Cisplatin-induced DNA Damage Response Research Articles

Myeloid Cell Leukemia 1 Small Molecule Inhibitor S63845 Synergizes with Cisplatin in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is an aggressive cancer that lacks specific molecular targets that are often used for therapy. The refractory rate of TNBC to broad-spectrum chemotherapy remains high; however, the combination of newly developed treatments with the current standard of care has delivered promising anti-tumor effects. One mechanism employed by TNBC to avoid cell death is the increased expression of the anti-apoptotic protein, myeloid cell leukemia 1 (MCL1). Multiple studies have demonstrated that increased MCL1 expression enables resistance to platinum-based chemotherapy. In addition to suppressing apoptosis, we recently demonstrated that MCL1 also binds and negatively regulates the transcriptional activity of TP73. TP73 upregulation is a critical driver of cisplatin-induced DNA damage response, and ultimately, cell death. We therefore sought to determine if the coadministration of an MCL1-targeted inhibitor with cisplatin could produce a synergistic response in TNBC. This study demonstrates that the MCL1 inhibitor, S63845, combined with cisplatin synergizes by inducing apoptosis while also decreasing proliferation in a subset of TNBC cell lines. The use of combined MCL1 inhibitors with cisplatin in TNBC effectively initiates TAp73 anti-tumor effects on cell cycle arrest and apoptosis. This observation provides a molecular profile that can be exploited to identify sensitive TNBCs.

Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B), the human homologue of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares high sequence homology with Mad2, the mitotic checkpoint protein. Previously, we demonstrated the involvement of Mad2B in the cisplatin-induced DNA damage response. In this study, we extend our findings to show that Mad2B is recruited to sites of DNA damage in human cancer cells in response to cisplatin treatment. We found that in undamaged cells, Mad2B exists in a complex with Polζ-Rev1 and the APC/C subunit Cdc27. Following cisplatin-induced DNA damage, we observed an increase in the recruitment of Mad2B and Cdc20 (the activators of the APC/C), to the complex. The involvement of Mad2B-Cdc20-APC/C during DNA damage has not been reported before and suggests that the APC/C is activated following cisplatin-induced DNA damage. Using an in vitro ubiquitination assay, our data confirmed Mad2B-dependent activation of APC/C in cisplatin-treated cells. Mad2B may act as an accelerator for APC/C activation during DNA damage response. Our data strongly suggest a role for Mad2B-APC/C-Cdc20 in the ubiquitination of proteins involved in the DNA damage response.

A Recurrent ADPRHL1 Germline Mutation Activates PARP1 and Confers Prostate Cancer Risk in African American Families.

This study highlights a potential strategy for ADPRHL1 mutation detection in prostate cancer-risk assessment and a potential therapeutic application for individuals with prostate cancer in AA families.

FC 132: Deficiency of Proximal Tubular Cyclin-Dependent Kinase 12 Exacerbates Kidney Injury Through DNA-Damage Response

Abstract BACKGROUND AND AIMS Acute kidney injury (AKI) is a frequent, serious condition characterized by acute tubular necrosis involving the destruction of tubular epithelial cells. Recent studies have shown that DNA damage contributes to renal tubular cell death during kidney injury. Cyclin-dependent kinase 12 (CDK12) is a member of the cyclin-dependent kinase family that phosphorylates the carboxy-terminal domain of RNA polymerase II and selectively regulates transcription of DNA-damage response (DDR) genes. CDK12 inhibitor was reported to play an important role in a variety of cancers. So far, the functions of CDK12 deficiency in the pathogenesis of AKI have not been studied. METHOD Here, we established a cisplatin-induced AKI mouse model with CDK12 knockdown mice and created stable in vitro models with CDK12 knockdown tubular epithelial cells. Cisplatin nephrotoxicity was induced by intraperitoneal injection of cisplatin (20 mg/kg). After 2 days following cisplatin treatment, blood and kidney tissues were harvested. Renal function and histology were evaluated. DDR, tubular apoptosis, cell proliferation were evaluated by immunofluorescence assays, real-time PCR, flow cytometer and western blot analysis. RESULTS Here we found that CDK12 was downregulated in the renal tubular epithelial cells of AKI patients and murine AKI models and associated with kidney functions. RNA-sequencing of kidney cortex demonstrated increased transcripts involved in DDR and tubular apoptosis signaling pathways and decreased transcripts cell proliferation pathways, in knockout versus control mice. Moreover, more severe AKI occurred in tubular cell-specific knock-down of CDK12 mice (CDK12RTEC+/–) than age- and sex-matched wild type mice, as evidenced by increased levels of serum creatinine (Scr), blood urea nitrogen (BUN) and tubular injury score. Mechanistically, CDK12 deficiency accelerated cisplatin-induced DDR, mitochondrial apoptosis and abrogates cell proliferation of renal tubular cells in these mice. Similar results were observed after the human renal tubular epithelial cell line HK-2 cells exposure to CDK12 siRNA stimulation. In contrast, lentivirus-mediated CDK12 overexpression protected against DDR and mitochondrial apoptosis while attenuating renal dysfunction, morphological damage and renal tubular cell death in cisplatin-induced AKI models. Moreover, representative Integrative Genomics Viewer (IGV) profiles demonstrated a minimal impact of gene length-dependent elongation defects, a substantial proportion of which participate in the DDR and cell proliferation. CONCLUSION Thus, proximal tubule-specific deficiency of CDK12 exacerbates acute kidney injury and tubular apoptosis, likely due to affecting the expression of genes involved in the DDR and mRNA processing.

MicroRNA-191 modulates cisplatin-induced DNA damage response by targeting RCC2.

Cisplatin is a first-line chemotherapeutic agent for the treatment of many types of cancer, but the emergence of chemoresistance hinders its application. Thus, a better understanding of cisplatin-induced DNA damage response (DDR) would help to overcome this problem. Previously, we have identified a panel of microRNAs with altered expression after cisplatin treatment in HeLa cells. In the current study, we focused on one of them, miR-191, and investigated its function in cisplatin-induced DDR. We found that overexpression of miR-191 sensitized HeLa cells to the cytotoxic effects of cisplatin, resulted in decreased viable cells. However, overexpression of miR-191 did not cause changes in apoptotic cell ratio but rather induced significant G2/M arrest in HeLa cell treated with cisplatin. Additionally, enhanced cisplatin-induced DNA damage was observed. Through bioinformatic analysis and verified by dual-luciferase assay, it was demonstrated that the chromosome condensation 2 regulator (RCC2) gene is a target for miR-191 regulation. Furthermore, the downregulation of RCC2 by siRNA mimics the effects of miR-191, in which greater DNA damage was observed upon cisplatin treatment. Taken together, our study indicates that miR-191 may be an important player in cisplatin-induced DDR, and it elicits its function, at least partially, through the regulation of RCC2.

Pro-apoptotic functions of TRAF2 in p53-mediated apoptosis induced by cisplatin.

Tumor necrosis factor receptor-associated factor 2 (TRAF2) is an essential component of tumor necrosis factor-α (TNF-α) signaling that regulates nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK) pathways, and compelling evidence has demonstrated that TRAF2 suppresses TNF-α-induced cytotoxicity. On the other hand, it has been reported that oxidative stress-induced cytotoxicity is potentiated by TRAF2, indicating that TRAF2 both positively and negatively regulates stress-induced cytotoxicity in a context-specific manner. However, the causal role of TRAF2 in DNA damage response (DDR) remains to be explored. In this study, we assessed the function of TRAF2 in DDR induced by cisplatin, a representative DNA-damaging agent, and found that TRAF2 exerts pro-apoptotic activity through p53-dependent mechanisms at least in human fibrosarcoma cell line HT1080. TRAF2 deficient cells exhibit significant resistance to cell death induced by cisplatin, accompanied by the reduction of both p53 protein level and caspase-3 activation. Moreover, cisplatin-induced JNK activation was attenuated in TRAF2-deficient cells, and pharmacological inhibition of JNK signaling suppressed p53 stabilization. These results suggest that TRAF2 promotes p53-dependent apoptosis by activating the JNK signaling cascade in HT1080 cells. Thus, our data demonstrate a novel function of TRAF2 in cisplatin-induced DDR as a pro-apoptotic protein.

VE-822 mediated inhibition of ATR signaling sensitizes chondrosarcoma to cisplatin via reversion of the DNA damage response.

IntroductionCisplatin has been reported to elicit the DNA damage response (DDR) via activation of the ATR-Chk1 pathway, which in turn contributes to the induction of cisplatin resistance. Inhibition of ATR-Chk1 signaling reverses cisplatin resistance in some cancers. However, the influence of inhibiting ATR-Chk1 signaling on cisplatin resistance in chondrosarcoma cancer has not been reported.Materials and methodsWe compared the expression levels of ATR kinases in human nasopharyngeal carcinoma, choriocarcinoma and chondrosarcoma cell lines. We inhibited ATR kinase function with VE-822, a selective ATR inhibitor, and suppressed ATR kinase expression with shRNA. Western blotting, the CCK-8 assay, cell cycle distribution assay and apoptosis analysis were used to study the influence of inhibiting ATR-Chk1 signaling on reversing cisplatin resistance in chondrosarcoma cell lines.ResultsWe found that chondrosarcoma cells expressed very low basal levels of phosphorylated ATR, but cisplatin treatment induced the activation of ATR-Chk1 signaling in a dose- and time-dependent manner, suggesting the induction of DDR. As expected, ATR inhibition with VE-822 reversed cisplatin-induced DDR and enhanced cisplatin-induced activation of H2AX, which is an important marker of DNA damage. Meanwhile, ATR inhibition by RNA interference also reversed DDR and promoted DNA damage. Furthermore, both pharmacological and molecular inhibition of ATR accelerated cisplatin-induced inhibition of cell proliferation and cell death.ConclusionOur results suggested that inhibiting ATR activation promoted cisplatin-induced cell death via reversion of DDR, and VE-822 may be a valuable strategy for the prevention of cisplatin resistance in chondrosarcoma.

Triggering TLR3 pathway promotes tumor growth and cisplatin resistance in head and neck cancer cells

Over the last decades, significant advances in targeted therapies have helped provide more effective treatment for head and neck cancer patients. However, chemo-resistance to cisplatin significantly contributes to treatment failure in the clinical management of patients. In response to chemotherapeutic agents, certain molecules inside the cell are released or secreted from damaged or dead/dying cells, named damage-associated molecular patterns (DAMPs), thereby initiating an immune response through interaction with pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs).In present study, we investigated the link between cisplatin-induced DAMPs and TLR3 signaling. We found that cisplatin could be a potential activator of TLR3 and cisplatin treatment results in activation of PRRs’ signaling and down-stream associated cytokine/chemokine, IFNβ, and CCL5 in TLR3High OC2 cells, but not in TLR3Low FaDu cells. Furthermore, knockdown of the TLR3 gene attenuates the expression of IFNβ and CCL5 mRNA and enhances the cytotoxicity of cisplatin in TLR3High OC2 cells.To determine whether TLR3 status affects the stress response of OC2 cells to cisplatin, we generated TLR3 knockdown OC2 cells (psi-TLR3 cells) with a psiRNA-hTLR3 plasmid containing shRNA to TLR3 and control OC2 cells (psi-NT cells) expressing non-silencing shRNA. OC2 cells were more sensitive to cisplatin treatment after TLR3 knockdown. In our animal model, OC2 psi-NT cells were more tumorigenic than were OC2 psi-TLR3 cells. Together, our in vitro and in vivo data imply TLR3 may contribute to tumor development and protect cisplatin-induced DNA damage response leading to cisplatin resistance in head and neck cancer cells.

Depletion of Paraspeckle Protein 1 Enhances Methyl Methanesulfonate-Induced Apoptosis through Mitotic Catastrophe.

Previously, we have shown that paraspeckle protein 1 (PSPC1), a protein component of paraspeckles that was involved in cisplatin-induced DNA damage response (DDR), probably functions at the G1/S checkpoint. In the current study, we further examined the role of PSPC1 in another DNA-damaging agent, methyl methanesulfonate (MMS)-induced DDR, in particular, focusing on MMS-induced apoptosis in HeLa cells. First, it was found that MMS treatment induced the expression of PSPC1. While MMS treatment alone can induce apoptosis, depletion of PSPC1 expression using siRNA significantly increased the level of apoptosis following MMS exposure. In contrast, overexpressing PSPC1 decreased the number of apoptotic cells. Interestingly, morphological observation revealed that many of the MMS-treated PSPC1-knockdown cells contained two or more nuclei, indicating the occurrence of mitotic catastrophe. Cell cycle analysis further showed that depletion of PSPC1 caused more cells entering the G2/M phase, a prerequisite of mitosis catastrophe. On the other hand, over-expressing PSPC1 led to more cells accumulating in the G1/S phase. Taken together, these observations suggest an important role for PSPC1 in MMS-induced DDR, and in particular, depletion of PSPC1 can enhance MMS-induced apoptosis through mitotic catastrophe.

Abstract 2530: Bcl-2 and Bcl-xL dual inhibitor, ABT-737, circumvents JNK mediated upregulation of anti-apoptotic molecules in cisplatin treated non-small cell lung cancer models

Abstract Introduction; Non-small cell lung cancer (NSCLC), which does not have druggable driver oncogene, is treated with cytotoxic chemotherapy, but clinical outcome is suboptimal. Cisplatin is still one of the most commonly used drugs for the treatment of NSCLC, and, however, there are limitations of the therapeutic effect because of the multiple resistance mechanisms. In particular, elevated level of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1) triggered by cisplatin-induced DNA damage response correlate with the drug resistance. In this study, we investigated synergic effects of combined treatment with cisplatin and dual inhibitor of Bcl-2/Bcl-xL, ABT-737, in NSCLC models. Materials and Methods; H460 and A549 NSCLC cells, and LSL K-Ras G12D mouse model were used. Protein expression was evaluated by immunoblotting and cytotoxicity was measured by FACS followed by annexin-V and PI staining. LSL Kras G12D mouse were inhaled 5 × 107 PFU AdCre virus at 8 weeks after birth and, in 24 weeks after inhalation, were taken microCT, randomized into 4 groups, and then treated with ABT-737 (50mg/kg, i.p., daily) and/or cisplatin (5 mg/kg, i.p., weekly) for two weeks. Results; There was a dose dependent phosphorylation of JNK, which is most prominent between 6 to 24hr after cisplatin treatment. Expression of anti-apoptotic molecules, Bcl-2, Bcl-w, Bcl-xL, and Mcl-1 were increased by cisplatin treatment in a dose and time dependent manner. JNK specific activator, anisomycin, increased mRNA level of anti-apoptotic molecules. ABT-737 displaced BCL-xL to the cytoplasm and aggregated Bak. Furthermore, ABT-737 released cytochrome C from mitochondria to the cytoplasm. ABT-737 itself showed cytotoxic effects and combination of ABT-737 potentiates cytotoxic effects of cisplatin with strong synergism. Combination of ABT-737 with cisplatin treatment induced significant tumor regression in 2 weeks in LSL K-ras G12D mouse model when compared those treated with single agents or vehicle. Conclusion; Combination of ABT-737 with cisplatin showed synergistic cytotoxic effects and anti-tumor activities in NSCLC preclinical models. These findings suggest that clinical trials using combination with ABT-737 and cisplatin may be beneficial in advanced NSCLC patients. Citation Format: Eun Young KIM, Arum Kim, Yoon Soo Chang. Bcl-2 and Bcl-xL dual inhibitor, ABT-737, circumvents JNK mediated upregulation of anti-apoptotic molecules in cisplatin treated non-small cell lung cancer models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2530. doi:10.1158/1538-7445.AM2015-2530

Arsenic trioxide amplifies cisplatin toxicity in human tubular cells transformed by HPV-16 E6/E7 for further therapeutic directions in renal cell carcinoma

Human papillomavirus (HPV) DNA integrations may affect therapeutic responses in cancers through ATM network-related DNA damage response (DDR). We studied whether cisplatin-induced DDR was altered in human HK-2 renal tubular cells immortalized by HPV16 E6/E7 genes. Cytotoxicity assays utilized thiazolyl blue dye and DDR was identified by gene expression differences, double-strand DNA breaks, ATM promoter activity, and analysis of cell cycling and side population cells. After cisplatin, HK-2 cells showed greater ATM promoter activity indicating activation of this network, but DDR was muted, since little γH2AX was expressed, DNA strand breaks were absent and cells continued cycling. When HK-2 cells were treated with the MDM2 antagonist inducing p53, nutlin-3, or p53 transcriptional activator, tenovin-1, cell growth decreased but cisplatin toxicity was unaffected. By contrast, arsenic trioxide, which by inhibiting wild-type p53-induced phosphatase-1 that serves responses downstream of p53, and by depolymerizing tubulin, synergistically enhanced cisplatin cytotoxicity including loss of SP cells. Our findings demonstrated that HPV16 E6/E7 altered DDR through p53-mediated cell growth controls, which may be overcome by targeting of WIP1 and other processes, and thus should be relevant for treating renal cell carcinoma.

Paraspeckle protein 1 (PSPC1) is involved in the cisplatin induced DNA damage response--role in G1/S checkpoint.

Paraspeckle protein 1 (PSPC1) was first identified as a structural protein of the subnuclear structure termed paraspeckle. However, the exact physiological functions of PSPC1 are still largely unknown. Previously, using a proteomic approach, we have shown that exposure to cisplatin can induce PSPC1 expression in HeLa cells, indicating the possible involvement for PSPC1 in the DNA damage response (DDR). In the current study, the role of PSPC1 in DDR was examined. First, it was found that cisplatin treatment could indeed induce the expression of PSPC1 protein. Abolishing PSPC1 expression by siRNA significantly inhibited cell growth, caused spontaneous cell death, and increased DNA damage. However, PSPC1 did not co-localize with γH2AX, 53BP1, or Rad51, indicating no direct involvement in DNA repair pathways mediated by these molecules. Interestingly, knockdown of PSPC1 disrupted the normal cell cycle distribution, with more cells entering the G2/M phase. Furthermore, while cisplatin induced G1/S arrest in HeLa cells, knockdown of PSPC1 caused cells to escape the G1/S checkpoint and enter mitosis, and resulted in more cell death. Taken together, these observations indicate a new role for PSPC1 in maintaining genome integrity during the DDR, particularly in the G1/S checkpoint.

SET overexpression decreases cell detoxification efficiency: ALDH2 and GSTP1 are downregulated, DDR is impaired and DNA damage accumulates

Alcohol and tobacco consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Aldehyde dehydrogenase2 (ALDH2) and glutathioneS-transferase pi1 (GSTP1) are important enzymes for cellular detoxification and low efficiencies are implicated in cancer. We assessed the potential role of SET protein overexpression, a histone acetylation modulator accumulated in HNSCC, in gene regulation and protein activity of ALDH2 and GSTP1. SET was knocked down in HN13, HN12 and Cal27, and overexpressed in HEK293 cells; ethanol and cisplatin were the chemical agents. Cells with SET overexpression (HEK293/SET, HN13 and HN12) showed lower ALDH2 and GSTP1 mRNA levels and trichostatinA increased them (real-time PCR). Ethanol upregulated GSTP1 and ALDH2 mRNAs, whereas cisplatin upregulated GSTP1 in HEK293 cells. SET-chromatin binding revealed SET interaction with ALDH2 and GSTP1 promoters, specifically via SET NAP domain; ethanol and cisplatin abolished SET binding. ALDH2 and GSTP1 efficiency was assessed by enzymatic and comet assay. A lower ALDH2 activity was associated with greater DNA damage (tail intensity) in HEK293/SET compared with HEK293 cells, whereas HN13/siSET showed ALDH2 activity higher than HN13 cells. HN13/siSET cells showed increased tail intensity. Cisplatin-induced DNA damage response showed negative relationship between SET overexpression and BRCA2 recruitment. SET downregulated repair genes ATM, BRCA1 and CHEK2 and upregulated TP53. Cisplatin-induced cell-cycle arrest occurred in G(0) /G(1) and S in HEK293 cells, whereas HEK293/SET showed G(2) /M stalling. Overall, cisplatin was more cytotoxic for HN13 than HN13/siSET cells. Our data suggest a role for SET in cellular detoxification, DNA damage response and genome integrity.

Cisplatin treatment leads to changes in nuclear protein and microRNA expression

Using a proteomic approach, we have previously shown that exposure to different concentrations of cisplatin during a 12-h period can lead to changes in nuclear protein expression and alternative splicing in HeLa cells. To further shed light on the DNA damage response (DDR) induced by cisplatin, we examined the nuclear proteome profiles of HeLa cells treated with 5μM cisplatin for different times (2, 12, and 24h). Two-dimensional electrophoresis (2-DE) identified 98 differentially expressed proteins in cisplatin-treated cells as compared to control cells. Among them, 54 spots (55%) were down-regulated and 44 spots (45%) were up-regulated. 51 spots were subjected to Matrix-assisted-laser-desorption-ionization Time-of-flight/time-of-flight Mass spectrometry (MALDI-TOF/TOF MS) identification, and 40 spots were identified. Among these, 22 proteins were located in nucleus. These proteins were involved in stress response, cell cycle and division, apoptosis, mRNA processing, transport, splicing and microRNA (miRNA) maturation. The changed expression of Annexin A1 and Lamin B1 were confirmed by Western blot. The role of Annexin A1 in the response to cisplatin-induced DNA damage was further analyzed, and it was shown that after Annexin A1 knockdown, cisplatin-induced DNA damage was significantly increased. In addition, the changed expression of several miRNAs was also observed by quantitative real-time PCR (qRT-PCR). Taken together, these data indicate that cisplatin-induced DDR is a complex process, and that those proteins identified by proteomics can lead to new directions for a better understanding of this process.

MutS homologue hMSH5: role in cisplatin-induced DNA damage response

BackgroundCisplatin (cis-diamminedichloroplatinum (II), CDDP) and its analogues constitute an important class of anticancer drugs in the treatment of various malignancies; however, its effectiveness is frequently affected by mutations in genes involved in the repair and signaling of cisplatin-induced DNA damage. These observations necessitate a need for a better understanding of the molecular events governing cellular sensitivity to cisplatin.ResultsHere, we show that hMSH5 mediates sensitization to cisplatin-induced DNA damage in human cells. Our study indicates that hMSH5 undergoes cisplatin-elicited protein induction and tyrosine phosphorylation. Silencing of hMSH5 by RNAi or expression of hMSH5 phosphorylation-resistant mutant hMSH5Y742F elevates cisplatin-induced G2 arrest and renders cells susceptible to cisplatin toxicity at clinically relevant doses. In addition, our data show that cisplatin promotes hMSH5 chromatin association and hMSH5 deficiency increases cisplatin-triggered γ-H2AX foci. Consistent with a possible role for hMSH5 in recombinational repair of cisplatin-triggered double-strand breaks (DSBs), the formation of cisplatin-induced hMSH5 nuclear foci is hRad51-dependent.ConclusionCollectively, our current study has suggested a role for hMSH5 in the processing of cisplatin-induced DSBs, and silencing of hMSH5 may provide a new means to improve the therapeutic efficacy of cisplatin.