Poly(ADP-ribose) polymerase 1 is overexpressed in ...mct.aacrjournals.org/content/molcanther/early/2013/08/24/1535-7163... · Poly(ADP-ribose) polymerase 1 is overexpressed in nasopharyngeal

Poly(ADP-ribose) polymerase 1 is overexpressed in nasopharyngeal carcinoma and its

inhibition enhances radiotherapy

Jeremy P.H. Chow∆,1, Wing Yu Man∆,1, Mao Mao1, Han Chen1, Florence Cheung2, John

Nicholls3, Sai Wah Tsao4, Maria Li Lung5, and Randy Y.C. Poon*

(∆) These authors contributed equally

(1) Division of Life Science, Center for Cancer Research, and State Key Laboratory of

Molecular Neuroscience, The Hong Kong University of Science and Technology, Clear

Water Bay, Hong Kong

(2) Department of Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong

(3) Department of Pathology, (4) Department of Anatomy, (5) Department of Clinical

Oncology and Center for Cancer Research, The University of Hong Kong, Hong Kong

(*) Corresponding author: Division of Life Science, Hong Kong University of Science and

Technology, Clear Water Bay, Hong Kong

Tel: [852]-23588703

Fax: [852]-23581552

Email: [email protected]

Home page: http://ihome.ust.hk/~rycpoon

Key words: AZD2281; DNA damage; DNA repair; Olaparib; PARP1

Running title: Poly(ADP-ribose) polymerase in NPC

Grant information: This work was supported in part by the Research Grants Council grant

AOE-MG/M-08/06 to R.Y.C. Poon.

Conflict of interest: The authors declare no conflict of interest

on July 10, 2018. © 2013 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 26, 2013; DOI: 10.1158/1535-7163.MCT-13-0010

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2

ABSTRACT

Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer. As options of agents

for effective combination chemoradiotherapy of advanced NPC are limited, novel therapeutic

approaches are desperately needed. The ubiquitin ligase CHFR is known to target

poly(ADP-ribose) polymerase 1 (PARP1) for degradation and is epigenetically inactivated in

NPC. We present evidence that PARP1 protein is indeed overexpressed in NPC cells in

comparison to immortalized normal nasopharyngeal epithelial cells. Tissue microarray

analysis also indicated that PARP1 protein is significantly elevated in primary NPC tissues,

with strong correlation with all stages of NPC development. We found that the PARP

inhibitor AZD2281 (Olaparib) increased DNA damage, cell cycle arrest, and apoptosis in

NPC cells challenged with ionizing radiation or temozolomide. Isobologram analysis

confirmed that the cytotoxicity triggered by AZD2281 and DNA damaging agents was

synergistic. Finally, AZD2281 also enhanced the tumor-inhibitory effects of ionizing

radiation in animal xenograft models. These observations implicate that PARP1

overexpression is an early event in NPC development and provide a molecular basis of using

PARP inhibitors to potentiate treatment of NPC with radiotherapy and chemotherapy.




3

INTRODUCTION

Nasopharyngeal carcinoma (NPC) is a highly invasive cancer. Although NPC is relatively

rare in most parts of the world (annual incidence rate generally less than one per 100,000

population), high incidence rates are found in southern China and Southeast Asia (1). For

example, NPC among women and men in Hong Kong is 7 and 18 per 100,000, respectively

(2). The prevailing view is that infection by Epstein-Barr virus (EBV) is a significant

etiological factor for NPC (reviewed in (3)). Epidemiological evidence also links the

consumption of dietary nitrosamines (such as salted fish) to NPC (reviewed in (4)).

Radiotherapy has been used as the primary treatment of NPC (5). This reflects

both the inaccessible location of the nasopharynx as well as the radiosensitivity of NPC

cells. More recent advances in radiotherapy such as 3D conformal radiotherapy and

intension-modulated radiotherapy have helped to achieve good prognosis, in particular

for early stage NPC (6, 7). For advanced NPC, however, treatment outcomes have been

poor due to distant metastases and recurrence (8). Chemotherapy is used in combination

with radiotherapy to control advanced NPC. Commonly used chemotherapeutic agents

are restricted to standard compounds such as the DNA-damaging agent cisplatin and the

thymidylate synthase inhibitor 5-flurouracil. Effective agents for combination

chemoradiotherapy for NPC are still to be established.

One of the earliest responses to DNA damage is the binding of poly(ADP-ribose)

polymerase (PARP) to damaged sites. PARP catalyzes the transfer and polymerization of

ADP ribose units from NAD+ to form branched polymers of ADP ribose covalently

linked to heterologous acceptor proteins or PARPs themselves (reviewed in (9)). PARP-

catalyzed poly(ADP-ribosyl)ation plays pivotal roles in the processing and resolution of

DNA breaks. The binding of PARP to the site of DNA damage activates its catalytic




4

activity and promotes local poly(ADP-ribose)-dependent recruitment of DNA repair

enzymes. Poly(ADP-ribosyl)ation of histones and other proteins may also mediate the

decondensation of chromatin structure required for efficient DNA repair. PARP1 is the

most abundant and active enzyme of the PARP family; but roles of other members

including PARP2 and PARP3 in DNA damage responses are emerging (10).

PARP inhibitors have been evaluated in clinical trials either as single agents or in

combination with DNA damaging agents (reviewed in (11)). As stand alone agents,

PARP inhibitors have been used to induce synthetic lethality in homologous repair-

deficient tumors. For example, PARP inhibitors are effective in targeting BRCA1- or

BRCA2-deficient breast and ovarian cancer. The rationale of the approach is that

inhibition of PARP blocks the repair of spontaneous single-strand lesions, leading to the

formation of double strand breaks through mechanisms such as stalled replication forks.

These double-strand breaks become lethal in cells that are deficient in double-strand

break repair (such as those with mutations in BRCA1 or BRCA2). Synthetic lethality of

PARP inhibitors is not limited to BRCA1 or BRCA2 deficiency. Recently, it was

discovered that PARP inhibitors markedly sensitize Ewing’s sarcoma cells harboring the

EWS-FLI1 gene translocation (12). More importantly, PARP inhibitors could potentially

be used as agents that enhance chemotherapy- or radiation-induced DNA damage in

patients without defined gene mutations. This remains an emerging field with many

parameters to be deciphered (reviewed in (11)).

While PARP1 has been implicated as a therapeutic target in several types of

cancer, its role has not been vigorously investigated in NPC. Here we present evidence

that PARP1 is overexpressed in NPC in comparison to normal nasopharyngeal cells.

Importantly, NPC cell growth was inhibited synergistically by DNA damaging agents




5

and an inhibitor of PARP.




6

MATERIALS AND METHODS

Materials

Olaparib (AZD2281) (Selleck Chemicals, Houston, TX, USA) and temozolomide (TMZ)

(Sigma-Aldrich, St. Louis, MO, USA) were obtained from the indicated suppliers.

Cell culture

IMR90 (normal fibroblasts) were obtained from the American Type Culture Collection

(Manassas, VA, USA). NPC cell lines C666-1 (13), CNE2 (14), HNE1 (15), and HONE1

(15) were obtained from NPC AoE Cell Line Repository and were propagated in RPMI1640

(for C666-1) or DMEM (for other NPC cells) supplemented with 10% (v/v) fetal bovine

serum (Life Technologies, Carlsbad, CA, USA) and 50 U/ml penicillin-streptomycin (Life

Technologies). No authentication was done by the authors. Telomerase-immortalized

nasopharyngeal epithelial cell lines NP361, NP460, and NP550 (16) were propagated in

keratinocyte serum-free medium supplemented (Life Technologies, Carlsbad, CA, USA)

with 50% v/v Epilife (Sigma, St Louis, MO, USA). Cells were grown in humidified

incubators at 37°C in 5% CO2.

Trypan blue analysis was performed as described (17). For clonogenic survival

assays, 500 cells were seeded onto 60 mm-dishes. After 16 h, the cells were irradiated with

different doses of IR and either mock-treated or exposed to the different concentrations of

AZD2281 for another 24 h. After 10 days, colonies were fixed with methanol/acetic acid

(2:1 v:v) and visualized by staining with 2% (w/v) crystal violet in 20% methanol.




7

Flow cytometry

Flow cytometry analysis after propidium iodide staining was performed as described

previously (17).

Ionizing radiation

IR was delivered with a caesium137 source from a MDS Nordion Gammacell 1000 Elite

Irradiator. Unless stated otherwise, cells were irradiated with a dose of 15 Gy.

γ-H2AX staining

Cells grown on poly-L-lysine-treated coverslips were fixed by ice-cold methanol for 10 min.

The cells were then washed twice with PBS, 5 min each, before permeabilized and blocked

with 3% bovine serum albumin (BSA) and 0.2% Tween-20 in PBS at room temperature for

30 min. The cells were washed twice with wash buffer (0.2% Tween-20 in PBS) before

incubated with phosphor-histone H2AXSer139 antibody (Bethyl Laboratory, Montgomery, TX,

USA) (diluted in 3% BSA, 0.2% Tween-20 with PBS) at 4°C for 16 h. The cells were then

washed with wash buffer for four times, 5 min each, and incubated in dark with Alexa Fluor

594 goat anti-rabbit IgG secondary antibodies (Life Technologies) at 25°C for 2 h. After

washed four times with wash buffer for 5 min each, the cells were stained with Hoechst

33342 (5 µg/ml in wash buffer) for 5 min, washed three times with wash buffer, before

mounted with 100 mM N-propyl-gallate in 9:1 v/v glycerol:PBS.

Isobologram analysis

The combination effects of two treatments were analyzed according to the median-effect

method of Chou and Talalay (18). Each dose-response curve was used to calculate the




8

isobologram, the value of median-effect dose (Dm), the sigmoidicity of the dose-effect curve

(m), and the linear correlation coefficient of the median-effect plot (r). The combination

index (CI) at different effective dose (ED) ED50, ED75 and ED90 was calculated according to

Chou and Talalay (18) using Calcusyn Version 2.1 (Biosoft, Cambridge, UK). CI value < 1

indicates synergistic effect (0.1–0.5 strong synergism; < 0.1 very strong synergism); CI value

of 1 indicates additive effect; and CI value > 1 indicates antagonistic effect.

RT-PCR

Total RNA was extracted from ~3x106 cells using Nucleospin RNA (Macherey-Nagel,

Düren, Germany). The total RNA (1 µg) was used to synthesize cDNA using High-Capacity

cDNA Reverse Transcription Kit (Life Technologies) according to the manufacturer’s

instruction. Semi-quantitative real-time PCR was performed by mixing the cDNA with

SYBR Green Real-Time PCR Master Mixes in a 7500 Fast Real-Time PCR System (Life

Technologies) using the following primers for actin (5'-GGGAAATCGTGCGTGACATT-3'; 5'-

GGAACCGCTCATTGCCAAT-3'), CHFR (5'-GGCAACCAGAGGTTTGACAT-3'; 5'-

AGTCAGGACGGGATGTTACG-3'), and PARP1 (5'-GATGGGTTCTCTGAGCTTCG-3'; 5'-

ACACCCCTTGCACGTACTTC-3').

Antibodies and immunoblotting

Antibodies against β-actin (Sigma-Aldrich, St. Louis, MO, USA), phosphor-histone

H2AXSer139 (Bethyl Laboratory, Montgomery, TX, USA), cleaved PARP1(Asp214) (BD

Biosciences, Franklin Lakes, NJ, USA), and PARP1 (sc-7150, Santa Cruz Biotechnology,

Santa Cruz, CA, USA) were obtained from the indicated suppliers. Rabbit antibodies against




9

CHFR were raised against a bacterially expressed GST-CHFR(C∆427) protein.

Immunoblotting was performed as described (19).

NPC tissue microarray analysis

A tissue microarray containing normal nasopharyngeal epithelium and NPC specimens

from Hong Kong was constructed. The NPC specimens were obtained from NPC

patients prior to treatment at Queen Mary Hospital and Pamela Youde Nethersole Eastern

Hospital. Construction of the NPC tissue microarray and immunohistochemical staining

using the standard streptavidin-biotin-peroxidase complex method was similar to that

previously reported (20). Slides were incubated with antibodies against PARP1 at 1:200

dilution and counterstained with hematoxylin. The immunohistochemical stained tissue

microarray was reviewed by two independent pathologists. For evaluation of the

staining, the nonmalignant and malignant tissues were scored by assessing the PARP1

staining in the nucleus. A random four views were counted under 400x microscopy. The

intensity of staining was graded by an arbitrary scale that ranged from 0 to 3,

representing negative (“0”), weak (“1”), moderate (“2”) and strong (“3”) staining,

respectively. Staining values of 0 and 1 were classified as low expression, while 2 and 3

were classified as high expression. The Pearson χ2 test was used to assess the association

between PARP1 expression and several clinicopathologic variables. A p value <0.05 was

regarded as statistically significant.

Tumor xenografts

The experimental protocol was evaluated and approved by the Animal Care Committee,

HKUST. HONE1 cells (2x107) were injected subcutaneously into both sides of the dorsa of




10

4-6-week-old female BALB/c nude mice. Four animals (n=8) per group were used in control

and combination treatment and three animals were used in the single treatments. Tumors

were regularly measured using a Vernier caliper. Volume was calculated according to the

formula: π/6×length×width2. Day=0 was designated as when the tumor volume was ~100

mm3. AZD2281 (50 mg/kg) was administrated with intraperitoneal injection into the mice

daily from day 1 to day 5. IR (2 Gy) was delivered at day 1 and 4. In the combination

treatment, AZD2281 was administrated 30 min before IR delivery. Mice were killed when

the tumors in control group reached 1,000 mm3.




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RESULTS

Overexpression of PARP1 protein in NPC cell lines

Frequent alterations of gene expression have been reported in NPC. However, their

implication in tumorigenesis or treatment is not always immediately apparent. Among these,

transcription of the ubiquitin ligase CHFR was found to be epigenetically inactivated in NPC

through promoter methylation (21). To study if CHFR protein is altered in NPC cells,

antibodies against CHFR were generated. Lysates from different NPC cell lines (C666-1,

CNE2, HNE1, and HONE1) were prepared and analyzed with immunoblotting using

antibodies specific for CHFR. Normal nasopharyngeal (NP) epithelial cells that were

immortalized with telomerase were used as a comparison. CHFR was found to be

downregulated in all the NPC cell lines (Fig 1A). In agreement with this, CHFR was also

present at a relatively high level in three different immortalized NP epithelial cell lines

(NP361, NP460, and NP550) (Fig 1B). As a control, the normal human fibroblasts strain

(IMR90) also contained a high abundance of CHFR.

Recently, CHFR was reported to be able to regulate PARP1 by targeting PARP-1 for

ubiquitination and degradation (22). We also found that PARP1 expression could be reduced

by overexpression of CHFR and increased by knockdown of CHFR (our unpublished data).

This prompted us to examine the expression of PARP1 in NPC cells. Significantly, PARP1

displayed an inverse relationship with CHFR, expressing at high levels in all the NPC cell

lines and at low levels in the NP epithelial cell lines (Fig 1A and 1B). As expected, CHFR

mRNA was present at a lower level in NPC cell lines than in the NP epithelial cell lines (Fig

1C). Consistent with regulation by post-translational mechanisms, we found that PARP1

mRNA was expressed at similar levels between NPC and NP cell lines. Taken together,




12

these results indicate that PARP1 is present at a significantly higher level in NPC cell lines

than in normal NP epithelial cells.

Inhibition of PARP enhances the cytotoxicity induced by DNA damaging agents in

NPC cells

Given that PARP1 is overexpressed in NPC cells, we next addressed if this translates into

alterations in the sensitivity to PARP inhibitors. Olaparib (AZD2281) (23) is an orally active

PARP inhibitor undergoing Phase I and II clinical trials (11). Incubation of NPC and normal

NP epithelial cells with AZD2281 alone induced a stress response. In the NPC cells HONE1

and HNE1, a relatively high concentration of AZD2281 (10 µM) induced apoptotic cell

death (as indicated by the appearance of sub-G1 cells) (Fig 1D). An increase in 4N DNA

contents was also apparent, in particular for HONE1, suggesting a robust activation of the G2

DNA damage checkpoint. In contrast, no sub-G1 cells were detected when normal NP

epithelial cells were challenged with the same concentrations of AZD2281. In agreement

with this, trypan blue analysis indicated that a dose-dependent reduction of cell number for

NPC cells (Fig 1E). In contrast, only a marginal decrease in the number of viable cells was

observed after normal NP epithelial cells were incubated with AZD2281. Consistent with an

increase in apoptotic cell death in NPC cells, we found that AZD2281 stimulated an

accumulation of cleaved PARP1 and p53 in a dose-dependent manner (Fig 1F). The long

term clonogenic survival of HONE1 cells was likewise reduced by AZD2281 treatment (Fig

1G).

We next examined if inhibition of PARP could potentiate the cytotoxic effects of

DNA damaging agents. A partial G2 arrest was stimulated in both NPC and NP epithelial

cells after irradiated with 10 Gy of ionizing radiation (IR) (Fig 2A). Significantly more NPC




13

cells were arrested in G2 when they were irradiated in the presence of 1 µM of AZD2281. In

marked contrast, normal NP epithelial cells appeared to be impervious to AZD2281. As a

control, exposure of NPC cells to AZD2281 alone did not affect normal cell cycle

progression (see also Fig 1D). Moreover, an extensive sub-G1 population was induced when

HONE1 were treated with both IR and AZD2281. Although sub-G1 population was not

apparent for HNE1 and CNE2 cells, trypan blue exclusion analysis confirmed that

substantial cell death was triggered when the cells were exposed to both IR and AZD2281

(Fig 2B).

To ensure that the stimulation of NPC cytotoxicity by AZD2281 was not limited to

IR, cells were also treated with the DNA-methylating agent temozolomide (TMZ).

Incubation with either AZD2281 or TMZ (125 µM) alone did not significantly stimulate

apoptosis. Massive apoptosis was triggered when NPC cells were incubated with both TMZ

and AZD2281 together (Fig 3A). Although normal NP epithelial cells also responded to the

combined AZD2281 and TMZ treatment by displaying more G2 arrested cells, there was no

increase in sub-G1 population. Consistent with the results from the flow cytometry, high

level of γ-H2AX was induced after incubation with AZD2281 and TMZ in HONE1, but not

in NP460 (Fig 3B).

To see if the increase in DNA damage-mediated arrest and cell death in AZD2281-

treated cells was caused by defective DNA repair, we next examined γ-H2AX staining in the

presence or absence of AZD2281 (Fig 4A). As expected, irradiation with 5 Gy of IR

induced γ-H2AX foci formation in both NPC (HONE1) and NP epithelial (NP460) cells.

The γ-H2AX foci staining returned to a background level after 24 h, suggesting that the

damaged DNA was repaired in both NPC and NP cells (Fig 4B). Significantly, efficient




14

DNA repair in HONE1 cells was attenuated in the presence of AZD2281. In contrast, the

DNA repair in NP460 cells was resistant to AZD2281.

Collectively, these results indicate that inhibition of PARP enhances IR- or TMZ-

mediated DNA damage and apoptosis in NPC cells but not in normal NP epithelial cells.

PARP inhibition and ionizing radiation act synergistically to promote cytotoxicity

To investigate more vigorously if the effects of PARP inhibition and DNA damage on NPC

cells represent synergism, isobologram analysis was carried out according to the median-

effect method of Chou and Talalay (18). Using trypan blue exclusion assays to monitor

cytotoxicity, we found that AZD2281 and IR acted synergistically on NPC cells (HONE1

and HNE1) (Fig 5A and Table 1). The combination index (CI) was less than 1, which

indicated synergism, over a range of effective dose (ED50, ED75 and ED90). In contrast,

synergism between AZD2281 and IR were less effective on NP epithelial cells (NP460),

with the CI values actually exceeding 1 at ED90 (indicating antagonism). We also examined

the long term survival of HONE1 cells using clonogenic survival assays (Fig 5B and Table

2). Similar analysis indicated a synergism between AZD2281 and IR at ED75 and ED90 for

long term clonogenic survival. Collectively, these data demonstrated that AZD2281 and IR

act synergistically to induce cytotoxic effects on NPC cells.

Combined AZD2281 and ionizing radiation treatment reduces tumor growth in

tumor xenograft models

We next evaluated the effects of PARP inhibition on NPC growth in animal tumor models.

HONE1 cells were injected subcutaneously into nude mice. AZD2281, IR, or a combination

of both were delivered using a fractionated dose approach (Fig 6A). Treatment with




15

AZD2281 or IR individually reduced the rate of tumor growth. But more significant

reduction of tumor growth was induced in the group treated with both AZD2281 and IR.

More importantly, there was in fact a decrease in tumor volume during the period of

combined treatment (Fig 6A and 6B). Tumor size increased again only after the treatment

ceased. These results indicated that AZD2281 and IR exerted a strong tumor inhibitory

effect in xenograft mouse models.

Overexpression of PARP1 in primary human NPC

To confirm that the overexpression of PARP1 observed in NPC cell lines are relevant in

primary NPC tumors, we next examined the expression of PARP1 in primary human NPC

using tissue microarray analysis (Fig 6C). Normal NP and NPC tissues were scored for low

or high expression of PARP1 by assessing PARP1 staining in the nucleus (Table 3). High

expression of PARP1 was found in NPC tissues. Correlation was observed between PARP1

expression and several clinicopathologic parameters, including patient age and survival.

Importantly, an increase in PARP1 expression could be observed in all NPC stages,

suggesting that the alteration of PARP1 expression is an early event in NPC development.




16

DISCUSSION

In this study, we found that PARP1 protein is highly expressed in NPC cell lines and primary

NPC tissues in comparison to nasopharyngeal epithelial cells. The PARP inhibitor

AZD2281 increased DNA damage, cell cycle arrest, and apoptosis in NPC cell lines treated

with IR or temozolomide. Finally, AZD2281 also enhanced the tumor-inhibitory effects of

IR in mouse xenograft models.

A major focus of the clinical development of PARP inhibitors is to determine if they

can potentiate radio/chemotherapy-mediated DNA damage, especially for tumors that lack

intrinsic defects in DNA damage repair (reviewed in (11)). For NPC, we found that PARP

inhibition was indeed effective in potentiating the cytotoxicity of DNA damaging agents

using both cell line (Fig 2 and 3) and animal models (Fig 6). Isobologram analysis indicated

that at least for the cell line models, the effects of AZD2281 and IR were synergistic rather

than simply additive (Fig 5). AZD2281 and IR displayed synergism on NPC cells over the

range of effective doses in both short-term growth assay (Fig 5A and Table 1) and long-term

cell survival (Fig 5B and Table 2). Results from the xenograft studies further indicated that

combinational treatment of AZD2281 and IR could further lead to shrinkage of tumor size

(Fig 6). However, the treatment also has synergistic effects on normal NP cells (for ED50

and ED75) (Fig 5A and Table 1). Moreover, synergism was found at all effective doses for

long-term NPC cell growth (Fig 5B and Table 2). Furthermore, the combined AZD2281 and

IR treatment resulted in a ~15% weight loss of the animals (Supplemental Fig 1), suggesting

potential high toxicity to normal cells.

Hence although our findings provide a rationale to support testing of PARP inhibitors and

DNA damaging agents on NPC, further research is required to uncover the precise clinical

responses of these treatments.




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Conceptually, PARP inhibition should only enhance radio/chemotherapy if DNA

damage is selectively increased in tumor compared to normal tissues. Tumors that already

harbor DNA repair defects such as mutations in BRCA1 or BRCA2 genes serve as classic

examples. So far there is no evidence of impairment of the components of the DNA repair

pathways such as BRCA2 in NPC (24, 25). As implicated by our study, however, the

abundance of PARP1 in tumors may also play a role in selectively sensitizing the cells to

PARP inhibitors. NPC cell lines contained significantly more PARP1 than NP epithelial

cells (Fig 1A and 1B) were more sensitive to AZD2281 alone (Fig 1D and 1E) or when

AZD2281 was added together with DNA damaging agents (Fig 2 and 3).

The observations that PARP1 was overexpressed even at early stages of NPC

development (Table 3) imply that alteration of PARP1 could be a driver of tumorigenesis.

Consistent with our observations, overexpression of PARP1 has also been reported in other

tumors, including colorectal cancer (26), endometrial cancer (27), hepatocellular carcinoma

(28), and melanomas (27). Several studies have reported the association of PARP1 protein

expression with a worse prognosis. For example, PARP1 overexpression is associated with

overall poor prognosis, higher tumor grade, BRCA1-mutated and triple-negative phenotype

breast cancers (29-31). In NPC, PARP1 expression was correlated with increase patient age

and poorer survival (Table 3).

What are the mechanisms that lead to PARP1 overexpression in NPC and other

cancers? Transcription of the PARP1 gene relies on transcription factors such as Sp1 and

Sp3 (32) and NF1 (33). However, the increase in PARP1 protein in NPC was unlikely to be

due to an increase in transcription because no significant variation in PARP1 mRNA

between NPC and NP epithelial cell lines was observed (our unpublished data). This is also

consistent with the lack of report on an alteration of PARP1 mRNA expression from various




18

microarray studies. Instead, we believe that the protein level of PARP1 is usually kept at a

low level by CHFR in normal NP cells (22). The epigenetic inactivation of CHFR in NPC

(21) provides a molecular basis of how PARP1 is upregulated at the protein level (see Fig 6D

for a model). In this connection, it is possible that methylation of the CHFR promoter can be

used as a marker for PAPR1 expression, which in turn can be used as a prediction for

sensitivity to PARP inhibitors.

An interesting question is why overexpression of PARP1 would lead to an increase in

sensitivity to PARP inhibition. We believe that “PARP1 addiction” may play a role on the

reliance of NPC cells on PARP1 for DNA damage responses. As the DNA repair circuitry

gets re-wired by the constitutive overexpression of PARP1, the cell may become exquisitely

dependent on PARP1 activity. NPC cells were clearly more dependent on PARP activity for

effective repair than NP epithelial cells (Fig 4). The PARP1 addiction may be analogous to

the addiction to many oncogenes in cancer cells. Another possible reason why PARP1-

overexpressing cells were sensitive to PARP inhibitor is the recent finding that the

cytotoxicity of PARP inhibitors including AZD2281 is in part caused the trapping of PARP1

at damaged DNA complexes (34). Hence higher expression of PARP1 may allow larger

number of PARP1-DNA complexes to form.

Results from this study introduced further questions about PARP1 and NPC.

PARP inhibitors in development mimic the nicotinamide moiety of NAD+ and bind

PARP’s catalytic domain. In addition to AZD2281, several PARP inhibitors are in the

development pipeline. It would be interesting to see if they offer better efficacy when

combined with IR in treatment of NPC. Although PARP1 is believed to be the most

abundant isoform of the PARP family, it is possible that other family members may also

play a role in NPC. Inhibitors that target specific isoforms may be useful. Eventually,




19

randomized studies will be needed to validate PARP inhibitors as therapeutic agents for

NPC patients. Another unresolved issue is although we know that PARP1 is

overexpressed in NPC and other cancers, little is known about its activity. It would be

interesting to assay PARP1 activity in cell lines and tumor specimens from patients

treated with PARP inhibitors. In addition to DNA repair, PARP family also participates

in other processes, including remodeling of chromatin and the regulation of transcription

(reviewed in (35)). It is conceivable that the sensitization of NPC cells with PARP

inhibition was not just due to disruption of DNA repair.

In conclusion, PARP1 overexpression is an early event in NPC development.

Cell line and animal studies revealed that PARP inhibitors might be able to potentiate

radiotherapy and chemotherapy for NPC.




20

ACKNOWLEDGEMENTS

We thank Anita Lau and Kenji Nishiura for technical assistance, and Junyi Zhang for help in

generating the CHFR antibodies.




21

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TABLES Combination index (CI) Dm m r ED50 ED75 ED90 HONE1 0.510 0.633 0.786 1.037 0.940 0.998 HNE1 0.199 0.181 0.165 0.707 0.764 0.961 NP460 0.468 0.740 1.372 0.860 0.538 0.943

Table 1. AZD2281 and IR act synergistically to induce cytotoxicity in NPC cells. NPC (HONE1 and HNE1) and NP epithelial cells (NP460) were treated with IR and AZD2281 for 48 h and analyzed with trypan blue analysis. The combination index (CI) at ED50, ED75 and ED90 was calculated. The value of median-effect dose (Dm), the sigmoidicity of the dose-effect curve (m), and the linear correlation coefficient of the median-effect plot (r) are indicated. A CI value < 1 indicates synergism.




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Combination index (CI) Dm m r ED50 ED75 ED90 HONE1 1.142 0.830 0.606 1.072 3.940 1.000

Table 2. AZD2281 and IR act synergistically on clonogenic survival of NPC cells. HONE1 cells were treated with IR and AZD2281 and analyzed with clonogenic survival assays. The combination index (CI) at ED50, ED75 and ED90 was calculated. A CI value < 1 indicates synergism.




28

PARP1 expression Low High Variable n (%) n (%) Total p

Age ≥50 (median) 29 (58) 21 (42) 50 <50 11 (24) 34 (76) 45 0.003*

Sex Male 29 (43) 38 (57) 67 Female 13 (42) 18 (58) 31 0.900 Survival Alive 19 (54) 16 (46) 35 Dead 15 (29) 36 (71) 51 0.020*

NPC stage Normal 10 (91) 1 (9) 11

I 1 (20) 4 (80) 5 0.005*

II 12 (43) 16 (57) 28 0.006* III 7 (41) 10 (59) 17 0.008* IV 13 (39) 20 (61) 33 0.003* M 3 (38) 5 (62) 8 0.013* I,II,II,IV,M 36 (40) 55 (60) 91 0.001*

Table 3. PARP1 is overexpressed in human primary NPC. The expression of PARP1 in normal NP and NPC tissues was analyzed using tissue microarray and immunohistochemistry. Nonmalignant and malignant tissues were scored for low or high expression of PARP1 by assessing PARP1 staining in the nucleus. The

Pearson χ 2 test was used to assess the association between PARP1 expression and several clinicopathologic variables. Variables with statistical significant (p<0.05) by

Pearson χ 2 test are indicated by asterisks.




29

FIGURE LEGENDS

Figure 1. NPC cell lines overexpress PARP1 and are sensitive to PARP1 inhibition.

(A) Overexpression of PARP1 and downregulation of CHFR in NPC cell lines. Several NPC

cell lines (C666-1, CNE2, HNE1, and HONE1) and an immortalized normal nasopharyngeal

cell line (NP460) were analyzed. Cell-free extracts were prepared and the indicated proteins

were detected by immunoblotting. Actin analysis was included to assess protein loading and

transfer.

(B) The expression of PARP1 and CHFR in several immortalized normal nasopharyngeal

cell lines (NP361, NP460, NP550) and NPC cell lines (C666-1 and HONE1) were analyzed.

Normal human fibroblasts strain IMR90 was also included as a control. Actin analysis was

included to assess protein loading and transfer.

(C) CHFR but not PARP1 is regulated at the transcriptional level in NPC cell lines. The

relative expression of the mRNA of PARP1 and CHFR in NPC cell lines (CNE2, C666-1,

HNE1) and immortalized normal nasopharyngeal cell lines (NP361, NP460, NP550) was

quantified with semi-quantitative RT-PCR and normalized with actin mRNA (mean±SD of

three independent experiments).

(D) AZD2281 promotes apoptotic cell death in NPC cells. NPC (HONE1 and HNE1) and

normal NP epithelial cells (NP460) were incubated with either control buffer or the indicated

concentrations of AZD2281 for 48 h. The cells were harvested, fixed, and their DNA

contents were analyzed with flow cytometry. The positions of 2N and 4N DNA contents are

indicated.

(E) AZD2281 reduces cell viability in NPC cells. NPC and normal NP epithelial cells were

treated as in panel C. The cells were stained with trypan blue and the number of viable cells

was analyzed with a haemocytometer.




30

(F) AZD2281 promotes PARP1 cleavage in HONE1. HONE1 cells were treated as in panel

C. Lysates were prepared and subjected to immunoblotting analysis. Equal loading of

lysates was confirmed by immunoblotting for actin.

(G) AZD2281 reduces clonogenic survival in HONE1 cells. HONE1 cells were incubated

with different concentrations of AZD2281 for 48 h. The cells were washed and replated for

clonogenic survival assays.

Figure 2. AZD2281 enhances IR-mediated DNA damage responses in NPC cells.

(A) AZD2281 increases IR-mediated G2 arrest and apoptosis in NPC cells. NPC (HONE1,

HNE1, and CNE2) and normal NP epithelial cells (NP460) were treated with either control

buffer, AZD2281 (1 µM), IR (10 Gy), or a combination of IR and AZD2281. The cells were

harvested after 48 h and analyzed with flow cytometry. The positions of 2N and 4N DNA

contents are indicated.

(B) AZD2281 and IR reduce cell viability in NPC cells. NPC cells (HNE1 and CNE2) were

treated with either control buffer, AZD2281 (1 µM), IR (10 Gy), or a combination of IR and

AZD2281. The cells were harvested after 48 h and the viable cell number was analyzed

using trypan blue exclusion assay.

Figure 3. AZD2281 enhances TMZ-mediated DNA damage responses in NPC cells.

(A) AZD2281 increases TMZ-mediated G2 arrest and apoptosis in NPC cells. NPC (HONE1

and C666-1) and normal NP epithelial cells (NP460 and NP361) were incubated with either

control buffer, AZD2281 (1 µM), TMZ (125 µM), or a combination of TMZ and AZD2281.

The cells were harvested after 48 h and analyzed with flow cytometry. The positions of 2N

and 4N DNA contents are indicated.




31

(B) AZD2281 increases TMZ-mediated DNA damage in NPC cells. NPC cells (HNE1 and

CNE2) were incubated with either control buffer, AZD2281 (1 µM), TMZ (125 µM), or a

combination of TMZ and AZD2281 for 48 h. Lysates were prepared and analyzed with

immunoblotting. Equal loading of lysates was confirmed by immunoblotting for actin.

Figure 4. Inhibition of PARP disrupts DNA damage repair in NPC cells.

(A) AZD2281 inhibits repair of IR-mediated DNA damage in NPC but not in normal NP

epithelial cells. NPC (HONE1) and NP epithelial cells (NP460) were irradiated with IR (5

Gy), either the absence or presence of AZD2281 (1 µM). The cells were fixed at either 1 h

or 24 h after irradiation (cells treated with AZD2281 alone were also harvested after 24 h).

DNA damage and recovery were assessed by immunostaining with an antibody against γ-

H2AX. Nuclei were identified by staining with Hoechst 33342 dye. Representative images

are shown the various treatments.

(B) Quantification of γ-H2AX foci in the presence or absence of AZD2281. Cells were

treated as described in panel A. The percentage of cells positive for γ-H2AX was quantified

(n=75). Cells containing more than five γ-H2AX foci in the nucleus were considered as

positive. Mean±SD of three independent experiments.

Figure 5. AZD2281 and IR act synergistically to induce cytotoxicity in NPC cells.

(A) AZD2281 and IR act synergistically on NPC cells but not NP460 in reducing cell

number. NPC (HONE1 and HNE1) and NP epithelial cells (NP460) were treated with IR

and AZD2281 for 48 h and analyzed with trypan blue analysis. Isobologram plots of




32

effective dose (ED) ED90 are shown. Summary of the combination index (CI) is shown in

Table 1.

(B) AZD2281 and IR act synergistically on clonogenic survival of NPC cells. HONE1 cells

were treated with IR and AZD2281 and analyzed with clonogenic survival assays.

Isobologram plots of effective dose ED50, ED75, and ED90 are shown. Summary of the

combination index (CI) is shown in Table 2.

Figure 6. AZD2281 and IR inhibit tumor growth in mouse xenograft models.

(A) HONE1 cells were injected subcutaneously into nude mice. AZD2281 (closed arrow

head) and IR (opened arrow head) were delivered at the indicated time points as described in

Materials and Methods. The volume of the tumor was measured on different days (control,

white square, n=8; AZD2281, white circle, n=6; IR, grey circle, n=6; IR and AZD2281, dark

circle, n=8) (mean±SD).

(B) Representative mice with different treatments at day 28 are shown.

(C) PARP1 is overexpressed in human primary NPC. The expression of PARP1 in normal

NP and NPC tissues was analyzed using tissue microarray and immunohistochemistry.

Representative images of PARP1 and hematoxylin and eosin staining in normal NP and NPC

tissues are shown.

(D) Model of the roles of CHFR and PARP1 in NPC. In NPC, CHFR is epigenetically

inactivated by promoter hypermethylation. The decrease in CHFR protein lead to a

stabilization of PARP1 protein. As PARP1 is increased in early stages of NPC, the sustained

increase in PARP1 may contribute to tumor progression of this cancer. Moreover, PARP1

overexpression may lead to “PARP1 addiction”, in which DNA repair becomes more reliant

on PARP1 than in normal cells.






















Published OnlineFirst August 26, 2013.Mol Cancer Ther Jeremy P.H. Chow, Wing Yu Man, Mao Mao, et al. radiotherapynasopharyngeal carcinoma and its inhibition enhances Poly(ADP-ribose) polymerase 1 is overexpressed in

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