A Dissertation Entitled Identifying and Targeting Cellular Mechanisms To

A dissertation

Entitled

Identifying and Targeting Cellular Mechanisms to

Enhance Cisplatin Chemotherapeutic Response in Cancer

Sanjeevani Arora

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy

Degree in Biomedical Sciences

Stephen M. Patrick, PhD, Committee Member

William A. Maltese, PhD, Committee Member

Randall J. Ruch, PhD, Committee Member

Ivana de la Serna, PhD, Committee Member

James C. Willey, MD, Committee Member

Dr. Patricia R. Komuniecki, Dean

College of Graduate Studies

The University of Toledo

August 2012

This document is copyrighted material. Under copyright law, no parts of this document

may be reproduced without the expressed permission of the author

Abstract

Cisplatin is one of the most effective and widely used anticancer agents used especially in treating testicular, ovarian, head and neck and lung cancers. As in the case of many chemotherapeutic drugs, a clinical limitation is cancer recurrence and resistance. Cisplatin targets DNA and forms distinct lesions which block DNA replication and transcription. These lesions mediate the cisplatin cytotoxic response and their repair is detrimental to drug cytotoxicity. Cancers exhibit altered repair of cisplatin-DNA lesions leading to drug resistance.

Thus, targeting the DNA repair mechanisms is important for increasing cisplatin efficacy. This study validates XPF/ERCC1, a DNA repair enzyme complex, as an important molecular target to enhance cisplatin sensitivity in cancer cells globally. XPF/ERCC1 is vital to the repair of all forms of cisplatin-DNA damage and hence important in mediating clinical response to cisplatin. XPF/ERCC1 enhances cisplatin cytotoxicity by inhibiting the repair of DNA damage.

Our studies next identify small molecules that inhibit XPF/ERCC1 in primary and secondary in vitro screens. These compounds potentiate cisplatin sensitivity in cancer cells by inhibiting the repair of cisplatin-DNA damage. Further studies with these compounds could yield inhibitors which can clinically potentiate the effects of cisplatin and result in lower doses of cisplatin being administered while enhancing the cytotoxic effect. Our studies also identify gap junctions and their mediated intercellular communication as an important mechanism in maintaining cisplatin sensitivity. We show that gap junctional intercellular communication induces a bystander effect after cisplatin treatment in untreated bystander cells and hence further potentiates cisplatin‟s effect. The bystander effect elicits as DNA Double Strand Breaks

(DSBs) and further sensitizes XPF/ERCC1 knockdown cells to cisplatin. Further studies might iii

help identify the “signal” that induces DSBs in bystander cells and how unrepaired DNA damage enhances the bystander effect. Studies delineating mechanisms that mediate resistance or maintain sensitivity are important in improving platinum-based therapeutics.

Dedication

My first dedication is to cancer survivors and in memory of those who have succumbed to cancer. Next, I dedicate this thesis to my family – my parents (Dr. Harsukh Rai Arora and

Neelam Arora) and my sister (Swati Arora Majumdar) – for their love, support and understanding. I am thankful to them for standing by my choices and decisions and encouraging me in every aspect of my life. I am truly blessed to be a part of my family. I also dedicate this thesis to my boyfriend and dearest friend, Sumit Bhattacharya, who has been my partner in so many aspects in life and has gone through the trials and tribulations of graduate school with me. I thank him for making me laugh when things did not go the right way. I also dedicate this thesis to my friends – Namita Goel, Sawona Biswas, Ranjan Hebbar and

Chandrima Bhattacharya - who have made life fun and have been an ear I could chew off.

Acknowledgments

I would like to thank my major advisor, Dr. Steve M. Patrick, for giving me the opportunity to pursue graduate work with him. I want to thank him for giving me the creative freedom, independence and encouragement to pursue my dissertation work. I thank him for his suggestions, support and guidance during my graduate studies.

I would like to thank Dr. Randall J. Ruch, who was my first year advisor in graduate school and is also a part of my advisory committee. I thank him for his help and advice on the gap junction project described in this dissertation. I thank the other members of my advisory committee: Dr. William A. Maltese, Dr. Ivana de la Serna and Dr. James C. Willey for their help, time and valuable insights.

I want to thank Dr. Anbarasi Kothandapani for her valuable advice, help and friendship. I am truly fortunate to have received assistance and guidance from her. I have enjoyed working with Vivian Kalman-Maltese on the XPF/ERCC1 project. We have spent numerous hours talking about work and life in general during the past three years and I want to thank her for her friendship. I also want to thank Elaine C. Chalfin for working with me on the gap junction project and for her patience with connexin proteins. She has been an absolute pleasure to work with and has been an asset to this project. I am grateful to Kristin Tillison, former lab member, for teaching me various techniques relevant to my project and making my rotation fun in the Patrick lab. I am truly fortunate to have had a chance to come vi

across such phenomenal people and make so many friends during my time in the

Patrick lab. I also thank other Patrick lab members for their help and support.

I would also like to thank SURF students, Eleanor C. Cook and Glenn

Westphal, for helping with the gap junction project and bringing fresh insight and enthusiasm to this project. I also want to thank CD3 members – Dr. Paul Erhardt,

Dr. Jeffrey Sarver and Dr. Jill Trendel for their suggestions and help with the

XPF/ERCC1 inhibition project. I want to thank Jenny Zak and Ann Chlebowski for their help and assistance.

On a personal note, I would like to thank my parents for being a constant source of guidance, love and support during my graduate studies. I will always credit my parents, Dr. Harsukh Rai Arora and Neelam Arora, for all my accomplishments in life. I want to thank my sister, Swati Arora Majumdar, for being the best big sister in the world. I thank Sumit Bhattacharya for always being there for me and helping me accomplish my dreams. I also thank my closest friends- Namita Goel, Sawona Biswas, Ranjan Hebbar and Chandrima

Bhattacharya, for their love and support.

vii

Table of Contents

Title ...... i

Abstract ...... iii-iv

Dedication ...... v

Acknowledgements...... vi-vii

Table of Contents ...... viii

1. Literature Review & References ...... 1-64

2. Downregulation of XPF/ERCC1 enhances cisplatin cytotoxicity in cancer cells &

References ……………………………...... 65-114

3. Identification and characterization of small molecules to inhibit XPF/ERCC1 & potentiate cisplatin chemotherapy in cancer cells & References………………….….115-171

4. Gap junction intercellular communication increases cisplatin cytotoxicity by inducing

DNA damage through bystander signaling in cancer cells & References……….……172-222

Overall Summary...... 223-224

viii

Introduction

Cisplatin was first FDA (Food and Drug Administration) approved in 1978 for the treatment of testicular and bladder cancer. Currently, cisplatin and platinum-based chemotherapy is clinically active in a wide spectrum of solid cancer likes testicular, bladder, ovarian, colorectal, lung and head and neck cancers [1, 2]. Cisplatin chemotherapy is usually initially successful and stabilizes the patient‟s condition, but in case of cancers like colorectal, lung and prostate, there is also intrinsic resistance to platinum-based therapies. Another fraction of patients, especially in case of ovarian cancers, are initially sensitive but develop chemoresistance over time [3-5] .

Thus, drug resistance greatly limits drug success and clinical gains. Cisplatin chemotherapy is also limited to increased toxicity and morbidity in patients especially nephrotoxicity, neurotoxicity and ototoxicity [6, 7]. However, the main clinical limitation of cisplatin as an anticancer drug is the high incidence of chemoresistance. In the early 1980s, second generation platinum agents were developed to retain the same anticancer effects as cisplatin and to reduce the toxic side effects of cisplatin. Carboplatin was the first agent which did not cause any nephrotoxicity and neurotoxicity but also formed the same lesions with DNA as cisplatin but with reduced potency. Carboplatin was approved for use in ovarian cancer and it was seen that most cisplatin-resistant cancers failed to respond to carboplatin [8]. The next platinum agent, oxaliplatin, was used clinically in 2002 and there was still some degree of cross-resistance observed. There are new platinum agents that have entered clinical trials, but clinically cisplatin is the most prominent platinum agent [9, 10]. Thus, understanding the mechanisms that mediate cisplatin resistance and circumventing them is clinically very important.

The key molecular mechanism that mediates cisplatin resistance is the repair of cisplatin-

DNA damage which has been widely studied and reported clinically and experimentally. Inside

the cells, aquated cisplatin reacts with DNA and forms cisplatin-DNA lesions. Although only

~1% cisplatin binds DNA in the cells [11], it is a block to essential check references cellular processes like replication and transcription and hence causes cell death and cytotoxicity. Thus, cisplatin DNA-damage response and repair is an important mechanism leading to cellular cytotoxicity with occurrence of cisplatin resistance due to alterations in pathways that mediate

DNA repair. Persistence of cisplatin-DNA damage is important for cisplatin cytotoxicity [12,

13]. Hence, DNA repair is an important target to enhance the chemotherapeutic response of cisplatin-based and other platinum based therapies.

In these studies, the overall goal is identification of cellular mechanisms that can clinically enhance the cisplatin chemotherapeutic response.

In chapter 1 of this thesis, I provide the published proof-of-principle that

XPF/ERCC1 enzyme complex is an important molecular target to enhance cisplatin sensitivity.

XPF/ERCC1 is important in mediating cisplatin resistance and mediates the repair of all forms of cisplatin-DNA damage [14]. Cisplatin resistant cancers have enhanced expression of ERCC1 and patients with ERCC1 negative tumors respond better to platinum-based therapy [15]. We show using different cancer cell lines, that in terms of cisplatin-based treatment, XPF/ERCC1 is important in potentiating its response. We correlate increased cytotoxicity to decreased DNA repair on XPF/ERCC1 downregulation. We also show that XPF/ERCC1 downregulation enhances cisplatin response in cisplatin resistant cancer cells and these responses correlate to the levels of ERCC1. Thus, XPF/ERCC1 can globally potentiate cisplatin response in different cancer types and is a valid target for improving therapeutic response.

In chapter 2 of this thesis, following the proof-of-concept with XPF/ERCC1, I describe screening methods to identify small molecules that can inhibit XPF/ERCC1. We performed

primary and secondary screens with the small molecules to show specificity to XPF/ERCC1.

Cell culture experiments with small molecules show increased cisplatin cytotoxicity which correlates to decrease in repair of cisplatin- DNA damage. We also performed structure activity

(SAR) studies to further characterize these small molecules. Findings from this chapter in combination with future studies could yield inhibitors that could be used in clinical settings to improve cisplatin chemotherapeutic response.

In chapter 3 of this thesis, we explore novel mechanisms that can maintain cisplatin sensitivity in cancer cells. We identify gap junction and gap junction intercellular communication (GJIC) as an important mechanism in mediating cisplatin cytotoxic response.

Our studies indicate that expression and functional activity of gap junctions, especially gap junction protein Connexin43 (Cx43), in cancers is important in mediating a bystander killing response. This bystander response elicits as DNA DSBs in the bystander cells which left unrepaired can lead to cell death. Finally, we also show that cells targeted for the DNA repair enzyme, XPF/ERCC1, further sensitize in the presence of functional GJIC. Thus, our studies suggest that gap junctions could not only be an important biomarker for cisplatin chemotherapy as it maintains sensitivity but can increase toxicity in cancers deficient in the repair of cisplatin-

DNA damage. These above findings are significant not only for treatment of cancers that usually respond to cisplatin based chemotherapy but also for cancers that are intrinsically resistant to cisplatin and other platinum agents.

Chapter 1

Literature Review

1.1 Cisplatin

Cisplatin [cis-diammine-dichloroplatinum (II)] is one of the most widely used platinum containing chemotherapeutic agents. It forms cisplatin-DNA adducts (Figure 1) which includes monoadducts, intrastrand adducts and interstrand crosslinks (ICLs). These adducts inhibit and block DNA replication, which can lead to cell death. The major target of cisplatin is DNA, but it can also bind to other macromolecules like proteins and RNA. Platinum binds to the N (7) position of purines and predominantly gives rise to 1, 2-d(GpG) intrastrand adducts. Other adducts formed are 1, 2-d(ApG) and1,3-d(GpNpG) intrastrand adducts; ICLs and protein –DNA crosslinks [15, 16] . The intrastrand adducts comprise of ~80-85% of the cisplatin-DNA adducts, whereas, ICLs form around 5-10% frequency and are not a major result of cisplatin‟s reaction with DNA but are crucial in determining cisplatin cytotoxicity. The ICLs are highly toxic covalent links between both strands of DNA that distort the helix. These crosslinks physically prove an absolute block to DNA replication, recombination, and RNA transcription [17]. The downstream effect of failure to repair ICLs prior to DNA replication is induction of DNA breaks, chromosomal rearrangements, and eventually cell death. Apart from cisplatin, other agents that induce ICLs are mitomycin C (MMC), nitrogen mustards, and also endogenous products of lipid peroxidation [18].

The monoadducts and intrastrand adducts are repaired by nucleotide excision repair

(NER). ICL repair pathways remain poorly defined but are known to involve proteins from multiple DNA repair pathways like Homologous Recombination (HR), translesion polymerases,

Fanconi Anemia (FA) and NER [19].

How does cisplatin enter cells?

Cisplatin enters cells mainly through passive diffusion by losing the chloride groups from the cisplatin molecule. Outside the cell, chloride concentration is around 100 mM while it ranges between 2 and 30 mM inside the cells. Cisplatin gets aquated to [Pt(H2O)Cl (NH3)2]+ and

[Pt(H2O)2(NH3)2]2+ cations. These cations are highly reactive towards biomolecules [13, 20]

Recent evidence also suggests an active mechanism for cisplatin uptake and these can be modulated by pharmacological agents like the Na+/K+-ATPase inhibitor ouabain and membrane-interactive agents like amphotericin B and digitonin [21]. Recent studies also show a direct connection between the cellular concentrations of copper and platinum, suggesting an active transport mechanism along with passive diffusion [22].

Clinical Use

Cisplatin is approved for use alone or in combination according to NCI for the following cancers- testicular cancer, bladder cancer, cervical cancer, non-small cell lung cancer (NSCLC), malignant mesothelioma, ovarian cancer and squamous cell carcinoma of the head and neck.

Randomized clinical trials have confirmed that adjuvant chemotherapy with platinum-based

(carboplatin or cisplatin) drug combinations for NSCLC significantly increases survival [23].

Cisplatin has been the most successful in achieving over 90% cure rates for testicular cancer which was around 5 % prior to the discovery of cisplatin. At present cisplatin or its derivative carboplatin in combination with paclitaxel, is the standard treatment for patients with advanced – stage ovarian cancers with response rates of >70 %. Therapeutic benefit has been seen by combining cisplatin or carboplatin with paclitaxel (mitosis inhibitor) or etoposide (topoisomerase

II inhibitor) (NCI website). Recent research shows that cisplatin can also improve gemcitabine based chemotherapy [24]. Cisplatin is also combined with radiotherapy and has been used effectively for many tumor types especially for treating NSCLC and some squamous cell carcinoma [25, 26]. The effectiveness of all these treatment options depends especially on the cancer type and the treatment protocol.

Cisplatin is usually given intravenously which could be in cycles of once per week or cycles of continuous infusion with rest or sequentially with another drug or together etc. It may also be given locally to increase drug exposure and diminish side effects. The dose differs based on the patient height and weight, cancer type and the patient response. According to NCI, usually a dose of 50 mg/m2 is considered a lower dose while a high dose is usually 100 mg/m2.

However, these values are subject to change based on use alone or in combination. [27].

Despite the success of cisplatin, its effectiveness over time in the treatment of cancer is limited. This is due to the development of resistance. There are multiple mechanisms of cisplatin resistance but increased DNA repair is proposed to be one of the major reasons of resistance development [28]. Studies with a series of cisplatin-resistant cells in ovarian cancer cell lines show a clear relation between adduct removal and cisplatin cytotoxicity in a cisplatin-resistant model. Resistance to cisplatin can be acquired or intrinsic [29].

1.2 Mechanisms of Cisplatin Resistance

Resistance to cisplatin is a major clinical limitation of the drug and it is important to understand the mechanisms that mediate resistance. The cellular mechanisms that are well known are (a) diminished accumulation of cisplatin (b) increased detoxification by thiols like glutathione and metallothionein; (c) improved repair of/tolerance to DNA-drug lesions, leading to a concomitant reduction in apoptosis. Overall, these mechanisms can be grouped to reduced cisplatin-DNA damage. Another area leading to increased resistance is increased cell survival despite DNA damage. These defects can be in signaling pathways leading to decreased cell death

[30, 31].

a) Reduced cellular accumulation

It is well understood that cisplatin cytotoxicity is dependent on the formation of DNA adducts, hence decreased drug accumulation like decreased uptake or increased efflux can reduce the available cisplatin to form DNA lesions [11]. Studies with cisplatin resistant and sensitive parental cell lines show reduced accumulation of the drug suggesting a role for active transporters for reduced accumulation of cisplatin. Mutation or deletion of the copper transporter

(CTR1) gene is associated with cisplatin resistance. Ctr1 is involved in active transport of cisplatin into the cells with resistant cell lines showing reduced expression. Over expression of

CTR1 in cell lines showed increased uptake of cisplatin [22]. Another area of investigation is increased efflux of the drug, copper-transporting P-type adenosine triphosphate (ATP7B), regulates copper homeostasis and plays a role in cisplatin efflux. ATP7B has also been associated with cisplatin resistance in different cancer types [32]. Another protein implicated in cisplatin efflux is of the multidrug resistance (MRP) – associated family – MRP2 or cMOAT.

MRP2 also has been consistently shown to be increased in levels in resistant cells [33].

b) Binding to non-DNA targets

Thiol-containing molecules such as glutathione (GSH) and metallothionein (MT) detoxify cisplatin and make it unavailable to bind its cellular targets. Studies with cisplatin- resistant models as well as clinical studies show increased levels of GSH and MT. Reaction catalyzed by glutathione-S-transferase (GST) enzyme conjugates GSH with cisplatin and studies show a correlation between increased concentration of glutathione or GST with cisplatin resistance in different cancer cell lines. Despite this, no patient studies have to date been conclusive in correlating patient GSH or GST expression levels to response rate or outcome after cisplatin treatment [34, 35].

Cisplatin can also bind with components of the cell membrane, cellular proteins and RNA

[36]. Cisplatin binds to enzymes, receptors and proteins through coordination with sulfur atoms in cysteine and methionine residues as well as nitrogen atoms in histidine residues and alters their activity [22]. All these non-DNA targets reduce the available drug or inactivate it.

c) Increased/Altered DNA Repair

The major cytotoxic effect of cisplatin is produced from its reactions with DNA and thus increasing the DNA- bound cisplatin and/or retention of these cisplatin-DNA lesions increases

cytotoxicity [14]. Cisplatin forms a covalent bond with the N7 position of purine bases to primarily form 1, 2- or 1, 3-intrastrand crosslinks and a lower percentage of interstrand crosslinks (ICLs). Formation of these lesions interferes with DNA replication and transcription especially the DNA-ICLs which are an absolute block. These lesions distort the DNA structure and these alterations are recognized by cellular proteins which repair cisplatin-DNA damage.

Increased repair of cisplatin-induced DNA damage is associated with cisplatin resistance and is one of the first pathways initiated [11, 13].

d) Increased cell survival

Changes in expression or mutations in cell-cycle checkpoint proteins as well as proteins involved in apoptosis give the cancer cell an advantage to survive post DNA damage. These signaling mechanisms lie downstream of cisplatin - DNA damage and their aberrant signaling leads to cisplatin resistance [31]. In terms of cell cycle checkpoint proteins, p53 plays a central role in cisplatin mediated apoptosis. Tumor cells with null or mutated p53 become resistant to cisplatin by tolerating the DNA damage. Mutations in p53 inhibit the activation of p53- dependent genes and the pro-apoptotic response. However, not all cell lines gain resistance to cisplatin with a dysfunctional or absent p53, wherein some become sensitive [37].

Proteins involved in apoptosis may also influence the outcome post cisplatin treatment.

For example, p53 directly affects expression of downstream genes that regulate sensitivity to apoptosis, activating transcription of proapoptotic Bax and repressing transcription of antiapoptotic Bcl-2 proteins [11]. Studies with cisplatin sensitive and resistant ovarian cancer cell lines has shown an association between increased levels of Bax and Bak proteins with cisplatin-induced apoptosis [38]. Apoptotic inhibitor molecules like survivin and XIAP (X- linked inhibitor of apoptosis protein) are overexpressed in cisplatin resistant cancers and they

impact the caspase cascade involved in the apoptotic response. Antiapoptotic Bcl-xl is also overexpressed in cisplatin resistance and this is likely to be due to the increase in glutathione levels [39].

1.3 DNA damage repair mechanisms

DNA is frequently subjected to attack by endogenous and exogenous sources which constantly challenge DNA integrity. This damage if left unrepaired leads to genomic instability, cancer and aging. Cells have developed surveillance mechanisms that respond when there is

DNA damage: 1) DNA repair 2) Cell-cycle checkpoint control and 3) Apoptosis. While apoptosis is triggered when the cell cannot cope with the damage, the most effective cellular mechanism is repair of the DNA damage for which the cell has developed various ways to deal with multiple lesions [40]. In mammalian cells there are at least five mechanisms of DNA repair: a) Nucleotide excision repair (NER) b) Base excision repair (BER) c) Mismatch repair (MMR) d) Homologous recombination repair (HRR) and e) Non-Homologous End-joining (NHEJ) [41].

In the following paragraphs, I briefly describe these DNA repair mechanisms.

NER is a versatile pathway that deals with a variety of lesions by sensing the distortion in the DNA. The damaged strand is identified and a short region spanning the lesion is excised, leaving a gap that is filled by the replicative polymerases. Thus, a common set of enzymes can deal with different lesions through this pathway. NER mechanism is absolutely important for the repair of cisplatin - DNA intrastrand adducts [42].

BER deals with excision of damaged DNA bases mainly arising from cellular metabolism events like deamination, oxidative damage and alkylation. The core BER pathway requires the function of only four proteins: a DNA glycosylase to cleave the N-glycosidic bond between the damaged base and the sugar phosphate backbone of the DNA generating an

apyrimidinic/apurinic (AP) or abasic site, an AP endonuclease or AP DNA lyase to process the phosphodiester backbone, a DNA polymerase to fill the gap and a DNA ligase to seal the nick

[43, 44]. Cisplatin resistant cells usually have a reduced expression of BER proteins suggesting loss of BER may cause an up-regulation of effective cisplatin DNA repair or increase adduct tolerance. Recent evidence suggests that BER could interact or compete with other repair mechanisms to prevent actual repair processes and maintain cisplatin sensitivity [45].

MMR deals with the repair of base-pairing mismatches and insertion–deletion loops which are usually a result of cellular metabolism. These mismatches if left unrepaired can be highly mutagenic and can lead to mutations. The damage recognition complexes, MSH2-MSH6

(MutS) or MSH2-MSH3 (MutS) recognize the damage. Next, a heterodimer of MLH1 with

PMS2 (MutLa) binds to the MSH complex and to a single strand nick in the DNA. This region is then excised leading to a gap that is repaired by DNA polymerases delta and epsilon and the replication machinery [46]. It has been seen that MMR and NER could have some overlap in damage recognition, like MSH2 and MutS can recognize and bind to cisplatin-DNA adducts that are repaired by NER [47].

MMR proteins have been shown to play a role in cell death in response to DNA damage agents like cisplatin and carboplatin. MMR proteins are required for G2-checkpoint activation induced by cisplatin. Loss of MMR leads to low-levels of cisplatin resistance and cisplatin resistant cell lines usually have a deficiency in MLH1, MSH2 or hPMS2 [47, 48] . One of the explanations is that MMR proteins bind to cisplatin intrastrand adducts and attempt to repair the damaged DNA [48-50]. This results in a “futile cycle” of repair which could result in cell cycle arrest and/or apoptosis. Another possibility is that MMR proteins bind to cisplatin- intrastrand

adducts and directly signal apoptosis or inhibit replicative bypass and translesion synthesis [48,

49, 51, 52].

Repair of Double Strand Breaks (DSBs) is a unique problem to the cell as it affects both strands of the DNA duplex and leads to problems in cellular processes like replication, transcription and chromosomal segregation. Proper repair of DSBs is absolutely essential to maintain genomic stability. DSBs are repaired by: 1) Non-Homologous End Joining (NHEJ) which is a common but error-prone pathway and 2) Homologous Recombination (HR) which is an error-free recombination event between homologous DNA sequences [53].

In NHEJ, DNA is repaired by simply joining the two broken ends without the requirement of a homologous sequence, making this an error prone pathway. The final reaction may result in small insertions or deletions of DNA sequences at the DSB location. However,

NHEJ is a quick, energy saving process for the cells especially in cell cycle phases where a homologous sequence is unavailable [54].

NHEJ has the following steps: detection of the DSB, processing of DNA ends into ligatable substrates and end ligation. These activities are mediated by the Ku70, Ku80, DNA-

PKcs, Xrcc4, DNA Ligase IV, and Artemis proteins. The Ku70-Ku80 protein heterodimer binds to the DSBs and recruits DNA-PKcs. Artemis, binds to DNA-PKcs and processes DNA ends.

Artemis-DNA-PKcs complex process DNA ends by removing 5‟ ends and trim long 3‟ overhangs. Next, polymerases fill the gaps which are sealed by XRCC4-DNA ligase IV complex.

In yeast, absence of Ku70 accelerates DSB end degradation thereby suggesting a protective role of Ku for unligated DSB ends. Also, DNA 5‟-end resection is a prerequisite for repair by HR and thus Ku channels DSB repair to NHEJ [55].

Evidence suggests that the choice between HR and NHEJ is regulated through cell-cycle.

Cells prefer HR when a homologous sequence is available during the DNA –replicative phase.

HR mostly involves proteins encoded by genes of the RAD52 epistasis group composed of

RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and

XRS2. Classical HR is mainly characterized by three successive steps: 1) resection of the 5‟- ended DNA ends by nucleases, 2) strand invasion of a homologous un-damaged DNA duplex and strand exchange, and 3) resolution of recombination intermediates [56, 57].

Cisplatin does not directly induce DSBs but unrepaired damage in the S phase of the cell cycle obstructs replication, leading to replication fork collapse and resulting in the formation of replication-associated DSBs. Usually, processing of cisplatin-ICLs results in DSBs [58]. Another pathway that needs mention here in terms of ICL repair is the Fanconi anemia (FA) pathway which is required for resistance to DNA-ICL inducing agents. FA is an autosomal or X-linked recessive disorder characterized by chromosomal instability, bone marrow failure, cancer susceptibility, and increased sensitivity to DNA- ICL inducing agents. To date, 15 genes have been identiﬁed that when mutated, result in FA or an FA-like syndrome. Identiﬁcation of the 15

FA and FA-like genes [FANCA, B, C, D1 (BRCA2), D2, E, F, G, I, J (BACH1/BRIP1), L, M, N

(PALB2), P (SLX4/BTCD12) and O (RAD51C)] has led to a signiﬁcant progress in understanding of this disease. Mutations in FA-A, FA-C and FA-G are the most common and account for approximately 85% of patients [59].

Briefly, FANCM-associated histone-fold-containing protein complex, MHF1-2 and

FAAP24 recruit a large multi-subunit ubiquitin E3 ligase, termed the FA core complex, to DNA lesions. This core complex then mono-ubiquitylates FANCD2 and FANCI. Monoubiquitylated

FANCD2-FANCI recruit nuclease FAN1 to damage sites and colocalize with downstream FA

proteins (PALB2, BRCA2, FANCJ, RAD51C and SLX4), and facilitate DNA ICL repair.

Among the 15 FA or FA-like proteins, 8 are assembled into a nuclear complex (A, B, C, E, F, G,

L, and M), called FA core complex. The major function of the FA core complex is to mono- ubiquitylate, FANCD2 and FANCI forming a protein complex called the ID. This mono- ubiquitylation event results in the ubiquitin tagged ID complex assembling at the sites of DNA damage in the nucleus where they subsequently colocalize with downstream effectors of FA proteins, FANCD1 (BRCA2), FANCN (PLAB2), FANCJ (BACH1/BRIP1) and FANCO

(RAD51C). This suggests that there is a possible crosstalk between the FA pathway, HR and translesion synthesis (TLS) [59].

In response to ICL, FANCD2 is phosphorylated in an ATR-dependent manner and this phosphorylation promotes FANCD2 mono-ubiquitylation and enhances resistance to DNA crosslinking agents. Like FANCD2, phosphorylation of FANCI has also been shown to be necessary for the mono-ubiquitylation and localization of both FANCI and FANCD2 to DNA damage sites [59].

Nucleotide Excision Repair (NER)

A major pathway involved in the repair of cisplatin-DNA adducts is the NER pathway

(Figure 2). NER deals with bulky, helix-distorting lesions like those formed by UV, chemotherapeutic agents and environmental mutagens. NER has two different sub-pathways for damage recognition - transcription-coupled repair (TCR) and global genome repair (GGR). TCR deals with activation of NER when RNA – polymerase II progression is blocked by lesions on the transcribed strand. GGR as the name suggests, deals with lesions spanning the whole genome which are recognized by XPC-RAD23B heterodimer, in some cases with help from UV-DDB.

The later steps are similar for both the sub pathways where in different proteins sequentially

assemble at the lesion [60-62]. NER has been reconstituted in vitro and has the following sequential steps: lesion sensing, opening of a denaturation bubble, incision of the damaged strand, displacement of the lesion-containing oligonucleotide, gap filling, and ligation. In GGR, the XPC complex requires a distortion in the DNA and a chemical modification to be activated.

In mammals there are two orthologs of the yeast

protein Rad23, HR23A and HR23B. Both can interact

with XPC and increase its activity but normally XPC

is associated with HR23B [63]. Another issue is

lesions that do not distort DNA for example UV-

induced lesions such as cyclobutane pyrimidine

dimers (CPDs). In such cases, the DDB complex, a

damaged DNA-binding heterodimer consisting of

DDB1 and DDB2/XPE, comes into play. The DDB

complex has a high affinity for DNA damage, such as

CPDs and it is believed to induce a kink in the DNA

which helps XPC sense this damage [63-65].

Once the damage is sensed and recognized, it

sets the stage for the opening of the denaturation bubble around the lesion which is achieved by the transcription factor TFIIH. TFIIH has XPB and XPD helicases; XPD helps in opening up the bubble of denaturation around the lesion while the ATPase activity of XPB is also required for this function [63, 64].

XPA preferentially binds to the damaged DNA and is associated with the three subunits of the RPA heterotrimer, which is a single-strand DNA binding protein. As XPA is recruited

after TFIIH, it is not thought to be involved in damage recognition anymore. Recent evidence suggests that XPA is important in identifying the damaged strand of DNA. It is important to note here that XPA is absolutely required for NER [63, 64, 66].

Next, the damaged strand is incised on the 3‟ side of the lesion by XPG endonuclease and on the 5‟ side by the XPF-ERCC1 heterodimer, which releases a 24–32 nucleotide damaged segment. The presence of XPG is necessary for XPF-ERCC1 to incise the other end of the bubble, but XPG does not necessarily cut first. Presence of XPG stabilizes TFIIH to set the stage for the incision reaction [42]. The resulting gap is likely filled by either of the replicative DNA polymerases delta and epsilon and the nick is sealed by a ligase, most likely ligase III. In TCR, bulky cisplatin lesions like the 1, 2- or 1, 3- intrastrand adduct are a strong block to RNAPII.

These lesions probably affect the RNA: DNA hybrid and promote the polymerase arrest and initiate TCR. The CSB and CSA (Cockayne syndrome type B and Cockayne syndrome type A) proteins are involved in TCR, with CSB coupling repair by recruiting NER factors and chromatin remodelers and CSA for E3-ubiquitin ligase complex to the stalled RNA polymerase

[65]. The later steps of TCR are same as those for GGR.

The importance of NER is seen from the diseases associated with mutations in the NER genes. Xeroderma Pigmentosum (XP) is caused by an absence or greatly reduced level of NER.

This disease is caused due to mutations in any of seven genes, from XP-A to XP-G, predisposing patients to cancer due to defects in NER [67]. It has been shown that XP cells are sensitive to UV light as well as to chemotherapeutic agents like cisplatin. Cockayne syndrome (CS) has five complementation groups. Two result from deficiencies in the CSA and CSB genes, which are specifically required for TCR but not for GGR. The remaining three, are due to mutations in XP genes: XPB, XPD, and XPG. COFS (cerebro-occulo-facio-skeletal syndrome) is closely related

to CS. COFS is indistinguishable from CS, and often caused by mutations in the same genes.

COFS patients carry mutations in the CSB gene, XPD or XPG genes. The only patient observed with a mutation in the ERCC1 gene also suffered from COFS. The mutations prevented the dimer formation between XPF and ERCC1 but only moderately affected NER activity. As the symptoms were extremely severe, this suggested a separate role for XPF/ERCC1 outside NER.

UV-sensitive syndrome (UVSS) is characterized by mild photosensitivity in sun-exposed areas of the skin without the high propensity to skin cancer. Patients with UVSS have deficiencies in

TCR. Trichothiodystropy (TTD) is characterized by brittle hair, due to a deficiency in high molecular weight sulfur-rich proteins. People with TTD also present with photosensitivity and have mutations in subunits of TFIIH [63, 64].

NER activity is absolutely important for the repair of cisplatin - DNA adducts. Increased

NER activity has been reported in cisplatin- resistant cancer cells and this activity is detrimental to cisplatin efficacy as a chemotherapeutic agent. For example, testicular tumor cell lines have low levels of NER activity and hence are sensitive to cisplatin. Low levels of NER protein expression was found in testicular tumor cell lines as compared to cell lines derived from other cancer types [68]. Also, addition of NER proteins to protein extracts of testicular tumor cell lines increased their DNA-repair capacity to normal levels [69]. The XPF/ERCC1 complex is required for the processing of the NER pathway and its role is essential to the repair of ICLs. Thus,

XPF/ERCC1 is required in all aspects of cisplatin-DNA repair [70].

1.4 XPF/ERCC1

XPF/ERCC1 functions by forming a heterodimer in a 1: l stoichiometry [71]. The heterodimer forms a structure-specific endonuclease which excises at a junction of single strand and double strand DNA on the 5‟ side. In yeast, the Radl-Rad10 complex is the counterpart of

XPF/ERCCl. In vitro reconstitution experiments suggest that XPF/ERCC1 is the last subunit to arrive and even though the reaction occurs in a concerted manner, normally the 3‟ incision may precede the 5‟ incision [72]. Although XPF/ ERCCI function is critical in the NER pathway, this is not its only role. Mutations in XPF or ERCC1 in mice as well as humans show extreme sensitivity to ICL-inducing agents as compared to other XP mutants. This means that

XPF/ERCC1 has a central role in ICL repair but the exact mechanisms that occur during ICL repair are still speculative [73-75].

XPF/ERCC1 Structure

XPF/ERCC1 forms a heterodimer through the C-terminal, tandem helix–hairpin–helix

(HhH2) domains of both proteins (Figure 3) and small deletions at the C terminus of either protein result in a loss of activity [76].

Figure 3. XPF/ERCC1 domains and heterodimer formation.

XPF

XPF harbors the nuclease active site and consists of three domains (Figure 3) [77]. The first domain is the N-terminal helicase-like domain which lacks several key residues required for

DNA unwinding activity. This domain may lead to DNA binding activity which is still speculative. The central nuclease domain has conserved metal-binding residues as well as functionally important basic residues that can possibly interact with DNA substrates. The C-

terminal HhH2 domain of XPF specifically dimerizes with ERCC1 to form the functional nuclease and may additionally contribute to DNA binding [78, 79].

ERCC1

The ERCC1 subunit consists of only two domains (Figure 3) – a conserved central domain and the C-terminal HhH2 domain. The central domain is structurally similar to XPF nuclease domain but lacks residues to make it a characteristic nuclease [80]. The C-terminal domain dimerizes with the equivalent domain in XPF and may also bind DNA. ERCC1 binding stabilizes XPF nuclease and presumably also contributes to specific selection of DNA substrates for the nuclease to act on. ERCC1 also mediates an interaction with XPA, which is also essential to NER [76].

XPF/ERCC1 heterodimer is recruited for DNA repair predicatively through specific protein-protein interactions. The interaction between XPA and ERCC1 is essential for NER and involves a region of XPA encompassing three consecutive glycines (residues 72–74). A short and unstructured peptide of XPA, XPA67–80, undergoes a disordered-to-ordered transition on binding to the central domain of ERCC1. This 14- amino acid stretch of XPA is necessary and sufficient to mediate its interaction with ERCC1, and the XPA67–80 peptide can inhibit NER activity in vitro. Mutations in the central domain of ERCC1 severely impact NER activity in vitro and in vivo but do not affect the function of XPF/ERCC1 in other DNA repair pathways

[81]. These studies show that based on the protein-protein interactions not only can the function of XPF-ERCC1 be allocated to different pathways but also suggests that XPF-ERCC1 can be specifically targeted to inhibit different DNA repair pathways.

XPF/ERCC1: Interstrand crosslinks (ICLs) and more

ICL repair encompasses NER, HR and translesion synthesis (TLS). Elucidating the mechanisms that lead to ICL repair is important and highly relevant to chemotherapeutic drugs that induce

DNA crosslinks. In yeast it has been shown that loss of several NER proteins leads to ICL sensitivity but that is not the case in mammalian cells. XP-F patients‟ cells were shown to proficiently process ICLs but failed to repair NER- specific damage. Exquisite ICL sensitivity is only seen on loss of XPF or ERCC1 as compared to other proteins important for NER [18, 82].

This suggests XPF/ERCC1 has a role independent of NER in ICL repair. It has been shown that

XPF/ERCC1 facilitates the repair of DSBs induced by cisplatin-ICL processing and it has been proposed that the key role of XPF/ERCC1 is the unhooking of the cross-link in ICL repair.

Recent evidence suggests that XPF/ERCC1 plays a role in the completion of Homologous

Recombination (HR) possibly in the unhooking of ICLs and not in resolving the HR intermediates. XPF reportedly interacts with hRad52, which might recruit the complex to sites of homologous recombination or single-strand annealing [83-85].

It is known that ERCC1 interacts with the mismatch repair protein MSH2, and it has been suggested that MSH2 and ERCC1 work together in ICL repair. However, MSH2- deficient cells do not show highly pronounced sensitivity as ERCC1-deficient cells suggesting MSH2 might be dispensable for certain ICL repair events. [47]

MEI-9, the Drosophila homolog of XPF interacts both with ERCC1 and MUS312.

Mus312 has roles in ICL repair. Recently SLX4 was identified as the human homolog of

MUS312. SLX4 is important in HR and interacts with endonucleases, including XPF/ERCC1,

MUS81-EME1, and SLX1, suggesting very likely that it could be a major player in the coordination of endonucleases recruited for ICL repair [81].

Cells deficient in ERCC1 and XPF accumulate replication associated DSBs upon ICL induction. Accumulation of DSBs could be due to defective ICL processing or a defect in unhooking during S phase thus preventing replication fork restoration. Recently it was shown that XPF/ERCC1 works with hSNM1A (a protein important for ICL repair) to initiate ICL repair. XPF/ERCC1 incises and provides the starting point for hSNM1A to digest and help in

ICL resolution [86]. Recent evidence also suggests that ERCC1 plays a role in mitotic progression, a novel role independent of XPF. In hepatocellular carcinoma cells lines, it was shown that ERCC1 knockdown cells were delayed in their cell cycle and became multinucleated

[87]. Studies also suggest that XPF/ERCC1 participate in the same pathway of ICL repair as the

Fanconi anemia (FA) proteins. Cells lacking XPF/ERCC1 have monoubiquitylated FANCD2 with impaired translocation to the chromatin. Next, it was shown that FA protein, FANCG through its tetratricopepetide repeats binds to the central domain of ERCC1 and is involved in the unhooking step of the ICL repair process with ERCC1/XPF [88, 89].

Mutations in XPF and ERCC1 orthologs, in yeast suppress HR between sequence repeats.

As in yeast, HR and end-joining of DSBs is attenuated in XPF/ERCC1 deficient cells.

XPF/ERCC1 is also required for efficient single-strand annealing (SSA) and gene conversion in mammals [83, 90]. Thus, XPF/ERCC1 is involved in both HR and Non-Homologous End

Joining (NHEJ) mechanism of DSB repair.

XPF/ERCC1 deficiency is associated with accelerated ageing; but cells deficient in them do not show any differences in telomere length and sister chromatid exchanges (SCEs) at telomeres. It has been shown that ERCC1 co-localizes with TRF2 at telomeres and in the absence of TRF2, XPF/ERCC1 makes chromosomes more susceptible to end-to-end fusions.

Thus, XPF/ERCC1 overexpression or correction in XP-F cells leads to telomere shortening [90].

Thus, aging associated with XPF/ERCC1 is at least not thought to be associated with telomere- dependent replicative senescence.

Mutations in ERCC1 cause cerebro-oculo-facio-skeletal syndrome, whereas mutations in

XPF that severely affect protein expression cause accelerated aging (XFE progeroid syndrome).

Mice completely deficient in XPF/ERCC1 age rapidly and have a dramatically reduced life span suggesting additional roles outside NER. In ERCC1 knockout mice, postnatal growth is severely retarded and they die at ~ 3 weeks. These mice spontaneously develop symptoms of progressive neurodegeneration [75]. Although the hematopoietic system develops normally, by the end of their life these mice are leukopenic and thrombocytopenic. There is also extensive adipose transformation of the bone marrow which is a hallmark of normal aging in mice. They are also impaired in proliferation of multi-potent and lineage-specific progenitors [91]. Their bone marrow progenitors are extremely sensitive to crosslinking agents like mice models of Fanconi

Anemia [92]. There is severe liver function impairment, musculoskeletal and nervous system defects [93]. There is growth suppression and these mice are very small in size. These mice have a lot of overlap in genetic profile when compared to wild-type mouse models of aging [94]. The

XPF mutant mice have severe phenotype identical to ERCC1 null mice suggesting their exclusive function as a complex. Another study showed that XPF/ERCC1 could be important in repair of reactive oxygen species (ROS)-induced DNA damage by trimming 3‟-blocked ends

[95]. This suggests that unrepaired ROS-induced DNA damage could contribute to accelerated ageing in the absence of XPF/ERCC1.

XPF- ERCC1 and platinum-based therapy in cancer

It has been suggested that ERCC1 levels influence and correlate with DNA repair capacity [96]. ERCC1 expression has been associated with cellular and clinical resistance to

platinum-based chemotherapy. There is much speculation about the importance of ERCC1 as a prognostic indicator of cisplatin response in different cancer types like gastric, ovarian, colorectal, esophageal cancer and in NSCLC [97]. Testicular cancer is generally responsive to cisplatin and has low levels of XPF-ERCC1, raising the possibility that XPF-ERCC1 levels influence the response to platinum therapy [68].

There is an inverse correlation between ERCC1 mRNA levels from tumor samples taken from patients in small retrospective clinical trials of ovarian, colorectal, and NSCLC and response to platinum therapy or survival. The trial by the International Adjuvant Lung Cancer taking into account immuno-histochemical analysis of ERCC1 showed patients with ERCC1- negative tumors were shown to benefit from cisplatin-based adjuvant chemotherapy, whereas patients with ERCC1-positive tumors did not. [98].

ERCC1 polymorphisms, codon 118C/T and C8092A, are shown to be associated with response to platinum-based chemotherapy [98]. Triple negative (TN) breast tumors that are clinically more aggressive rely only on chemotherapy due to the lack of molecular targets. For these tumor types, it has also been recently suggested that ERCC1 expression would be a beneficial tool to identify patients that can benefit from platinum therapy [99]. Thus, it would be important to establish the contribution of levels of ERCC1 to platinum therapy.

1.5 DNA Repair as a molecular target

DNA damaging agents like cisplatin preferentially targets cells in a rapid dividing state hence eliminating tumor cells. The toxicity of cisplatin is reduced by enhanced or altered DNA repair mechanisms that remove the cytotoxic lesion or tolerate the cisplatin-induced DNA damage thereby decreasing cisplatin efficacy. Thus, the toxicity of a DNA-damaging agent can be enhanced by targeting the repair pathways for cancer therapy.

Some of the repair mechanisms are already downregulated or inactivated in some cancers making this an attractive molecular target. Defects in one pathway could be compensated by another and these pathways can be identified in synthetic lethality screens and targeted specifically in DNA-repair defective tumors. For example, breast and ovarian tumors defective in homologous recombination mechanisms (BRCA-1 and BRCA-2) are being tested in phase II clinical trials for synthetic lethality with poly (ADP-ribose) polymerase (PARP) inhibitors. DNA repair inhibitors can also prevent repair of replication lesions in constantly dividing tumors and convert them to fatal replication lesions to induce death [99-101].

DNA damaging agents usually cause replication block or obstruct transcription, cell- cycle arrest, and induce cell death. Cisplatin-DNA adducts mediate cytotoxicity and furthermore, cisplatin-DNA ICL processing leads to the formation of replication-associated DSBs. DSBs are generally considered to be the most toxic of all DNA lesions. In case of cisplatin and its analogs,

XPF/ERCC1 shows promise as it is involved in all aspects of cisplatin-DNA damage repair and can be targeted to further sensitize cisplatin to tumor cells. Currently, such sensitizing therapies in clinical trials are a combination of pseudosubstrates of O-6-methylguanine-DNA methyltransferase (MGMT) and temozolomide. These pseudosubstrates would deplete cellular

MGMT thereby sensitizing the cells to temozolomide. Such therapies have shown promise in preclinical models but a therapeutic index needs to be established in clinical trials. [102, 103]

Studies with the anticancer agent UCN-01 (7-hydroxystaurosporine) shows potentiation of cisplatin and carboplatin toxicity in preclinical and phase I clinical trials. UCN-01 has been shown to interfere with the protein-protein interaction of ERCC1 and XPA [104]. But, disrupting the interaction of ERCC1 and XPA only affects the repair of cisplatin-DNA intrastrand lesions repaired through NER. However, by disrupting the protein-DNA interaction between

XPF/ERCC1 and DNA, will prevent the repair of all types of cisplatin-DNA lesions, thus having the potential to dramatically increase cisplatin efficacy as well as that of other platinum analogues. XPF/ERCC1 is central to cisplatin-DNA repair and its inhibitor can be used in clinical therapies that use cisplatin alone or in combination with other drugs. An XPF/ERCC1 inhibitor could also be tested in synthetic lethality screens for cancers defective in DSB repair pathways and other mechanisms that overlap with XPF/ERCC1 function to effectively kill cancer cells. An XPF/ERCC1 inhibitor can also be used alone to overwhelm tumor cells with unrepaired DNA damage especially for tumors with defects in other repair mechanisms. A therapy in current trials is PARP inhibitor as a monotherapy in HR-defective tumors. PARP inhibitors sensitize these tumors by most likely inducing single-strand breaks that can result in

DSBs due to stalled replication forks and remain unrepaired in HR-defective tumors [101].

1.6 The bystander effect (BE) in cancer therapeutics

The radiation induced “bystander effect” (RIBE) is communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. This effect has been well studied and has been shown both in vitro and in vivo. It has been studied in a wide range of cell types like lymphocytes, fibroblasts, endothelial cells and cancer cells. This effect has also been observed with ultraviolet radiation, photodynamic therapy, gene therapy, heat and chemotherapeutic agents [105].

Studies with different agents and especially with radiation, shows that the Bystander

Effect (BE) involves direct cell-cell communication mediated by Gap Junctions (GJ) especially the ubiquitously expressed connexin, Connexin43 (Cx43). Much of the early work characterizing the BE through Gap Junction Intercellular Communication (GJIC) was performed in confluent cell monolayers that were selectively irradiated with some cells left unirradiated. Little and

colleagues showed that in conditions when only 1% cells are irradiated, 30% of the rest of the cells showed chromosomal aberrations [106, 107].

Studies with inhibitors of GJIC like lindane prevented the bystander signaling in primary human fibroblasts. Another area way through which the BE is communicated is through the release of soluble factors from irradiated cells which can be taken up by un-irradiated cells [108,

109]. Some of these factors under study for mediating the bystander effect are lipid peroxide products, inosine nucleotides and cytokines like tumor necrosis factor-α (TNF-a), interleukin 6 and 8, transforming growth factor-β1 (TGFβ1), reactive oxygen species (ROS) like superoxide radicals, and reactive nitrogen species [108-110].

One of the important points to keep into consideration in terms of the RIBE is that unlike the direct effects of radiation, this effect does not follow a dose-response relationship. In fact, the bystander response becomes saturated at relatively low doses (typically less than 1 Gy). Studies with low-dose exposure show that the BE is as effective as direct exposure to radiation. It is speculated that there is a saturation of the BE at least in vitro as there could be a limit to the signals produced by the treated cells and thus increasing doses might not show further effect. In some studies, it has been shown that there is a decreased or no effect at high doses and BE reaches a plateau at low doses [111]. As it is still not clearly understood what the “actual” signal for the bystander response is, it is difficult to generalize the effect of low and high dose as well as difficult to extrapolate these responses to humans.

A study in 1922 showed that the serum from irradiated animals stimulated the growth of lymphoid cells up to 2 hours after irradiation while the serum from control group caused cell degradation [112]. It was also reported in the 1960s, blood from patients under radiotherapy has mutagenic factors [105]. Many studies show close parallels of the bystander signaling to

inflammatory responses. In macrophages, there are increased levels of oxidative stress after radiation exposure under bystander conditions. Cyclooxygenase 2 (CoX2) is a central player in inflammatory responses and also mediates bystander signaling. CoX2 downstream signaling leads to activation of nitric oxide (No) synthase leading to the production of reactive nitrogen species. Inhibition of these pathways in bystander cells leads to inactivation of BE. Using microbeams; it was shown that just triggering ROS production in the cytoplasm can lead to the

BE too. This also brings in mitochondrial signaling as a mediator in this response [113].

Three-dimensional human skin reconstructs were locally irradiated with helium ions from a microbeam and 72 hours later the skin was sectioned, and scored at different distances away from the target site. Significant numbers of damaged cells were observed up to 1 mm away. In lung reconstruct models, levels of γH2AX, a marker of DNA DSBs, as well as DNA methylation and senescence were increased [114]. Khan et al found that partially irradiating rat lung led to micronucleus formation in non-irradiated areas of the lung, indicating DNA damage at these non-irradiated sites. However, exposure of animals with Cu–Zn superoxide dismutase (SoD) or the Nitric Oxide signaling (NoS) inhibitor before irradiation showed a reduced BE in the non- targeted area [115]. Camphausen et al. found that there was reduced tumor growth when they irradiated the legs of mice that had tumors transplanted at the dorsal midline. This response was prevented by blocking p53 by the drug α-pifithrin [116]. Recently it was shown that NO- dependent signaling is required in tumor cells to undergo RIBE response hence approaches that could introduce NOS2 (Nitric oxide synthase 2) in such cancer cells could lead to greater cell killing [117].

The DNA damage response and repair mechanisms are emerging as key processes occurring in bystander cells. When cells are treated to radiation, there is direct damage to the

DNA producing DSBs. As already discussed, DSBs are absolutely lethal to the cells if not repaired, residual damage can also lead to genetic instability and cell death. RIBE elicits in bystander cells as DSBs basically leading to an end-point of DNA damage, mutations, chromosomal aberrations and cell death [106, 118]. So modulating these responses could have therapeutic benefits for cancer therapeutics. In irradiated cells, inhibition of DNA damage sensors like Ataxia Telangiectasia Mutated (ATM), Ataxia Telangiectasia and Rad3-related

(ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PK) proteins increases radiosensitivity. But in bystander cells inhibition of ATR or ATM prevents the killing of bystander cells. Also the ATR-dependent bystander foci induction was restricted to S-phase cells

[119]. In such a case it would be important to understand the predominant role of ATR in the bystander response.

Studies have shown that bystander cells deficient in Non Homologous End Joining

(NHEJ) - repair mechanism show increased mutations and chromosomal aberrations. It is believed that transfer of ROS from treated cells to bystander cells produces oxidative DNA damage which in these repair deficient cells may lead to stalled replication forks and production of DSBs [106, 118]. Mutations in NHEJ-deficient bystander cells were primarily deletions, consistent with unrepaired DNA damage. It was also shown by Nagasawa H et al, that HR is unable to repair the damage in the NHEJ-deficient cell lines leading to SCEs and chromosomal aberrations. Thus, cells deficient in different DNA repair processes could show an enhanced BE

[107, 120]. Consistent with the theory on oxidative damage, in another study, there was an increase in protein levels of AP endonuclease, a redox and DNA Base Excision Repair protein in the bystander cells and not in the irradiated cells [118].

In another study, the impact of senescent cells on normal cells with intact DNA damage response and repair mechanisms was evaluated. The authors showed pro-oxidant and pro- inflammatory signals from the senescent cells trigger DNA damage in the surrounding bystander normal cells leading to propagation of senescence. In the same cells, blocking GJIC with a general gap junction blocker or blocking ROS blocked the bystander response demonstrating their importance [121]. In another study, there was an up-regulation of the p53 damage-response pathway in the bystander cells exposed to low fluences of alpha particles. The p53 in these bystander neighboring cells was phosphorylated on serine 15 suggesting a consequence of DNA damage [109].

1.7 Gap junction proteins: Connections with connexins

Gap junctions are specialized intercellular channels that connect the cytoplasm of connecting cells (Figure 4). These channels can be gated. Two connexons or hemichannels form a GJ channel from two contributing cells. Each connexon is composed of six connexins that assemble to form a central pore through which there is cell-to-cell communication (Figure 5).

Cells through this way exchange amino acids,

secondary metabolites, chemicals, ions etc,

however allowing only hydrophilic molecules

with molecular weights of ~1 kDa are allowed.

These molecules usually diffuse passively

through the pore. Recent evidence shows that

small interfering RNA and small peptides can

also traverse these pores. Thus, cells maintain Figure 4 Structure of a Gap Junction tissue homeostasis, organ function and

development by gap junction intercellular communication (GJIC) [122].

Gap junctions are present in all cell types of vertebrates, except very few cases such as red blood cells, platelets, some neurons, mature skeletal muscle fibers and spermatozoids [123].

There are 21 known human connexin (Cx) proteins which are usually expressed in cell and a development specific manner. A cell can express more than one type of connexin so that connexons can be homomeric or heteromeric. A gap junction channel can also be heterotypic with 2 different types of connexons. The most widely studied Cxs are Cx43, Cx32, and Cx26 because of their relative abundance. Cx43 knockout mice die early in development due to heart malformations. Cxs are tightly regulated and their expression depends on tissue specificity, developmental stage, intracellular and extracellular stimuli, and cell cycle phase [124, 125].

Each Cx protein spans the

membrane four times (Figure 5), and is

oriented with cytoplasmic amino- and

carboxy-termini, while the extracellular

domains are involved in connexon–

connexon interactions to form a pore

between cells. Cxs are regulated via the Figure 5 Formation of Gap Junction by Connexin proteins carboxy terminus and this region contains consensus sites for phosphorylation, calmodulin binding and channel gating. Cxs not only create channels to interact with cells but they also interact with the extracellular environment through hemichannels. Cxs also interact with cellular signaling mechanisms and function beyond their role as a GJ protein [126].

Normal progenitor and many terminally differentiated cells exhibit functional GJIC. It is also known that coupled cells show contact inhibition. It has also been seen that normal stem cells do not exhibit functional GJIC till they start the process of differentiation. Studies with normal fertilized eggs (totipotent stem cells) and several human pluripotent stem cells do not express connexins or couple. Growth factors, cytokines, hormones, cell adhesions and extracellular molecules trigger different cellular functions like transcription, activation of kinases, calcium signaling etc which in turn modulates gap junction function [122].

In most cells, GJIC is involved in cell cycle progression and is reduced in late G1, S and

M phases. In proliferating murine neocortex cells, Cx26 expression increases from S to the early

G1 phase while Cx43 expression is highest in S/G2 phases and decreases towards the G1 phase.

Cx phosphorylation is an important event that regulates GJIC during cell cycling. Some of the kinases involved are PKC, Cdk1/cyclin B complex, Cdk1/cyclin B. Cell growth requires the activation of numerous signaling cascades like MAPK and the PI3K/Akt cascades that negatively regulate gap junction. Thus, the diminished GJIC or gap junction expression could be a part of cellular proliferation. Forced expression of Cx43 and Cx32 in neoplastic lung and liver cells restored G1 growth control and was associated with increased p27 (kip-1) and decreased cyclin

D1 expression. Similarly, overexpression of Cx43 in osteosarcoma cells inhibited cell cycle transition from G1 to S phase by increasing the expression of p27 [122, 127]. Connexins can regulate growth through GJIC and GJIC –independent effects as shown by Zhang et al. In human osteosarcoma cells, forced expression of Cx43 suppresses cellular proliferation which is associated with increased expression of p27. This is in part due to the GJIC- mediated flux of cAMP that enhances p27 and in part due to Cx43‟s promotion of Skp2 degradation that is involved in p27 ubiquitination, a function independent of GJIC [128].

Cxs can also directly control gene expression as seen by modification in gene expression patterns of cells transfected with Cxs. For example, expression of growth enhancing or repressing genes is modulated. Stains et al showed the existence of a “connexin response element” (CxRE) in the promoter region of the rat osteocalcin gene, which is regulated by Sp transcription factors. In a GJIC dependent manner, growth stimulation signals traverse the gap junctions and activate downstream signaling cascades for ERK and PI3/Akt pathways. This activation leads to phosphorylation of Sp1 which binds to the CxRE resulting in the transcription of the osteocalcin gene [129]. In HeLa and Neuro2A cells, transfection of the carboxy-terminal tail of Cx43 is associated with growth inhibition showing specific regions of Cxs effect gene expression. However, mutants of Cx32 and Cx43 lacking the C-terminal tail can also inhibit growth showing other structural regions can also do the same [130].

Cells primed for apoptosis show drastic alterations in Ca2+ concentration, an ion that is intercellularly exchanged via gap junctions. Thus, Ca2+ ions or its induced signaling events are thought to be the “death signals” spreading in a wave from cell to cell [131, 132]. On progression of cell death, GJIC activity declines with increased removal of GJs from the membrane. GJs can also limit the transfer of toxic metabolites or healthy cells can send rescue signals like glucose,

ATP and ascorbic acids to dying neighboring cells. Cx43 expression was shown to inversely correlate with cell death. Several studies show Cxs control expression of apoptosis related genes, a function which is GJIC-independent [131].

Studies show that chemicals that promote tumorigenesis inhibit GJs or GJIC. Oncogenic activation is also another reason for downregulation of GJIC. Some agents are known to either suppress or upregulate GJIC. Downregulating connexin genes can also have such effects. Studies

show that a connexin 32 knockout mouse can develop both spontaneous and induced liver cancers [133].

1.8 Gap junction intercellular communication (GJIC) mediates the bystander response

Mitchell et al showed that cell contact was necessary for the bystander response induced cell killing and transmission of bystander signal was more efficient through gap junctions than through culture medium [134]. Recent studies also showed that in cells exposed to high or low

LET radiation increased expression of Cx43 in irradiated cells which correlated with enhanced cell to-cell communication. The same was not seen with ultraviolet light (UV), where there was a decrease in the Bystander Effecr (BE) as there was a down regulation of Cx43 in UV treated cells [135].

In a study by Harada K et al, heavy-ion microbeam apparatus was used to investigate the impact of the bystander effect. They showed that there was a marked reduction in colony survival of cells treated at confluence thus showing the importance of cell-to-cell communication in bystander effect induced cell killing. The authors also used a GJIC stimulator, cAMP, which enhanced bystander cell killing while lindane, an inhibitor of GJIC, suppressed this effect [136].

Azzam et al. have shown that cells expressing Cx43 GJs, increased radiation stress with increased expression of p21waf–1 expression which correlated with the induction of DNA damage.

These cells were compared to GJ compromised cells (chemically or genetically) which did not show this BE [109]. Previously, these authors also showed that when GJIC is blocked in confluent cells by lindane or when low density cultures are used there is a reduction in the induction of TP53 and CDKN1A levels post irradiation. Thus, implicating gap junction proteins and GJIC in the bystander response [109].

Suicide genes in combination with suitable pro-drugs may be used to kill cancer cells.

The suicide gene codes for an enzyme that catalyzes the transformation of a harmless prodrug into a toxic metabolite. Such combinations are currently under investigation, for example, cytosine deaminase (CD)/5-fluorocytosine, thymidine phosphorylase/5'-deoxy-5-fluorouridine

(TP/5'-DFUR), herpes simplex virus thymidine kinase (HSV-tk) in combination with the prodrug ganciclovir (GCV) etc. Cancer cells transfected with the HSV-tk gene become sensitive to GCV, a guanosine analog. The transfected cells produce the viral enzyme thymidine kinase (TK), which phosphorylates GCV into a monophosphate (GCV-MP). Subsequently, cellular kinases phosphorylate GCV-MP to the corresponding diphosphate (GCV-DP) and toxic triphosphate

(GCV-TP) which inhibits DNA polymerases and blocks DNA replication. In in vitro experiments with tumor cell lines, this suicide/gene therapy also exhibits a BE described by

Moolten et al. Not only the HSV-tk positive cells die, but their neighboring HSV-tk negative cells are also killed after administration of GCV. In vitro experiments show that a transfection percentage of only 1% is sufficient to kill virtually the complete cell population. For this BE, cell-cell contact was essential and the extent of cell killing was different in different tumor types.

Wygoda et al showed that bystander cells through GJIC could also protect HSV-tk transfected cells. The bystander cells through GJIC take up the phosphorylated GCV metabolites and protect the transfected cells while dying in the process. This has been called the “Good-Samaritan effect”. Widel et al showed that in response to signals from irradiated human melanoma cells, non-irradiated normal human dermal fibroblasts trigger unknown “rescue” signals that modify the redox status of the irradiated melanoma cells and hence protect them [137].

Mesnil et al. showed that HeLa cells (which have poor GJIC) co-transfected with HSV-tk positive cells and Cx43, displayed 100% cell killing at a transfection percentage of 10% [123].

Cx43 is an important player in GJIC, with other studies showing that the expression and membrane localization of Cx43 GJ necessary for the occurrence of the BE in human colon tumor cells. The BE was also observed with Cx26 and Cx37. Dilber et al. transfected Cx43 in rat gliomas and showed an increase in in vivo bystander induced cell killing with HSV-tk therapy.

Similarly in another study, two rat glioma cell lines with differential expression of Cx43 were mixed and the BE in vitro and in vivo was enhanced with high expression of Cx43 while no BE was seen when the Cx43 level was low [138]. Meritxell Carrió‟s group has shown that introduction of Cx26 in pancreatic tumor cells improves GJIC and thus increases gemcitabine

BE in vivo as well the HSV-tk gene therapy BE [139].

1.9 Cancer and the Gap

Loss of functional gap junctions has been implicated as a causative factor in numerous diseases, including heart failure, neuropathology, deafness, skin disorders and cataracts. There is also significant evidence that dsyregulation of GJIC is important in carcinogenesis. There is a large body of evidence in the literature showing downregulation or deficiency of connexins and the development of the cancer phenotype [123].

It was first demonstrated by Loewenstein and Kanno, a lack of electrical coupling in rat hepatomas. This phenomenon was observed in both chemically-induced and transplanted hepatomas [140, 141]. In humans, it was first seen in stomach carcinoma cells that failed to couple [142]. Studies show the difference in expression of connexins in different stages of cancer progression and the loss of gap junctions and coupling. Cx43 has been found to be highly reduced in dysplastic regions of the human cervix compared to normal region. Similarly, endometrial hyperplastic cells have low expression for Cx26 and Cx32 [143-145]. Patients at end-stage renal disease have high incidence of renal cell carcinoma and a hypermethylation of

the Cx32 gene is observed in the cancerous regions of the kidney in such patients [146]. Cx43 negative HeLa cells when treated with 5-aza-2V-deoxycytidine start expressing Cx43 implicating DNA methylation [147]. In some cancers, decrease in Cx level is only evident in later stages of the disease. For example in prostate cancer, Cx43 levels decrease in later stages.

Studies also show progressive decrease in expression of Cx43 with progression of glioma [148-

150].

In rat liver cancer, there is a progressive decrease in the Cx32 expression which is accompanied with a decrease in GJIC [151]. In some cancers, the levels of Cxs are not affected but they are localized sub-cellular or nuclear. In hepatocellular carcinomas, Cx32 is mostly localized in the cytoplasm. Cx26 was found to be cytoplasmic in human breast invasive carcinomas and in some human bladder tumors. A cytoplasmic localization for Cx is usually associated with highly invasive tumors. Growth inhibition in HeLa cells was induced by Cx43 localized in the nucleus [123].

Studies with mice and human skin tumors show a decrease of Cx26 and Cx43 expression progressively from papillomas to well-, moderately- and poorly differentiated squamous cell carcinomas of the skin [152].

It has been demonstrated that Cx43 is expressed in normal breast tissue while it disappears during the development of ductal and lobular carcinomas. Functional restoration of

Cx43 restores GJIC and subsequently the bystander effect. These studies concluded that Cx43 expression could be a biomarker for breast oncogenesis [153].

In mouse and human lung cancer cell lines, there is often a decreased expression of Cx43

[154]. However, in some stages of cancer, the Cx expression is actually required as seen for

Cx26 which is upregulated in invasive lesions of breast carcinomas [155].

In some cases, there are discrepancies in the in vitro and in vivo data. Hepatoma cells that don‟t express Cx32 in culture, express once transplanted in vivo but post transplantation

Cx32 is downregulated [156]. These changes in expression of gap junction proteins in the tumor eventually lead to a reduction in GJIC as evidenced by reduced capacity for dye-transfer as compared to normal regions of the tissue. Several mutations have been reported for Cx with most correlation between Cx43 mutations and cancer. Cx43 mutations are associated with advanced stages of tumor progression with most at the carboxy terminal of Cx43 leading to a shift in the reading frame of the gene. Mutated Cx43 was expressed in invasive tumors [157].

Cx43 mRNA was not detected in a highly metastatic human lung carcinoma cell line and in breast cancer. Restoration of Cx43 expression, GJIC and normal breast tissue phenotype was observed in a metastatic human breast cancer cell line when transfected with the breast metastasis suppressor 1 (BRMS1) cDNA [158].

2.0 Cisplatin as a bystander effect inducing agent

Recent studies have reported cisplatin-induced cell killing also has a bystander component which could have potential in clinical therapeutics as well as could be developed into a biomarker for cisplatin chemotherapy.

Jensen and Glazer reported that cisplatin induced cytotoxicity may be transferred to untreated neighboring cells through GJIC. They found that a deficiency in the NHEJ complex

Ku70/80 could lead to enhanced cisplatin sensitivity. They found out that this effect was dependent on cell density. Ku-deficient cells plated at high density were sensitive to the drug while the same cells plated at low density did not exhibit a difference in sensitivity compared to parental cells. In contrast, MMR-deficient cells show moderate resistance regardless of density, suggesting distinct responses through Ku 70/80. They next showed that the Ku-initiated death

signal required DNA-PK activation and was transmitted from cell to cell through gap junctions

[159].

Cx43 is a phosphorylation target of src. In another publication by the same group, using mouse embryonic fibroblasts they showed that the activation of src produced tyrosine phosphorylation of Cx43 which decreased GJIC and increased survival in response to cisplatin.

This effect was abrogated using inhibitors of src or by knockdown of Cx43 or increased by overexpression of Cx43 [160]. Surprisingly in another study, Cx43 gene expression and protein levels were found highly upregulated in a cisplatin resistant ovarian cancer cell line and its downregulation led to a further increase in cisplatin resistance [161].

Kalvelyte et al have shown that functional gap junctions enhance apoptosis induced by cisplatin. In bladder cancer cells, gap junction expression promotes cisplatin induced cell cycle arrest, downregulation of BCL-2 and cell death [162] Andrew Harris„s group showed that cisplatin and oxaliplatin directly interact with connexin protein and inhibit their activity as well as reduce their expression levels, thus decreasing cytotoxicity. Their study used HeLa cells stably transfected to express heteromeric Cx32/Cx26 channels [163]. Harris‟s group published another study where they showed that analgesics, Tramadol and flurbiprofen but not morphine, reduced cisplatin cytotoxicity by altering gap junction communication in HeLa cells over expressing Cx32. These drugs inhibited dye-coupling through gap junctions but did not affect connexin expression [164].

The Harris group in another study showed that downregulation of GJIC decreased cisplatin cytotoxicity in testicular cancer cells but enhanced it in normal testicular cells.

Enhanced toxicity due to GJIC downregulation in normal testicular cells correlated with increased cisplatin-DNA ICLs. In the cancer cells, modulation of GJIC had no effect on ICL

formation suggesting a different mechanism for GJIC-toxicity in cancer cells. This study speculated a protective role of GJIC in normal cells from cisplatin induced toxicity while killing of tumor cells [165].

Thus, from these studies it can be seen that cisplatin cytotoxicity could have a gap junction dependent component which could enhance the cytotoxic response. This is important to evaluate as it adds to the understanding that interaction of cancer cells with each other in a tumor and its environment could predict chemotherapeutic outcomes.

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Chapter 2

Downregulation of XPF/ERCC1 enhances cisplatin efficacy in cancer cells

Sanjeevani Arora, Anbarasi Kothandapani, Kristin Tillison, Vivian Kalman-Maltese, and Steve

M. Patrick1*

This work is published DNA Repair (Amst). 2010 Jul 1; 9(7):745-53 and additional data were added that support results in the published article for this dissertation.

Department of Biochemistry & Cancer Biology, University of Toledo Health Science Campus,

Toledo, OH

*To Whom Correspondence should be addressed:

Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus,

405 Block Health Sciences Building, 3000 Arlington Avenue, Toledo, OH, 43614

Telephone: 419-383-4152

Fax: 419-383-6228

Email address: [email protected]

Conflict of Interest: None declared

Short Title: XPF-ERCC1 mediates cisplatin sensitivity

Financial Support: American Cancer Society- (ACS, RSG-06-163-01-GMC) to S.M.P

ABSTRACT

Bulky cisplatin lesions are repaired primarily by nucleotide excision repair (NER), in which the structure specific endonuclease XPF–ERCC1 is a critical component. It is now known that the XPF–ERCC1 complex has repair functions beyond NER and plays a role in homologous recombination (HR). It has been suggested that expression of ERCC1 correlates with cisplatin drug resistance in non-small cell lung cancer (NSCLC). In our study, using NSCLC, ovarian, and breast cancer cells, we show that the XPF–ERCC1 complex is a valid target to increase cisplatin cytotoxicity and efficacy. We targeted XPF–ERCC1 complex by RNA interference and assessed the repair capacity of cisplatin intrastrand and interstrand crosslinks by ELISA and alkaline comet assay, respectively. We also assessed the repair of cisplatin-ICL-induced double-strand

Breaks (DSBs) by monitoring -H2AX focus formation. Interestingly, XPF protein levels were significantly reduced following ERCC1 downregulation, but the converse was not observed. The transcript levels were unaffected suggesting that XPF protein stability is likely affected. The repair of both types of cisplatin-DNA lesions was decreased with downregulation of XPF,

ERCC1 or both XPF–ERCC1. The ICL-induced DSBs persist in the absence of XPF–ERCC1.

The suppression of the XPF–ERCC1 complex significantly decreases the cellular viability which correlates well with the decrease in DNA repair capacity. A double knockdown of XPF–ERCC1 displays the greatest level of cellular cytotoxicity when compared with XPF or ERCC1 alone.

The difference in cytotoxicity observed is likely due to the level of total protein complex remaining. These data demonstrate that XPF–ERCC1 is a valid target to enhance cisplatin efficacy in cancer cells by affecting cisplatin-DNA repair pathways.

Keywords: XPF–ERCC1, Cisplatin, DNA repair and cancer

Abbreviations: NER, nucleotide excision repair; NSCLC, non-small cell lung cancer;

ICL, interstrand crosslink; DSBs, double-strand breaks; ERCC1, excision repair cross- complementation group 1; XPF, xeroderma pigmentation group F; siRNA, small interfering

RNA; StaRT-PCR, standardized reverse transcription-polymerase chain reaction; ACTB, - actin; ELISA, enzyme linked immuno-absorbent assay; SEM, standard error of the mean.

1. Introduction

Cisplatin [cis-diammine-dichloroplatinum (II)] is one of the most widely used platinum containing chemotherapeutic agents. It is an alkylating agent that acts by forming cisplatin-DNA adducts which include monoadducts, intrastrand DNA adducts and DNA interstrand crosslinks

(ICLs). These adducts inhibit and block DNA replication, which leads to cell death [1, 2].

Cisplatin is used clinically to treat a wide variety of tumors such as ovarian, testicular, head and neck, and NSCLC. Randomized clinical trials have confirmed that adjuvant chemotherapy with platinum-based (carboplatin or cisplatin) drug combinations for NSCLC significantly increases survival [3]. Despite its success with testicular cancer, its effectiveness in the treatment of other cancers is limited. This is due to the development of resistance over time. There are multiple mechanisms of cisplatin resistance but increased DNA repair is proposed to be one of the major reasons of resistance development [4]. Studies with a series of cisplatin-resistant cells in ovarian cancer cell lines show a clear relation between adduct removal and cisplatin cytotoxicity in a cisplatin-resistant model [5]. A major pathway involved in the repair of cisplatin-DNA adducts is the nucleotide excision repair (NER) pathway [6]. While the intrastrand DNA lesions and monoadducts are repaired by NER, the exact mechanism and events occurring during ICL repair are poorly understood [7]. The NER pathway has several steps: DNA damage recognition, dual incision/excision, repair synthesis and ligation. An important member of NER, the excision repair cross-complementing group 1 protein (ERCC1), forms a heterodimer with XPF and together they perform a critical incision step in the NER reaction. The XPF–ERCC1 complex is responsible for the incision 5‟ to the lesion to cleave the damaged strand during NER [8]. It has been suggested that ERCC1 levels influence and correlate with DNA repair capacity [9–11].

ERCC1 expression has been associated with cellular and clinical resistance to platinum-based

chemotherapy. There is much speculation about the importance of ERCC1 as prognostic indicator of cisplatin response in different cancer types [12]. Testicular cancer is generally responsive to cisplatin and has low levels of XPF–ERCC1, raising the possibility that XPF–

ERCC1 levels influence the response to platinum therapy [13]. XPF–ERCC1 also has specific roles in ICL repair, recombination and regulates telomere integrity [14–19]. It has been shown that XPF–ERCC1 facilitates the repair of DSBs induced by cisplatin-ICL processing, and it has been proposed that the key role of XPF–ERCC1 is unhooking the crosslink in ICL repair [14]. It has also been shown that the complex plays a role in completion of homologous recombination during ICL repair [20]. Thus, we rationalize that XPF–ERCC1 complex is an important molecular target in cancer chemotherapy to potentiate cisplatin cytotoxicity as decreased levels of XPF–ERCC1 can increase responses to cisplatin treatment. In our study, we used RNA interference to downregulate the XPF–ERCC1 complex in addition to individual suppression of

XPF and ERCC1 proteins in cancer cell lines to compare their DNA repair capacity and test the effect on cisplatin sensitivity.

2. Methods

2.1. Chemicals

Cisplatin [cis-diammine-dichloroplatinum (II)] was purchased from Sigma–Aldrich. For

StaRT-PCR, primers that amplify ERCC1, XPF and -actin (control) were obtained from Gene

Express (Toledo, OH). ERCC1 forward primer 5‟-CTGGAGCCCCGAGGAAGC -3‟; reverse primer 5‟-CACTGGGGGTTTCCTTGG-3‟. The XPF forward primer 5‟-

AGTGCATCTCCATGTCCCGCTACTA-3‟; and the reverse primer is 5‟-

CGATGTTCTTAACGTGGTGCATCAA-3‟. For β-actin, the forward primer is 5‟-

CCCAGATCATGTTTGAGACC-3‟ and the reverse primer is 5‟- CCATCTCTTGC

TCGAAGTCC -3‟. TRIzol Reagent was from Invitrogen. We used the Qiagen DNeasy blood and tissue kit for DNA isolation and Profoldin DNA binding plates for the ELISA. ICR4 antibody was kindly provided by Michael J. Tilby, University of New Castle, UK. All other reagents and chemicals were from standard suppliers.

2.2. Antibodies

The antibodies were polyclonal ERCC1-fl-297 (sc-10798, Santa Cruz), monoclonal XPF

(MS-1381-PIABX, Neomarker), monoclonal -tubulin (T5168, Sigma), monoclonal anti- phosopho -H2AX (clone JBW301, Millipore), and Alexa 488-conjugated goat anti-mouse

(Molecular Probes). ICR4 antibody was kindly provided by Michael J. Tilby, University of New

Castle, UK.

2.3. Cell culture

NSCLC cell lines, H1299 (provided by Dr. Randall Ruch, University of Toledo) and

H1355 (provided by Dr. James C. Willey, University of Toledo) and 2008, 2008/C13, A2780,

A2780/C30, ovarian cancer cell lines (provided by Dr. Stephen Howell, University of California,

San Diego), were maintained in RPM1 1640 supplemented with 10% FBS in the presence of penicillin (100 IU/ml) and streptomycin (100 g/ml). MDA-MB-231, which is a triple negative breast cancer cell line (provided by Dr. Manohar Ratnam, University of Toledo) was maintained in DMEM high glucose with 10% FBS in the presence of glutamine and sodium pyruvate and antibiotics. Cells were grown at 37 ◦C in a 5% CO2 incubator.

2.4. siRNA sequence

siRNA smart pools designed to target human ERCC1 and XPF were purchased from

Dharmacon RNA technologies, catalogue numbers L-006311-00 and L-019946-00-10,

respectively. A nontargeting siRNA pool was used in control experiments (catalogue number D-

001810-10-20).

2.5. Transfection with siRNAs

H1299, H1355, 2008, 2008/c13, A2780, A2780/C30, MDA-MB-231 cells were seeded in six well plates (density 2.5×105 cells/well) in antibiotic free media. Two transfections were done at 24 h interval in each cell line to knockdown ERCC1, XPF or XPF–ERCC1 together (wherever mentioned) according to the manufacturer‟s protocol. Addition of lipid reagent (Dharmafect,

Dharmacon) without the siRNA is described as mock transfection.

2.6. Western blot

At each indicated time points of 72, 96 and 120 h post transfection one, the cells were centrifuged, washed with PBS, and lysed on ice for 30 min in lysis buffer (10mM Tris, pH

8.0,120mM NaCl, 0.5% NP-40, 1mM EDTA) with protease inhibitors (0.5M phenyl methyl sulfonyl fluoride (PMSF), 1mg/ml leupeptin, 1mg/ml pepstatin A). Equal amounts of protein were loaded and electrophoresed on 10% SDS–polyacrylamide gels. The proteins were transferred onto PVDF membrane (Immobilon transfer membrane, Millipore Corporation). After electroblotting, the membranes were blocked with Tris-buffered saline with Tween-20 (1M Tris–

HCl, pH 7.5, 150mM NaCl, and 0.5% Tween-20) containing 2% non-fat dry milk. Primary antibodies, recognizing ERCC1, XPF or - tubulin were diluted in blocking buffer and incubated for 30 min. The membranes were then washed, incubated with the appropriate secondary antibodies in a blocking buffer for 30 min, and washed again. The blotted proteins were detected using chemiluminescence detection system. The level of protein knockdown was determined using an Alpha Innotech Fluoro Chem HD2.

2.7. RNA isolation, reverse transcription and transcript abundance

Cells were lysed with 1ml of TRIzol reagent and the total RNA was extracted following the manufacturer‟s protocol. The total RNA extracted from each cell line was reverse transcribed with oligo dT primer and M-MLV-RT as described previously [21, 22]. Transcript levels were quantified using the previously described StaRT-PCR protocol [21, 22]. Briefly, a mixture of internal standard competitive template (SYSTEM 1, Gene Express, Inc.) was included in a master mix with cDNA and PCR reagents (dNTPs, etc.). The use of internal standards allows comparing data from different experiments giving a highly reproducible, standardized, quantitative measurement of transcript levels. In these studies, -actin (ACTB) was used as a loading control gene. The master mix was aliquoted into tubes containing each gene-specific primer (ACTB, ERCC1, and XPF). PCR was carried out in a Rapidcycler (Idaho Technology

Inc.) with each reaction mixture subjected to 35 cycles each of 5 s denaturation at 94 ◦C, 10 s of annealing at 58 ◦C and 15 s of elongation at 72 ◦C. PCR products were separated and quantified electrophoretically by the Agilent 2100 Bioanalyzer (Agilent Technologies Inc.) using the DNA

1000 Assay kit. Following electrophoresis, a ratio of the endogenous PCR products (or native template, NT) to the internal standard (competitive template) was taken to calculate molecules of

NT in the reaction. Each transcript abundance value was normalized to ACTB and values are reported as target gene mRNA/106 ACTB mRNA. All experiments were performed in triplicate.

2.8. Cisplatin intrastrand adduct measurement by ELISA

Repair of intrastrand adducts was assessed by ELISA as described with some modifications [23, 24]. Cells were treated with cisplatin for 2 h in serum free medium. Cells were then washed with PBS and fresh medium was added. At various time points between 0 and

48 h after drug treatment, genomic DNA was isolated and sonicated for 30–60 s in a Cole Palmer

ultrasonic processor. Equal amounts of DNA were coated on 96-well DNA binding ELISA plates in binding buffer (1M sodium chloride, 50mM sodium phosphate buffer, pH 7.4, 0.02% sodium azide) and incubated at 4 ◦C overnight. The wells were blocked with 1% BSA in PBS for 1 h at room temperature. ICR4 antibody diluted 1:2000 in dilution buffer (0.2% BSA, 90mM sodium chloride, 0.2% Tween-20 in PBS) was added to the wells and incubated at 37 ◦C for 1 h.

Following three washes with washing buffer (0.1% Tween-20 in PBS), HRP conjugated goat anti-rat antibody diluted 1:2500 (1% BSA, 0.2% Tween-20 in PBS) was added to the wells and incubated at 37 ◦C for 30–60 min. After five washes with washing buffer, TMB (1 step ultra

TMB-ELISA, Thermo Scientific) was added and kinetics of absorbance was measured at 650nm in a Spectramax M5 plate reader (Molecular Devices) for 15 min. The reaction was stopped by adding 1M sulfuric acid and absorbance was measured at 450 nm. All samples were assayed in triplicates. The mean background (antibody blank) was subtracted from all the readings and the

% intrastrand adducts were calculated using OD 450nm where the 0 h time point was used as

100% intrastrand adducts in each cell line.

2.9. Cisplatin interstrand crosslink measurement by comet assay

Repair of interstrand crosslinks was assessed by alkaline comet assay with some modifications [25, 26]. Cells were treated with cisplatin for 2 h in serum free medium. At the end of treatment, cells were washed with PBS and incubated in fresh medium for the required post- incubation time or assayed immediately (time 0 h). Cells were further treated with 100 mM hydrogen peroxide for 15 min to induce DNA strand breaks. Cells were then trypsinized, pelleted and resuspended in 1% low melting point agarose. Cell samples (~10,000 cells) were embedded on a microscopic slide precoated with 1% normal melting point agarose. Another layer of 0.5% low melting point agarose was added and allowed to solidify. The slides were then incubated in

lysis solution (2.5M NaCl, 10mM Tris, 100mM EDTA, pH 10, containing 1%, v/v Triton X-100) for 1 h at 4 ◦C in the dark. The slides were then transferred to an electrophoresis tank containing ice-cold alkaline solution (300mM NaOH, 1mM EDTA, pH> 13), incubated for 20 min to allow

DNA unwinding to occur and electrophoresis was carried out for 30 min at 0.7 v/cm, 300 mA.

Slides were removed and kept in neutralizing solution (0.4M Tris–HCl, pH 7.5) for 10 min.

Slides were then stained with SYBR green (Trevigen) and comets were analyzed using a Nikon epifluorescence microscope at 200× magnification. Fifty cells were analyzed per slide using

Komet Assay Software 5.5F (Kinetic Imaging, Liverpool, UK). The degree of DNA interstrand crosslinking present in the cisplatin-treated sample was determined as described by comparing the tail moment of cisplatin +H2O2 treated samples with H2O2 treated samples and untreated control samples [24]. The level of interstrand crosslinking was calculated by the following formula: [1 − (TMpt − TMctl)/(TMH2O2 − TMctl)] × 100, where TMpt is the mean tail moment of the cisplatin + H2O2 treated sample, TMctl is the mean tail moment of the untreated control sample and TMH2O2 is the mean tail moment of H2O2 treated sample. The data was expressed as the percent of ICLs that remained at a particular time point where 0 h was normalized to 100%.

2.10. Immunofluorescence

H1299, H1355, 2008 and MDA-MB-231 untransfected and siRNA transfected (double knockdown cells at 48 h post-initial transfection) cells were trypsinized and seeded onto glass coverslips at 25% confluency. The next day the cells were treated with cisplatin in serum free medium and 2 h later fresh complete medium was added and the cells were incubated for the indicated time points. For all experiments, control and knockdown cells were treated with equitoxic dose of cisplatin, with doses yielding 50% survival for all cell lines. The cells were washed with Hank‟s balanced salt solution, fixed and permeabilized with ice-cold methanol for

15 min and blocked with 10% goat serum in PBS. For detecting the phosphorylated form of -

H2AX, cells were incubated for 1 h with the monoclonal anti-H2AX (1:500, Millipore) antibody followed by incubation with Alexa-488 goat anti-mouse antibody (1:1000, Molecular

Probes) diluted in 10% goat serum in PBS. Cells were washed and counterstained with DAPI for

5 min. Coverslips were mounted with DAKO mounting medium onto slides and the edges were sealed with nailpolish. Images were visualized using a Nikon Eclipse T2000-U microscope at

100× oil immersion objective. Foci were counted in 250 cells at each time point per condition in each cell line and results are expressed as % -H2AX foci per nuclei. The data was collected from two individual experiments.

2.11. Colony survival assay

H1299, H1355, 2008, 2008/C13, A2780, A2780/C30 and MDA-MB-231 cells following two transfections were split and seeded at a density of 300–600 cells in 60mm plates and incubated overnight. The next day, the cells were treated with different concentrations of cisplatin for 2 h, and after treatment, fresh complete medium with antibiotics was added and the cells were then allowed to grow for 7–14 days. Fresh medium was added when needed. Colonies were fixed with 95% methanol and stained with 0.2% crystal violet. Colonies with ≥50 cells were counted using a light microscope. Cell survival was expressed as the ratio of the average number of colonies in drug treated cells versus control cells×100. The experiment was done in triplicate for each drug concentration.

3. Results

3.1. siRNA mediated knockdown of ERCC1, XPF and XPF/ERCC1 in cancer cells

We have chosen different NSCLC, ovarian and breast cancer cells to downregulate XPF–

ERCC1. NSCLC cells, H1299 and H1355 cells were transiently transfected with smartpool

siRNA‟s either directed individually against XPF and ERCC1 or at XPF–ERCC1 simultaneously

(Fig. 1, Supplemental Fig. 2 and Table 1). Protein extracts from 72 to 120 h post-initial transfection were analyzed for ERCC1 and XPF expression with -tubulin as a loading control in each cell line (Fig. 1, Supplemental Fig. 2 and Table 1). The results demonstrate that we achieve protein knockdown between 72 and 96 h. The knockdown for each protein is sustained past 120 h. Quantification of the protein levels indicate that we achieve >90% knockdown for

ERCC1 and >80% knockdown for XPF. When both XPF and ERCC1 are knocked down simultaneously, we achieve the greatest level of protein knockdown for each. We also observed that when ERCC1 protein is knocked down, the XPF protein was also significantly reduced in both cell lines (Fig. 1A and Supplemental Fig. 2A). However, ERCC1 protein levels showed a modest decrease as compared to controls when XPF is knockdown (Fig. 1B and Supplemental

Fig. 2B).

Next, the changes in protein expression were correlated with the change in transcript level in all cell lines tested (Fig. 2, Supplemental Fig. 1 and Table 1). StaRT-PCR [21, 22] was employed to assess the basal target gene expression as well as the XPF and ERCC1 knockdowns in H1299 and H1355 cells (Fig. 2 and Supplemental Fig. 1). The 2008 and MDA-MB-231 cell lines are summarized in Table 1 after double knockdown. The mRNA levels were measured 48 h post-first transfection. The cells were either mock transfected, transfected with non-targeting

(NT)-, XPF-, ERCC1- or XPF–ERCC1 siRNA. Experimental results show that ERCC1 siRNA

(Fig. 2A, Supplemental Fig. 1A and Table 1) decreased the mRNA levels by ≥95% on individual and double knockdown, respectively, compared to the non-targeting control siRNA. XPF siRNA

(Fig. 2B, Supplemental Fig. 1B and Table 1) reduced mRNA levels by ~80–85% on individual knockdown and ≥90% on double knockdown, as compared to the controls. The gene

expression is expressed as a ratio of target gene mRNA/106 ACTB mRNA. To further assess the result that we observed with protein levels, we monitored XPF transcript levels when ERCC1 was knocked down and vice versa and show that the transcript levels are not affected (Fig. 2 and

Supplemental Fig. 1). This shows that the decreased XPF protein levels are not due to non- specific effects caused by the siRNA transfection.

3.2. Repair kinetics of cisplatin intrastrand crosslinks

Considering that XPF–ERCC1 is required for NER and the cisplatin intrastrand adducts are repaired via NER [27], we assessed the repair efficiency of intrastrand adducts in cell lines following XPF, ERCC1 and XPF–ERCC1 downregulation. Fig. 3 shows the repair kinetics of cisplatin intrastrand adducts in H1299 and H1355 cell lines. We utilized an ELISA method to assess the repair of cisplatin 1, 2 dGpG intrastrand adducts over time. This assay utilizes a monoclonal antibody specific for the major cisplatin dGpG intrastrand adduct [23, 24]. The repair kinetics of cisplatin intrastrand adducts was calculated as the percent of adducts remaining over time, relative to the percent of adducts present at the 0 h treatment (100%). In untransfected cells, the intrastrand adducts were repaired gradually from 24 to 48 h, with ~75% of adducts were removed at 48 h in both cell lines. In XPF or ERCC1 individual siRNA transfected cells, the removal rate of these adduct was decreased. The double knockdown of XPF–ERCC1 results in the highest level of complex downregulation (summarized in Table 1) and concomitantly, results in the lowest level of cisplatin intrastrand adduct repair. This data illustrates the importance of the XPF–ERCC1 complex in the repair and removal of the major cisplatin-DNA adduct and is consistent with previous results, where NER mutants are defective in the repair of intrastrand adducts compared to wild type cells [24].

3.3. Repair of cisplatin interstrand crosslinks (ICLs)

After demonstrating the role of XPF and ERCC1 on the repair of cisplatin intrastrand adducts, we then investigated the effect of silencing these two proteins on cisplatin-ICL DNA repair by alkaline comet assay. Comet assay has been used to evaluate DNA interstrand crosslink induction and repair in vivo at the single cell level [28]. The repair kinetics of cisplatin-ICLs in each cell line was evaluated after 0, 24, 48 and 72 h post-treatment with cisplatin and was expressed as the percentage of crosslinks remaining at the time points assessed. Fig. 4 shows the percentage of cisplatin-ICLs with increasing time in untransfected and siRNA transfected H1299

(Fig. 4A) and H1355 (Fig. 4B) NSCLC cells. Fig. 4C and D shows the percent of ICLs with increasing time in 2008 and MDA-MB-231, respectively in untransfected and double knockdown cells. Cisplatin treatment induced a similar extent of ICL formation at 0 h in untransfected and transfected cells for both cell lines. Cisplatin-ICLs were removed efficiently in untransfected cells with ~25% of the ICLs remaining at 72 h, whereas in transfected cells, significantly greater levels of ICLs still remained. Increased formation of cisplatin-ICLs in transfected cells at 24, 48 and 72 h indicates a possible conversion of monoadducts or intrastrand adducts to interstrand crosslinks. Of significance, no cisplatin-ICL repair is observed out to 72 h in the siXPF, siERCC1 or siXPF–ERCC1 (siX + siE) transfected cells. These data support previous reports of

ICL repair in mammalian cells and show a requirement for XPF–ERCC1 in cisplatin-ICL repair

[24, 29].We speculate that there is a direct relation between the time required to repair a cisplatin-DNA lesion and the cytotoxic effect of the drug.

3.4. Kinetics of -H2AX focus formation and repair of DSBs post-cisplatin-DNA damage

To further investigate and corroborate the results of the comet assay, we investigated the repair of ICL-induced DSBs in untransfected and XPF–ERCC1 double knockdown cells. The

histone variant H2AX is phosphorylated at serine 139 upon exposure to ionizing radiation and forms distinct nuclear foci at sites of DSBs [30]. - H2AX foci also form upon exposure to cisplatin, although detection of DSBs in certain instances has been shown to be limited [31]. The nuclease processing at the sites flanking the ICLs leads to the generation of the DSBs [14].

Cisplatin-treated cells were categorized as having 0–5, 6–10 and >10 foci/nuclei (Fig. 5).

Elevated levels of spontaneous endogenous - H2AX foci has been previously observed in cancer cells [32], and it is believed that these cryptic foci are a consequence of chromatin instability [33]. In comparison to untransfected cells, XPF–ERCC1 knockdown cells showed a higher frequency of - H2AX foci formation as well as more nuclei with >10 foci. This suggests that in the absence of the XPF–ERCC1 complex the cells retain a state of DNA damage which remains unrepaired. Fig. 5 shows kinetics of - H2AX focus formation in H1299 (Fig. 5A and B) and H1355 (Fig. 5C and D) untransfected and double knockdown cells. Under both conditions, cells with more than 10 foci peak at 12 h post-cisplatin treatment (data not shown).

Untransfected cells consistently repaired DSBs in all the cell lines and the cells with a maximum number of foci started declining between 12 and 24 h (Fig. 5A and C and Supplemental Fig. 3).

In contrast, DSBs were sustained even at 72 h in XPF/ERCC1 knockdown cells with persistence of >10 foci (Fig. 5B and D and Supplemental Fig. 3). This data demonstrates that the damage appears to be more pronounced in cancer cells that lack XPF/ERCC1. Foci in MDA-MB-231 cells were divided into 0, 1–10 and >10 foci/nuclei and the results for both the 2008 and MDA-

MB-231 cells are in Supplementary Fig. 3. Overall, we show that in the absence of XPF–

ERCC1, there is an accumulation of ICL induced DSBs which remain persistent as the crosslinks are not “unhooked”/uncoupled. These results parallel results observed on Mitomycin C damage in Ercc1−/− null cells [14].

3.5. Downregulation of the XPF/ERCC1 complex sensitizes NSCLC cells to cisplatin

To assess the effect of XPF/ERCC1 downregulation on cell viability in response to cisplatin, clonogenic assays were performed in transfected and control cells. The results of the colony survival assays are shown in Fig. 6 and summarized in Table 1. XPF and ERCC1 individual knockdowns increased cisplatin cytotoxicity by 1.5–2-fold and 3–4-fold, respectively in H1299 (Fig. 6A) and H1355 (Fig. 6B) NSCLC cell lines. XPF/ERCC1 simultaneous downregulation results in 6-fold and 4-fold changes in IC50 values for H1299 and H1355 (Fig.

6A and B), respectively when compared to mock (data not shown) and non-targeting siRNA treated cells. 2008 cells showed ~2-fold change while MDA-MB-231 cells showed ~3.5-fold change in IC50 values following double knockdown (Table 1). The fold changes observed in

IC50 values are likely a result of the level of protein downregulation (summarized in Table 1), where XPF–ERCC1 downregulation results in the best protein/transcript knockdown. This data supports our hypothesis that XPF–ERCC1 is a valid target for enhancing cisplatin efficacy for cancer chemotherapy.

In figure 7, we downregulated XPF/ERCC1 together in the cisplatin sensitive and their daughter derived cisplatin resistant ovarian cancer cells. We analyzed ERCC1 levels (Fig. 7D and E) in both the cisplatin sensitive and resistant cell line sets. We show that when we target

XPF/ERCC1 in the resistant 2008/C13 cells (Fig. 7A); we bring back the IC50 levels to the original sensitive cells and show a ~5 fold increase in toxicity which correlates to the increased levels of ERCC1 in the resistant cells (Fig. 7D). In the A2780/C30 cisplatin resistant cells (Fig.

7C), the ERCC1 levels are upregulated ~2-fold (Fig. 7E) as compared to the original sensitive line which correlates to a ~2-fold increase in toxicity when we target XPF/ERCC1 in these cells

(Fig. 7C). The cisplatin sensitive 2008 and A2780 (figure 7A and table 1 for 2008 and 7C for

A2780) do not show much further increase in toxicity when we knockdown XPF/ERCC1 in these cells.

4. Discussion

Cisplatin cytotoxicity is believed to be attributed to the formation of platinum-DNA adducts which are primarily repaired by NER [5, 7]. As NER is a major player in the mechanism of cisplatin induced DNA repair, targeting key NER components represents a molecular approach to enhance cisplatin efficacy for cancer treatment. Clinical investigations suggest that

ERCC1 expression is a useful marker or predictor of response to cisplatin chemotherapy [34].

This has led to our evaluation of the XPF–ERCC1 complex, which could eventually mediate platinum resistance. Low levels of ERCC1 correlate with better clinical outcome and this provides a strong rationale for our knockdown studies [35, 36]. In our study, we targeted the

XPF–ERCC1 complex through RNA interference to assess effects on DNA repair and cisplatin cytotoxicity in different cancer types. The set of timecourse experiments (Fig. 1) delineate time points to treat cells with cisplatin and also define optimal conditions to perform later experiments to assess DNA repair and cisplatin cytotoxicity in vivo. We also looked at the transcript levels which complement the protein expression studies. The XPF mRNA levels are relatively similar in all cell lines tested. However, the transcript levels for ERCC1 differ, wherein the cell line most sensitive to cisplatin had decreased abundance as compared to others in the panel (data not shown). Studies with more cell lines would be required to fully address this phenomenon.

Interestingly, when we knock down ERCC1, a significant reduction in XPF protein expression is observed. However, we observed no changes in XPF mRNA levels. Previous literature shows that in eukaryotic cells, formation of the heterodimeric complex is required for the stability of both components [37–41]. A recent study showed that XPF forms HhH homodimers in vitro

which are more stable than the heterodimer with ERCC1. However, the same results are not observed in vivo with a low amount or no XPF present in the absence of ERCC1 [42]. In our study, we observed minimal loss of ERCC1 protein following XPF knockdown. The degradation or elimination of the binding partner must require an extremely low level or complete absence of the other. Studies with XPF cases and the first case of human ERCC1 deficiency show reduced but not complete absence of XPF–ERCC1, which showed only a mild impairment of NER in opposition to what we might expect [41]. It is possible that low levels of XPF–ERCC1 are still sufficient to perform critical NER functions. A previous study showed that ERCC1 mRNA is transcribed normally in XP2YO (XPF) cells [43], so the effect involves XPF protein stability due to the lack of complex formation in the absence of ERCC1. XPF-deficient and ERCC1 knockout mice have identical phenotypes which supports their function as a complex rather than separate functions in repair [44–46]. To address the effects on DNA repair, we studied the repair of the cisplatin 1, 2-dGpG intrastrand adduct and ICLs. The intrastrand adducts represent the most abundant lesions and are repaired by the NER pathway [7]. In all cell lines, double knockdown of XPF–ERCC1 showed maximum reduction in repair of cisplatin- DNA intrastrand adducts

(Fig. 3 and data not shown). ICL repair encompasses multiple mechanisms with important roles from the XPF–ERCC1 complex. ICLs are believed to play a significant role in the cytotoxicity and antitumor activity of anticancer drugs [47, 48]. A recent study showed that there is an increase in the repair of cisplatin-DNA ICLs in ovarian cancer patients‟ post-cisplatin treatment and this is an important factor that leads to acquired clinical resistance [49]. This strengthens the importance of increased DNA repair which is detrimental to drug cytotoxicity in the context of cancer chemotherapy. Our data reveals that ICL repair is significantly inhibited in a double knockdown of XPF–ERCC1. The data shows that there is no repair of cisplatin-ICLs. This has a

major impact on platinum therapy as it increases the potential for the cisplatin lesion to persist longer on DNA and hence, can have a better cytotoxic effect. We also studied the kinetics of -

H2AX foci formation in response to ICL-induced DSB formation. In the absence of XPF–

ERCC1, the DSBs persist and remain unrepaired. DSB formation is detrimental to cell survival, and if they remain unrepaired, it can lead to cell death. Collectively, our data shows that XPF–

ERCC1 mediates the repair of cisplatin-DNA adducts, and its absence significantly inhibits cisplatin-DNA repair. The cellular capacity of the cells to repair cisplatin-DNA adducts is an important way for tumor cells to survive and resist the effects of chemotherapy. Hence, cells that are defective in uncoupling ICLs and cisplatin intrastrand adducts should show extreme sensitivity upon exposure to cisplatin. Also, ERCC1 and XPF–defective cells show UV sensitivity and high sensitivity to DNA crosslinking agents and are the most sensitive to crosslinking agents in comparison to other NER mutants [50, 51]. Results from the clonogenic assay demonstrate a correlation between the decrease in DNA repair capacity and the enhancement of cellular cytotoxicity to cisplatin (Fig. 6 and Table 1). An XPF–ERCC1 double knockdown shows a particularly pronounced increase in cellular cytotoxicity in both H1299 and

H1355. When we target XPF–ERCC1 simultaneously in H1299, we see a 6-fold change in cisplatin IC50 values while we observe a 4-fold change in H1355. It is important to note here that these two cell lines also differ in their p53 status. H1299 is p53 deficient while H1355 is a heterozygous mutant of p53. Following DNA damage, p53 is normally activated to induce cell cycle arrest and facilitate DNA repair [52]. The differences in p53 expression could affect cell survival and DNA repair efficiency following XPF–ERCC1 knockdown. Our studies with the cisplatin sensitive and their derived cisplatin resistant cell lines (Fig. 7) further show that

XPF/ERCC1 is essential to cisplatin cytotoxicity and cancer cells that acquire resistance or are

resistant to cisplatin chemotherapy probably increase levels of XPF/ERCC1 leading to resistance. Studies in mice have shown that a ~2- fold change in IC50 value is significant in regard to affecting tumor growth in response to cisplatin treatment [53]. The results suggest that targeting XPF–ERCC1 could bring about changes in chemotherapeutic regimens where decreased drug concentrations could have a greater cytotoxic effect. Overall our data contributes to the evidence that XPF–ERCC1 is vital to mediate the response against cisplatin-induced DNA damage. These results are consistent with previous work on ERCC1 disruption in cancer cell lines including ovarian, colon, and prostate [5, 54, and 55].

In conclusion, we are the first to show that disrupting the XPF–ERCC1 complex instead of ERCC1 alone through a targeted mechanism results in significant enhancement in cisplatin cytotoxicity in cancer cell lines. XPF–ERCC1 is a broader therapeutic target than its counterparts in the NER pathway due to its repair roles in both intrastrand and ICL lesions. These studies support the idea that XPF–ERCC1 is a valid therapeutic target for enhancing cisplatin effectiveness not only in lung cancer, but other cancer types. Identification of small molecule inhibitors of the XPF–ERCC1 endonuclease complex could potentiate the effectiveness of cisplatin and yield better patient outcome and increase survival rates in lung cancer and other cancers which are quickly becoming resistant to conventional treatment regimens.

Acknowledgements

We gratefully acknowledge Erin L. Crawford and Dr. James C. Willey (University of

Toledo) for helping with technical procedures for StaRT-PCR/the transcript abundance data. We also thank Akshada Sawant for helping with the  -H2AX foci images.

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FIGURE LEGENDS

Fig. 1. Timecourse showing siRNA mediated downregulation of ERCC1 and XPF protein in

H1299 cells. Cells were transiently transfected twice at 24 h intervals, each time with smartpool siRNAs (100 nM) directed against ERCC1 (A), XPF (B) and both proteins (C, double knockdown). Control cells were left untransfected (UT), or mock (M) – and non-targeting (NT) siRNA (100 nM) – transfected. Proteins were extracted at the indicated time points of 72, 96 and

120 h post-transfection, and probed with XPF, ERCC1 and -tubulin as a loading control. The percent of knockdown was quantified using the Alpha Innotech HD2 and is represented for all cell lines tested in Table 1. Blots from (A) and (B) were also probed for XPF or ERCC1 on individual knockdown of ERCC1 or XPF, respectively.

Fig. 2. Transcript levels on siRNA mediated downregulation. (A) Represents ERCC1 transcript levels. H1299 cells were mock (M) treated or non-targeting (NT)-, XPF-, ERCC1-, and XPF–

ERCC1 (denoted as siX + siE) siRNA transfected, twice at 24 h intervals each and harvested at

48 h post-transfection. Total RNA was extracted from cells and analyzed using StaRT-PCR, as described in Section 2. Each PCR was run in triplicate. The transcript levels are represented as

ERCC1 mRNA/106 ACTB mRNA. The values are represented as mean ± SEM from triplicate

PCRs. (B) represents XPF transcript levels. H1299 cells were mock (M) treated or non-targeting

(NT)-, XPF-, ERCC1-, and XPF–ERCC1 (denoted as siX + siE) siRNA transfected, twice at 24 h intervals each and harvested at 48 h post-transfection. Total RNA was extracted from cells and analyzed using StaRT-PCR, as described. Each PCR was run in triplicate. The transcript levels are represented as XPF mRNA/106 ACTB mRNA. The values are represented as mean ± SEM from triplicate PCRs.

Fig. 3. Repair of cisplatin intrastrand adducts in H1355 (A) and H1299 (B) NSCLC cell lines.

Untransfected (UT), siXPF, siERCC1 and both siXPF–siERCC1 (denoted as siX + siE) transfected cells were treated with cisplatin for 2 h and genomic DNA was isolated at different time intervals (0, 24, and 48 h). ELISAs were performed as described using cisplatin intrastrand adduct antibody and the percentage of intrastrand adducts remaining was calculated for H1355

(A) and H1299 (B) at the denoted times. The results are represented as mean ± SEM of three independent experiments.

Fig. 4. Repair of cisplatin interstrand crosslinks in H1299 (A) and H1355 (B) 2008 (C) and

MDA-MB-231 (D) cell lines. Untransfected (UT, open squares), siXPF (filled circles), siERCC1

(open circles) and siXPF–siERCC1 siRNA (filled triangles, denoted as siX + siE) transfected cells were treated with cisplatin for 2 h and the comet assay was performed as described at different time intervals (0, 24, 48 and 72 h). The percentage of interstrand crosslinks remaining was calculated using olive tail moment. Results are represented as mean ± SEM of three independent experiments.

Fig. 5. Repair kinetics of -H2AX foci post-cisplatin treatment. H1299 (A and B) and H1355 (C and D) cell lines and quantitation of  -H2AX focus formation at various time points post- cisplatin treatment in untransfected (A and C) and XPF + ERCC1 (B and D) double knockdown cells, respectively. The cells were seeded onto glass coverslips at 25% confluency. The next day they were treated with cisplatin for 2 h and then fresh complete medium was added. The cells were fixed and immunostained for  -H2AX at the indicated time points post-cisplatin treatment.

For each data point, foci were counted in 250 cells per condition in each cell line. The foci have been categorized as having 0–5, 6–10, or >10 foci per nucleus. The results are expressed as %  -

H2AX foci per nuclei. The data was collected from two individual experiments.

Fig. 6. Colony survival in H1299 (A) and H1355 (B) cell lines. Non-targeting (NT, filled squares), siXPF (filled circles), siERCC1 (open circles) and siXPF–siERCC1 (filled triangles, denoted as siX + siE) siRNA transfected cells were treated with increasing doses of cisplatin for

2 h, and cell viability was determined by a clonogenic assay. The mock treated cells were left off of the graph for clarity reasons, but resulted in cytotoxicity nearly identical to non-targeting siRNA (data not shown). Calculated cisplatin IC50s for transfected cells are summarized in

Table 1. Table 1 also summarizes the IC50s for control and knockdown cells for all other cell lines tested. Values are represented as mean ± SEM from three independent experiments.

Fig. 7. Colony survival in ovarian cancer cells -2008 and 2008/C13 cell lines (A), A2780 cells

(B), A2780/C30 (C). Non-targeting siRNA (siC, filled squares for 2008s and filled circles for

2008/c13 cells; filled circles for A2780 and/or A2780/C30 cells) and siXPF–siERCC1 (open squares for 2008, open circles for 2008/c13 cells; open circles for A2780 and/or A2780/C30 cells denoted as siX + siE) siRNA transfected cells were treated with increasing doses of cisplatin for

2 h, and cell viability was determined by a clonogenic assay. Calculated cisplatin IC50s are shown in the figure. Values are represented as mean ± SEM from three independent experiments.

(D and E) ERCC1 transcript levels in ovarian cancer cells (D – 2008 and 2008/C13 and E-

A2780 and A2780/C30). Total RNA was extracted from cells and analyzed using StaRT-PCR, as described in methods section. Each PCR was run in triplicate. The transcript levels are represented as ERCC1 mRNA/106 ACTB mRNA. The values are represented as mean ± SEM from triplicate PCRs.

Supplemental Data:

Fig. S1. Transcript levels on siRNA mediated downregulation. (A) represents ERCC1 transcript levels. H1355 cells were mock (M) treated or Non-targeting (NT) - , XPF-, ERCC1- , and XPF-

ERCC1 (denoted as siX+siE) - siRNA transfected, twice at 24 h intervals each and harvested at

48 h post-transfection. Total RNA was extracted from cells and analyzed using StaRT PCR, as described in the Methods section. Each PCR was run in triplicate. The transcript levels are represented as ERCC1 mRNA /106 ACTB mRNA. The values are represented as mean ± S.E.M from triplicate PCRs. (B) represents XPF transcript levels. H1355 cells were mock (M) treated or Non-targeting (NT) - , XPF-, ERCC1-, and XPF-ERCC1 (denoted as siX+siE) - siRNA transfected, twice at 24 h intervals each and harvested at 48 h post-transfection. Total RNA was extracted from cells and analyzed using StaRT PCR, as described. Each PCR was run in triplicate. The transcript levels are represented as XPF mRNA /106 ACTB mRNA. The values are represented as mean ± S.E.M from triplicate PCRs.

Fig S2. Timecourse showing siRNA mediated downregulation of ERCC1 and XPF protein in

H1355 cells. Cells were transiently transfected twice at 24 h intervals, each time with smartpool siRNAs (100 nM) directed against XPF (A), ERCC1 (B) and both proteins (C - double knockdown). Control cells were left untransfected (UT), or mock (M) - and Non-targeting (NT) siRNA (100 nM) - transfected. Proteins were extracted at the indicated time points of 72, 96 and

120 h post-transfection, and probed with XPF, ERCC1 and α-tubulin as a loading control. The percent of knockdown was quantified using the Alpha Innotech HD2 and is represented for all cell lines tested in Table 1. Blots from (A) and (B) were also probed for ERCC1 or XPF on individual knockdown of XPF or ERCC1, respectively. α- Tubulin is shown as a loading control.

Fig. S3. Repair kinetics of γ-H2AX foci post cisplatin treatment in (A and B) 2008 (C and D)

MDA-MB-231 show quantitation of γ-H2AX focus formation at various time points post cisplatin treatment in untransfected (3 A and C) and XPF+ERCC1 (3 B and D) double

knockdown cells respectively. The cells were seeded onto glass coverslips at 25% confluency.

The next day they were treated with cisplatin for 2h and then fresh complete medium was added.

The cells were fixed and immunostanined for γ-H2AX at the indicated time points post cisplatin treatment. For each data point foci were counted in 250 cells at each time point per condition in each cell line. The foci have been categorized as zero to 5 (2008) or zero (MDA-MB-231), six to ten (2008) or one to ten (MDA-MB-231), and greater than ten foci per nucleus. The results are expressed as % γ- H2AX foci per nuclei, and the data was collected from two individual experiments.

A ERCC1 Knockdown in H1299

P M NT 72 96 120 XPF ERCC1 a-tubulin 5 1 2 3 4

B XPF Knockdown H1299

UT M NT 72 96 120 XPF ERCC1 α-tubulin 1 2 3 4 5 6

C Double Knockdown in H1299 UT M NT 72 96 120 XPF ERCC1 α-tubulin 1 2 3 4 5 6

Fig. 1

ERCC1 Levels - H1299

A N

R 30000

C A

20000

A 10000

C 0

C s s s M N iE iX iX

R T R + P E C F C s 1 iE

XPF Levels - H1299 A

N 400

B 300

0 200

N 100

F 0 P N s s s M T iX iX iE X P + R F C si C E 1

Fig. 2

100

A Intrastrand Repair in H1355 120 0 h

s 24 h t

c 100 48 h

u d

d 80

n 60

s a

r 40

n I

20 %

0 s s s U iX iE iX T P R F C + C s 1 iE B Intrastrand Repair in H1299 120 0 h

s 24 h t

c 100 48 h

u d

d 80

n 60

t s

a 40

n I

20 %

0 s s s U iX iE iX T P R F C + C s 1 iE

Fig. 3

This experiment was done with Dr. Anbarasi Kothandapani

101

A Comet assay in H1299 180

160 UT siXPF

k 140 siERCC1

s 120

d 100

r t 80

n 60

f o 40

% 20 0 0 20 40 60 80 Post-incubation time (hr)

B Comet assay in H1355

180 UT 160 siXPF

s k siERCC1

i l 140 siXPF+siERCC1

r 120

n 100

r 80

n i 60

o 40

% 20 0 0 20 40 60 80 Post-incubation time (hr)

Fig. 4

This experiment was done with Dr. Anbarasi Kothandapani

102

C Comet assay in 2008 180 160 UT

k 140 siXPF+siERCC1

s 120

d 100

r t 80

n 60

f o 40

% 20

0 0 20 40 60 80 Post-incubation time (hr)

D Comet assay in MDA MB231

180 UT 160 siXPF+siERCC1

i l 140

r 120

n 100

t s 80

n i 60

o 40

% 20 0 0 20 40 60 80 Post-incubation time (hr)

Fig. 4

This experiment was done with Dr. Anbarasi Kothandapani

103

A Repair of Gamma-H2AX in Untransfected H1299 cells

80 0 to 5 6 to 10 > 10

/ 40

0 0 h 24 h 48 h 72 h Time post cisplatin treatment

B Repair of Gamma-H2AX in XPF-ERCC1 knockdown H1299 cells

100 0 to 5 6 to 10 > 10 80

c 60

F 40

0 0 h 24 h 48 h 72 h Time post cisplatin treatment

Fig. 5

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C Repair of Gamma-H2AX in Untransfected H1355 cells

100 0 to 5 6 to 10 > 10 80

c 60

F 40

0 0 h 24 h 48 h 72 h Time post cisplatin treatment

D Repair of Gamma-H2AX in XPF-ERCC1 knockdown H1355 cells

100 0 to 5 6 to 10 > 10 80

c 60

F 40

0 0 h 24 h 48 h 72 h Time post cisplatin treatment

Fig. 5

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Colony survival in H1299 A 100 NT siXPF siERCC1 siXPF+siERCC1

10 0 1 2 3 4 Colony survival in H1355 B 100

S NT 10 % siXPF siERCC1 siXPF-siERCC1

0 2 4 CISPLATIN CONCENTRATION ( M)

Fig. 6

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Fig. 7

*Figure contributed by Vivian Kalman-Maltese

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Fig. 7

*Figure contributed by Vivian Kalman-Maltese

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Fig. 7

109

ERCC1 Levels - H1355

R m

1.5e+5

6 1.0e+5

N 5.0e+4

C 0.0 C M N si si si

R E X X T R P E C + F C s 1 iE B

XPF Levels - H1355 A

N 400

B 300

0 200

N 100

P 0 M N s s s X iX iX iE T P R F + C s C iE 1

Supplementary Fig. S1

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A XPF Knockdown in H1355 UT M NT 72 96 120 XPF ERCC1

a-tubulin 1 2 3 4 5 6 B ERCC1 Knockdown in H1355 UT M NT 72 96 120 XPF ERCC1 α-tubulin

1 2 3 4 5 6 C Double Knockdown in H1355

M NT 72 96 120 XPF ERCC1 α-tubulin 1 2 3 4 5

Supplementary Fig. S2

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A Repair of Gamma-H2AX in Untransfected 2008 cells

120 0 to 5 6 to 10 100 > 10

/ 60

% 40

0 0 h 24 h 48 h 72 h Time post cisplatin treatment

Repair of Gamma-H2AX in XPF-ERCC1 knockdown 2008 cells

100 0 to 5 6 to 10 > 10 80

c 60

F 40

0 0 h 24h 48h 72h Time post cisplatin treatment

Supplementary Fig. S3

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C Repair of Gamma-H2AX in Untransfected MDA-MB-231 cells

100 0 1 to 10 > 10 80

c 60

F 40

0 0 h 24h 48h 72h Time post cisplatin treatment D Repair of Gamma-H2AX in XPF-ERCC1 knockdown MDA-MB-231 cells

80 0 1 to 10 > 10 60

/ 40

0 0 h 24h 48h 72h Time post cisplatin treatment

Supplementary Fig. S3

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Table 1- Summary of cisplatin IC50s (represented as mean values from three independent experiments ± SEM) for knockdown and control cells with quantification of protein knockdown at the time of cisplatin treatment using the Alpha Innotech HD2 and transcript levels using StaRT PCR represented as percent knockdown.

Cell siRNA Protein Transcript Knockdown Fold line knockdown knockdown Knockdown IC50 (M)± siControl change quantification (~ %) SEM IC50 (~) (~ %) (M)± SEM

H1299 1. XPF (X) X - 81% X - 80% 1.54 ± ~1.62 2. ERCC1 E - 95% E - 93% 0.068 2.5 ± ~3.5 (E) X- 90% & E- X- 88% & 0.74 ± 0.059 ~6.0 3. XPF (X) 90 % E- 99% 0.042 +ERCC1 0.42 ± (E) 0.013

H1355 1. XPF (X) X - 87% X- 84% 2.96 4.32 ± ~1.5 2. ERCC1 E - 94% E- 95% ±0.036 0.028 ~3.0 (E) X - 90% & E X- 92% & 1.44 ± 4.0 3. XPF (X) - 91% E- 99% 0.476 +ERCC1 1.04 ± (E) 0.040

2008 1. XPF X- 83% & E- X - 88% & 0.87 ± 0.03 1.61 ± ~2.0 (X) 80% E- 92% 0.06 +ERCC1 (E)

MDA- 1. XPF X- 83% & E- X- 90 % & 3.03 ± 0.50 10.5 ± * MB- (X) 90% E - 89% 0.94 ~3.5 231 +ERCC1 (E)

*MDA-MB-231 experiments done with Vivian Kalman-Maltese

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Chapter 3

Identification and characterization of small molecules to inhibit XPF/ERCC1 and potentiate cisplatin chemotherapeutic efficacy

Sanjeevani Arora, Vivian Kalman-Maltese, Kristin Tillison, Elaine C. Chalfin and Steve M.

Patrick*

1Department of Biochemistry & Cancer Biology, University of Toledo Health Science Campus,

Toledo, OH

*To Whom Correspondence should be addressed:

Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus,

405 Block Health Sciences Building, 3000 Arlington Avenue, Toledo, OH, 43614

Telephone: 419-383-4152

Fax: 419-383-6228

Email address: [email protected]

Conflict of Interest: None declared

Short Title: Small molecule inhibition of XPF/ERCC1

Financial Support: American Cancer Society- (ACS, RSG-06-163-01-GMC) to S.M.P and

National Institutes of Health - (1R01-GM088249) to S.M.P

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ABSTRACT

Cisplatin is a DNA-damaging agent which is limited clinically through tumor resistance. DNA repair mechanisms or proteins involved in DNA repair limit cisplatin cytotoxicity leading to resistance. DNA repair protein complex, XPF/ERCC1, has been shown to be important in the repair of platinum-DNA damage and its specific inhibition has been shown to enhance cisplatin cytotoxicity in cancer cells. ERCC1 has been extensively studied as a biomarker and been reported to have prognostic value in determining outcome of platinum-based chemotherapy. The incision and removal of the DNA damage is an important step mediated by

XPF/ERCC1 in all types of cisplatin-DNA repair, making it an ideal target. In this study, we describe a high throughput screen (HTS) used to identify small molecules that inhibit the endonuclease activity of XPF/ERCC1. Primary screening identified 2 compounds that inhibit

XPF/ERCC1 activity in the nM range. These compounds were validated in secondary screens against two other non-related endonucleases for specificity to XPF/ERCC1. The results from the primary and secondary screens were validated using an in vitro gel-based nuclease assay. Using electrophoretic mobility shift assay (EMSA), we show that these compounds do not inhibit the binding of XPF/ERCC1 to DNA. Next, using colony survival assays, we show that these identified compounds potentiate cisplatin cytotoxicity in lung cancer cells. The identified compounds reduce survival by inhibiting DNA repair shown by comet assay and an ELISA- based assay for the repair of cisplatin-interstrand crosslinks (ICLs) and cisplatin-intrastrand adducts, respectively. Structure activity relationship (SAR) studies identify related compounds for at least one of the original hits which also potentiates cisplatin cytotoxicity in cancer cells.

This study is the first that identifies compounds that inhibit XPF/ERCC1 and further

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development of these is expected to display cytotoxic activity by sensitizing cells to cisplatin therapy.

ABBREVIATIONS: NER, nucleotide excision repair; NSCLC, non-small cell lung cancer; ICL, interstrand crosslinks; DSBs, double strand breaks; ERCC1, excision repair cross- complementation group 1; XPF, xeroderma pigmentation group F; siRNA, small interfering

RNA ; StaRT–PCR, standardized reverse transcription–polymerase chain reaction; ACTB, β- actin; ELISA, enzyme linked immuno-absorbent assay; SAR, Structure activity relationship;

HTS, High throughput screen; Pt, Platinum; SMI, small molecule inhibitor; EMSA, electrophoretic mobility shift assay.

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INTRODUCTION

Cisplatin chemotherapy is the first line of chemotherapy for several cancers including testicular, ovarian, non-small cell lung, cervical and head and neck cancers. Clinically, cisplatin is used alone or in combination with other chemotherapeutic agents. Cisplatin is also used in adjuvant therapy after radiation or surgery. However, a majority of patients present with either intrinsic resistance or they acquire resistance after treatment, thus limiting clinical response [1,

2]. Cisplatin imparts its antitumor activity by its interaction with DNA forming different lesions that are repaired by DNA repair mechanisms [3, 4]. Majorly cisplatin forms the intrastrand Pt-

DNA adducts which are repaired by the nucleotide excision repair (NER) pathway for which the

XPF/ERCC1 endonuclease complex is essential for removal of the damage. Cisplatin also generates interstrand crosslinks (ICLs) with DNA which also relies on the XPF/ERCC1 endonuclease. These lesions when not repaired result in inhibition of replication and transcription leading to the induction of apoptosis, which ultimately results in cancer regression.

Increased DNA repair capacity of cancer cells is an important mechanism of cisplatin resistance and it is now well known that DNA repair is important in determining sensitivity to cisplatin and its analogues [5, 6].

Testicular cancers are highly responsive to platinum agents and it has been shown that they have low level of NER capacity due to low levels of NER proteins [7]. Another study has shown that testicular tumor cell lines are limited in their capacity to repair cisplatin ICLs due to low levels of XPF/ERCC1, thus maintaining cisplatin sensitivity [8]. Studies with malignant ovarian cancer that does not respond to Pt chemotherapy shows increased levels of DNA repair protein, ERCC1 [9]. Another study has shown that ERCC1 is essential for melanoma growth and

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resistance to cisplatin in xenograft models. Mice with tumors that have ERCC1 disrupted survive longer than controls after cisplatin treatment [10, 11].

Our studies show that XPF/ERCC1 is essential to all aspects of cisplatin-DNA repair and thus targeting this enzyme complex could enhance cisplatin‟s cytotoxic effect. Studies with

ERCC1 disruption in various cancer types are consistent with the idea that targeting DNA repair improves Pt efficacy. Our studies show that specifically targeting the whole complex can significantly enhance cytotoxicity in cancer cell lines [12]. Clinically, ERCC1 has been extensively studied as a potential biomarker and a prognostic indicator in determining outcome as well as predicting response to Pt-based therapy [13]. Thus, novel agents that can inhibit the

XPF/ERCC1 nuclease activity hold the potential to enhance cisplatin efficacy in patients with high ERCC1 expression. Additionally, further increase in cisplatin sensitivity could be achieved in patients with cancers sensitive to cisplatin thus reducing tumor progression.

Protein-DNA interactions have been targeted in a small number of studies. Peptide based molecules were used to target the sequence specific Notch transcription factor [14]. Small molecules were identified for targeting the sequence specific DNA-binding transcription factor

HOXA13 by a high throughput screen (HTS) using fluorescence polarization (FP) [15]. Small molecules have also been identified for damage specific DNA binding proteins, RPA and XPA, by fluorescent based HTSs to identify small molecule inhibitors (SMIs) [16-18].

In this study, we describe a fluorescence based HTS of chemical compounds to identify molecules that target XPF/ERCC1-DNA interaction by inhibiting the nuclease activity. Our screens identify a new class of molecules that could be developed to target XPF/ERCC1 for therapeutic benefit in enhancing cisplatin chemotherapy.

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METHODS

Chemicals

Cisplatin [cis-diammine-dichloroplatinum (II)] was purchased from Sigma –Aldrich.

The antibodies were polyclonal ERCC1 -fl-297 (sc-10798, Santa Cruz) and monoclonal XPF

(MS-1381-PIABX, Neomarker). We used the Qiagen DNeasy blood and tissue kit for DNA isolation and Profoldin DNA binding plates for the ELISA. ICR4 antibody was kindly provided by Michael J. Tilby, University of New Castle, UK. All other reagents and chemicals were from standard suppliers. Hit 1 and 2 were from the NCI-DTP diversity set. Compound CD3-669 is from the CD3 library at University of Toledo and all other compounds were purchased from standard suppliers.

Cell culture

NSCLC cell line, H460 (provided by Dr. James C. Willey, University of Toledo) was maintained in RPM1 1640 supplemented with 10 % FBS in the presence of penicillin (100

IU/ml) and streptomycin (100 g/ml). Cells were grown at 37 °C in a 5 % CO2 incubator. The wt-CHO cells were obtained from Dr. Rodney S. Nairn (MD Anderson) and were maintained in

DMEM supplemented with 10% FBS and antibiotics.

XPF/ERCC1 protein purification

The ERCC1 and XPF cDNA were co-overexpressed in SF9 cells using a baculovirus expression system (obtained from Aziz Sancar, University of North Carolina) as previously described [19]. Briefly, 48 hours post-infection virus expression, cells were collected, washed and cell free extracts were prepared as described previously. Initial fractionation was performed on a 40 ml phosphocellulose column which was initially loaded and washed with a low salt buffer. Bound protein fraction containing XPF/ERCC1 was eluted in high salt (500mM NaCl)

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and supplemented with imidazole to 10 mM. The eluted protein was directly applied to a 5 ml

Ni-NTA agarose column and washed with buffer containing 50 mM imidazole. XPF/ERCC1 was eluted with 250 mM imidazole and active fractions were determined via nuclease activity using a fluorescence assay as described in the methods section for HTS fluorescence assay and fractions with greater than 50 % maximum eluted activity were pooled and dialyzed in low salt buffer. Further fractionation was performed by S-Sepharose and Q-Sepharose chromatography to make sure all nuclease activity was a function of XPF/ERCC1 catalyzed cleavage. The final pool of protein was stored at -80°C. The highly purified XPF/ERCC1 is very stable and displays no loss of activity over 6 months. Typical yields are 1 mg/ L of infected cells.

Supplemental Figure 1A shows the different fractions from the purification process and the highly purified XPF/ERCC1 enzyme.

Design and methodology of the HTS fluorescence assay

To screen for XPF/ERCC1 inhibition, an existing in vitro assay was optimized to a

96-well plate format to allow for screening of compound libraries. The assay was optimized for

DNA, MgCl2 concentrations as well as the incubation time to be used in the fluorescence assay in a 96 well plate format (Supplemental Figure 1B). The assay consists of a 10nM DNA (Q+Fl substrate annealed) and 7.5nM XPF/ERCC1 protein in buffer containing 50mM Tris-HCl pH8.0,

2mM MgCl2, 0.1mM BSA, and 0.5mM β-mercaptoethanol. For preliminary screening, 150 L of DNA was added to each well and the fluorescence signal was measured at 525 nm following excitation at 485 nm. Compounds or controls were then added to individual wells for a total of

80 compounds on each plate and the fluorescent signal was re-measured (Supplemental Figure

2A). 16 controls were used per plate which consisted of buffer alone, DNA alone, XPF/ERCC1 alone, 10 nM fluorescein ssDNA (positive signal), and 10 nM Q-Fl DNA with 7.5 nM

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XPF/ERCC1 (positive signal). 7.5nM XPF/ERCC1 is added to each well containing the compounds and DNA and incubated for 30 minutes at 37°C after which the fluorescent signal was measured again. In the 96 well platform, the Z‟ factor was calculated to be 0.87 indicating a highly robust assay.

The substrate is a synthetic DNA substrate that mimics the native DNA substrate of

XPF/ERCC1 (Figure S1B). This DNA substrate is a forked DNA, with a double-stranded DNA region (14 bases) and a region containing two single-stranded DNA flaps (12 bases each). A

DNA oligonucleotide that contains a site specific fluorescein modification at the position depicted (*) was purchased from Midland DNA technologies and HPLC and gel purified to ensure that the DNA contains a fluorescein modification. The DNA substrate is the same for gel- based and the HTS screen but the HTS assay substrate has modifications as described in the HTS assay methods section. The semi-complementary strand was synthesized containing a DABCYL quencher (Q) molecule at the depicted position (Figure S1B). The fluorescein (*) molecule has an excitation peak at centered at 485 nm and an emission peak centered at 525 nm. The quencher (Q) molecule is able to quench the fluorescence signal of the fluorescein when it is in close proximity. The design of the semi-duplex DNA substrate is such that the fluorescein and

DABCYL quencher are directly opposite one another at the double-stranded and single-stranded

DNA junction (Supplemental Figure 1B). This results in a significantly quenched signal when excited at 485 nm measuring emission at 525 nm. On cleavage by XPF/ERCC1, (4 bases from the dsDNA-ssDNA) the fluorescein label on the cleaved DNA is released into the solution and results in a significant increase in fluorescence as the quencher is no longer in close proximity

(Supplemental Figure 1B). The DNA substrate is also incorporated with a restriction enzyme site (Hha I) within the duplex DNA that will result in a cleavage pattern similar to XPF/ERCC1.

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This serves as our positive control and allows optimizing conditions for XPF/ERCC1 cleavage.

This DNA substrate is used in the solution based fluorescence screening assay to screen for inhibitors of XPF/ERCC1 endonuclease activity.

“Hit” validation in secondary gel-based assay

Hits from the HTS fluorescence screen were validated in a gel based nuclease activity assay as previously described [19]. This assay is robust and highly quantitative. Briefly, the 26- mer DNA substrate (Figure 1) was labeled on the 5‟-terminus with [32P] ATP using T4 polynucleotide kinase (NEB) for 30 min at 37°C. The 5‟-labeled DNA substrate was annealed to its complementary strand by heating to 95°C for 5 min, followed by 65°C and then 37°C for a total of 2 h. This substrate was gel-purified on a 10% native polyacrylamide gel in TBE (Tris, borate, EDTA electrophoresis buffer), developed, cut and kept for overnight gel elution overnight at 4°C. Post gel elution, the substrate was ethanol precipitated and the counts were determined. The final substrate was stored at -20 °C.

Reactions were carried out in a volume of 8 l at 37 for 30 min in reaction buffer containing 50mM Tris pH 8, 0.5mM b-mercaptoethanol, 0.1 mg/ml bovine serum albumin

(BSA) and 0.75mM MgCl2. 10 femtomoles of the DNA substrate was added to the reaction with

15 femtomoles of the purified XPF/ERCC1 enzyme or 20U/ml of Hha1 and the compound was titrated in a 8 l reaction. The reaction was stopped by adding formamide/EDTA and the samples were heated for 5 min at 95°C before loading on to the gel. Incision products were separated on a 12% sequencing gel for 2 h. The gel was removed and dried and products visualized were by autoradiography, or on a STORM phosphorimager (Molecular Dynamics).

For each experiment we used the following controls: DNA alone, DNA with XPF/ERCC1 and with vehicle control.

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Analysis of binding

Rapid dilution experiments as described previously [20] were used to demonstrate reversible or irreversible binding of the identified compounds – hit 1/143099, hit2/16168, CD3-

669-01, 4233-96-9 to XPF/ERCC1. Briefly, in a 10 l reaction, XPF/ERCC1 concentration was increased 100-fold from normal reaction conditions of 7.5 nM and mixed with 10 times the IC90 concentration of the compound or vehicle control (9:1 DMSO-glycerol or DMSO). After incubation at 37°C for 30 minutes, 2 µL of the pre-incubate was diluted into a 198 µL solution containing the DNA substrate and reaction buffer described in the HTS assay in a 96 well plate.

The 96-well plate was read in a Spectramax M5 repeatedly for 60 minutes. The data is plotted as increase in the fluorescent incision product showing the activity of XPF/ERCC1 against the

DNA substrate.

DNA Binding-Electrophoretic mobility shift assay (EMSA)

The labeled DNA substrate as already described in the methods section of the secondary in-vitro gel based assay was used for testing the effect of compounds on the DNA binding activity of XPF/ERCC1 to DNA. As XPF/ERCC1 requires metal for DNA incision, MgCl2 was excluded from the reaction buffer. 0.1 M EDTA was added to the reaction buffer and the reactions were carried out in a similar manner as described for the gel-based incision assay. Post incubation, glutaraldehyde was added to a final concentration of 0.25% and samples were incubated for 5 min at 37°C. The reaction was run on a 10% native polyacrylamide gel in TBE at

4°C (Tris, borate, EDTA electrophoresis buffer). The gel was removed and dried and products visualized were by autoradiography, or on a STORM phosphorimager (Molecular Dynamics).

For each experiment we used the following controls: DNA alone, DNA with XPF/ERCC1 and

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with vehicle control. Hit 1 or Hit 2 were titrated were titrated into the reaction with Hit 1 titrated from 10 nM, 50 nM, 250 nM , 500 nM, 1 M, 15M and 50 M. Hit 2 was titrated at 1 M, 10

M, 50 M and 100 M.

Cisplatin Intrastrand adduct measurement by ELISA

Repair of intrastrand adducts was assessed by ELISA as described with some modifications [12]. Cells were treated to compound (hit 1 or hit 2) and cisplatin or just cisplatin alone in serum free media. For treatment with compound and cisplatin, cells were treated with compound at 15M for 2h and then cisplatin was added (at IC90 concentration for the cell line used) to the media and these were incubated for another 2 h. Cells were then washed with PBS and fresh medium was added. At various time points between 0 and 72 h after drug treatment, genomic DNA was isolated and sonicated for 30 – 60 s in Cole Palmer ultrasonic processor.

Equal amounts of DNA was coated on 96 well DNA binding ELISA plates in binding buffer (1M sodium chloride, 50 mM sodium phosphate buffer, pH 7.4, 0.02% sodium azide) and incubated at 4ºC overnight. The wells were blocked with 1% BSA in PBS for 1 h at room temperature.

ICR4 antibody diluted 1:2000 in dilution buffer (0.2% BSA, 90 mM sodium chloride, 0.2%

Tween-20 in PBS) was added to the wells and incubated at 37ºC for 1 h. Following three washes with washing buffer (0.1% Tween-20 in PBS), HRP conjugated goat anti-rat antibody diluted

1:2500 (1% BSA, 0.2% Tween-20 in PBS) was added to the wells and incubated at 37ºC for 30-

60 min. After five washes with washing buffer, TMB (1 step ultra TMB-ELISA, Thermo

Scientific) was added and kinetics of absorbance was measured at 650nm in a Spectramax M5 plate reader (Molecular Devices) for 15 min. The reaction was stopped by adding 1 M sulfuric acid and absorbance was measured at 450 nm. All samples were assayed in triplicates. The mean background (antibody blank) was subtracted from all the readings and the % intrastrand

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adducts were calculated using OD 450 nm where the 0 h time point was used as 100% intrastrand adducts in each cell line.

Cisplatin Interstrand crosslink measurement by comet assay

Repair of interstrand crosslinks was assessed by alkaline comet assay with some modifications [12]. Cells were treated to compound (hit 1 or hit 2) and cisplatin or just cisplatin alone in serum free media. For treatment with compound and cisplatin, cells were treated with compound at 15M for 2h and then cisplatin was added (at IC90 concentration for the cell line used) to the media and these were incubated for another 2 h. At the end of treatment, cells were washed with PBS and incubated in fresh medium for the required post-incubation time or assayed immediately (time 0 h). Cells were further treated with 100 M of hydrogen peroxide for 15 min to induce DNA strand breaks. Cells were then trypsinized, pelleted and resuspended in 1% low melting point agarose. Cell samples (~10,000 cells) were embedded on a microscopic slide precoated with 1% normal melting point agarose. Another layer of 0.5% low melting point agarose was added and allowed to solidify. The slides were then incubated in lysis solution (2.5

M NaCl, 10 mM Tris, 100 mM EDTA, pH 10, containing 1% v/v Triton X-100) for 1 h at 4 ºC in the dark. The slides were then transferred to an electrophoresis tank containing ice-cold alkaline solution (300 mM NaOH, 1 mM EDTA, pH > 13), incubated for 20 min to allow DNA unwinding to occur and electrophoresis was carried out for 30 min at 0.7v/cm, 300 mA. Slides were removed and kept in neutralizing solution (0.4 M Tris–HCl, pH 7.5) for 10 min. Slides were then stained with SYBR green (Trevigen) and comets were analyzed using a Nikon epifluorescence microscope at 200x magnification. Fifty cells were analyzed per slide using

Komet Assay Software 5.5F (Kinetic Imaging, Liverpool, UK). The degree of DNA interstrand cross-linking present in cisplatin-treated sample was determined as described by comparing the

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tail moment of cisplatin + H2O2 treated samples with H2O2 treated samples and untreated control samples. The level of interstrand cross-linking was calculated by the following formula: [1 –

(TMpt – TMctl)/(TMH2O2 – TMctl)] x 100, where TMpt is the mean tail moment of the cisplatin +

H2O2 treated sample, TMctl is the mean tail moment of the untreated control sample and TM H2O2 is the mean tail moment of H2O2 treated sample and was expressed at the percent of ICLs that remained at that particular time point.

Colony survival assay

Cells were split and seeded at a density of 300-400 cells in 60 mm plate and incubated overnight. The next day, the cells were treated with a fixed concentration of the compound (hit 1 or hit 2) with cisplatin titration, after treatment, fresh complete medium with antibiotics was added and the cells were then allowed to form colonies. In another method, the cells were seeded onto 60mm plates and the next day, compound (hit 1 or hit 2) was titrated alone or titrated with a fixed concentration of cisplatin (IC50 for the cell line used) was added.

Post treatment, the cells were allowed to form colonies. Colonies were fixed with 95% methanol and stained with 0.2 % crystal violet. Colonies with ≥50 cells were counted using a light microscope. Cell survival was expressed as the ratio of average number of colonies in drug treated cells versus control cells x 100. The experiment was done in triplicate for each drug concentration.

RESULTS

High-throughput and secondary screening results

In order to identify compounds that have the potential for activity against

XPF/ERCC1, the NCI-DTP diversity set of ~1990 compounds was screened. Using the DNA substrate and the HTS assay as described above in the methods section, we screened for the

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ability to inhibit XPF/ERCC1 endonuclease activity. In the primary screens against

XPF/ERCC1, 28 „hits‟ inhibited the enzyme (1.4% hit rate). In secondary screens with two other non-related endonucleases (HhaI and XPG), the hits were narrowed to 12 small molecules that inhibit XPF/ERCC1 activity, but display no inhibitory effect on the other two endonucleases. 5 of the 12 „Hits‟ that were identified inhibit XPF/ERCC1 enzyme activity by >90% at low M or nM concentrations (Table 1).

Supplemental Figure 2A shows a typical screening assay illustrating the low background signal of the DNA alone, the dynamic range of the positive signal with XPF/ERCC1 protein and the inhibitory response observed with “hits” in particular wells of a 96-well plate against XPF/ERCC1 enzyme. Supplemental Figure 2B and 2C shows titration of a compound in the HTS assay, the compound in supplemental figure 2B specifically inhibits XPF/ERCC1 but the compound in supplemental figure 2C inhibits in both the primary and secondary screens and hence is non-specific. Hit 1 and 2 have a very potent inhibitory activity with 50% inhibition at ~

25 nM and 500 nM, respectively (Table 1). Importantly, cleavage of the DNA substrate by

(Table 1) Hha 1 and XPG (data not shown) is unaffected demonstrating good specificity of hit 1 and 2.

In vitro inhibition of XPF/ERCC1- DNA incision activity by SMIs

The gel-based in vitro incision assay has been described and extensively used previously [19]. In this assay, we incubated the titrated hit 1 (Figure 1A) or 2 (Figure 1B) with

XPF/ERCC1 on ice and reactions were initiated by the addition of the 5‟ [32P] radiolabeled DNA substrate. The reaction was terminated by the addition of EDTA, the reaction is heated for 5 min at 95°C and the products are separated by a 12% denaturing polyacrlyamide gel electrophoresis.

The products are visualized via phosphorimager analysis and the incised product migrating faster

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in the gel is consistent with the reduced size following XPF/ERCC1 cleavage (Figure 1A and

1B). The data demonstrates effective inhibition of the XPF/ERCC1 incision activity and correlates with our HTS data. The 50% inhibition value from the gel-based assay for hit 1 is ~ 25 nM (Figure 1A) and for hit 2 is ~500 nM (Figure 1B). In Figure 1A, lane 4-9 represents Hit 1 titration, 4- 1nM, 5- 25 nM, 6- 50 nM, 7-150 nM, 8- 500 nM, and 9-1 M. In Figure 1B, lane 4-8 represents Hit 2 titration, 4- 150 nM, 5- 400 nM, 6- 800 nM, 7-5 M, and 8- 15 M.

Next, we used the gel-based assay for compound titration with Hha 1 endonuclease (Figure 1C) and show that the addition of the compound does not inhibit the nuclease activity for both hit 1 and 2 (Figure 1B and data not shown, respectively). In Figure 1C, lane 4-8 represents hit 1 titration, 4- 1nM, 5- 25 nM, 6- 50 nM, 7-150 nM, and 8- 500 nM. Taken together, these data validate our HTS screening results and also demonstrate the ability to identify hits with low nM activity and provides an excellent platform to screen for more SMIs.

SMIs don’t inhibit the DNA binding ability of XPF/ERCC1

In order to study if the compounds affect the enzyme activity by affecting the binding ability of the complex to the DNA, we performed EMSAs [21]. In this assay, we incubated the titrated hit 1 or 2 respectively with XPF/ERCC1 on ice and reactions were initiated by the addition of the

5‟ [32P] radiolabeled DNA substrate. The samples were separated on a 10% native gel and the products were visualized via phosphorimager analysis. Figure 2A shows EMSA for hit 1, wherein the titration of the compound does not affect the binding of XPF/ERCC1 to DNA. In

Figure 2A, hit 1 titration is from lane 4-9, 4- 50 nM, 5- 250 nM, 6- 500 nM, 7-1M, 8- 15M, and 9-50 M. Figure 2B shows EMSA analysis for hit 2 which shows similar results as hit 1. Hit

2 at higher concentration could affect some of the DNA binding but concentrations that are used in further experiments do not affect DNA binding. In Figure 2B, hit 2 titration is from lane 4-7,

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4- 1M, 5- 10 M, 6- 50 M, and 7-100 M. These data demonstrate that at least these compounds do not affect the activity of XPF/ERCC1 by preventing binding of the enzyme complex to DNA.

Structure-activity relationship (SAR) studies for SMIs

Hit/Compound 1 (NSC 143099, see Table 2), identified from the screens, is although a good inhibitor in vitro, it has a very high molecular weight (578g/mol) and possesses more than

5 hydrogen donors and acceptors. All of these properties violate Lipinski‟s rules for proper drug absorption thus making it difficult for it to become a “traded” drug. The best way to develop

SAR for this molecule would be to remove/substitute the dihydroxy benzene rings and see if it affects potency. Another issue is the original compound would require a large synthetic effort to prepare synthetically. Hence, we tested commercially available compounds with similarities to the original hit 1 that fell into the guidelines of Lipinski‟s rules for drug development. We screened commercially available as well as CD3 (Centre for Drug design and development,

University of Toledo) compounds, results for which are summarized in Table 2. Another aspect of hit 1 is that it has 5 stereocenters; making drug development difficult. We also wanted to test if a particular stereochemistry affects the activity of hit 1. Thus, we tested all commercially available stereoisomers of hit 1 summarized in Table 2. The CD3 compound CD3-669-01, inhibited XPF/ERCC1 at ~150 nM in the HTS assay and we decided to further pursue SAR with this compound, results for which are also summarized in Table 2.

Hit 2 (NSC 16168, see Table 2), has a molecular weight of 473 and follows Lipinski‟s rules and has phenyl and biphenyl hydrophobic groups that help in absorption. However, sulfonic acid esters could potentially be toxic due to alkylation. These groups can also hinder absorption, thus if these groups can be replaced it could be a good potential lead molecule. We

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screened commercially available core structures of hit 2 or with sulphonic substitutions. The results from these studies are also summarized in Table 2.

SMIs potentiate cisplatin cytotoxicity by reducing colony survival

We have previously shown that knockdown of XPF/ERCC1 increases cisplatin cytotoxicity by decreasing colony survival [12]. Thus, we next wanted to assess whether these

SMIs impact colony survival. From our previous studies we know that treating XPF/ERCC1 knockdown cells with cisplatin makes a huge impact on cytotoxicity [12], thus for studies with

SMIs, we decided to pre-treat the cells with SMIs before any cisplatin treatment wherever mentioned. We used H460, NSCLC cells, to first assess the impact on colony survival by knocking down XPF/ERCC1 in these cells. XPF/ERCC1 knockdown cells show a ~ 3 fold change in IC50 (Figure 3A, filled squares). Next, we wanted to assess if colony survival is affected if we titrate cisplatin in these cells against a particular concentration of hit 1(50 M) or hit 2 (25 and 50 M). As seen in figure 3A and 3B for hit 1 and 2 respectively, colony survival is reduced further when compounds are used. Next, we assessed cisplatin potentiation by treating cells at a fixed cisplatin concentration (cisplatin IC50 in H460 cells) with hit 1 or 2 titration

(Figure 3C, filled symbols). Here, the compound is treated for a total of 4 h while cisplatin is added to the media after 2h of the compound treatment, 2h later media is changed. We do not observe any additional effects if the compound containing media was left for over 24h, indicating the compounds must have a short half-life (data not shown). As seen by colony survival graphs in Figure 3C, both the compounds potentiate cisplatin‟s effect. We also titrated hit 1 or hit 2 alone in these cells and treated for 4 h. Figure 3C (open symbols) shows that these compounds alone have no effect on colony survival. We also tested wild type CHO cells with hit

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1 or 2 with or without cisplatin and did not observe any effect on colony survival thus concluding these SMIs must be specific for human XPF/ERCC1 (Figure 3D).

SMIs potentiate cisplatin cytotoxicity by targeting DNA repair

XPF/ERCC1 is vital to the repair of cisplatin-DNA intrastrand lesions and ICLs. We have previously shown this using DNA repair assays for the two lesions – intrastrand adducts and ICLS in XPF/ERCC1 knockdown cancer cells [12]. We utilized an ELISA method (Figure

4A) to assess the repair of cisplatin 1, 2 dGpG intrastrand adducts over time using a monoclonal antibody specific for the major cisplatin dGpG intrastrand adduct. The repair kinetics of cisplatin intrastrand adducts at various time intervals was calculated as the percent of adducts remaining over time, relative to the percent of adducts present at the 0 h treatment (100%). In cisplatin alone treated cells, the intrastrand adducts were repaired gradually from 24 to 48 h, with

~15 % of the adducts remaining at 72 h (Figure 4A). However, when we treated cells to hit 1 or 2

(Figure 4A), the removal rate of these adducts was decreased. Hit 1 had ~65 % of adducts still remaining at the last time point tested and similarly for hit 2 had ~60% of adducts present at the last time point tested (Figure 4A). This data illustrates that these compounds inhibit the repair of the major cisplatin-DNA adduct by targeting the role of XPF/ERCC1 in the NER pathway.

Next, we looked for effects on ICL repair using the alkaline comet assay. We have previously described and used comet assay to show that ICLs are not repaired and increase over time when we knockdown XPF/ERCC1 in cancer cells [12]. We show (Figure 4B) the repair kinetics of cisplatin ICLs in H460 cells with hit 1 or 2 after 0, 24, 48 and 72 h post treatment and compare it to treatment with cisplatin alone. The data is expressed as the percentage of crosslinks remaining at the time points assessed. Cisplatin treatment induced a similar extent of ICL formation at 0 h in cisplatin alone or when cisplatin was used in combination with either of the

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compounds (Figure 4B). In only cisplatin treated cells, cisplatin ICLs were removed efficiently with ~1-5 % of the ICLs remaining at 72 h, whereas when combined with hit 1 ICL repair was significantly reduced, with increase in ICLs till 48h time point. At 72 h, we see a sudden drop and repair of the ICLs which could be attributed to the short half-life of the compound.

Interestingly, with hit 2 we saw similar kinetics of ICL repair as cisplatin alone showing hit 2 does not have any effect on ICL repair.

Hit/Compound 1 SAR identifies a class of compounds

SAR studies with compounds similar to hit 1 revealed an epicatechin class of compounds which are components of green tea (Table 2). These components have been shown to be anti-cancerous and have several targets in the cellular environment [22]. We tested these compounds in colony survival assays in H460 lung cancer cells. In H460 cells, CD3-669-01

(Table 2) potentiates cisplatin cytotoxicity but is also very cytotoxic when used alone. A similar compound to the CD3 compound, 4233-96-9, (Table 2) shows similar results. When we tested these compounds in cisplatin resistant ovarian cancer cells (data not shown), these compounds were highly toxic alone in the low M range indicating non-specificity. In view of a very good in vitro activity in the HTS screen, further development of this class of compounds could yield specific and potent inhibitors of XPF/ERCC1. In Table 2, a half-molecule of Hit 2, 5460-09-3, was tested for activity against XPF/ERCC1. This compound inhibited XPF/ERCC1 in the fluorescence screen in low micromolar range but had no effect in cell culture.

Analysis of reversible or irreversible binding

To determine reversible or irreversible binding we used a rapid dilution method. Here, the amount of enzyme (XPF/ERCC1) is increased 100-fold than normally used in a reaction and is preincubated for 30 min with a concentration of the SMI 10-times greater than the IC90

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concentration or with vehicle control. After incubation, the enzyme/inhibitor mixture is diluted

100-fold (i.e., to normal reaction conditions) in reaction buffer with the DNA substrate at normal reaction conditions. A reversible inhibitor dissociates quickly, allowing immediate recovery of enzymatic activity, whereas a slowly reversible inhibitor allows a gradual increase in activity. In contrast, an irreversible inhibitor prevents recovery of any enzymatic activity. In figure 5A, for hit 1 we see virtually no recovery of the enzymatic activity while hit 2 recovers slowly. Figure

5B, the CD3-669-01 compound also shows no recovery while compound 4233-96-9 recovers slowly. These results show how these compounds bind to XPF/ERCC1 and further studies determining the binding constants would give us more detailed information on the pharmacokinetic and pharmacodynamic properties of these compounds.

DISCUSSION

Targeted therapies are becoming popular in cancer owing to the development of resistance or no response from conventional chemotherapeutic agents [17]. Cisplatin is a main- stay chemotherapeutic agent for several different cancers and enhancing its clinical efficacy will benefit patient outcome as well as overall survival [23]. Both laboratory and clinical data indicate that inhibition of XPF/ERCC1 has the potential to modulate repair of cisplatin induced

DNA damage and impact patient response to platinum based therapies. Our idea is to inhibit the repair of cisplatin-DNA damage and prevent the removal of cisplatin-DNA lesions that mediates cisplatin cytotoxicity. The importance of DNA repair has been established in the treatment response of ovarian cancer as well as in lung cancers [6, 12, 24, 25]. NER is an ideal target for cisplatin-DNA damage and previous reports have described screens and identification of a class of inhibitors for RPA and XPA [16-18]. However, XPF/ERCC1 is involved in roles beyond NER

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which encompass all types of cisplatin-DNA lesions and hence makes it an ideal target to enhance cisplatin efficacy [12, 26].

In this study, we describe a fluorescent screen to identify compounds that specifically inhibit XPF/ERCC1. From the primary and secondary screens with the HTS assay we selected the top 2 hits that inhibited XPF/ERCC1 in the nanomolar range. These hits were further validated in vitro using a gel based nuclease incision assay. Inhibition values from both the HTS assay and the gel assay were the same (Table 1). Secondary screens with two non-related endonucleases further validate specificity of these two hits to XPF/ERCC1 (Table 1). While XPF has the nuclease domain, ERCC1 has the central domain that binds the complex to DNA. The

DNA binding ability is absolutely important for the function of the complex [27]. EMSA results for DNA binding activity show that these hits do not inhibit the binding of XPF/ERCC1 to DNA

(Figure 2). This could also mean that these compounds could potentially be binding to the active site of XPF/ERCC1.

Owing to the great activity in vitro, we next tested these hits in cell culture assays (Figure

3). An inhibitor of XPF/ERCC1 would potentiate cisplatin efficacy in cancer cells which can be observed by colony survival. We tested the two hits in H460 lung cancer cell line, where the hits alone do not show any cytotoxic or cytostatic effects. When we treated them in combination with cisplatin, we observed a 2-fold change in cisplatin IC50 with 15 M and 4 M concentrations of hit 1 and 2, respectively. A 2-fold change in IC50 in cell culture has been shown to be clinically relevant.

Next, we performed DNA repair assays to measure the repair of cisplatin-DNA lesions

(Figure 4) and correlated that to decreased colony survival. We used the ELISA based assay for

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determining the repair of cisplatin-DNA intrastrand adducts. Intrastrand adducts are the major product of cisplatin‟s reaction with DNA and they are believed to be formed around 65-85%.

These adducts are normally repaired by NER in which XPF/ERCC1 plays a central role as an endonuclease [12]. Compounds that target the repair of these lesions could potentially also be used for other agents that utilize this pathway like repair of UV-DNA damage. From figure 4A, we see that both the compounds inhibit the repair of these lesions and they are inhibited till the last time point studied (72 h). We next used comet assay (Figure 4B) to study the repair of ICLs, which are believed to be the most cytotoxic lesion as they are an absolute block to DNA replication. We show that hit 1 is a good inhibitor of ICL repair but probably due its short half- life is not able to inhibit repair past 48h. Interestingly, hit 2 does not inhibit ICL repair and has the same repair kinetics as cisplatin alone. This could potentially mean that hit 2 could be inhibiting a specific area of the enzyme that is responsible for interaction domains used in the

NER pathway. Thus, hit 2 possibly disrupts repair of intra-adducts by disrupting NER interactions but does not affect the domains which may be required for ICL repair. Further testing would yield answers whether this is a property of hit 2.

We next performed SAR studies for both hit 1 and hit 2 (Table 2). SAR studies with stereoisomers of hit 1 show that, stereochemistry plays an important role in the activity of the compound towards inhibition of XPF/ERCC1. We also tested these different isomers in cell culture and found activity for only of the hits (Procyanidin B3 in Table 2). Unfortunately, we were unable to test all the known stereoisomers due to limited commercial availability. Next we also tested core structure molecules of hit 1 and 2. Core structures or half structures of hit 1 were unable to show any activity in any of our assays. Only one core structure molecule of hit 2 (Cas

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no 5460-09-3, Table 2) showed activity in the HTS assay but had no activity in cell culture. This could potentially mean that for activity, structure beyond the half-molecule is essential.

Next, we identified a class of epicatechin compounds found in green tea that inhibited

XPF/ERCC1 in the low nanomolar range and we performed SAR studies for them (Table 2).

From table 2, we can see that stereochemistry and position of -OH groups plays an important role in potency and specificity to XPF/ERCC1. We identified CD3-669-01 and 4233-96-9 as potential hits that could be developed further to specifically inhibit XPF/ERCC1 with high potency. Owing to the high toxicity in cell culture with the epicatechin compounds, they would need to be selectively developed to get good specificity in cells. Next, we also studied how these selected compounds bind to XPF/ERCC1 using the rapid dilution method. This technique gives important information on the reversibility or irreversibility of the interaction between the compound and the enzyme. We identified compound/hit 1 and CD3-669-01 as an irreversible inhibitor of XPF/ERCC1 while compound/hit 2 and 4233-96-9 were slowly reversible.

In conclusion, we are the first to identify compounds that target XPF/ERCC1 and decrease DNA repair thereby increasing cisplatin sensitivity. These identified compounds will serve as lead structures for drug development efforts towards targeting XPF/ERCC1 for use in combination with cisplatin in the treatment of lung as well as ovarian cancers where

XPF/ERCC1 plays a critical role in the determining the efficacy of Pt-based chemotherapy.

ACKNOWLEDGMENT

We thank members of the Patrick lab for critical reading of the manuscript. We also thank members of the CD3 and Dr. Paul Erhardt‟s group at the University of Toledo for their helpful suggestions and providing compounds. This study was supported by grants from the

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American Cancer Society (RSG-06-163-01) and the National Institutes of Health (GM088249) both awarded to SMP.

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FIGURE LEGENDS

Fig. 1. Gel based assay for XPF/ERCC1 (A and B) and Hha1 (C) activity. The 5‟ [32P] labeled- forked DNA substrate was used which is cleaved by XPF/ERCC1 and also has a Hha1 recognition site. Denaturing gel electrophoresis of the products allows the separation of the substrate and products. A) Gel-based assay demonstrating Hit 1 titration and inhibition of

XPF/ERCC1. Lane 1 represents DNA substrate alone, lane 2 is XPF/ERCC1, lane 3 is

XPF/ERCC1 with vehicle control, and lane 4-9 represents Hit 1 titration, 4- 1nM, 5- 25 nM, 6-

50 nM, 7-150 nM, 8- 500 nM, and 9-1 M. B) Gel-based assay demonstrating Hit 2 titration and inhibition of XPF/ERCC1. Lane 1 represents DNA substrate alone, lane 2 is XPF/ERCC1, lane 3 is XPF/ERCC1 with vehicle control, and lane 4-8 represents Hit 2 titration, 4- 150 nM, 5- 400 nM, 6- 800 nM, 7-5 M, and 8- 15 M. C) Demonstrates compound titration and no effect on

Hha1 activity. Lane 1 represents DNA substrate alone, lane 2 is XPF/ERCC1, lane 3 is

XPF/ERCC1 with vehicle control, and lane 4-8 represents hit 1 titration, 4- 1nM, 5- 25 nM, 6- 50 nM, 7-150 nM, and 8- 500 nM.

Fig. 2. Gel based EMSA analysis for Hit 1 (A) and 2 (B). The 5‟ [32P] labeled forked DNA substrate and XPF/ERCC1 was added to the reaction buffer (buffer is without metal to prevent the nuclease function) and the compound was titrated. Native gel electrophoresis allows the separation of the free DNA and that bound by XPF/ERCC1. A) Lane 1 shows free DNA, lane 2 shows XPF/ERCC1 bound DNA and free DNA, lane 3 with vehicle control, and lane 4-9 with hit

1 titration, 4- 50 nM, 5- 250 nM, 6- 500 nM, 7-1M, 8- 15M, and 9-50 M. B) Lane 1 shows free DNA, lane 2 shows XPF/ERCC1 bound DNA and free DNA, lane 3 with vehicle control, and lane 4-7 with hit 2 titration, 4- 1M, 5- 10 M, 6- 50 M, and 7-100 M.

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Fig. 3. Colony survival in H460 cells (A- Hit1/NSC143099, B-Hit2/NSC16168, and C- Hit 1,

Hit 2). In A and B, H460 cells were treated at a constant concentration of the compound and then cisplatin was titrated and treated for 2h. After treatment, medium was changed and cells were allowed to form colonies. A) Shows comparison of clonogenic survival for non-targeting siRNA

(siC, open circles) and XPF/ERCC1 (filled squares denoted as siX + siE) siRNA transfected cells with Hit1 treated cells (closed circles). Calculated IC50s are shown in the figure. B) Shows clonogenic survival of cisplatin titration alone (open circles), Hit 2 at 25 M (closed squares) and Hit 2 at 50 M (closed circles). C) Shows titration of Hit 1 or hit 2 alone (hit 1 -open circles and hit 2 – open squares) and Hit 1 or hit 2 titration with cisplatin at IC50 for H460 cell line (hit 1

- closed circles and hit 2 – closed squares). The plot for cisplatin at IC50, 100% is survival at IC50 value. Calculated cisplatin IC50s are shown in the figure. Values are represented as mean ± SEM from three independent experiments. D) Titration of Hit 1 or hit 2 alone (open circles and squares respectively) and Hit 1 or hit 2 titration with cisplatin at IC50 in CH0 cells (closed circles and squares respectively). Values are represented as mean ± SEM from two independent experiments.

Fig. 4. Repair of cisplatin intrastrand adducts (A) and interstrand crosslinks (B) in H460 cells.

Cells were either treated to cisplatin alone for 2h at IC90 value for H460 cells or treated with Hit

1 or Hit 2 for 2h at 15 M and then cisplatin at IC90 was made up in the medium and incubated for another 2h. A) After the treatment time, genomic DNA was isolated at different time intervals (0, 24, 48 and 72h). ELISAs were performed as described using cisplatin intrastrand adduct antibody and the percentage of intrastrand adducts remaining was calculated at the denoted times. The results are represented as mean ± SEM of three independent experiments. B)

After treatment, comet assay was performed as described at different time intervals (0, 24, 48

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and 72 h). The percentage of interstrand crosslinks remaining was calculated using olive tail moment. Results are represented as mean ± SEM of three independent experiments. In A and B, black bars denote cisplatin alone, light blackish-grey denotes Hit 2 and light grey denotes Hit 1.

Fig. 5. Rapid Dilution for enzyme and compound binding analysis (A and B). XPF/ERCC1 was increased 100-fold and preincubated for 30 min with 10 times the IC90 of the compound or with vehicle control. This reaction is diluted to normal conditions and substrate is added and read in

Spectramax M5 plate reader for 60 mins. The values are plotted over time as % increase in fluorescent incision product or increase in fluorescent incision product over time directly correlated to XPF/ERCC1 activity. Results are represented as mean ± SEM of three independent experiments. A) Hit 1 and 2 against vehicle control. B) CD3669 and 4233969 compound against vehicle control.

Supplemental Figure:

Fig S1 A) Overexpression and purification of XPF/ERCC1. Lane 1, cell-free extract; lane 2, nickel-NTA agarose pool and lane 3, S-Sepharose pool. B) Fluorescence assay measuring the nuclease activity of XPF/ERCC1. The position of the fluorescent modification is depicted by

(Fl.) and quencher by (Q).

Fig. S2. (A) 96-well plate assay for ERCC1/XPF activity. Pre-read of the plate with DNA only is indicated by the blue surface. Fluorescence measured following the addition of ERCC1/XPF is indicated by the yellow. “Hits” are indicated by the downward deflection in individual wells.

(B and C) Selection of compounds from HTS by testing activity in a primary screen with

XPF/ERCC1 and secondary screen with Hha1 and XPG (data not shown). Briefly, the reaction is setup in a tube with 10nM DNA, 7.5 nM XPF-ERCC1/ 20 U/ml of Hha1/7.5 nM XPG (data not shown) in nuclease reaction buffer with compound titration and incubated at 37 ˚C for 30 min.

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The reaction is stopped by chelating the metal in the buffer and read in Spectramax M5. The graph represents inhibition of fluorescence by compounds against fluorescent product from protein/DMSO control. In B) Compound titration shows a compound that specifically inhibits

XPF/ERCC1 and has no effect on the other endonuclease while C) shows compound non- specifically inhibits both endonucleases.

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Fig. 1A

146

Fig. 1B

147

Fig. 1C

148

Fig. 2A

149

Fig. 2B

150

Fig. 3A

*Contributed by Vivian Kalman-Maltese

151

Fig. 3B

152

Fig. 3C

153

Fig. 3D

154

Fig. 4A

155

Fig. 4B

156

Fig.5A

157

Fig.5B

158

Supplementary Fig. S1A

*Figure contributed by Elaine C. Chalfin

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Supplementary Fig. S1B

160

Supplementary Fig. S2A

*Figure contributed by Kristin Tillison

161

Supplementary Fig. S2B

*Figure contributed by Vivian Kalman-Maltese

162

Supplementary Fig. S2C

*Figure contributed by Vivian Kalman-Maltese (VKM)

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*Values contributed by both Sanjeevani Arora (SA) and VKM

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Table 2 - Summary of compounds tested with structure and the results from different assays.

Compound Structure Assay result

HTS assay I50% = 23.4 nM Radio. assay I50% = 25 nM

NSC 143099/Hit 1 Cell culture = Require 15  M

to change cisplatin Ic50 by 2-fold

HTS assay I50% = 900 nM

Procyanidin B1 Cell culture = No effect

HTS assay I50% = 470 - 500 nM Procyanidin B2

IC40 = ~50  M

HTS assay I50% = 250 nM

Procyanidin B3 Cell culture = Require 19.5 M

to change cisplatin Ic50 by 2-fold

*Data points for HTS with 143099/hit 1, Procyanidin B1 and B2 also contributed by VKM.

Additional data points to these were added by SA

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HTS assay = No effect Cas no 215257-15-1 till 25  M

HTS assay I50% = 19  M

Cas no 154-23-4 Cell culture = No effect

OH HTS assay = No effect AG-690/1288317 HO O OH till 25  M

OH OH O

O CH 3 CH3 O O HTS assay = No effect AO-079/15259072 O till 25  M CH OH 3 O H3C

*Above contributed by VKM. Additional data points to compound 154-23-4 and cell culture was

done by SA

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HTS assay = No effect Cas no 490-46-0 till 25  M

HTS assay = No effect Cas no 18829-79-4 till 25  M

Cs no 35323-91-2 HTS assay = No effect till 25  M

HTS assay I50% = ~8.3  M Amentoflavone

Cell culture = No effect

*Contributed by VKM except Amentoflavone data is by SA.

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HTS assay I50% =4-7  M

Cas no 224434-07-5 Cell culture = Require 20  M

to change cisplatin Ic50 by 2-fold

Cas no 108907-44-4 HTS assay I20% =25  M

Cas no 298700-56-8 HTS assay I50% =9-17  M

Cas no 298700-57-9 HTS assay I50%=12-17  M

*Data contributed by VKM

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HTS assay I50% = 150 nM

CD3-669-01 Cell culture = Require 7-8  M

to change cisplatin Ic50 by 2-fold

Ic50 alone = 18  M

HTS assay I50% =50 nM

 Cas no 4233-96-9 Cell culture = Require 20 M to change cisplatin Ic50 by 2-fold

Ic50 alone = 41  M

Cas no 1257-08-5 HTS assay I50% = 10  M Cell culture = No effect

HTS assay I50% =100-150 nM

Cas no 130405-40-2 Inhibits Hha1

*VKM also contributed additional data points to SA‟s data with compound HTS for 1257-08-5,

CD3-669-01, 4233-96-9. VKM tested 1257-08-5 in cell culture.

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HTS assay I50%=100-300nM Cas no 83104-87-4

HTS assay I50%=500-600 nM

NSC 16168 Cell culture = Require 4  M to change cisplatin Ic50 by 2-fold

HTS assay I50%=3.2-3.6 M

Cas no 5460-09-3 Cell culture = No effect

HTS assay I20%=25 M AG-690/36490010

*VKM contributed HTS data for AG-690/36490010 and 83104-87-4. VKM added

additional data points to SA‟s HTS data for NSC 16168/Hit2 and 5460-09-3.

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AG-698/40847863 HTS assay I20%=25 M

AG-548/43179567 HTS assay I20%=25 M

p-Toluene sulphonic acid HTS assay = No effect till 25 M

*VKM contributed the above data

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Chapter 4

Gap junction intercellular communication increases cisplatin cytotoxicity in cancer cells by inducing DNA damage through bystander signaling

Sanjeevani Arora, Elaine C. Chalfin, Randall J. Ruch, and Steve M. Patrick*

Department of Biochemistry & Cancer Biology, University of Toledo Health Science Campus,

Toledo, OH

*To Whom Correspondence should be addressed:

Department of Biochemistry and Cancer Biology, University of Toledo Health Science Campus,

405 Block Health Sciences Building, 3000 Arlington Avenue, Toledo, OH, 43614

Telephone: 419-383-4152

Fax: 419-383-6228

Email address: [email protected]

Conflict of Interest: None declared

Short Title: Gap junctions maintain cisplatin sensitivity

Financial Support: American Cancer Society- (ACS, RSG-06-163-01-GMC) to S.M.P and

National Institutes of Health - (1R01-GM088249) to S.M.P

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ABSTRACT

Radiation induced bystander effect (RIBE) has been well documented leading to increased toxicity in a gap junction dependent manner in unirradiated bystander cells. Recent reports show that cisplatin toxicity is also dependent on functional gap junction intercellular communication (GJIC). These studies suggest a novel mechanism by which, cisplatin exerts a

“bystander effect” and induces cell death via GJIC. The aim of this study is to identify the role of gap junction modulation on cisplatin cytotoxicity. Using lung and ovarian cancer cells, we show that there is a density dependent increase in cisplatin cytotoxicity. This effect is not seen when we target an important gap junction protein, Connexin 43, leading to cisplatin resistance but only at high or gap junction forming density. We also show that the cisplatin-mediated bystander effect elicits as DNA Double Strand Breaks (DSBs) with positive -H2AX formation, an indicator of DNA DSBs. These DSBs are not observed when we prevent gap junction formation.

We also show that cisplatin is not the “death” signal traversing the gap junctions by using the cisplatin-GG intrastrand adduct specific antibody. Finally, we also show that cells deficient in

XPF/ERCC1, an endonuclease complex important in mediating cisplatin resistance via repair of cisplatin-DNA damage, further sensitize in the presence of functional gap junctions. These data demonstrate the positive effect of gap junction intercellular communication on cisplatin cytotoxicity.

Keywords: Connexin 43, gap junctions, gap junction intercellular communication, cisplatin, cisplatin

Abbreviations: NSCLC, non-small cell lung cancer; GJ, gap junction; GJIC, gap junction intercellular communication; ICL, interstrand crosslinks; DSBs, double strand breaks; Cx43, connexin-43; ERCC1, excision repair cross-complementation group 1; XPF, Xeroderma

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pigmentation group F; siRNA, small interfering RNA ;StaRT–PCR, standardized reverse transcription–polymerase chain reaction; ACTB, β-actin;. RIBE, Radiation induced bystander effect; BE, Bystander effect.

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INTRODUCTION

Cisplatin chemotherapy is highly effective and currently active against testicular, lung, ovarian, cervical and head and neck cancers [1, 2]. Resistance or variable responses limit its use, thus making it important to understand the mechanisms by which cisplatin kills cells as well as becomes resistant. Cisplatin produces a variety of platinum-DNA adducts, including intra- and inter-strand crosslinks (ICLs), and it is well accepted that these lesions mediate cisplatin‟s cytotoxic effect [3]. It is well known that resistance to cisplatin and its analogues can arise through multiple mechanisms broadly divided into: 1) Mechanisms that reduce formation of platinum-DNA adducts like decreased drug uptake, increased drug efflux, detoxification, or increased/altered DNA repair; and 2) Increased cell survival in the presence of DNA damage, for example alterations in the signaling mechanisms affecting apoptosis [4].

Recent literature suggests a novel mechanism through which cisplatin can also kill cells.

It was shown that cisplatin-induced cytotoxicity can be passed onto neighboring untreated bystander cells through gap junctions (GJs) [5]. There is a large body of evidence showing enhanced radiation toxicity with gap junctional intercellular communication (GJIC) [6]. It has been shown that this increased or enhanced toxicity is due to toxic or “death” signals traversing the gap junction channels although it has not been shown as yet what these actual toxic/”death” signals are. In experiments blocking GJIC or gap junctions, pharmacologically or by transcript downregulation respectively, it was shown that this “bystander effect” (BE) is dependent on the level of GJIC, that even low levels of GJIC can exhibit this bystander toxicity [7, 8].

Gap junctions form a direct connection from cell to cell to transfer molecules like ions, cyclic AMP, cyclic GMP, phosphoinositides, nucleotides, amino acids, glutathione or electrical charges through GJIC. Communication from cell to cell maintains tissue and organ homeostatis

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and helps in diverse processes like growth, development and differentiation. GJ channels are made of connexin proteins with 6 connexin monomers forming a hemi channel which docks onto a hemi channel from a neighboring cell to complete the gap junction channel [9] Studies with connexin proteins or GJIC in cancer agree that loss of connexin expression or GJIC is a marker of tumorigenesis. There is reduced GJIC or GJs from normal tissue to tumor tissue. However, some studies show that some cancers retain GJIC or can upregulate GJIC especially when transitioning to a metastatic phenotype [10].

Jensen and Glazer reported that cells deficient in the non-homologous end joining mechanisms (NHEJ) showed resistance to cisplatin only when treated at gap junction forming high density [5]. Kalvelyte et al have shown that functional GJs enhance apoptosis induced by

Cisplatin [11]. Another study by Glazer‟s same group showed that activated src phosphorylates

Cx43 which decreases GJIC and increases survival in response to Cisplatin [12]. Thus, formation of GJ channels and GJIC could be viewed optimistically as a potential biomarker or a target to enhance cisplatin cancer therapy. Hence in our study, using lung and ovarian cancer cell lines, we wanted to test if cisplatin cytotoxicity has a gap junction mediated component and what happens in the bystander cells. We also tested the effect of GJIC on cisplatin cytotoxicity in

DNA repair deficient cells.

METHODS

Chemicals

Cisplatin [cis-diammine-dichloroplatinum (II)] was purchased from Sigma –Aldrich. The antibodies were monoclonal α- tubulin (T5168, Sigma), Connexin43 antibody from Invitrogen, monoclonal anti-phosopho -H2AX (clone JBW301, Millipore), Alexa 488- conjugated goat anti-mouse and anti-rat (Molecular Probes). For StaRT-PCR, primers that amplify Cx43/GJA1

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and β-actin (control) were obtained from Gene Express (Toledo, OH). β-actin forward primer 5‟-

CCCAGATCATGTTTGAGACC-3‟; reverse primer 5‟-CCATCTCTTGC TCGAAGTCC-3‟.

Connexin43/GJA1 forward primer 5'-AGCAGTCTTTTGGAG TGACCAGCAACTTTG-3'; reverse primer - 5'-CATGCAATGAAGCTGAACATG ACCGTAGTT- 3'.

Cell culture

NSCLC cell lines, H1299 (provided by Dr. Randall Ruch, University of Toledo), H1355,

H460 (provided by Dr. James C. Willey, University of Toledo) and ovarian cancer cell lines,

2008, 2008/C13, A2780, A2780/C30 (provided by Dr. Stephen Howell, UCSD) were maintained in RPM1 1640 supplemented with 10 % FBS in the presence of penicillin (100 IU/ml) and streptomycin (100 g/ml). MDA-MB-231, which is a triple negative breast cancer cell line,

(provided by Dr. Manohar Ratnam, University of Toledo) was maintained in DMEM high glucose with 10% FBS in presence of glutamate and sodium pyruvate and antibiotics. Cells were grown at 37 °C in a 5 % CO2 incubator. siRNA sequence and transfections

SiRNA smart pools designed to target human ERCC1, XPF and Cx43 were purchased from Dharmacon RNA technologies, catalogue numbers L-006311-00, L-019946-00 and L-

011042-00 respectively. A non-targeting siRNA pool was used in control experiments

(catalogue number D-001810-10-20). Cells were seeded in six-well plates (density 2.5 × 10 5 cells /well) in antibiotic free media. Two transfections were done at 24 h interval in each cell line to knockdown Cx43 or XPF/ERCC1 according to the manufacturer‟s protocol.

Western blot

At indicated time points post transfection one, the cells were centrifuged, washed with

PBS, and lysed on ice for 30 min in lysis buffer (10mM Tris , pH 8.0, 120 mM NaCl, 0.5% NP-

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40, 1 mM EDTA) with protease inhibitors (0.5 M phenyl methyl sulfonyl fluoride (PMSF),

1mg/ml leupeptin, 1mg/ml pepstatin). Equal amounts of protein were loaded and electrophoresed on 10% SDS–polyacrylamide gel. The proteins were transferred onto PVDF membrane (Immobilon transfer membrane, Millipore Corporation). After electroblotting, the membranes were blocked with Tris-buffered saline with Tween 20 (1M Tris–HCl, pH 7.5, 150 mM NaCl, and 0.5% Tween 20) containing 2% non-fat dry milk. Primary antibodies recognizing Cx43 or α- tubulin were diluted in blocking buffer and incubated for 30 min. The membranes were then washed, incubated with the appropriate secondary antibodies in a blocking buffer for 30 min, and washed again. The blotted proteins were detected using chemiluminescence detection system.

RNA isolation, Reverse Transcription and Transcript abundance

Cells were lysed with 1 mL of TRIzol reagent and the total RNA was extracted following the manufacturer‟s protocol. The total RNA extracted from each cell line was reverse transcribed with oligo dT primer and M-MLV-RT as described previously [13, 14]. Transcript levels were quantified using the previously described StaRT-PCR protocol. Briefly, a mixture of internal standard competitive template (SYSTEM 1, Gene Express, Inc.) was included in a master mix with cDNA and PCR reagents (dNTPs etc.). The use of internal standards allows comparing data from different experiments giving a highly reproducible, standardized, quantitative measurement of transcript levels. In these studies, β-actin (ACTB) was used as a loading control gene. The master mix was aliquoted into tubes containing each gene-specific primer (ACTB and Cx43). PCR was carried out in a Rapidcycler (Idaho Technology Inc.) with each reaction mixture subjected to 35 cycles each of 5 s denaturation at 94 °C, 10 s of annealing at 58 °C and 15 s of elongation at 72 °C. PCR products were separated and quantified

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electrophoretically by the Agilent 2100 Bioanalyzer (Agilent Technologies Inc.) using the DNA

1000 Assay kit. Following electrophoresis, a ratio of the endogenous PCR products (or native template, NT) to the internal standard (competitive template) was taken to calculate molecules of

NT in the reaction. Each transcript abundance value was normalized to ACTB and values are reported as target gene mRNA/106 ACTB mRNA. All experiments were performed in triplicate.

Colony survival assay

Colony formation was assessed by a colony-forming assay adapted from Jensen and

Glazer [5] for high and low cell density corresponding to conditions in which gap junction formation is permitted or not, respectively. Cells were left untransfected, transfected with control siRNA or transfected with siRNA for Cx43 or XPF/ERCC1 wherever mentioned. For the high- density condition, cells were seeded such that they were between 95 to 100% confluent monolayer at the time of drug exposure. Cells were treated with cisplatin for 2h, washed with phosphate buffered saline (1X PBS), trypsinized, counted and 300-500 cells were seeded into 60 mm dishes and allowed to form colonies. Fresh medium was added when needed. For the low- density condition, 300-500 cells were seeded onto 60mm dishes and the next day treated with cisplatin for 2h, after treatment they were washed and fresh medium was added and they were allowed to form colonies. There was no significant difference in plating efficiency between the low- and high-density cultures (data not shown). Colonies were fixed with 95% methanol and stained with 0.2 % crystal violet. Colonies with ≥50 cells were counted using a light microscope.

Cell survival was expressed as the ratio of average number of colonies in drug treated cells versus control cells x 100. The experiment was done in triplicate for each drug concentration.

To make sure the trypsinization process post treatment at high density does not affect survival, we plated 5000-10,000 cells at colony forming density. The next day we treated them to

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cisplatin and after treatment, trypsinized and counted and seeded 300-500 cells in 60mm dishes for colony survival. The results obtained from this survival assay corresponded to the low- density survival results (Supplemental Figure S1).

Lucifer yellow dye-transfer assay

The Lucifer yellow (Invitrogen) dye transfer assay was performed as previously described [15]. Briefly, cells at complete monolayer were treated with 0.05% Lucifer yellow in 2 mL of DMEM + 10% FBS, linear scrapes were made through the monolayer using a scalpel blade. Treated cells were incubated at 37°C for 5 min to allow dye uptake, and then dye containing media was removed and cells were washed 2-3 times thoroughly with PBS, fixed with formalin and resuspended in PBS. Dye transfer from cells at the edge of the scrape to the neighboring cells (as a measure of gap junction communication) was visualized using Nikon

Eclipse T2000-U microscope at 20X (Supplementary Figure/Table 2C).

Gamma-H2AX phosphorylation for DNA DSB measurement

H1355 and A2780 (Cx43 knockdown, control siRNA or untransfected wherever mentioned) cells were trypsinized and divided into 3 populations per condition per cell line – 1)

P1C: Labeled with vital cell tracker dye and cisplatin treated, 2) P1: Labeled with vital cell tracker dye, 3) P2: Untreated and no label. The cells were labeled by a vital cell tracker dye called cell tracker orange according to the manufacturer‟s protocol (Invitrogen). It was determined that the H1355 and A2780 cells used for these experiments retained the cell tracker label at least 120 h post treatment (data not shown). The P1C cells were treated to cisplatin for

2h at doses giving 50% survival for all cell lines tested, washed, trypsinized and mixed with trypsinized P2 cells plated onto coverslips. Similarly, the P1 cells were left untreated and mixed

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with P2 cells plated onto coverslips. The cells were mixed such that P1C or P1 cells were in a ratio of 1:9 with P2 cells and these cells were 85 to 100 % confluent for the assay.

The next day, the cells were washed with Hank‟s balanced salt solution, fixed with freshly prepared 3.7% methanol-free paraformaldehyde for 15 min on ice and permeabilized with 0.3 % Triton-X-100 in PBS and blocked with 10% goat serum in PBS. For detecting phosphorylated form of -H2AX, cells were incubated for 1 hour with the monoclonal anti -

H2AX (1:1000, Millipore) followed by incubation with Alexa-488 goat anti-mouse antibody

(1:1000, Molecular Probes) diluted in 10% goat serum in PBS. Cells were washed and counterstained with DAPI for 5 min.

Coverslips were mounted with DAKO mounting medium onto slides and the edges were sealed with nail polish. Images were visualized using a Nikon Eclipse T2000-U microscope at

60X or 100X (wherever needed) oil immersion objective. Foci were counted in 100 randomly chosen cells per condition per cell line per experiment and results are expressed as % -H2AX foci per nuclei. Error bars indicate standard deviation and the data were collected from three individual experiments.

Immunofluorescence with cisplatin-DNA intraadduct specific antibody

Immunofluorescence with cisplatin-DNA intrastrand adduct specific antibody was performed as follows: H1355 cells were trypsinized and divided into 3 populations per cell line as described above for -H2AX immunofluorescence– P1C, P1 and P2. The labeled cisplatin treated P1C or labeled untreated P1 cells were mixed with P2 cells respectively and plated onto

60 mm dishes on coverslips for 80 to 100 % confluence. The next day, these cells were washed with PBS and fixed with freshly prepared 3.7% methanol-free paraformaldehyde for 15 min on ice and permeabilized with 0.3 % Triton-X-100 in PBS and the DNA was denatured using 2N

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HCl for 20 min at room temperature. The denatured cells were blocked for 1-2 h at room temperature with 20% fetal bovine serum (FBS) in washing buffer (0.1% Triton X-100 in PBS).

For detecting cisplatin-DNA intrastrand adducts we used the ICR4 (provided by Mike Tilby) antibody diluted in 1% BSA (1:500) and incubated for 1 h at room temperature followed by incubation with the secondary (1:1000, Sigma) diluted in 1% BSA for 2 h at room temperature.

Coverslips were mounted with DAKO mounting medium onto the slides. Images were captured by Confocal Microscopy at the Advanced Microscopy and Imaging center at University of

Toledo with help from Dr. Andrea Kalinoski (Supplemental Figure 3).

RESULTS

Cisplatin treatment at high density sensitizes lung and ovarian cancer cells

Lung and ovarian cancer cells were tested for density dependent cisplatin cytotoxicity. A high density confluent monolayer of cells promotes formation of gap junctions while at low colony forming density cells are dispersed with no contact to promote gap junction formation.

This procedure has been used extensively to study the role of gap junction mediated effects [5].

Figure 1, shows the survival of cells exposed to cisplatin for 2h at low- and high-density conditions. Clonogenic survival is reduced in both conditions however when cells are treated at high density there is a further decrease in survival in a concentration-dependent manner. Thus, increased cisplatin cytotoxicity is observed in conditions where there is an opportunity to form gap junction channels that could allow GJIC. Figure 1, HI355 and A2780 cells show the maximum fold change in toxicity ( ~4 fold and ~2.5 fold respectively) with a minimal change in

H1299 and H460 cells (~1.8 fold and ~1.5 fold change respectively). Next, the trypsinization process post cisplatin treatment could also affect survival at high density; hence we treated cells to cisplatin at colony forming density and replated them for colony formation after treatment.

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Supplemental Figure 1A and 1B, shows that there is no difference in IC50 values when cells are trypsinized and replated post treatment.

Connexin expression in lung and ovarian cancer cells

Connexins 43 (Cx43) has been widely studied and shown to be dsyregulated in cancer

[16, 17]. We studied the expression of Cx43 in non-small cell lung cancer cells (NSCLC) -

H1355, H1299 and H460. Figure 2A shows that H1355 cells highly express Cx43 while H1299 and H460s have a very reduced expression. In figure 2C, RNA expression analysis by StaRT

PCR confirms the protein expression analysis and shows that H1355 cells highly express Cx43.

Figure 2B and D shows connexin expression in ovarian cancer cells. Parental cisplatin sensitive and their resistant counterpart ovarian cancer lines – 2008 and 2008/C13 and A2780 and A2780/C30 were tested. From 2D, Cx43 expression is significantly increased in cisplatin sensitive ovarian cancer cells and decreases in resistant cell lines. We also tested protein expression and show that the A2780 cells (figure 2B) have a high expression of Cx43 with the resistant cell line having a very low expression. We also tested the triple negative breast cancer cell line, MDA-MB-231, and observed no expression for Cx43 (data not shown).

The Lucifer yellow dye transfer is a commonly used technique to detect the presence of functional gap junctions and has been extensively used previously [15]. We performed Lucifer yellow dye-transfer analysis and show that all the cell lines tested were able to communicate the gap junction permeant dye, Lucifer yellow (Supplemental Figure/Table 2C). For H1299 and

H1355 cells, we also observed that dye transfer is not affected on cisplatin treatment. This shows that at least in these cell lines, cisplatin treatment does not affect gap junction activity.

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Cx43 knockdown cells leads to cisplatin resistance at high density treatment

Increased cisplatin cytotoxicity at high density is consistent with results seen with radiation and recent reports on cisplatin [5, 6, 11, 18-20]. This density-dependent cytotoxicity results implicate gap junction formation and GJIC. To test the role of gap junctions in this enhanced cytotoxicity, we knocked down Cx43 in H1355, H460 and A2780 cells. From Figure 2,

H1355 cells highly express Cx43 while H460 express at much reduced levels. Supplemental

Figure 2A and 2B shows Cx43 knockdown in some of the cell lines with a time course analysis.

Figure 3, when Cx43 downregulated cells are treated with cisplatin at high density there is resistance to cisplatin and an increase in survival with no effect at low density. Figure 3A, H1355 highly express Cx43 and show a greater increase in resistance to cisplatin at high density treatment in comparison to H460 cells (Figure 3C). These results contribute to the evidence that

GJIC mediates cisplatin cytotoxicity at high density which promotes gap junction formation. The ovarian cancer cell line, A2780, also shows increased resistance at high density on Cx43 knockdown (Figure 3B). In Supplemental Figure 2D, we show that Cx43 levels are not affected when cells are plated at low or high density, thus it is their function rather than levels that potentially lead to differences in toxicity at low and high density cisplatin treatment.

Cisplatin treatment produces DNA DSBs in Bystander cells

The radiation-induced "bystander effect" (RIBE) has been shown experimentally in vitro and in vivo and has been shown to manifest by GJIC from irradiated cells to non-irradiated cells

[6, 19]. RIBE leads to DNA DSB formation in neighboring cells. A recent study showed that cell stresses other than radiation can also lead to the formation of DNA DSBs. UVC damage was also shown to cause DNA DSBs in bystander cells [21]. These reports prompted us to study if cisplatin toxicity in bystander cells is due to the formation of DNA DSBs.

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DSBs are a very lethal form of DNA damage and if it persists can lead to genomic instability and cell death. Bystander effects are usually point mutations, chromosomal abnormalities, micronuclei formation and apoptosis. The histone variant -H2AX phosphorylates at serine 139 on exposure to ionizing radiation and forms distinct nuclear foci at sites of DSBs.

The disappearance of the same is correlated to the repair of DSBs. -H2AX foci also form on exposure to cisplatin, although cisplatin does not directly induce DSBs. It is known that it is the processing of ICLs that results in the formation of DSBs [22].

In radiation treated cells, different methodologies have been used to study this effect, like individual cells are targeted using a single particle microbeam accelerator which can specifically targets cells in culture with defined number of charged particles (usually -particles), and this way biological effects can be recorded in irradiated and unirradiated bystander cells. In another case, cells populations were irradiated and immediately mixed with unirradiated cell [21]. Since cisplatin cannot be targeted to specific cells like irradiation, we decided to vitally label cells and then treat with cisplatin and then mix them immediately post treatment with untreated unlabeled cells to study the biological effects. In Figure 4, H1355 (4A) and A2780 (4B) cells were labeled with 5 M of vital cell tracker dye, cell tracker orange. Both the cell lines stably carry the dye till at least 5 days from the first treatment which is within the experimental time frame (data not shown). The cells were divided into two labeled populations: P1C and P1 for each cell line. P1C cells were treated with cisplatin for 2h and P1 cells left untreated, trypsinized and mixed with the unlabeled and untreated P2 cells on coverslips respectively. 24 h later, these cells were fixed and stained for the phosphoepitope of -H2AX.

In both cases, H1355 and A2780, when cisplatin treated P1C cells were mixed with untreated P2 cells, there was a high frequency of -H2AX foci formation as well as more nuclei

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with foci above the background levels (1-5 foci for H1355 and 1-3 foci for A2780). However, when untreated P1 cells were mixed with untreated P2 cells, there is just a regular distribution of foci or no foci. We studied the 24 h time point post treatment as cisplatin induced foci usually peak at 12-24 h period post treatment. This data suggests that in the presence of cisplatin treated cells, the bystander cells get some “toxic” signal or damage which elicits as DNA DSBs.

Overall, we show that the bystander effect manifests as DNA DSBs in neighboring untreated cells which when not properly repaired could lead to cell death and hence might be the reason for the pronounced sensitivity to cisplatin at high density. These results parallel results observed with radiation or UVC bystander effect (BE).

DSB production in bystander cells depends on functional gap junctional communication

Next, we wanted to further confirm the role of GJIC in DSB formation in bystander cells.

We either knocked down Cx43 in all the 3 populations: P1C, P1 and P2 or mixed these populations at a density where they could not form functional gap junctions to evaluate the contribution of GJIC. In figure 4C, Cx43 was knocked down in H1355 cells and in both cases, we see similar distribution pattern of foci/DSBs. We chose H1355 cells as we achieve the best

Cx43 knockdown in these cells. Previous reports show that residual levels might be able to contribute to a substantial level of bystander effect.

In figure 4D, H1355 cisplatin treated (P1C) or untreated (P1) cells were plated at colony forming density with the P2 bystander cells. We again observed the same results as seen with

Cx43 knockdown. These data suggests that GJIC plays a significant role in the manifestation of the BE. Figure 5 shows immunofluorescence images (H1355) of cisplatin treated or untreated labeled cells co-cultured with bystander cells.

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Cisplatin does not diffuse to bystander cells to induce DNA DSBs

Cisplatin has a molecular weight of 300 Da and is small enough to diffuse through GJ channels and spread to the untreated cells. In our studies, we have observed that cisplatin treatment from 2 h to 24h (data not shown) shows progressive increase in cytotoxicity suggesting at least in cultured cells cisplatin is in active form for this time period. Thus, to rule out the possibility that cisplatin could be the molecule diffusing through GJs, we performed immunofluorescence with the cisplatin-DNA GG intrastrand specific antibody. If cisplatin is diffusing through gap junctions, then the unlabeled untreated bystander P2 cells would also form the major cisplatin-DNA intrastrand adducts in addition to the treated P1C cells. In H1355 cells, we treated P1C cells with cisplatin for a period of 2 h to induce adducts or left them untreated

(P1 cells) and then trypsinized and mixed with the P2 cells. The next day, the cells were trypsinized and fixed and stained with the antibody specific to the GG intrastrand adducts. In

Supplementary Figure 3, only H1355 cisplatin treated cells have the damage-specific foci and not the bystander cells, showing that cisplatin does not cross GJs and induce BE in the neighboring cells.

Functional gap junction communication further sensitizes DNA repair deficient cells to cisplatin

As DNA repair is an important target to increase cisplatin cytotoxicity, we next wanted to test how cisplatin cytotoxicity is affected in DNA repair deficient cells in the presence of functional GJIC. Jensen and Glazer [5] have shown that a deficiency in the NHEJ complex

Ku70/80 could lead to cisplatin resistance only when cells are treated at gap junction forming density. They also showed that the Ku-initiated death signal required DNA-PK activation and was transmitted from cell to cell through gap junctions. This study was the first to show that

DNA repair status in one cell could affect the survival in another cell.

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For cisplatin-DNA damage repair, XPF/ERCC1 is an important enzyme and has been shown to be important in all aspects of cisplatin-DNA repair [23]. Thus, we wanted to test whether positive gap junction interactions further sensitizes cisplatin-DNA repair deficient,

XPF/ERCC1 knockdown cells. In previous studies, we have downregulated XPF/ERCC1 at ~90-

95% levels and shown that this highly sensitizes cisplatin treated cancer cells [23]. In figure 6A,

XPF/ERCC1 knockdown A2780 cells; when treated to cisplatin at high density there is a further increase in toxicity as compared to XPF/ERCC1 cells at low density. These results suggest that positive gap junction formation and communication could increase cisplatin cytotoxicity in cancer cells targeted with DNA repair inhibitors or DNA repair deficient cells. When compared to the original IC50 of A2780 cells at low density, there is a ~10 fold increase in toxicity. H1299 cells (Figure 6B), show a modest increase in toxicity at high density and have reduced levels of

Cx43. On XPF/ERCC1 knockdown they do not show a difference in the IC50 values when treated at high or low density with cisplatin. However, survival at higher concentrations post high cell density treatment is further reduced. H1299 is p53 deficient cell line and differences in p53 expression could also affect cell survival and DNA repair efficiency following XPF/ERCC1 knockdown. From these experiments we observe that there is an increase in bystander killing in cells deficient in cisplatin-DNA damage repair and synergism if any needs to be further explored between the bystander effect and DNA repair mechanisms.

DISCUSSION

Our study evaluates the gap junction dependent component of cisplatin cytotoxicity and its impact on the bystander cancer cells. Lung and ovarian cancer patients receive a platinum doublet combined (cisplatin or carboplatin) with a second chemotherapeutic. Some of these patients will be non-responders or will eventually develop resistance to this therapeutic option

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[24, 25]. Thus, making it important to understand how platinum agents can be made more effective or resistance can be reversed. Different mechanisms of cisplatin resistance have been explained over the years, of which repair of cisplatin - DNA repair is considered to be the most important mechanism of resistance [23]. Targeting DNA repair mechanisms is considered an important target for increasing platinum efficacy. This knowledge also makes it important to understand if other mechanism or pathways can enhance cisplatin toxicity in DNA repair targeted/deficient cells.

Recent reports suggest cisplatin cytotoxicity can be enhanced by modulating gap junction intercellular communication [5, 11, 12, 18]. Some studies have also shown that in normal cells,

GJIC can exert a protective effect in response to specific types of stress/chemotherapeutic drugs by probably propagating survival signals among cells in response to treatment. A study also showed that there was GJIC-mediated decrease in cisplatin-DNA ICL formation in normal cells when treated with cisplatin suggesting that the survival “signal” reduces DNA damage in normal cells [20]. Thus to better understand the contribution of gap junction mediated effects on cisplatin cytotoxicity we evaluated different cancer cell lines in our study. We observed that cisplatin cytotoxicity increases with an increase in cell density in lung and ovarian cancer cells

(Figure 1). It was also observed (Figure 1) in most of the cell lines used, that at lower cisplatin concentrations a greater change in cytotoxicity is observed at high density while the effect is similar at higher concentrations of cisplatin. This suggests that the positive effect of density could be limited to certain concentrations of the drug and needs to be further explored. We studied the expression of Cx43, which is the most well studied and understood connexin protein, which was especially elevated in expression in cisplatin sensitive ovarian cancer cell lines and reduced in the daughter-derived cisplatin resistant cells.

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Next, we also show that the increase in density dependent toxicity depends on the formation of functional gap junctions and not just the expression of Cx43 (Figure 2 and 3 and supplemental figure 2D). It has been previously reported that irradiation of cells induces -H2AX focus formation in bystander cells. Thus, we performed co-culturing studies and observed that mixing untreated cells with cisplatin treated cells induces DNA DSBs in the bystander cells

(Figure 4). Also, DSBs are not induced when Cx43 is downregulated or cells are at a low density where they cannot form gap junctions (Figure 4). Studies also show that media collected from irradiated cells can also induce DNA DSBs in bystander cells, showing that gap junctions also form hemichannels that could release toxic signals into the media which can be picked up by the GJ hemichannels in the untreated cells. In our studies, we did not observe any DSB formation in cells at a low density thus requiring further studies to understand the contribution of connexin hemichannels (Figure 4). Further studies are needed to elucidate why the bystander cells accumulate DSBs and timecourse analysis would show if these DSBs get resolved.

We also show that cisplatin (Supplemental Figure 3) is not the molecule that traverses the gap junctions to induce the DNA damage in bystander cells. The “death” signal has been under a lot of debate and investigation; several molecules have been implicated as the toxic molecule bringing in the “kiss” of death. Glutathione (GSH) is a likely candidate (307 Da) to protect against cisplatin induced damage by detoxification of cisplatin [20]. Another report showed that the death signal passing through gap junction in cells treated with cisplatin may be produced by

DNA-dependent protein kinase/Ku70/Ku80 signaling [5]. Reactive oxygen species (ROS), or other signaling molecules, including ATP, cAMP, IP3 and calcium could likely be the death signals too. Even inflammatory stress signals have also been implicated in being the bystander

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signal. Thus, further studies which inhibit some of these molecules could indicate the contribution from these factors [26].

Lastly, we also show that functional GJIC can further enhance sensitivity in cells deficient in XPF/ERCC1 DNA repair enzyme. XPF/ERCC1 is an important target to increase cisplatin cytotoxicity and mechanisms that can further potentiate cytotoxicity of cells targeted for

XPF/ERCC1 would be clinically beneficial for platinum chemotherapy. In figure 6, we show that positive gap junction formation further sensitizes XPF/ERCC1 deficient cells to cisplatin. These results make it important to further explore how DNA damage by chemotherapy and the DNA damage response pathway interact or function with GJIC.

Thus, gap junction and functional GJIC enhances cisplatin cytotoxicity by spreading the

“toxic” signals among coupled cancer cells that never received the chemotherapeutic agent. In a tumor, it would be essential to have functional gap junction interactions to yield a greater chemotherapeutic response. Several cancers mutate or down regulate connexins which means gap junction expression can be developed as a biomarker for assessing the response of chemotherapeutic agents that have a GJ dependent cytotoxicity component. Several cancers increase/decrease GJs or GJIC when they become invasive or metastatic thus showing gap junctions proteins can be differentially regulated in cancer cells [26].

There are various known biological agents that are known to upregulate gap junctions like caroteniods, green tea components like epicatechin, vitamin D, lycopene (a component of tomatoes), resveratrol (antioxidant in red wines) etc. Supplementing cisplatin chemotherapy with these agents could further enhance cytotoxicity or targeted overexpression of gap junction proteins in tumor cells can also further benefit platinum therapy [17]. Overall, this work highlights the importance of understanding mechanisms that mediate cisplatin cytotoxicity and

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how these can be used to our advantage to increase outcome and overall survival in cancer patients.

ACKNOWLEDGMENT

We thank members of the Patrick lab for critical reading of the manuscript. We thank

SURF students at University of Toledo – Eleanor C. Cook and Glen Westphal. We also thank

Erin Crawford and Dr. James C. Willey (University of Toledo) for help with StaRT PCR protocols. We would like to thank Dr. Andrea Kalinoski (University of Toledo) for help and suggestions for confocal microscopy. This study was supported by grants from the American

Cancer Society (RSG-06-163-01) and the National Institutes of Health (GM088249) both awarded to SMP.

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FIGURE LEGENDS

Fig. 1. Clonogenic survival after cisplatin treatment at low and high density of cells: A) H1355,

B) H1299, C) H460, D) A2780 cells. Clonogenic survival was performed at high density and low density cisplatin treatment as described in the methods section. Calculated IC50 values are represented in each figure for each cell line. Values are represented as mean ± SEM from three independent experiments.

Fig. 2. Cx43 expression in NSCLC and ovarian cancer cells: protein (A and B) and transcript levels (C and D). A and B) Proteins were extracted and probed with antibody for Cx43 with - tubulin as a loading control. C and D) Total RNA were extracted from cells and analyzed using

StaRT-PCR, as described in methods section. Each PCR was run in triplicate. The transcript levels are represented as ERCC1 mRNA/106 ACTB mRNA. The values are represented as mean

± SEM from triplicate PCRs.

Fig. 3. Clonogenic survival at high and low density post Cx43 knockdown – A) H1355, B)

A2780, C) H460 cells. Non-targeting siRNA transfected (siC) and siCx43 transfected cells were treated to cisplatin at high density and low density and plated for colony survival as described in the methods section. Calculated IC50 values are represented in each figure for each cell line.

Values are represented as mean ± SEM from three independent experiments.

Fig. 4. Positive -H2AX foci indicating DNA- DSB formation in bystander cells post-cisplatin treatment. A) H1355 and B) A2780 cells were divided into 2 populations - labeled with vital cell tracker dye or left unstained. Labeled cells were either treated or left untreated (control) to cisplatin and then mixed with unstained untreated cells and then visualized for positive Gamma

H2AX foci by immunostaining. Values are represented as percent above background ± S.D. from

3 independent experiments. C and D) DSB formation in H1355, Cx43 knockdown cells (C) and

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cells plated at colony density (D). In both cases, cell were divided into 2 populations - labeled with vital cell tracker dye or left unstained. Labeled cells were either treated or left untreated

(control) to cisplatin and then mixed with unstained untreated cells and then visualized for positive Gamma H2AX foci by immunostaining. Values are represented as percent above background ± S.D. from 3 independent experiments.

Fig.5. Immunoflourescene images from the experiment in figure 4. The representative images are from H1355, Blue- DAPI, red/orange – cell tracker orange, green – -H2AX foci and merge.

From A-D) No treatment and E-H) Cisplatin treated.

Fig.6. Clonogenic survival in XPF-/ERCC1 knockdown cells on cisplatin treatment at low and high density of cells: A) A2780 and B) H1299 cells. Non-targeting siRNA (siC) and

XPF/ERCC1 siRNA transfected cells (siX+siE) transfected cells were treated to cisplatin at high density and low density and plated for colony survival as described in the methods section.

Calculated IC50 values are represented in each figure for each cell line. Values are represented as mean ± SEM from three independent experiments.

Supplemental Data:

Fig. S1. Clonogenic survival: Cisplatin treatment at low density, splitting and replating cells for colony survival - A) H1355 and B) A2780 cells. Clonogenic survival was performed at low density cisplatin treatment and then cells were split, counted and replated to 60mm dishes for colony formation. Calculated IC50 values are represented in each figure for each cell line.

Values are represented as mean ± SEM from two independent experiments.

Fig. S2. Cx43 knockdown timecourse in A) H1355 and B) A2780 cells. Proteins were extracted at each timepoint post transfection for knockdown and control cells. Non-targeting siRNA denoted as siCtr, Untransfected denoted as UT (data not shown) and Cx43 knockdown

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timepoints from 48 to 120h post transfection one as described in the methods section. A and B)

Proteins were extracted and probed with antibody for Cx43 with -tubulin as a loading control.

C) Summary of Lucifer yellow dye transfer assay for NSCLC and A2780 ovarian cancer cells with or without cisplatin treatment. D) Cx43 protein expression at high and low density cell plating for NSCLC cells – H460, H1299 and H1355.

Fig. S3. Immunofluorescence with cisplatin-intrastrand adduct specific antibody. In H1355 cells,

A) green is the cisplatin-intrastrand adduct specific antibody staining the cisplatin treated cell, B) red labels the cell tracker orange labeled cisplatin treated cells, C) Brightfield.

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Fig. 1A

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Fig. 1B

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Fig. 1C

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Fig. 1D

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Fig. 2A

Fig. 2B

*2A and B contributed by Elaine C. Chalfin.

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Fig. 2C

Fig. 2D

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Fig. 3A

Fig. 3B

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Fig. 3C

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Fig. 4A

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Fig. 4B

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Fig. 4D

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Fig. 7A

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Supplementary Fig. S1A

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Supplementary Fig. S1B

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Supplementary Fig. S2A

Supplementary Fig. S2B

*S2A and S2B contributed by Elaine C. Chalfin.

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Supplementary Fig. S2C

Supplementary Fig. S2D

*S2C was done by both Sanjeevani Arora and Glenn Westphal.

*S2D contributed by Elaine C. Chalfin.

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Supplementary Fig. S3A

Supplementary Fig. S3B

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Supplementary Fig. S3C

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Overall Summary

Cancer chemotherapeutics usually work by targeting rapidly diving cells i.e. cancer cells.

Cancer cells can become resistant to the drugs directed at them and overcoming this resistance is an important research focus. Another important aspect in chemotherapeutics is the additional toxicity associated with them that affects patient‟s overall quality of life. Another area is targeted and individualized therapy to better manage and stabilize condition to get better therapeutic response.

In cisplatin therapeutics, recognition and the repair of cisplatin damaged DNA results in cancer resistance. Recent studies with the DNA repair enzyme, XPF/ERCC1, which is vital to the repair of cisplatin and platinum-DNA damage, suggest this protein can be a potential biomarker of cisplatin resistance. In this dissertation, I have described the validation of

XPF/ERCC1 as a global target to enhance cisplatin response in cancers. This also means that specific targeting of XPF/ERCC1 can also reduce the cisplatin dose required to kill cancer cells.

These findings show that by targeting XPF/ERCC1, we can enhance clinical response and also prevent the side effects associated with high dose cisplatin chemotherapy. We also show that cisplatin resistant cancers over express XPF/ERCC1 and thus by individually selecting patients with platinum resistant cancers which over express XPF/ERCC1 further sensitization can be clinically achieved. Many cancers acquire mutations in DNA repair pathways and hence specifically targeting XPF/ERCC1 in these cancers could induce synthetic lethality and achieve better clinical response.

Next, we approached XPF/ERCC1 as an important therapeutic target for platinum drugs for a molecular-based treatment. In our studies we identify small molecules that specifically

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inhibit XPF/ERCC1 in our screens. These compounds were tested in cancer cells and potentiate cisplatin toxicity. The increase in toxicity was correlated to decreased repair of cisplatin- damaged DNA. These molecules do not inhibit the binding of XPF/ERCC1 to DNA but prevent its nuclease incision activity. Structure activity studies further characterize the action of these molecules and further studies might yield clinically useful inhibitors of XPF/ERCC1.

In this dissertation, I also identify other cellular mechanisms that are important in maintaining cisplatin sensitivity. Gap junctions form between cells and mediate intercellular communication to maintain cellular and tissue homeostatis. Our studies show that cisplatin toxicity is also dependent on functional GJIC which induces a bystander effect. This bystander effect has been studied in terms of radiation and UV damage and it further amplifies their cytotoxic response. Cancers usually downregulate gap junction expression or activity and the finding that a chemotherapeutic response can be amplified by them are important in determining clinical benefit. Our studies show that the bystander effect elicits as DNA DSBs and further sensitizes cancer cells targeted for DNA repair (XPF/ERCC1). Our studies suggest that gap junctions could be biomarkers for platinum therapy and important in determining therapeutic response.

These studies highlight the importance of the interactions between cancer cells in a tumor. Tumors with functional gap junctions could help maintain cisplatin sensitivity via the bystander effect. Such tumors can be further sensitized by targeting DNA repair pathways.

Natural agents can also induce gap junction proteins which could be supplemented with the chemotherapeutics. Overall these studies show that understanding the molecular mechanisms of chemotherapeutic cytotoxicity and chemoresistance can improve chemotherapeutic response, overall patient survival and quality of life.

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