examination of both basal and temporal gene expression levels and siRNA knockdown. A

pathway-based approach, comprising examination of 92 genes in 5FU transport,

metabolic and downstream activity (Figure 1), was adopted as the candidates identified

were more likely to have a rational basis for influencing 5FU sensitivity. Real-time PCR

was used to achieve optimal detection sensitivity, dynamic range and analytical precision,

as opposed to a hybridization array approach (Wong et al., 2005). Interrogation of cell

lines, xenografts and primary tumours were used to best identify candidates that were

74 CONCLUSION

robustly linked to 5FU sensitivity (Figure 10). However, candidate genes were not

derived from the cell lines as their chemosensitivity did not correspond well with sample

groupings. The lack of correlation between cell lines and clinical specimens has been described previously (Andreotti et al., 1994). The specific 5FU/LV regimen undertaken by the patients in this study makes it more valuable as studies which combine patients receiving different types of 5FU administration introduce greater variation between patients and thus, their findings do not strictly correspond to drug effect (Gustavsson et

al., 2008). Finally, siRNA knockdowns were used to confirm that reduced CTPS2

expression directly leads to 5FU resistance (Figure 13). Although the use of one CTPS2 siRNA sequence might raise concern with off-target effects, it was shown using BLAST analysis that the designed siRNA sequence has 100% specificity to the gene of interest, i.e. CTPS2, thus minimizing off-target effects. Not more than one siRNA sequence is introduced as high concentration of siRNA could potentially lead to induction of alpha-

and beta-interferon pathway via protein kinase K pathway (Khabar et al., 2003; Garcia et

al., 2006). Taken together, these numerous lines of evidence suggest a role for CTPS2 in

determining 5FU sensitivity.

A major feature of this study is that we have also identified determinants through

temporal gene expression analysis following 5FU treatment. On the premise that drug

sensitivity is likely to be influenced by cell responses to treatment, we characterized the

expression patterns of the 92 pathway-based genes at 6 timepoints over 48 hours in

extremely sensitive (SW837) and resistant (RKO) cell lines. Multiple timepoints were measured as we considered significant information could be missed between traditional

75 CONCLUSION

two (pre- and post-treatment) timepoint assessments. STEM software has been useful for

clustering genes of similar regulation patterns across a short time-series (Lulko et al.,

2007; Martinez et al., 2007), and we took this further by identifying the most tightly regulated expression patterns (and component genes) in respective cell lines as determinants of 5FU sensitivity and resistance. CTPS2 was one of four genes (the others were DTYMK, ITPA, UPP1) in the top gene groups of both sensitive and resistant cell lines (Figure 12). Interestingly, the level of CTPS2 went up in the sensitive cell line, and decreased in the resistant line, consistent with the baseline expression trends.

CTPS2 is one of 2 genes (the other is CTPS) encoding human cytidine triphosphate synthase (van Kuilenberg et al., 2000). The enzyme catalyses the rate-limiting conversion of uridine triphosphate (UTP) to CTP, that is important for biosynthesis of RNA, DNA and lipids. Hence, one possible mechanism for 5FU resistance observed with low CTPS2 expression could be UTP accumulation occurring due to reduced conversion to CTP. The

UTP may compete with the incorporation of FUTP into RNA, thereby reducing the RNA- directed cytotoxic effects of 5FU. However, other scarce data on CTPS2 neither supports nor refutes this mechanism, even more so its relationship to 5FU sensitivity. Increased activity of human CTP synthase has been observed in leukemias and tumors of liver, lung and colon (Kizaki et al., 1980). Whelan et al. showed treatment of CHO cells with ultra- violet radiation and ethylmethanesulfonate generates CTPS mutations, eliminating its feedback regulation by CTP, altering its synthase activity and increasing intracellular pools of CTP and dCTP and resistance to 5FU (Whelan et al., 1993). Somatic mutation of neither CTPS nor CTPS2 in tumors has been reported so far. It is indeed possible that

76 CONCLUSION

other mechanisms like loss of heterozygosity or methylation-induced transcriptional

silencing of CTPS2 to cause inactivation of this gene and lead to tumor resistance to 5FU.

The second potential candidate, NME4, is identified from the basal gene expression

analysis but not from the timepoint temporal analysis. NME4 is one of the isoforms of

nucleoside diphosphate kinase (NDPK/Nm23) and encodes for human mitochondrial

NDPK-D. NDPK is important for the regulation of intracellular nucleotide homeostasis.

NME4 was found to be overexpressed in colorectal cancer (Hayer et al., 2001).

Furthermore, high expression of NME4 has been shown to be associated with poor

prognosis in patients with acute myeloid leukemia (AML) (Kracmarova et al., 2008).

However, here we observed that relatively high NME4 expression was associated with

5FU response. This conflicting observation could be explained by the differences in

treatment and tumor characteristics of colorectal cancer and AML and the different

endpoint of response and survival. To further validate the role of NME4, functional

investigations will be carried out.

As compared to earlier studies, CTPS2 has not been identified as a determinant of 5FU

response, though its role in 5FU sensitivity has been suggested for via the gene

expression analyses and in-vitro experiments in our study. This difference in observations

could be due to the use of different types of samples (cell lines/ xenografts/ human

paraffin/ frozen tissues; tumor content) and various experimental approaches (DNA/

RNA/ protein analyses; RNA analysis using microarray/ real-time PCR). One limitation

of our study is sample bias, with only few 5FU-resistant patient tumors being analyzed.

77 CONCLUSION

Continual effort in developing tumor banks of fresh frozen tissue and maintain proper documentation of clinical follow up is of utmost importance in order to complement the currently available profiling technologies. When more tumors become available, these samples could contribute to the validation set for future profiling experiments to validate the role of CTPS2.

As in some previous studies, TYMS expression did not associated with 5FU sensitivity in our study. In the xenograft series, DPYD expression was observed to be higher among non-responders. This is in concordance with the findings by Yoshinare et al. that high

DPYD expression is associated with low 5FU sensitivity (Yoshinare et al., 2003).

However, this trend was not observed in the primary tumors. This could be possibly due to the under representation of the non responders in this series.

In summary, this is the first study which identified CTPS2 as a pharmacologically relevant marker which could be predictive for 5FU response, through a convergence of pathway-based, baseline and temporal, and in-vitro siRNA investigations. Future investigation of CTPS2 protein levels and activity will help further elucidate the potential role of CTPS2 in determining sensitivity to 5FU.

78 CONCLUSION

CONCLUSION

The antimetabolite, 5-fluorouracil (5FU) is the mainstay for the treatment of many cancer types. Inter-patient response to 5FU is however extremely variable, making it desirable to identify factors that influence 5FU sensitivity. Here, we describe the identification of cytidine triphosphate synthase II (CTPS2) as a significant determinant of 5FU sensitivity through an exhaustive search, involving real-time PCR analysis of 92 genes involved in

5FU and folate transport, metabolism and downstream effects in three cancer models

(cell lines, xenografts and primary tumors), with examination of both baseline and temporal data, and siRNA functional assessment. The identification of CTPS2 presents a potentially sensitive factor that could be used in the clinic for predicting response to 5FU, or devising strategies to potentiate drug activity. In addition, the pathway-based, multi- model data integration, and multi-timepoint temporal analysis approaches used in this study presents useful alternative strategies to discovering determinants of drug

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APPENDIX This research was supported by an academic research fund from National Medical

Research Council (1123/2007), Singapore.

The data in this manuscript was presented in the Poster Discussion session of the 20th

EORTC-NCI-AACR Symposium, Switzerland, October 2008.

PUBLICATIONS

Soong R, Shah N, Salto-Tellez M, Tai BC, Soo RA, Han HC, Ng SS, Tan WL, Zep N, Joseph D, Diasio RB, Iacopetta B. Prognostic significance of thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase protein expression in colorectal cancer patients treated with or without 5-fluorouracil-based chemotherapy. Annals of Oncology 19: 915-919 (2008).

Tan WL, Dong DF, Loh M, Soo R, Fichtner I, Wong LS, Soong R. Pathway determinants of 5-fluorouracil Sensitivity. (In submission)