DNA damage-induced S and G2/M cell cycle arrest requires mTORC2-dependent regulation of Chk1

mTOR signalling is commonly dysregulated in cancer. Concordantly, mTOR inhibitors have demonstrated efficacy in a subset of tumors and are in clinical trials as combination therapies. Although mTOR is associated with promoting cell survival after DNA damage, the exact mechanisms are not well understood. Moreover, since mTOR exists as two complexes, mTORC1 and mTORC2, the role of mTORC2 in cancer and in the DNA damage response is less well explored. Here, we report that mTOR protein levels and kinase activity are transiently increased by DNA damage in an ATM and ATR-dependent manner. We show that inactivation of mTOR with siRNA or pharmacological inhibition of mTORC1/2 kinase prevents etoposide-induced S and G2/M cell cycle arrest. Further results show that Chk1, a key regulator of the cell cycle arrest, is important for this since ablation of mTOR prevents DNA damage-induced Chk1 phosphorylation and decreases Chk1 protein production. Furthermore, mTORC2 was essential and mTORC1 dispensable, for this role. Importantly, we show that mTORC1/2 inhibition sensitizes breast cancer cells to chemotherapy. Taken together, these results suggest that breast cancer cells may rely on mTORC2-Chk1 pathway for survival and provide evidence that mTOR kinase inhibitors may overcome resistance to DNA-damage based therapies in breast cancer.     

Response of subtype-specific human breast cancer-derived cells to poly(ADP-ribose) polymerase and checkpoint kinase 1 inhibition

When DNA damage is detected, checkpoint signal networks are activated to stop the cell cycle, and DNA repair processes begin. Inhibitory compounds targeting components of DNA damage response pathways have been identified and are being used in clinical trials, in combination with chemotherapeutic agents, to enhance cancer therapy. Inhibitors of checkpoint kinases, Chk1 and Chk2, have been shown to sensitize tumor cells to DNA damaging agents, and treatment of BRCA1/2-deficient tumor cells, as well as triple negative breast cancers, with poly(ADP-ribose) polymerase (PARP) inhibitors has shown promise. But systematic studies to determine which tumor subtypes are likely to respond to these specific inhibitors have not been reported. The current study was designed to test sensitivity of specific breast cancer subtype-derived cells to two classes of these new inhibitory drugs, PARP and Chk1 inhibitors. Luminal, HER2 overexpressing, and triple negative breast cancer-derived cells were tested for sensitivity to killing by PARP inhibitors, ABT-888 and BSI-201, and Chk1 inhibitor, PF-00477736, alone or in combination with gemcitabine or carboplatin. Each of the triple negative breast cancer cell lines showed strong sensitivity to the Chk1 inhibitor, but only the BRCA1-deficient breast cancer cell lines showed sensitivity to the PARP inhibitors, suggesting that in vitro testing of cancer cell lines of specific subtypes, with panels of the different PARP and Chk1 inhibitors, will contribute to stratification of patients for clinical trials using these classes of inhibitors.     

Therapeutic potential of PARP inhibitors for metastatic breast cancer

Increasing understanding of the cellular aberrations inherent to cancer cells has allowed the development of therapies to target biological pathways, an important step towards individualization of breast cancer therapy. The clinical development of poly(ADP-ribose) polymerase (PARP) inhibitors, with their novel and selective mechanism of action, are an example of this strategy. PARP plays a key role in DNA repair mechanisms, particularly the base excision repair pathway. Initially developed as inhibitors able to enhance the cytotoxicity of radiation and certain DNA-damaging agents, they have more recently been shown to have single-agent activity in certain tumors. Inhibition of PARP in a DNA repair-defective tumor can lead to gross genomic instability and cell death by exploiting the paradigm of synthetic lethality. Several studies have evaluated the role of PARP inhibitors for treatment of breast cancer, particularly in the context of BRCA-mutated and triple-negative breast cancers. In addition, inhibition of PARPs repair functions for chemotherapy-induced DNA lesions has been shown to potentiate the effect of some chemotherapy regimens. This article discusses the current understanding of PARP inhibition as a treatment for metastatic breast cancer, evidence from clinical trials and addresses its future implications.     

PARP抑制剂治疗晚期乳腺癌的作用机制及相关研究进展

多聚二磷酸腺苷核糖聚合酶（poly ADP-ribose polymerase，PARP）抑制剂可使乳腺癌细胞的单链DNA损伤修复受阻，而BRCA突变可造成乳腺癌细胞的双链DNA损伤修复功能缺失，因此PARP抑制剂治疗乳腺癌易感基因（breast cancer susceptibility gene，BRCA）突变乳腺癌是通过同时阻断单链DNA和双链DNA损伤修复，导致细胞的DNA损伤修复失败，使癌细胞死亡。目前已研发出多种敏感性和特异性较高的PARP抑制剂，该类药物主要抑制PARP1和PARP2两种亚型。本文总结PARP抑制剂用于治疗BRCA突变乳腺癌的作用机制，并对多种PARP抑制剂单用或联合化疗药物治疗晚期乳腺癌的研究进展进行综述。      

BRCA1/2突变和同源重组修复缺陷(HRD)检测在乳腺癌中的临床研究进展

乳腺癌已成为发病率最高的癌症。DNA修复缺陷是乳腺癌最重要的特征之一。先前的研究表明,乳腺癌易感基因1/2(breast cancer susceptibility gene 1/2,BRCA1/2)突变是预测乳腺癌同源重组修复缺陷(homologous recombination deficiency, HRD)最主要的生物标志物,能识别铂类药物和多腺苷二磷酸核糖聚合酶(poly ADP ribose polymerase, PARP)抑制剂治疗的获益人群。美国食品药品监督管理局(FDA)已批准Olaparib和Talazoparib两种PARP抑制剂,用于BRCA1/2突变的早期和晚期乳腺癌的辅助治疗。但中国尚未获批。现有研究表明,一部分非BRCA1/2突变的乳腺癌患者也具有HRD特征,可以从铂类药物或PARP抑制剂中获益。本综述总结了涉及到BRCA1/2突变、同源重组修复(homologous recombination repair, HRR)基因突变和HRD状态检测的临床研究。阐明了各种检测方法在识别乳腺癌患者HRD状态和预测疗效方面的价值,并提出应尽快开发用于中国乳腺癌HR...     

Nuclear translocation of the receptor tyrosine kinase c-MET reduces the treatment efficacies of olaparib and gemcitabine in pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that lack effective therapeutic strategies. The response rate of PDAC for treatment with gemcitabine, a current first-line chemotherapeutic for this tumor, is lower than 20%. Identifying key targetable molecules that mediate gemcitabine resistance and developing novel strategies for precision PDAC medicine are urgently needed. Most PDACs have either intratumoral hypoxia or high reactive oxygen species (ROS) production; cytotoxic chemotherapy can elevate ROS production in PDACs. Although excessive ROS production leads to oxidative damage of macromolecules such as DNA, pancreatic cancer cells can survive high DNA damage stress levels. Therefore, identifying molecular mechanisms of overcoming ROS-induced stress in pancreatic cancer cells is important for developing novel therapeutic strategies. ROS-induced DNA damage is predominantly repaired via poly (ADP-ribose) polymerase 1 (PARP1)-mediated DNA repair mechanisms. A recent clinical trial reported that PARP inhibitors are effective in treating pancreatic patients carrying BRCA mutations. However, only less than 10% of pancreatic cancer patients bearing BRCA mutated tumors. Activation of the receptor tyrosine kinase c-MET positively correlates with poor prognosis for PDAC, and our previous study showed that nuclear c-MET can phosphorylate PARP1 at tyrosine 907 under ROS stimulation to promote DNA repair. As described herein, we proposed to expand PARP inhibitor-targeted therapy to more pancreatic cancer patients regardless of BRCA mutation status by combining olaparib, a PARP inhibitor, with c-MET inhibitors as we demonstrated in our previous studies in breast cancer. In this prospective study, we found that ROS-inducing chemotherapeutic drugs such as gemcitabine and doxorubicin stimulated nuclear accumulation of c-MET in BxPC-3 and L3.6pl pancreatic cancer cells. We further showed that combining a c-MET inhibitor with gemcitabine or a PARP inhibitor induced more DNA damage than monotherapy did. Moreover, we demonstrated the synergistic antitumor effects of c-MET inhibitors combined with a PARP inhibitor or gemcitabine in eliminating pancreatic cancer cells. These data suggested that accumulation of ROS in pancreatic cancer cells promotes nuclear localization of c-MET, resulting in resistance to both chemotherapy and PARP inhibitors. Our findings suggest that combining c-MET inhibitors with PARP inhibitors or gemcitabine is a novel, rational therapeutic strategy for advanced pancreatic cancer.     

Formation of the eIF4F translation-initiation complex determines sensitivity to anticancer drugs targeting the EGFR and HER2 receptors

Elucidating how cancer cells respond to antagonists of HER receptor family members is critical to understanding mechanisms of therapeutic resistance that arise in patients. In large part, resistance to such agents appears to arise from deregulation of the phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway. mTOR-dependent phosphorylation of the translation repressor 4E-BP1 leads to its dissociation from eIF4E, thereby causing an increase in the formation of the eIF4F complex, which also comprises eIF4G and eIF4A. In this study, we show that trastuzumab, cetuximab, and erlotinib all decrease the formation of the eIF4F complex in breast, colon, and head and neck cancer cells, respectively. Ectopic expression of eIF4E restores the trastuzumab-dependent defect in eIF4F formation, renders cells resistant to the trastuzumab-mediated decrease in cell proliferation, and rescues breast cancer xenografts from inhibition by trastuzumab. In breast tumor specimens, the level of eIF4E expression is associated with the therapeutic response to a trastuzumab-based regimen. Together, our findings suggest that formation of the eIF4F complex may be a critical determinant of the response to anticancer drugs that target HER2 and epidermal growth factor receptor.     

AKT inhibition overcomes rapamycin resistance by enhancing the repressive function of PRAS40 on mTORC1/4E-BP1 axis

The mTORC1 inhibitors, rapamycin and its analogs, are known to show only modest antitumor activity in clinic, but the underlying mechanisms remain largely elusive. Here, we found that activated AKT signaling is associated with rapamycin resistance in breast and colon cancers by sustained phosphorylation of the translational repressor 4E-BP1. Treatment of tumor cells with rapamycin or the AKT inhibitor MK2206 showed a limited activity in inhibiting 4E-BP1 phosphorylation, cap-dependent translation, cell growth and motility. However, treatment with both drugs resulted in profound effects in vitro and in vivo. Mechanistic investigation demonstrated that the combination treatment was required to effectively inhibit PRAS40 phosphorylation on both Ser183 and Thr246 mediated by mTORC1 and AKT respectively, and with the combined treatment, dephosphorylated PRAS40 binding to the raptor/mTOR complex was enhanced, leading to dramatic repression of mTORC1-regulated 4E-BP1 phosphorylation and translation. Knockdown of PRAS40 or 4E-BP1 expression markedly reduced the dependence of tumor cells on AKT/mTORC1 signaling for translation and survival. Together, these findings reveal a critical role of PRAS40 as an integrator of mTORC1 and AKT signaling for 4E-BP1-mediated translational regulation of tumor cell growth and motility, and highlight PRAS40 phosphorylation as a potential biomarker to evaluate the therapeutic response to mTOR/AKT inhibitors.     

Therapeutic potential of PARP inhibitors for metastatic breast cancer

Increasing understanding of the cellular aberrations inherent to cancer cells has allowed the development of therapies to target biological pathways, an important step towards individualization of breast cancer therapy. The clinical development of poly(ADP-ribose) polymerase (PARP) inhibitors, with their novel and selective mechanism of action, are an example of this strategy. PARP plays a key role in DNA repair mechanisms, particularly the base excision repair pathway. Initially developed as inhibitors able to enhance the cytotoxicity of radiation and certain DNA-damaging agents, they have more recently been shown to have single-agent activity in certain tumors. Inhibition of PARP in a DNA repair-defective tumor can lead to gross genomic instability and cell death by exploiting the paradigm of synthetic lethality. Several studies have evaluated the role of PARP inhibitors for treatment of breast cancer, particularly in the context of BRCA-mutated and triple-negative breast cancers. In addition, inhibition of PARPs repair functions for chemotherapy-induced DNA lesions has been shown to potentiate the effect of some chemotherapy regimens. This article discusses the current understanding of PARP inhibition as a treatment for metastatic breast cancer, evidence from clinical trials and addresses its future implications.     

DNA损伤应答缺陷作为乳腺癌治疗靶点的研究进展

DNA损伤应答(DNA damage response,DDR)缺陷是近年来乳腺癌治疗研究的热门靶点之一.DDR通路负责DNA损伤后的识别、信号转导和修复,其功能异常可导致细胞的凋亡或基因组不稳定性的增加.目前进入临床研究阶段的乳腺癌DDR靶向药物主要包括多聚腺苷二磷酸核糖聚合酶[poly(ADP-ribose)pol...     

Combination treatment using DDX3 and PARP inhibitors induces synthetic lethality in BRCA1-proficient breast cancer

Triple-negative breast cancers have unfavorable outcomes due to their inherent aggressive behavior and lack of targeted therapies. Breast cancers occurring in BRCA1 mutation carriers are mostly triple-negative and harbor homologous recombination deficiency, sensitizing them to inhibition of a second DNA damage repair pathway by, e.g., PARP inhibitors. Unfortunately, resistance against PARP inhibitors in BRCA1-deficient cancers is common and sensitivity is limited in BRCA1-proficient breast cancers. RK-33, an inhibitor of the RNA helicase DDX3, was previously demonstrated to impede non-homologous end-joining repair of DNA breaks. Consequently, we evaluated DDX3 as a therapeutic target in BRCA pro- and deficient breast cancers and assessed whether DDX3 inhibition could sensitize cells to PARP inhibition. High DDX3 expression was identified by immunohistochemistry in breast cancer samples of 24% of BRCA1 (p = 0.337) and 21% of BRCA2 mutation carriers (p = 0.624), as compared to 30% of sporadic breast cancer samples. The sensitivity to the DDX3 inhibitor RK-33 was similar in BRCA1 pro- and deficient breast cancer cell lines, with IC50 values in the low micromolar range (2.8–6.6 μM). A synergistic interaction was observed for combination treatment with RK-33 and the PARP inhibitor olaparib in BRCA1-proficient breast cancer, with the mean combination index ranging from 0.59 to 0.62. Overall, we conclude that BRCA pro- and deficient breast cancers have a similar dependency upon DDX3. DDX3 inhibition by RK-33 synergizes with PARP inhibitor treatment, especially in breast cancers with a BRCA1-proficient background.     

Synergistic Effect of Trabectedin and Olaparib Combination Regimen in Breast Cancer Cell Lines

Purpose

Trabectedin induces synthetic lethality in tumor cells carrying defects in homologous recombinant DNA repair. We evaluated the effect of concomitant inhibition of nucleotide-excision repair and poly (ADP-ribose) polymerase (PARP) activity with trabectedin and PARP inhibitors, respectively, and whether the synthetic lethality effect had the potential for a synergistic effect in breast cancer cell lines. Additionally, we investigated if this approach remained effective in BRCA1-positive breast tumor cells.

Methods

We have evaluated the in vitro synergistic effect of combinations of trabectedin and three different PARP inhibitors (veliparib, olaparib, and iniparib) in four breast cancer cell lines, each presenting a different BRCA1 genetic background. Antiproliferative activity, DNA damage, cell cycle perturbations and poly(ADP-ribosyl)ation were assessed by MTT assay, comet assay, flow cytometry and western blot, respectively.

Results

The combination of trabectedin and olaparib was synergistic in all the breast cancer cell lines tested. Our data indicated that the synergy persisted regardless of the BRCA1 status of the tumor cells. Combination treatment was associated with a strong accumulation of double-stranded DNA breaks, G2/M arrest, and apoptotic cell death. Synergistic effects were not observed when trabectedin was combined with veliparib or iniparib.

Conclusion

Collectively, our results indicate that the combination of trabectedin and olaparib induces an artificial synthetic lethality effect that can be used to kill breast cancer cells, independent of BRCA1 status.     

RAD50 targeting impairs DNA damage response and sensitizes human breast cancer cells to cisplatin therapy

In tumor cells the effectiveness of anti-neoplastic agents that cause cell death by induction of DNA damage is influenced by DNA repair activity. RAD50 protein plays key roles in DNA double strand breaks repair (DSBs), which is crucial to safeguard genome integrity and sustain tumor suppression. However, its role as a potential therapeutic target has not been addressed in breast cancer. Our aim in the present study was to analyze the expression of RAD50 protein in breast tumors, and evaluate the effects of RAD50-targeted inhibition on the cytotoxicity exerted by cisplatin and anthracycline and taxane-based therapies in breast cancer cells. Immunohistochemistry assays on tissue microarrays indicate that the strong staining intensity of RAD50 was reduced in 14% of breast carcinomas in comparison with normal tissues. Remarkably, RAD50 silencing by RNA interference significantly enhanced the cytotoxicity of cisplatin. Combinations of cisplatin with doxorubicin and paclitaxel drugs induced synergistic effects in early cell death of RAD50-deficient MCF-7, SKBR3, and T47D breast cancer cells. Furthermore, we found an increase in the number of DSBs, and delayed phosphorylation of histone H2AX after cisplatin treatment in RAD50-silenced cells. These cellular events were associated to a dramatical increase in the frequency of chromosomal aberrations and a decrease of cell number in metaphase. In conclusion, our data showed that RAD50 abrogation impairs DNA damage response and sensitizes breast cancer cells to cisplatin-combined therapies. We propose that the development and use of inhibitors to manipulate RAD50 levels might represent a promising strategy to sensitize breast cancer cells to DNA damaging agents.     

Tumor growth is suppressed in mice expressing a truncated XRCC1 protein

Tumor progression depends on the support of cells in the microenvironment, and is driven in part by the generation of reactive oxygen species (ROS). ROS can damage DNA, and the repair of damaged DNA is a well-known process involved in tumor initiation and promotion, but the role of DNA repair in tumor progression is not fully understood. In this regard the X-ray cross complementing 1 (XRCC1) protein is known to orchestrate the assembly of repair complexes at sites of DNA single strand breaks either directly or indirectly through repair of damaged bases, largely as the result of ROS-induced damage. XRCC1 polymorphisms have been shown to be associated with increased cancer. It was therefore of interest to investigate the effect of XRCC1 gene mutations on cancer progression. In an attempt to make XRCC1 point mutant mice, we generated a truncated protein (XRCC1tp) by the insertion of a neomycin cassette in intron12 of the XRCC1 gene. This unique finding allowed us to investigate cellular and tumor progression phenotypes in mice associated with expression and function of an altered XRCC1 protein on one allele. XRCC1tp cells showed increased toxicity to MMS, enhanced MMS-induced depletion of NADH suggesting increased PARP activity, and normal functional repair of MMS-induced DNA damage. Six months following treatment with the alkylating carcinogen azoxymethane (AOM) at 10 mg/kg once a week for 6 weeks, XRCC1tp mice had a decrease in average colon tumor volume of 14±3 mm(3) compared to 34±4 mm(3) in WT littermates (p ≤ 0.03, N= 20/genotype). XRCC1tp mice had a 72 per cent decrease in B16 melanoma tumor burden compared to wt littermates. Average tumor volume in transgenic PyMT metastatic breast cancer mice expressing XRCC1tp was 359 cubic mm in PyMT mice expressing XRCC1tp compared to 730 cubic mm in PyMT mice expressing XRCC1wt (p ≤ 0.001, N= 20/genotype). These data suggest that the presence of an XRCC1 truncated protein alters XRCC1 function independent of DNA repair, and is associated with anti-tumor activity.     

Pyrosequencing analysis of BRCA1 methylation level in breast cancer cells

BRCA1 and BRCA2 genes are crucial for double-strand break repair by homologous recombination, and mutations in these genes are responsible for most familial breast carcinomas. Cells with inactivating mutations of the BRCA1 or BRCA2 tumor suppressor genes are sensitive to poly (ADP-ribose) polymerase-1 (PARP1) inhibitors. Already in 2010, it has been predicted, that BRCA1 hypermethylation might be sensitive to PARP1 inhibitor. However, till today, a statistically significant proof has been missing, and the effectiveness of PARP1 inhibitors for breast cancer caused by BRCA1 promoter hypermethylation remained elusive. Pyrosequencing has been proposed as an optimal method to investigate the methylation status of the BRCA1 genes. Here, we show for the first time that BRCA1 CpG island hypermethylation is sensitive to PARP1 inhibitors. In clinical settings, this might improve treatment response and provide a more personalized therapy for breast cancer patients. Furthermore, the determination of methylation status of BRCA1 and other genes of the BRCA/homologous recombination (HR) pathway may be an important predictive classifier of response to PARP inhibitor therapy.     

Homologous recombination deficiency and ovarian cancer

The discovery that PARP inhibitors block an essential pathway of DNA repair in cells harbouring a BRCA mutation has opened up a new therapeutic avenue for high-grade ovarian cancers. BRCA1 and BRCA2 proteins are essential for high-fidelity repair of double-strand breaks of DNA through the homologous recombination repair (HRR) pathway. Deficiency in HRR (HRD) is a target for PARP inhibitors. The first PARP inhibitor, olaparib, has now been licensed for BRCA-mutated ovarian cancers. While mutated BRCA genes are individually most commonly associated with HRD other essential HRR proteins may be mutated or functionally deficient potentially widening the therapeutic opportunities for PARP inhibitors. HRD is the first phenotypically defined predictive marker for therapy with PARP inhibitors in ovarian cancer. Several different PARP inhibitors are being trialled in ovarian cancer and this class of drugs has been shown to be a new selective therapy for high-grade ovarian cancer. Around 20% of high-grade serous ovarian cancers harbour germline or somatic BRCA mutations and testing for BRCA mutations should be incorporated into routine clinical practice. The expanded use of PARP inhibitors in HRD deficient (non-BRCA mutant) tumours using a signature of HRD in clinical practice requires validation.     

Novel splice‐switching oligonucleotide promotes BRCA1 aberrant splicing and susceptibility to PARP inhibitor action

Tumors carrying hereditary mutations in BRCA1, which attenuate the BRCA1 DNA damage repair pathway, are more susceptible to dual treatment with PARP inhibitors and DNA damaging therapeutics. Conversely, breast cancer tumors with nonmutated functional BRCA1 are less sensitive to PARP inhibition. We describe a method that triggers susceptibility to PARP inhibition in BRCA1‐functional tumor cells. BRCA1 exon 11 is a key for the function of BRCA1 in DNA damage repair. Analysis of the BRCA1 exon 11 splicing mechanism identified a key region within this exon which, when deleted, induced exon 11 skipping. An RNA splice‐switching oligonucleotide (SSO) developed to target this region was shown to artificially stimulate skipping of exon 11 in endogenous BRCA1 pre‐mRNA. SSO transfection rendered wild‐type BRCA1 expressing cell lines more susceptible to PARP inhibitor treatment, as demonstrated by a reduction in cell survival at all SSO concentrations tested. Combined SSO and PARP inhibitor treatment increased γH2AX expression indicating that SSO‐dependent skipping of BRCA1 exon 11 was able to promote DSBs and therefore synthetic lethality. In conclusion, this SSO provides a new potential therapeutic strategy for targeting BRCA1‐functional breast cancer by enhancing the effect of PARP inhibitors.