Purpose: Triple-negative breast cancer (TNBC) is a highly heterogeneous disease and has the worst outcome among all subtypes of breast cancers. Although PARP inhibitors represent a promising treatment in TNBC with BRCA1/BRCA2 mutations, there is great interest in identifying drug combinations that can extend the use of PARP inhibitors to a majority of TNBC patients with wild-type BRCA1/BRCA2. Here we explored whether mTOR inhibitors, through modulating homologous recombination (HR) repair, would provide therapeutic benefit in combination with PARP inhibitors in preclinical models of BRCA-proficient TNBC.
Experimental Design: We have studied the effects of mTOR inhibitors on HR repair following DNA double-strand breaks (DSB). We further demonstrated the in vitro and in vivo activities of combined treatment of mTOR inhibitors with PARP inhibitors in BRCA-proficient TNBC. Moreover, microarray analysis and rescue experiments were used to investigate the molecular mechanisms of action.
Results: We found that mTOR inhibitors significantly suppressed HR repair in two BRCA-proficient TNBC cell lines. mTOR inhibitors and PARP inhibitors in combination exhibited strong synergism against these TNBC cell lines. In TNBC xenografts, we observed enhanced efficacy of everolimus in combination with talazoparib (BMN673) compared with either drug alone. We further identified through microarray analysis and by rescue assays that mTOR inhibitors suppressed HR repair and synergized with PARP inhibitors through regulating the expression of SUV39H1 in BRCA-proficient TNBCs.
Conclusions: Collectively, these findings strongly suggest that combining mTOR inhibitors and PARP inhibitors would be an effective therapeutic approach to treat BRCA-proficient TNBC patients. Clin Cancer Res; 22(7); 1699–712. ©2015 AACR.
Novel targeted therapies are urgently needed for triple-negative breast cancer (TNBC) that have limited therapeutic options and poor prognosis. PARP inhibitors provide benefit for TNBC with BRCA1/BRCA2 mutations. To expand the spectrum of TNBC patients who would benefit from PARP inhibitors, especially those that are BRCA-proficient, rational combination therapies that lead to homologous recombination (HR) defects and consequently synergize with PARP inhibitors should be evaluated. Here, we report that mTOR inhibitors are highly effective in suppressing HR repair. In addition, combination treatment of BRCA-proficient TNBCs with mTOR inhibitors and PARP inhibitors synergistically inhibits proliferation in vitro and exhibits enhanced efficacy in vivo. Finally, we found that mTOR inhibitors suppress SUV39H1, a novel mechanism by which they regulate HR repair and synergize with PARP inhibitors. These findings provide rationales for translation of the therapeutic combination of mTOR inhibitors with PARP inhibitors into clinical trials targeting BRCA-proficient TNBC patients, and potentially patients with tumors of other lineages.
Triple-negative breast cancer (TNBC) is characterized by the lack of estrogen receptors (ER) and progesterone receptors (PR), in addition to the absence of overexpression of HER2. It is a highly heterogeneous disease that accounts for approximately 15% of all breast cancers (1, 2). Because TNBC cannot be treated with specific hormone or anti-HER2 therapy, systematic conventional chemotherapy is usually the first-line therapy for patients with TNBC. However, TNBC, especially those with advanced-stage disease, generally shows the worst prognosis among all subgroups of breast cancer patients (3), making it an extremely challenging disease in clinical oncology. Hence, one of the major focuses to direct clinical trials of TNBC is identifying targeted therapies in sensitive TNBC subsets by the presence of predictive biomarkers. Inhibitors of PARP have demonstrated activity in breast cancers with mutations in BRCA1 or BRCA2 with low toxicity profiles (4). TNBC is generally associated with significant genomic instability due to DNA repair defects. Depending on different populations and studies, up to 10% to 20% of TNBC patients carry germline or sporadic mutations in BRCA1 or BRCA2 (5, 6), which primarily affect the ability to repair DNA double-strand breaks (DSB) through error-free homologous recombination (HR) repair (7). PARP is one of the key players in base excision repair (BER) for repairing DNA single-strand breaks (SSB). It directly binds to DNA SSBs and is catalytically activated to mediate PARylation of itself and other proteins to recruit DNA damage repair factors (8). Failure to repair SSBs leads to DSBs during DNA replication. Thus, PARP inhibition results in HR dependency for repairing DSBs. This vulnerability provides the rationale for synthetic lethal therapy with PARP inhibitors in patients with HR-incompetent cancers, such as TNBCs with BRCA1/2 mutations (9). In addition, PARP inhibitors have also been used in BRCA-proficient TNBCs (10, 11), which currently have limited therapeutic options, albeit with minor evidence for activity. Therefore, discovery of agents that would cause HR defects and synergize with PARP inhibitors is urgently needed to provide preclinical evidence to direct clinical trials of rational combinations in BRCA-proficient TNBCs.
Previously, our group applied a transcriptional profiling–based approach to systematically generate an HR-defect gene signature, which robustly predicts PARP inhibitor response and HR repair status in vitro (12). We then queried the Connectivity Map, which is a collection of genome-wide transcriptional expression data and shows connections among bioactive small molecules, gene expression, and diseases (13, 14), to screen compounds against the HR-defect gene expression profiles and identified those compounds that would potentially lead to HR defects and sensitize cancer cells to PARP inhibitors. Interestingly, mTOR inhibitors were among the top candidates, in terms of suppressing HR repair and possibly synergizing with PARP inhibitors. mTOR is a key downstream regulator of the PI3K pathway, and PI3K inhibitors have been reported to compromise HR repair and sensitize breast cancer cells to PARP inhibitors (15, 16). Deregulation of mTOR has been found in various human cancers (17), including TNBCs (18, 19). Therefore, inhibition of mTOR signaling pathway is an attractive clinical strategy for this disease. In addition to regulating cell growth, proliferation, and metabolism in response to environmental and nutritional stimuli (20) through phosphorylating p70 ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1), mTOR has been implicated in the DNA damage response in human cancers, possibly through modulating proteins that are essential in chromosomal integrity and DNA damage response (21–23). Recently, several studies found that mTOR regulates the DNA damage response through the NF-κB–mediated FANCD2 pathway in leukemia and rhabdomyosarcoma (24–26). However, the association between mTOR and DNA damage response in TNBCs remains largely unknown. Therefore, it was important to investigate whether mTOR inhibitors modulate HR repair and enhance the cytotoxic effects of PARP inhibitors in BRCA-proficient TNBCs.
We demonstrate herein that mTOR inhibitors suppress HR repair efficiency and synergize with PARP inhibitors in BRCA-proficient TNBCs cell lines. These observations were further confirmed in vivo in BRCA-proficient TNBC xenografts. Furthermore, this study demonstrates that mTOR inhibitors modulate HR repair through suppressing the expression of SUV39H1, a key histone methyltransferase (27). Our results thus provide evidence for the translation of rationale combination strategies with mTOR inhibitors and PARP inhibitors in BRCA-proficient TNBCs to the clinic and disclose a novel molecular mechanism by which the mTOR inhibitors modulate HR repair and synergize to PARP inhibitors.
Materials and Methods
Cell culture and chemicals
The U2OS, MDA-MB-231, BT-549, and MCF-12A cell lines were purchased from the ATCC and the cell lines were authenticated by short tandem repeat (STR) profiling by ATCC. U2OS cells were maintained in McCoy 5A medium supplemented with 10% FBS. MDA-MB-231 and BT-549 cells were cultured in DMEM or RPMI1640 medium supplemented with 10% FBS, respectively. MCF-12A cells were cultured in mammary epithelial growth medium [1:1 DMEM:DMEM/F12 medium, 20% horse serum, hydrocortisone (0.5 mg/mL), insulin (10 μg/mL), recombinant EGF (20 ng/mL), cholera toxin (100 ng/mL), and 1:100 penicillin/streptomycin (Invitrogen)]. mTOR inhibitors everolimus and KU-0063794 (KU) and PARP inhibitors olaparib (AZD2281) and talazoparib (BMN673) were purchased from Selleckchem. Myc-DDK-SUV39H1 plasmid was purchased from OriGene. Antibodies against SUV39H1, p-4EBP1 (Ser65), and Myc-Tag were purchased from Cell Signaling Technology. Vinculin antibody was purchased from Santa Cruz Biotechnology. An Annexin V FITC apoptosis detection kit was purchased from BD Biosciences, and an apoptosis assay was performed following the manufacturer's procedure. The transfection reagent FuGENE 6 and oligofectamine were purchased from Promega and Life Technologies, respectively.
Immunofluorescence staining and microscopy
To detect DNA damage–induced RAD51 foci formation, we pretreated MDA-MB-231 or MCF-12A cells with either the mTOR inhibitor everolimus (10 μmol/L) or KU (10 μmol/L) for 48 hours. To induce DSBs, we subjected the cells to ionizing radiation (IR, 10 Gy) and performed immunofluorescence staining as described previously (28). Cells were treated with cytoskeleton and stripping buffers, fixed with 4% paraformaldehyde, and subjected to permeabilization with 0.5% NP-40 and 1% Triton X-100. The cells were then incubated with primary antibody (rabbit anti-RAD51, 1:400; Abcam) for 2 hours at room temperature and were incubated with secondary antibody (Alexa Fluor 488 conjugated donkey anti-rabbit antibody, 1:400; Life Technologies) for 1 hour at room temperature. Slides were mounted in medium containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, H-1200) and were analyzed under a fluorescence microscope (Eclipse TE2000E, Nikon). We scored the percentage of cells with more than 10 RAD51 foci per cell in at least 50 cells per sample.
HR repair analysis
U2OS cells containing a single copy of the HR repair reporter substrate direct repeat GFP (DR-GFP) in a random locus were generated as previously described (12). GFP-expressing plasmid (pEGFP-C1) was used as a transfection efficiency control, and pCBASceI plasmid (gifts from Dr. Maria Jasin; Memorial Sloan-Kettering Cancer Center, New York, NY) was used for detecting HR repair efficiency. Everolimus (10 μmol/L) or KU (10 μmol/L) was added 6 hours after the transfection. After 48 hours, GFP-positive cells were detected via flow cytometric analysis using a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson). For HU (hydroxyurea)-synchronized HR repair assay (12), U2OS cells were treated with everolimus (10 μmol/L) or KU (10 μmol/L) after pCBASceI transfection and then treated with HU (2 mmol/L) to synchronize the cell cycle for 16 hours before flow cytometric analysis of cell-cycle distribution and GFP intensity. For the HR repair rescue assay, Myc-DDK-SUV39H1 plasmids were transiently transfected into U2OS cells. The next day, cells were transfected with pCBASceI plasmid. Everolimus (10 μmol/L) or KU (10 μmol/L) was added 6 hours after the transfection, and GFP intensity was detected after 48 hours.
Cells were suspended in staining solution [0.1% sodium citrate, 0.03% NP40, propidium iodide (0.05 mg/mL), and RNase A (0.02 mg/mL); ref. 29]. Cell-cycle analysis was performed at The University of Texas MD Anderson Cancer Center South Campus Flow Cytometry and Cellular Imaging Core Facility.
Colony formation assay
MDA-MB-231 and BT-549 cells were seeded in 6-well plates at low density. The indicated concentrations of mTOR inhibitors and PARP inhibitors or vehicle control (DMSO) were added the next day, and cells were left for 10 to 14 days to form colonies. Colonies were stained with 0.25% crystal violet and 25% methanol in PBS solution for visualization. Colonies with 50 or more cells were counted manually and digitally using ImageJ software with customized parameters that were optimized on the basis of three preliminary manual counts.
Cell proliferation assay
Cell proliferation was measured by PrestoBlue Cell Viability Reagent (Life Technologies). We seeded cells in 96-well plates in a total volume of 100 μL in triplicate in each experiment. The next day, cells were treated with DMSO or the indicated concentrations of mTOR inhibitors and PARP inhibitors. After 5 days, PrestoBlue Cell Viability Reagent (10 μL) was added to each well and incubated for optimized incubation time (4–6 hours) at 37°C. Then, the fluorescence was measured by a Fluoroskan Ascent FL Microplate Fluorometer (Thermo Scientific). After background subtraction, we calculated the cell viability as the percentage change relative to the control cells.
Drug combination studies
Drug combination treatments were designed according to the Chou–Talalay equation, which accounts for both the potency (median inhibitory concentration) and the shape of the dose–effect curve, and the combination index (CI) was determined using CompuSyn software (ComboSyn, Inc.). CI < 1 indicates synergism (30).
In vivo tumorigenesis assay
All animal studies were conducted in compliance with animal protocols approved by the MD Anderson Cancer Center Institutional Animal Care and Use Committee. Six-week-old female athymic nu/nu mice (NCI; average weight, 25 g) were used for all in vivo experiments. MDA-MB-231 or BT-549 cells (5 × 106) were mixed with BD Matrigel Basement Membrane Matrix (Becton Dickinson) and injected orthotopically into the lower left mammary fat pad of each mouse to establish xenografts. After the tumor volumes reached 50 mm3, the mice were randomized to groups treated daily by oral gavage with vehicle control, everolimus (3 mg/kg; ref. 31), BMN673 (BMN, 0.333 mg/kg; ref. 32), or a combination of both agents. The MDA-MB-231 groups (n = 10 per group) were treated for 6 weeks, and the BT-549 groups (n = 8 per group) were treated for 8 weeks. Body weights were monitored, and the perpendicular diameters of each tumor were measured twice a week with a digital caliper. Tumor volumes were calculated with the following formula: (length × width2)/2.
Microarray analysis, cDNA synthesis, and reverse-transcription PCR
Microarray analysis was conducted as described previously (12). This analysis was used to search for genes that were differentially expressed between cells treated with DMSO and those treated with the mTOR inhibitor everolimus. Specifically, MDA-MB-231 and BT-549 cells were seeded in 6-well plates and treated with DMSO or everolimus for 48 hours. Total RNA from the cells was extracted with a mirVana miRNA isolation kit (Illumina, AM156). Complementary RNA was generated using a TotalPrep RNA amplification kit (Illumina, AMIL1791). Complementary RNA (750 ng) was loaded onto a HumanHT-12 v4 Expression BeadChip (Illumina, BD-103-0204), and hybridization and labeling of streptavidin-Cy3 were performed using the manufacturer's procedure. The raw array data were processed using quantile normalization and log2 transformation with BRB-ArrayTools software (Biometric Research Branch, National Cancer Institute) for further analysis. A random-variance t test was used to identify genes that were differentially expressed in the DMSO and mTOR inhibitor everolimus-treated cells. Gene expression differences were considered significant if P < 0.005. Venn diagram was created by GeneSpring GX software (Agilent Technologies, Version 12.6), and Pathway Analysis was performed using the Ingenuity Pathway Analysis program (version 12710793). Raw data have been submitted to the GEO database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE73923. Reverse transcription was conducted using an iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer's instructions. Real-time PCR was conducted on the Mastercycler ep realplex (Eppendorf) using SYBR Green Real-Time PCR Master Mix (Life Technologies). Human SUV39H1 expression was quantified in real-time and was normalized to GAPDH. SUV39H1 protein expression was quantified by Western blotting. Total proteins were extracted for immunoblotting as previously described (33). Vinculin was used as an internal control, and p-4EBP1 (Ser65) was used to demonstrate the inhibition of mTOR signaling.
For transient transfection, human SUV39H1 siRNA was purchased from Thermo Scientific (SMARTpool, ON-TARGETplus), and the SUV39H1 target sequences were CUAAGAAGCGGGUCCGUAU (J-009604-07), GGUGAAAUGGCGUGGAUAU (J-009604-08), UCGAGUACCUGUGCGAUUA (J-009604-09), and CAAAUCGUGUGGUACAGAA (J-009604-10). Human BRCA1 siRNA was purchased from Thermo Scientific (SMARTpool, ON-TARGETplus), and the BRCA1 target sequence was CAACAUGCCCACAGAUCAA (J-003461-09), CCAAAGCGAGCAAGAGAAU (J-003461-10), UGAUAAAGCUCCAGCAGGA (J-003461-11), and GAAGGAGCUUUCAUCAUUC (J-003461-12). All siRNAs were transfected with oligofectamine for 48 to 72 hours for further analysis.
The statistical significance of differences between groups in the in vitro experiments was determined by the Student t test. Two-way ANOVA was used for statistical analysis for all in vivo experiments. P < 0.05 was considered as statistical significant.
mTOR inhibitors suppress HR repair
Previously, we used an established HR-defect gene signature and the Connectivity Map to predict compounds that would potentially inhibit HR repair (12). We proceeded to investigate the effects of mTOR inhibitor, one of the top compounds screened in terms of inducing HR-defect gene expression profiles, in regulating HR in BRCA-proficient TNBC. It is well-known that ionizing radiation (IR)-induced DSBs result in the formation of foci of RAD51, a key DNA recombinase that promotes homologous pairing and strand exchange in HR repair (34), and that RAD51 foci formation has been commonly recognized as a marker of HR competence (35). To determine whether mTOR inhibitors affect HR repair in BRCA-proficient TNBC, we treated MDA-MB-231 cells, a BRCA1/2 wild-type TNBC cell line, with two mTOR inhibitors with distinct mechanisms of action, including allosteric mTOR inhibitor everolimus (36) and catalytic mTOR inhibitor KU-0063794 (KU; ref. 37) and quantified formation of IR-induced RAD51 foci. We found that both everolimus and KU significantly decreased IR-induced RAD51 foci formation (Fig. 1A and B), suggesting impaired HR repair. It has been reported that mTOR inhibition is associated with G1 cell-cycle arrest (38). As HR repair occurs predominantly in S–G2 phase, when a sister chromatid is available as the template (7), it is important to clarify whether the observed HR defect in response to mTOR inhibitors is cell-cycle–dependent. We observed that the cell-cycle distribution in everolimus- or KU-treated cells did not change dramatically (Fig. 1B, bottom), which indicated that mTOR inhibitors directly suppressed HR repair in BRCA-proficient TNBC cells. Interestingly, we found no apparent inhibition of IR-induced RAD51 foci formation or altered cell-cycle distribution in a nontransformed breast epithelial cell line MCF-12A cells (Supplementary Fig. S1A and S1B), indicating that HR repair efficiency in normal cells is left intact after mTOR inhibitors treatment.
To further confirm this observation, we analyzed the effect of mTOR inhibitors on HR repair using a direct HR repair assay, which has been previously described (39). In this system, a single copy of the HR reporter DR-GFP is randomly incorporated into U2OS cells, a human osteosarcoma cell line that is frequently used in studies of DNA damage response and repair. Transient expression of pCBASceI plasmid leads to a DSB in the upstream GFP gene and the ability of cells to repair these DSBs through HR can be quantified by the percentage of GFP-positive U2OS cells through flow cytometry. Our results showed that the mTOR inhibitors everolimus or KU significantly reduced HR repair efficiency, as shown by the decreased percentage of GFP-positive U2OS cells (Fig. 1C). In addition, we found no apparent alteration of the cell-cycle distribution (Fig. 1D) after mTOR inhibitors treatment in these HR repair model cells. Indeed, everolimus or KU substantially decreased HR repair (Fig. 1E) even after hydroxyurea (HU) treatment, which is commonly used for cell-cycle synchronization (Fig. 1F; ref. 40). Therefore, consistent with the prediction by our HR-defect gene signature and the Connectivity Map, our results demonstrated that mTOR inhibitors suppressed HR repair in BRCA-proficient TNBC cells.
mTOR inhibitors synergize with PARP inhibitors in vitro
It is well-recognized that in the absence of HR repair via BRCA-dependent mechanisms, the loss of BER through PARP inhibition will lead to catastrophic genomic instability and cell death (41). Because mTOR inhibitors suppressed HR repair, we hypothesized that mTOR inhibitors would subsequently enhance the cytotoxicity of PARP inhibitors in BRCA-proficient TNBCs. We then evaluated the in vitro activity of two mTOR inhibitors, everolimus and KU, in BRCA-proficient TNBC cells treated with one of two PARP inhibitors in clinical trials for breast cancer therapy, talazoparib (BMN673, BMN, BioMarin) and olaparib (AZD2281, AZD, Astrazeneca). Colony formation assay results showed that mTOR inhibitors significantly increased the sensitivity of both MDA-MB-231 (Fig. 2A, top and B, top) and BT-549 cells to PARP inhibitors (Fig. 2A, middle and B, bottom). More importantly, compared with the single-agent treatment, combinations of mTOR inhibitors and PARP inhibitors did not further inhibit colony formation in MCF-12A cells (Fig. 2A, bottom), suggesting that mTOR/PARP inhibitor combination therapy may specifically target BRCA-proficient TNBC cells and spare normal cells.
Next, we performed a proliferation assay to further determine whether mTOR inhibitors synergize with PARP inhibitors in vitro. In MDA-MB-231 cells, combined treatment of mTOR inhibitors significantly decreased the IC50 for PARP inhibitors and a marked synergy was observed between mTOR inhibitors and PARP inhibitors based on the calculated CI values (ref. 30; Fig. 2C, left, mTOR inhibitors combined with BMN; right, mTOR inhibitors combined with AZD). Similar synergistic effect was observed in additional BRCA-proficient TNBC cells, BT-549 (Fig. 2D, left, mTOR inhibitors combined with BMN; right, mTOR inhibitors combined with AZD). In addition, combinations of mTOR inhibitors and PARP inhibitors markedly increased the percentage of apoptotic cells in both MDA-MB-231 (Fig. 2E, left) and BT-549 cells (Fig. 2E, right) when compared with cells treated with single agent alone.
mTOR inhibitor sensitizes PARP inhibitor in TNBC xenografts
We further assessed the effects of everolimus and BMN and their combination on tumor growth in BRCA-proficient TNBC in vivo. Everolimus or BMN as a single agent delayed tumor growth in MDA-MB-231 xenografts. Nonetheless, the BMN + everolimus combination was superior to BMN (P = 0.0143) or everolimus alone (P < 0.001) in reducing tumor growth (Fig. 3A and B). In addition, in BT-549 xenografts, the tumor volume in the BMN + everolimus combination group was also significantly decreased compared with that in the BMN (P < 0.001) or everolimus (P < 0.001) single-agent group (Fig. 3D and E). Most importantly, the combination induced tumor regressions in MDA-MB-231 model. No body weight loss was detected during the 6 weeks (MDA-MB-231 xenografts, Fig. 3C) or 8 weeks (BT-549 xenografts, Fig. 3F) of treatment, indicating that consistent with in vitro findings (Fig. 2A, bottom), combination therapy of mTOR inhibitor and PARP inhibitor may particularly target cancer cells. Taken together, the approach of mTOR inhibitors and PARP inhibitors combination therapy may specifically increase the therapeutic efficacy in BRCA-proficient TNBC without increasing the toxicity to normal cells.
mTOR inhibitors suppress SUV39H1 expression
Next, we searched for the underlying mechanisms that are responsible for mTOR inhibitor–induced suppression of HR repair and consequent sensitization to PARP inhibitors in BRCA-proficient TNBC. We found that mTOR inhibitors did not affect the mRNA or protein expression of key HR-related factors, including BRCA1, RAD51, and RPA2 (Supplementary Fig. S2A and S2B). To obtain a broad molecular understanding of this process, we applied a genome-wide expression profiling approach to systematically analyze cellular transcriptome alterations induced by mTOR inhibitor. Among the altered genes in response to mTOR inhibitor that are implicated in DNA replication, recombination, and repair by Ingenuity Pathway Analysis (IPA), we found that SUV39H1 was most significantly decreased in both MDA-MB-231 and BT-549 cells (Supplementary Fig. S3). SUV39H1 is a specific H3K9 histone methyltransferase (42) and has been implicated in DSB repair (43, 44). Loss of SUV39H1 decreases DSB repair mediated by HR and increases cell sensitivity to PARP inhibition (45). Hence, SUV39H1 might be a key molecule involved in the suppression of HR repair by mTOR inhibitors. Consistent with our microarray data, we found that MDA-MB-231 (Fig. 4A) and BT-549 (Fig. 4B) cells exposed to everolimus or KU had significantly decreased SUV39H1 mRNA than DMSO-treated control cells (P < 0.001). In addition, markedly reduced SUV39H1 protein levels were observed in response to everolimus or KU treatment when compared with DMSO treatment (Fig. 4C). The level of p-4EBP1 (Ser65) served as a marker for mTOR signaling activity. More importantly, cells lacking SUV39H1 demonstrated impaired HR repair (Fig. 4D) without changes in cell-cycle distribution (Fig. 4E). In the same experiment, BRCA1 knockdown was used as a positive control for defect in HR repair. Furthermore, recruitment of RAD51 to DSBs was significantly reduced following SUV39H1 loss in BRCA-proficient TNBC MDA-MB-231 cells (Fig. 4F). Consistently, depletion of SUV39H1 increased sensitivity of MDA-MB-231 cells to PARP inhibitors (Fig. 4G, left, AZD; right, BMN).
SUV39H1 overexpression partially rescues HR repair suppressed by mTOR inhibitors and blocks synergism between mTOR inhibitors and PARP inhibitors
To confirm that SUV39H1 plays an essential role in the suppression of HR repair by mTOR inhibitors, we performed rescue assays to evaluate HR repair function and sensitivity to PARP inhibitors after restoration of SUV39H1. We found that in U2OS-DR-GFP cells, overexpression of SUV39H1 significantly increased HR repair efficiency that had been reduced by mTOR inhibitors everolimus or KU (Fig. 5A). Furthermore, overexpression of SUV39H1 in MDA-MB-231 and BT-549 cells treated with BMN alone did not change cell proliferation; however, when these cells were treated with the combination of BMN and KU (Fig. 5B) or BMN and everolimus (Fig. 5C), cells with SUV39H1 overexpression were less sensitive to the drug treatments than the vector-transfected cells. This indicated that SUV39H1 overexpression suppressed, at least partially, the synergy between PARP inhibitors and mTOR inhibitors in BRCA-proficient TNBC cells. Taken together, these data strongly support the hypothesis that mTOR inhibitors suppress HR repair and synergize with PARP inhibitors in BRCA-proficient TNBCs by regulating SUV39H1 expression.
The search for combination therapies that would result in impaired HR with subsequent sensitization to PARP inhibitors in cells with competent HR repair has been an area of great research interest. To identify more precise biomarkers to predict patients' response to PARP inhibitors, our group previously identified an HR-defect gene signature to predict HR repair status and sensitivity to PARP inhibitors (12). Using the Connectivity Map as an effective drug discovery platform, we were able to predict small molecules that could potentially suppress HR repair and sensitize cells to PARP inhibitors. We found that mTOR inhibitors were at the top of the list in terms of inducing HR-defect gene expression profiles. Our current study demonstrated that, as the Connectivity Map predicted, mTOR inhibitors significantly impaired RAD51 foci formation in response to DSBs in BRCA-proficient TNBC cells. In addition, using U2OS cells with stable DR-GFP as an HR repair model, we found that mTOR inhibitors directly decreased HR repair efficiency, even after cell-cycle synchronization. Notably, our results also showed that mTOR inhibitors synergized with PARP inhibitors in BRCA-proficient TNBC cells in vitro and also significantly enhanced the cytotoxicity of PARP inhibitors in BRCA-proficient TNBC xenografts in vivo. Our findings suggest that mTOR inhibitors can suppress HR repair in BRCA-proficient TNBCs, therefore making these cancers vulnerable to PARP inhibitors. Importantly, although comparatively high concentrations of mTOR inhibitors are used in the current study, our observations are biologically relevant, as treatment of mTOR inhibitors do not affect HR repair (Supplementary Fig. S1) and their combinations with PARP inhibitors do not further reduce cell viability (Fig. 2A) in nontransformed breast epithelial cells. In addition, mTOR inhibitor and PARP inhibitor combination is well tolerated in mice (Fig. 3C and F). All these preclinical evidences provide rationales for application of dual mTOR and PARP inhibitions in BRCA-proficient TNBC cells. Furthermore, mTOR inhibitors could potentially extend the clinical application of PARP inhibitors to a wider spectrum of cancers. Of note, PI3K inhibitors, histone deacetylase (HDAC) inhibitors, and Hsp90 inhibitors, were also on our list with regard to impairing HR repair (12). This is indeed consistent with several recent studies. For example, two research groups independently reported (15, 16) that PI3K inhibition compromised HR repair, which led to downregulation of BRCA1, decreased RAD51 foci formation, and increased sensitization of BRCA1/2 wild-type TNBC cells or TNBC patient–derived primary tumor xenografts to PARP inhibitors. In addition, PI3K-mediated downregulation of BRCA1 has been found to be ERK-dependent, through the involvement of the ETS1 transcription factor (15). It is also reported that Hsp90 inhibition suppressed BRCA1-dependent HR repair (46) and altered the sensitivity of BRCA1-mutant cells to PARP inhibitors (47). Another study found that HDAC inhibitor treatment depleted BRCA1 and other HR proteins and synergized with PARP inhibitors in human TNBC cells (48). Together, our studies hold great promise to expand the clinical treatment options for TNBCs in the near future.
It has been reported that PI3K inhibition downregulates BRCA1 in TNBCs (15). In our study, mTOR inhibitors everolimus and KU-0063794 did not affect the expression of major HR-related factors, including BRCA1, RAD51, and RPA2 (Supplementary Fig. S2). Our exploration of the molecular mechanisms underlying suppression of HR by mTOR inhibitors through systematic analysis of cellular transcriptome alterations induced by these agents revealed downregulation of SUV39H1 expression in two BRCA-proficient TNBC cell lines. SUV39H1 is the most studied methyltransferase of lysine 9 of histone 3 (H3K9; ref. 27). It forms a multimeric complex with other histone H3K9 methyltransferases and is likely involved in pericentric heterochromatin formation and regulation of gene expression. SUV39H1 is rapidly loaded onto the chromatin at DSBs and methylates H3K9. The resulting H3K9me3 activates Tip60 histone acetyltransferase, which in turn acetylates ataxia telangiectasia mutated kinase (ATM) and promotes ATM-dependent phosphorylation of DSB repair proteins (45). Inhibition of SUV39H1 dramatically decreases HR repair efficiency and the recruitment of BRCA1 and replication protein A to DSBs (45). In this study, we found that SUV39H1 inhibition impaired RAD51 foci formation in MDA-MB-231 cells (Fig. 4F) and reduced HR repair in the U2OS-DR-GFP HR repair model (Fig. 4D). Importantly, we showed that the overexpression of SUV39H1 partially restored HR repair efficiency that had been suppressed by mTOR inhibitors, indicating that the regulation of SUV39H1 by the mTOR pathway contributes to HR repair. In addition, exogenous expression of SUV39H1 suppressed the synergy between PARP inhibitors and mTOR inhibitors, which provides a rational explanation of how mTOR inhibitors modulate HR repair in BRCA-proficient TNBCs. SUV39H1 is known to be specifically methylated by SET domain–containing protein 7 (SET7/9), and this methylation subsequently facilitates genome instability and eventually inhibits cell proliferation (49). Interestingly, we showed that the decrease of SUV39H1 protein expression was comparable with the decrease of SUV39H1 mRNA expression in both MDA-MB-231 and BT-549 cells (Fig. 4A–C), indicating that mTOR signaling might regulate SUV39H1 predominantly at the transcriptional level, which need to be further investigated for a thorough understanding in our future work. Also, whether these observations apply to TNBC with BRCA1/2 mutations or other breast cancer subgroups requires further investigations.
It is well known that TNBC is a highly heterogeneous disease with variable prognosis, which has added increased complexity to the development of personalized therapeutics for TNBCs. In our current study, we have focused on exploring combination therapies that would expand the spectrum of TNBC patients who would benefit from PARP inhibitors, especially those with intact HR. Therefore, we have used two TNBC cell line models, MDA-MB-231 and BT-549, to represent specific groups of TNBC that are BRCA1/2 wild-type (BRCA-proficient) and are categorized as HR-intact through our HR-defect gene signature. However, it remains unknown whether or how heterogeneous HR activity would exist in individual TNBC tumors, which may be answered in the future by single-cell transcriptome analysis. In addition, we have demonstrated the therapeutic benefit from the combination of mTOR inhibitors and PARP inhibitors on BRCA-proficient TNBC using both in vitro cultured cells and in vivo mouse xenograft model, which are two standard models in cancer biology. In the future, we will further verify these intriguing results on patient-derived xenograft (PDX) model, which more closely resemble the original tumors (50), to promote novel and effective personalized clinical trials for TNBC patients.
In summary, our results demonstrate that mTOR inhibitors suppress HR repair and further show that mTOR inhibitors dramatically enhance the cytotoxicity of PARP inhibitors in BRCA-proficient TNBCs in vitro and in vivo, without affecting HR repair or the proliferation of nontransformed breast epithelial cells. We have also revealed a novel mechanism of how mTOR signaling regulates HR repair and PARP inhibitor sensitivity in BRCA-proficient TNBC. These results present an effective approach for therapeutic combination of mTOR inhibitors with PARP inhibitors in BRCA-proficient TNBCs and might also benefit patients with other types of cancer.
Disclosure of Potential Conflicts of Interest
G.B. Mills reports receiving commercial research grants from Adelson Medical Research Foundation, AstraZeneca, Critical Outcome Technologies, Komen Research Foundation, and Nanostring; speakers bureau honoraria from AstraZeneca, ISIS Pharmaceuticals, Nuevolution, and Symphogen; has ownership interest (including patents) in Catena Pharmaceuticals, Myriad Genetics, PTV Ventures, and Spindletop Ventures; and is a consultant/advisory board member for Adventist Health, AstraZeneca, Blend, Catena Pharmaceuticals, Critical Outcome Technologies, HanAl Bio Korea, ImmunoMET, Millenium Pharmaceuticals, Nuevolution, Precision Medicine, Provista Diagnostics, Signalchem Lifesciences, and Symphogen. No potential conflicts of interest were disclosed by the other authors.
Conception and design: W. Mo, C.C.-J. Lin, G. Peng, G.B. Mills, K. Li, S.-Y. Lin
Development of methodology: W. Mo, C.C.-J. Lin, H. Dai, Y. Peng, F. Meric-Bernstam, K. Li
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Mo, Q. Liu, H. Dai, Y. Liang, G.B. Mills, K. Li
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): W. Mo, Q. Liu, C.C.-J. Lin, F. Meric-Bernstam, G.B. Mills, K. Li, S.-Y. Lin
Writing, review, and/or revision of the manuscript: W. Mo, Q. Liu, F. Meric-Bernstam, G.B. Mills, K. Li, S.-Y. Lin
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): W. Mo, Q. Liu, H. Dai
Study supervision: W. Mo, K. Li, S.-Y. Lin
This work was supported by The Department of Defense (DOD) Breast Cancer Research Program (BCRP) Era of Hope Scholar Award (W81XWH-10-1-0558; to S.-Y. Lin).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).
- Received July 24, 2015.
- Revision received October 12, 2015.
- Accepted October 26, 2015.
- ©2015 American Association for Cancer Research.