['lizbeth]

HER2 overexpression renders human breast cancers sensitive to PARP inhibition independently of any defect in homologous recombination DNA repair

Somaira Nowsheen, Tiffiny Cooper, [...], and Eddy S. Yang
Additional article information

Abstract

HER2 overexpression in breast cancer confers increased tumor aggressiveness. Although anti-HER2 therapies against have improved patient outcome, resistance ultimately occurs. Poly(ADP-Ribose) polymerase (PARP) inhibitors target homologous recombination (HR)-deficient tumors, such as the BRCA-associated breast and ovarian cancers. In this study, we show that HER2+ breast cancers are susceptible to PARP inhibition independent of a HR deficiency. HER2 overexpression in HER2 negative breast cancer cells was sufficient to render cells susceptible to the PARP inhibitors ABT-888 and AZD-2281 both in vitro and in vivo, which was abrogated by HER2 reduction. In addition, ABT-888 significantly inhibited NFκB (p65/RelA) transcriptional activity in HER2+ but not HER2 negative breast cancer cells. This corresponded with a reduction in phosphorylated p65 and total IKKα levels, with a concomitant increase in IκBα. Overexpression of p65 abrogated cellular sensitivity to ABT-888, while IκBα overexpression reduced cell viability to a similar extent as ABT-888. Therefore, susceptibility of HER2+ breast cancer cells to PARP inhibition may be due to inhibition of NF-kB signaling driven by HER2. Our findings indicate that PARP inhibitors may be a novel therapeutic strategy for sporadic HER2 positive breast cancer patients.

Keywords: breast cancer, HER2, PARP inhibitors, NFκB, homologous recombination repair

Introduction

The human epidermal growth factor receptor 2 (HER2) is a proto oncogene that belongs to a family of four transmembrane receptor tyrosine kinases that mediate the growth, differentiation, and survival of cells. Overexpression of the HER2 protein, amplification of the HER2 gene, or both, occur in approximately a third of breast cancers and are associated with aggressive behavior in the tumor (1). This may be, in part, due to activation of the NFκB signaling pathway by HER2, which enhances cell proliferation, invasion, and resistance to therapies (2–4). HER2 activation of NFκB requires IKKα, and this activation leads to an increase in cytokine and chemokine expression, as well as an increase in invasive phenotype (3). Additionally, activation of NFκB depends on poly(ADP-ribose) polymerase (PARP) (5, 6). Targeted therapy against HER2 has been shown to benefit patients with HER2-positive breast cancer (1). However, a significant number of patients either do not respond or quickly relapse and exhibit resistance to therapy. Thus, novel therapeutic strategies are needed.
PARP inhibitors have shown initial promise in clinical trials for their single-agent activity in BRCA-associated breast and ovarian cancers, based on synthetic lethal interactions between PARP inhibition and homologous recombination (HR) repair defects (7–9). This is due to the conversion of single strand DNA breaks into double strand breaks in PARP inhibited cells, which is normally repaired by HR. However, because BRCA-associated tumors are deficient in HR, the double strand break persists and is lethal to the tumor cell. Normal tissues still possess intact HR, which explains why minimal side effects have been observed in these patients. However, very few studies report the efficacy of PARP inhibition alone in sporadic cancers, presumably due to their intact HR repair. Instead, numerous trials incorporate PARP inhibitors because of their ability to enhance the action of other DNA damaging agents. Thus, the use of PARP inhibitors as part of novel therapeutic combinations is currently under extensive investigation.
In this study, we unexpectedly observed exquisite susceptibility of HER2+ breast cancer cells to PARP inhibitors alone independent of an inherent HR deficiency. HER2 overexpression itself was sufficient to render breast tumor cells susceptible to PARP inhibition. The mechanistic insight of this intriguing sensitivity is described in this report and involves attenuation of NFκB signaling driven by HER2. Our results suggest that PARP inhibitors may be useful for sporadic HER2+ breast cancer patients.

Materials and Methods

Cell culture

The human breast cancer cell line MDA-MB-361 was obtained courtesy of Dr. Andra Frost (University of Alabama at Birmingham, AL) while SKBR3 and HCC 1954 were obtained courtesy of Dr. Donald Buchsbaum (University of Alabama at Birmingham, AL). The human breast cancer cell lines BT-474 (HTB-20) and MCF7 (HTB-22) and T47D (HTB-133) were obtained from ATCC. HER2 overexpressing MCF7 cells (MCF7 HER2) and its isogenic control (MCF7 NEO) were obtained courtesy of Drs. Rachel Schiff and Kent Osborne (Baylor College of Medicine, TX) (10, 11). MCF7 DRGFP cells were a kind gift from Dr. Fen Xia (Ohio State University, OH) (12). The human breast cancer cell lines MDA-MB-361, SKBR3, and HCC 1954 were maintained in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals). The human breast cancer cell line BT-474 (HTB-20) was cultured in RPMI-1640 supplemented with 10% FBS, 0.1% insulin (Sigma) and 4 mM L-glutamine (Invitrogen). MCF7 (HTB-22) was maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum. MCF7 HER2 and MCF7 NEO were maintained in DMEM supplemented with 10% FBS, 0.4% G418 (Cellgro) and 15 μg/ml insulin. MCF7 DRGFP was cultured in DMEM supplemented with 10% FBS and 2 μg/mL puromycin (Sigma). T47D (HTB-133) was maintained in RPMI (Invitrogen) supplemented with 10% FBS. All cells were obtained in August 2010. The genetic background, including expression and function of key DNA repair and NF-κB signaling proteins, such as BRCA1, p53, p21, HER2, PAR, PARP, EGFR, p65, IκBα, and IKKα, as well as the growth characteristics and their response to genotoxic agents, was tested most recently on February 2012 using western blot analysis, immunohistochemistry, and colony formation assays. All experiments were performed within 10 passages, and no cell lines were kept in culture for more than 3 months after receipt or resuscitation. Cells obtained from the ATCC were also initially tested by ATCC via cytogenetic analysis and STR analysis.

Clonogenic survival assay

Cell survival was evaluated by the colony formation assay in the breast cancer cell lines as previously described (13, 14). Refer to SI Materials and Methods for details.

Cell Viability

Cell viability was measured using the ATP Lite 1 step luminescence assay (Perkin Elmer) following the manufacturer’s directions. Details are provided in SI Materials and Methods.

Immunofluorescence

To assay HR-mediated DSB repair capacity in breast cancer cell lines immunohistochemistry for radiation-induced rad51 foci was evaluated as previously described (13, 14). Immunofluorescence for HER2 expression in MCF7 HER2 tumor xenografts and H&E staining was performed as previously described (15).

Drugs, plasmids, and transfection

ABT-888 was obtained from Enzo Life Sciences (catalog # ALX-270-444) while Lapatinib (catalog # L-4804), Iniparib (catalog # I-5432) and Olaparib (catalog # O-9201) were obtained from LC Laboratories. The P65-DsRed and IκBα GFP plasmids were obtained courtesy of Dr. Markus Bredel (University of Alabama at Birmingham, AL). pDsRed and peGFP controls were obtained from Clontech. DR-GFP to measure chromosomal HR repair capacity, ISce-1 and the empty vector were gifts from Dr. Fen Xia (Ohio State University, OH) and has been described previously (12). All transfections were performed using Lipofectamine according to the manufacturer’s recommendations (Invitrogen).

Chromosomal homologous recombination mediated repair analysis

MDA-MB-361 cells were transfected with DRGFP substrate and stable integrant were selected with 2 μg/mL of puromycin (Sigma) for 3 weeks. Puromycin-resistant colonies were isolated and expanded. Chromosomal HR-mediated repair capacity was determined as described previously (12). Cells were transfected with either an empty vector, ISce-1 expression vector to measure HR-mediated repair capacity, or a GFP expression vector to measure transfection efficiency. % GFP positive cells were detected by flow cytometry. HR relative to total transfected cells was determined by division of the % GFP + cells from each ISce-1 transfection by the % GFP + cells from a parallel GFP transfection. Details are provided in SI Materials and Methods.

Immunoblotting

Immunoblotting was performed as described previously (13). Antibodies used are described in SI Materials and Methods.

HER2 knockdown

Endogenous HER2 was knocked down using SignalSilence HER2/ErbB2 siRNA I (catalog # 6283, Cell Signaling Technology). Scrambled siRNA (catalog # 6568, Cell Signaling Technology) was used as a control.

Luciferase reporter assay

NFκB transcriptional activity was measured using the NFκB Secreted Luciferase Reporter System (catalog # 631728, Clontech) according to the manufacturer’s instructions.

Tumor growth delay

3–4 week old female NOD.CB17-Prkdcscid/J mice were obtained from Jackson Labs and allowed a 1 week acclimatization period. Mice were anesthetized and supplemented with 0.72 mg 17β estradiol pellets from Innovative Research. Following recovery from pellet implantation, 2.5 × 10⁶ cells were injected into the mammary fat pad. Once the tumors were palpable (~5 mm) mice were randomized into groups (n=8): control and ABT-888 (100 mg/kg). ABT-888 was administered twice daily by oral gavage for 34 consecutive days. Tumor size was measured using digital calipers on alternate days and tumor volume was calculated using the following formula: ½ × length × width². Tumor size was measured for 35 days following which mice were sacrificed. All animal procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (Animal protocol no. 101109241).

Statistical analysis

The data were analyzed via analysis of variance (ANOVA) followed by a Bonferroni post-test using GraphPad Prism version 4.02 (GraphPad Software). Data presented as average +/− standard error of mean.


Results

HER2 positive breast cancers are susceptible to PARP inhibitors alone independent of steroid receptor or p53 status

We recently reported that cetuximab, which inhibits the EGFR (HER1) signaling pathway, can generate a DNA repair defect in head and neck cancer cells and subsequently induce a contextual synthetic lethality with the PARP inhibitor ABT-888 (Veliparib) (13). We thus hypothesized that lapatinib, a dual tyrosine kinase inhibitor which interrupts the HER1/HER2 growth receptor pathways, would generate a similar DNA repair deficit and induce susceptibility to ABT-888 in human HER2+ breast cancer cells. Consistent with our hypothesis, lapatinib indeed significantly reduced HR-mediated repair capacity in the well characterized BT-474 human HER2+ breast cancer cells (Supplemental Figure 1A) (16). However, unexpectedly, ABT-888 alone caused similar levels of cytotoxicity as the combination treatment of lapatinib and ABT-888 (Figure 1A). A sub-therapeutic dose of lapatinib (10 nM) alone, which was chosen to test for synergy with PARP inhibition, produced a 30% reduction in the survival fraction of these cells.
Figure 1
HER2 overexpressing breast cancer cells are susceptible to PARP inhibition alone


To further confirm our observations that ABT-888 alone was cytotoxic to HER2+ breast cancer cells, the effects of ABT-888 on cell viability (Figure 1B) were assayed using ATP Lite 1 step luminescence assay on four other HER2 overexpressing cell lines of various steroid receptor (estrogen and progesterone, ER/PR respectively) and p53 status (Supplemental Table 1), including MDA-MB-361, HCC1954, SKBR3, and ZR-7530 (16). Intriguingly, all HER2+ breast cancer cell lines demonstrated exquisite susceptibility to ABT-888.
Since assays of cell viability are often a measure of cellular metabolic activity and not necessarily cytotoxicity, and because PARP activity can change cellular levels of NAD+ and ATP (17), the effects of PARP inhibition in our assays may be due to alterations in metabolic processes rather than cell death itself. Thus, to validate PARP inhibitor-mediated susceptibility of HER2+ breast cancer cells, we used the gold standard cytotoxicity assay, the colony formation assay. As shown in Figure 1C, ABT-888 also significantly reduced the survival fraction of all HER2 overexpressing cells tested in a dose dependent manner but, as expected, demonstrated minimal cytotoxicity in non-HER2 overexpressing MCF7 and T47D cells (p<0.01). These results confirmed our cell viability data that HER2+ breast cancer cells are susceptible to PARP inhibition. Thus, for the remainder of our susceptibility experiments, we utilized the ATP Lite 1 step luminescence cell viability assay.
To further assess whether susceptibility of HER2+ breast cancer cells to ABT-888 can be generalized to other PARP inhibitors, we next tested the efficacy of BSI-201 (Iniparib) and AZD-2281 (Olaparib), currently used in clinical trials, in BT-474 and MDA-MB-361 HER2+ breast cancer cells. Similar to results observed with ABT-888, BT-474 (Figure 1D) and MDA-MB-361 (Figure 1E) cells were susceptible to AZD-2281 alone. However, consistent with recent reports that BSI-201 is not a bona fide PARP inhibitor, HER2+ breast cancer cells did not exhibit significant cytotoxicity following BSI-201 treatment (18, 19). These novel and intriguing results suggested that indeed, human HER2+ breast cancer cells are susceptible to PARP inhibitors alone independent of ER, PR, and p53 status.