Impaired DNA Damage Repair Research Articles

Abstract 5729: The Wnt transcription factor TCF4 mediates resistance of colorectal cancer cells to (chemo-) radiotherapy in a β-catenin-independent manner

Abstract Background: We recently demonstrated that the Wnt transcription factor TCF4 is over-expressed in rectal cancers that were resistant to preoperative chemoradiotherapy. Because the association between Wnt signaling and chemoradioresistance is a novel finding, we aimed to investigate whether this over-expression is functionally relevant. Methods: Three colorectal cancer cell lines were transfected with two shRNA-vectors targeting TCF4. Stable single-cell clone (SCC) populations were established, and selected clones were irradiated at 0, 1, 2, 4, 6 and 8 Gy. γH2AX staining was used to evaluate DNA double strand repair after irradiation, and changes in cell cycle distribution were analyzed before and after radiation. Finally, β-catenin activity was inhibited using both siRNAs and the small molecule inhibitor XAV939. Results: Silencing of TCF4 led to a significant radiosensitization in SW837 and SW480, whereas there was no effect in HT-29. Well-fitting, we observed significantly more γH2AX foci 24 hrs after radiation in SW837 SCCs compared to HT-29 SCCs, pointing to an impaired DNA damage repair in SW837. Furthermore, in SW837 SCCs, but not in HT-29 SCCs, we noticed a change in the cell cycle distribution towards radiosensitive cell cycle phases before radiation, and compromised cell cycle control after radiation. Finally, both siRNA- and small molecule-mediated inhibition of β-catenin activity did not influence the sensitivity to chemoradiotherapy. Conclusion: Our results indicate a novel mechanism through which the Wnt transcription factor TCF4 mediates chemoradioresistance in a β-catenin-independent manner. Moreover, they suggest that TCF4 is a potential molecular target to sensitize resistant tumor cells to (chemo-) radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5729. doi:1538-7445.AM2012-5729

Heritability of radiation response in lung cancer families.

Radiation sensitivity is assumed to be a cancer susceptibility factor due to impaired DNA damage signalling and repair. Relevant genetic factors may also determine the observed familial aggregation of early onset lung cancer. We investigated the heritability of radiation sensitivity in families of 177 Caucasian cases of early onset lung cancer. In total 798 individuals were characterized for their radiation-induced DNA damage response. DNA damage analysis was performed by alkaline comet assay before and after in vitro irradiation of isolated lymphocytes. The cells were exposed to a dose of 4 Gy and allowed to repair induced DNA-damage up to 60 minutes. The primary outcome parameter Olive Tail Moment was the basis for heritability estimates. Heritability was highest for basal damage (without irradiation) 70% (95%-CI: 51%–88%) and initial damage (directly after irradiation) 65% (95%-CI: 47%–83%) and decreased to 20%–48% for the residual damage after different repair times. Hence our study supports the hypothesis that genomic instability represented by the basal DNA damage as well as radiation induced and repaired damage is highly heritable. Genes influencing genome instability and DNA repair are therefore of major interest for the etiology of lung cancer in the young. The comet assay represents a proper tool to investigate heritability of the radiation sensitive phenotype. Our results are in good agreement with other mutagen sensitivity assays.

(212)Pb-radioimmunotherapy induces G(2) cell-cycle arrest and delays DNA damage repair in tumor xenografts in a model for disseminated intraperitoneal disease.

In preclinical studies, targeted radioimmunotherapy using (212)Pb-TCMC-trastuzumab as an in vivo generator of the high-energy α-particle emitting radionuclide (212)Bi is proving an efficacious modality for the treatment of disseminated peritoneal cancers. To elucidate mechanisms associated with this therapy, mice bearing human colon cancer LS-174T intraperitoneal xenografts were treated with (212)Pb-TCMC-trastuzumab and compared with the nonspecific control (212)Pb-TCMC-HuIgG, unlabeled trastuzumab, and HuIgG, as well as untreated controls. (212)Pb-TCMC-trastuzumab treatment induced significantly more apoptosis and DNA double-strand breaks (DSB) at 24 hours. Rad51 protein expression was downregulated, indicating delayed DNA double-strand damage repair compared with (212)Pb-TCMC-HuIgG, the nonspecific control. (212)Pb-TCMC-trastuzumab treatment also caused G(2)-M arrest, depression of the S phase fraction, and depressed DNA synthesis that persisted beyond 120 hours. In contrast, the effects produced by (212)Pb-TCMC-HuIgG seemed to rebound by 120 hours. In addition, (212)Pb-TCMC-trastuzumab treatment delayed open chromatin structure and expression of p21 until 72 hours, suggesting a correlation between induction of p21 protein and modification in chromatin structure of p21 in response to (212)Pb-TCMC-trastuzumab treatment. Taken together, increased DNA DSBs, impaired DNA damage repair, persistent G(2)-M arrest, and chromatin remodeling were associated with (212)Pb-TCMC-trastuzumab treatment and may explain its increased cell killing efficacy in the LS-174T intraperitoneal xenograft model for disseminated intraperitoneal disease.

Tumor suppressor BRCA1 epigenetically controls oncogenic microRNA-155

BRCA1, a well-known tumor suppressor with multiple interacting partners, is predicted to have diverse biological functions. However, so far its only well-established role is in the repair of damaged DNA and cell cycle regulation. In this regard, the etiopathological study of low-penetrant variants of BRCA1 provides an opportunity to uncover its other physiologically important functions. Using this rationale, we studied the R1699Q variant of BRCA1, a potentially moderate-risk variant, and found that it does not impair DNA damage repair but abrogates the repression of microRNA-155 (miR-155), a bona fide oncomir. Mechanistically, we found that BRCA1 epigenetically represses miR-155 expression via its association with HDAC2, which deacetylates histones H2A and H3 on the miR-155 promoter. We show that overexpression of miR-155 accelerates but the knockdown of miR-155 attenuates the growth of tumor cell lines in vivo. Our findings demonstrate a new mode of tumor suppression by BRCA1 and suggest that miR-155 is a potential therapeutic target for BRCA1-deficient tumors.

Suppression of Autophagy by FIP200 Deletion Impairs DNA Damage Repair and Increases Cell Death upon Treatments with Anticancer Agents

Autophagy is a lysosomal bulk degradation process for intracellular protein and organelles. FIP200 (200 kDa FAK-family interacting protein) is an essential component of mammalian autophagy that is implicated in breast cancer in recent studies. Here we show that inactivation of FIP200 resulted in deficient repair of DNA damage induced by ionizing radiation and anticancer agents in mouse embryonic fibroblasts (MEF). The persistent DNA damage correlated to increased apoptosis and reduced survival of FIP200 knockout (KO) MEFs after treatments with camptothecin (CPT), a topoisomerase I inhibitor and chemotherapeutic agent. Reexpression of FIP200 in FIP200 KO MEFs restored both efficient DNA damage repair and cell survival. Furthermore, knockdown of the increased p62 expression in FIP200 KO MEFs rescued the impaired DNA damage repair and CPT-induced cell death. In contrast, treatment of cells with N-acetyl cysteine did not affect these defects in FIP200 KO MEFs. Finally, FIP200 KO MEFs also showed deficient DNA damage repair and increased cell death compared with control MEFs, when treated with etoposide, a topoisomerase II inhibitor and another anticancer agent. Together, these results identify a new function for FIP200 in the regulation of DNA damage response and cell survival through its activity in autophagy and suggest the possibility of FIP200 or other autophagy proteins as a potential target for treatment to enhance the efficiency of cancer therapy using DNA damage-inducing agents.

Abstract 2490: Silencing of TCF7L2 sensitizes Wnt/β-catenin signaling-dependent colorectal cancer cells to radiation

Abstract Background: The clinical response of locally advanced rectal cancers to preoperative chemoradiotherapy is very heterogeneous. We recently profiled a series of responsive and resistant rectal carcinomas using gene expression microarrays and identified TCF7L2, the main downstream effector of the Wnt/β-catenin pathway, as over-expressed in the resistant tumors. The aim of this study was to explore the relevance of TCF7L2 for resistance to radiation and to study the underlining mechanisms. Methods: We transfected three microsatellite-stable colorectal cancer cell lines (SW837, SW480 and HT-29) with two different shRNA-vectors targeting TCF7L2 and a non-silencing. Next, we established single cell clone (SCC) populations, and after confirming the reduction of TCF7L2 protein levels by Western blotting, we irradiated the SCCs at 0, 1, 2, 4, 6 and 8 Gy and calculated survival fractions. To gain functional insight, we chose SW837 and HT-29 for further experiments. Using γH2AX staining we evaluated DNA double strand repair after irradiation at 2 Gy and analyzed cell cycle changes before and after radiation. Furthermore, we studied Wnt/β-catenin pathway activity using the TOPFLASH/FOPFLASH reporter assay. Results: RNAi-mediated silencing of TCF7L2 led to a pronounced reduction in TCF7L2 protein levels. In radiation experiments we observed a highly significant radiosensitization in SW837 and SW480 SCCs, whereas no effect was observed in HT-29 SCCs. Well-fitting, we observed significantly more γH2AX foci 24h after radiation in SW837 SCCs compared to HT-29 SCCs, pointing to impaired DNA damage repair in the SCCs from SW837. Furthermore, in SW837 SCCs but not in HT-29 SCCs we noticed a change in the cell cycle distribution towards radiosensitive cell cycle phases before radiation and compromised cell cycle control after radiation. Finally, using the TOPFLASH/FOPFLASH reporter assay we found SW837 wild-type cells to show high Wnt/β-catenin pathway activity, while HT-29 wild-type cells only showed very low activity. Conclusion: TCF7L2 was found to be over-expressed in resistant rectal carcinomas, and its RNAi-mediated silencing caused a significant radiosensitization, mediated by DNA damage repair deficiencies and impaired cell cycle control. Interestingly, radiosensitization effetcts were only observed in cell lines showing Wnt/β-catenin pathway activity. Thus, we have uncovered a novel role of Wnt/β-catenin signaling for mediating resistance to multimodal cancer therapy. Moreover, these data suggest that TCF7L2 is a potential molecular target for sensitizing Wnt/β-catenin-dependent tumor cells to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2490. doi:10.1158/1538-7445.AM2011-2490

Sonic Hedgehog Signaling Protects Human Hepatocellular Carcinoma Cells Against Ionizing Radiation in an Autocrine Manner

Sonic hedgehog (Shh) signaling is critical to embryogenesis and resistance to chemotherapy. We aimed to examine the role of Shh signaling in the response to radiation of human hepatocellular carcinoma (HCC) cells. Response to ionizing radiation therapy (RT) was evaluated by clonogenic assay. Quantitative RT-polymerase chain reaction for patched-1 (PTCH-1) expression was performed. Cytosolic accumulation of Shh and nuclear translocation of Gli-1 were assessed by immunofluorescence. Gli-1 knockdown was done by RNA interference (RNAi). Immunoprecipitation was performed to detect Shh ligand in conditioned medium. Immunofluorescent stain for γ-H2AX was used as an index of DNA double strand breaks (DSB). Expression of proteins related to DNA damage repair was assessed by Western blotting. We found that Shh ligand could protect human HCC HA22T and Sk-Hep1 cells against RT. In HA22T cells, Shh ligand activated the Shh signaling with upregulation of Shh, PTCH-1, and Gli-1 expression. The nuclear translocation of Gli-1 further supports the activation of Gli-1. The radioprotection by Shh ligand was partly blocked by Shh antibody neutralization and was abolished by Gli-1 RNAi, suggesting a critical role of Shh signaling in radiation resistance. Furthermore, we noted that soluble factors secreted into conditioned medium, either constitutively or responding to radiation, by HA22T or Sk-Hep1 cells protected subsequent culturing cells against RT. Immunoprecipitation shows the presence of Shh peptide in conditioned medium. Intriguingly, antibody neutralization of Shh ligand or knockdown of Gli-1 reversed the radioprotective effect of conditioned medium. Furthermore, Shh ligand reduced the RT-induced phosphorylation of checkpoint kinase 1 and impaired the repair of DNA DSB. Activation of Shh signaling protects HCC cells against ionizing radiation in an autocrine manner. Impairment of DNA damage repair might involve mechanism of Shh-induced radioresistance. Targeting Shh signaling pathway may be a novel strategy to enhance the radioresponse of human HCC cells.

DNA repair and neurodegeneration: a common pathology shared by polyglutamine diseases

Nuclear dysfunctions have been implicated in the pathology of polyglutamine diseases, while the details remain unclear. By employing various omics approaches, we have identified target molecules of mutant polyglutamine proteins in the nucleus. Proteome analysis of soluble nuclear proteins identified decreased HMGB1/2 in vulnerable neurons of Huntington's disease (HD) and spinocerebellar ataxia type 1 (SCA1). Interactome analysis unraveled Ku70 as a direct binding partner of mutant huntingtin. DNA damage repair is significantly impaired by interaction of mutant polyglutamine proteins with HMGB or Ku70, and the functional rescue of these molecules alleviated symptoms and pathologies of HD and SCA1. These results strongly suggest that impairment of DNA damage repair is a common pathology shared by multiple polyglutamine diseases.

Bortezomib Induced BRCAness Sensitizes Multiple Myeloma Cells to PARP Inhibitors

AbstractAbstract 789 Background:Poly-ADP-ribose-polymerase (PARP) inhibitors are cytotoxic to tumor cells with impaired DNA damage repair machinery (DRR), in particular those with a deficient homology directed repair (HR) of DNA double stranded breaks (DSB). Multiple Myeloma (MM) cells are characterized by a highly unstable genome and while the exact mechanisms for this karyotypic instability is largely unknown, their DDR machinery is thought to be highly stressed. The ubiquitin-proteasome system (UPS) is involved in the regulation of several cellular functions including DDR and in particular HR. In addition proteasome inhibitors are reported to induce an unfolded protein response (UPR) in MM cells resulting in their apoptotic death. We have postulated that inhibition of the 26S proteasome also alters the DNA-DSB repair machinery leading to a BRCAness state in MM cells, sensitizing them to PARP inhibitors. Methods and results:In order to biochemically inhibit PARP in MM cells, we used a novel selective inhibitor of PARP1 and PARP2, 2-(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide or ABT-888. We first demonstrated inhibition of PARP activity as measured by a reduction in poly-ADP-ribose (PAR) polymer levels (western blotting) in human MM cell lines (MM1S, U266, H929, RPMI8226, KMS-11, OPM2, INA-6) treated with ABT-888 (5 μM). PARP inhibition and the reduction of PAR levels resulted in DNA damage as evidenced by ATM phosphorylation and induced DNA-DSBs with increased γH2AX (phospho-Ser139-H2AX) levels within 6–12 hours of MM cells treatment with ABT-888. Increased γH2AX foci formation was also detected by immunofluorescent staining within 6–12 hours of ABT-888 treatment and nearly fully resolved by 24 hours, consistent with repair of resultant DNA-DSBs. As expected treatment with ABT-888 alone had no effect on the viability of MM cells consistent with their ability to repair DNA-DSBs resulting from PARP inhibition.We then examined the effect of bortezomib on HR-mediated repair of DNA-DSBs, in particular on the BRCA/FA pathway. A significant reduction of MM cells' FANCD2, BRCA1, BRCA2 and RAD51 mRNA levels (qRT-PCR) was observed within 6–12 hours of bortezomib treatment (10 nM). Similar results were observed at the protein level indicating that bortezomib impedes homology-directed DNA-DSBs repair and results in an operational BRCAness state in MM cells. Therefore, we next tested whether this bortezomib-induced BRCAness was sufficient to sensitize MM cells to PARP inhibition with ABT-888. Consistent with our hypothesis, we observed that co-treatment of MM cell lines with bortezomib and ABT-888 lead to persistent and increased γH2AX foci at 24 hours compared to treatment with ABT-888 alone. Co-treatment also significantly potentiated cell death (Annexin V/PI staining) compared to treatment with bortezomib alone. Similar results were observed in CD138+ primary MM cells (n=8) with strong synergistic effect (CI < 1) between bortezomib and ABT-888. Importantly, no impaired viability (Annexin/PI staining) or function (colony forming unit assay) was noted for CD138− cells or CD34+ peripheral blood stem cells after bortezomib and ABT-888 co-treatment. Mechanistic studies have also shown that apoptotic events (caspase 3, caspase 8 and PARP cleavage) are markedly enhanced by this combination.Based on our in vitro data, we evaluated in vivo the activity of ABT-888 in combination with bortezomib in a Scid murine xenograft model of human MM. Significant inhibition of tumour growth (p<0.005) was noted in mice treated with the combination of bortezomib and ABT-888 compared to bortezomib alone or control-treated mice. This tumour growth inhibition also resulted in a significant increase in survival (p<0.05) of the animals. No toxicity (e.g. weight loss, ruffled coats, paralysis, etc.) was observed in mice treated with the combination. Induction of DNA-DSBs was also confirmed in vivo as shown by an increase in 53BP1 and γH2AX foci formation in tumors of mice treated with the combination compared to bortezomib alone. Conclusion:Our studies indicate that bortezomib induces a BRCAness state in MM cells by impairing HR-mediated repair of DNA-DSBs and results in a contextual synthetic lethality when combined with the PARP inhibitor ABT-888. These data provide the scientific basis for the future clinical testing of PARP inhibitors in combination with proteasome inhibitors for the treatment of MM. Disclosures:No relevant conflicts of interest to declare.

Abstract 1394: Pro-apoptotic and anti-tumorigenic effects of a naturally occurring proteasome inhibitor on hormone-refractory prostate cancer

Abstract A major mode by which cancer cells evade death is by an acquired ability to suppress apoptosis. In hormone refractory prostate cancer, the overexpression of pro-survival/anti-apoptotic proteins results in resistance to chemo- and radio-therapy. Hence, developing co-therapies that sensitize cells to standard treatment regimens by overcoming this apoptotic block could improve treatment outcomes in advanced prostate cancer. The naturally occurring proteasome inhibitor celastrol has been previously reported to inhibit DNA damage processing and synergize with radiotherapy to kill PC-3 cells. To further explore the mechanism by which celastrol might potentiate cell death, several lines of questioning were explored. First, the open question regarding whether or not celastrol inhibits DNA damage repair, not simply processing, was tested in the comet assay. The tail moments measured in cells treated with celastrol, irradiated and allowed to recover were significantly greater than those in untreated cells, indicating that DNA damage repair was inhibited. In other experiments, cells treated with celastrol displayed an increased expression of the pro-apoptotic, BH3 only protein Noxa. This correlated with an increase in apoptotic cell death which was further augmented by ionizing radiation treatment. The pro-survival protein NFkB is tightly controlled by IkB-alpha binding. Proteasome inhibition can suppress the pro-survival activity of NFkB by decreasing the turnover of IkB-alpha. Indeed, IkB-alpha was found to accumulate in celastrol treated cells. Further, NFkB target gene activation was markedly reduced. To test the feasibility of celastrol treatment in animals, the PC-3 xenograft model in the athymic nude mouse was employed. When celastrol was combined with radiotherapy, tumor volume decreased, apoptosis increased, angiogenesis decreased, and NFkB target gene mRNA levels were found to be lower. Taken together, these data show that celastrol sensitized PC-3 cells to radiation both in vitro and in vivo by impairing DNA damage repair and augmenting apoptosis through suppressing NFkB activation and increasing the protein levels of Noxa. Celastrol may hold promise as a co-therapy with radiation for the treatment of hormone-refractory prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1394.

In vitro and In vivo Radiation Sensitization of Human Tumor Cells by a Novel Checkpoint Kinase Inhibitor, AZD7762

Inhibition of checkpoint kinase 1 has been shown to enhance the cytotoxicity of DNA-damaging targeted chemotherapy through cell cycle checkpoint abrogation and impaired DNA damage repair. A novel checkpoint kinase 1/2 inhibitor, AZD7762, was evaluated for potential enhancement of radiosensitivity for human tumor cells in vitro and in vivo xenografts. Survival of both p53 wild-type and mutant human cell lines was evaluated by clonogenic assay. Dose modification factors (DMF) were determined from survival curves (ratio of radiation doses for control versus drug treated at 10% survival). Flow cytometry, Western blot, and radiation-induced tumor regrowth delay assays were conducted. AZD7762 treatment enhanced the radiosensitivity of p53-mutated tumor cell lines (DMFs ranging from 1.6-1.7) to a greater extent than for p53 wild-type tumor lines (DMFs ranging from 1.1-1.2). AZD7762 treatment alone exhibited little cytotoxicity to any of the cell lines and did not enhance the radiosensitivity of normal human fibroblasts (1522). AZD7762 treatment abrogated radiation-induced G(2) delay, inhibited radiation damage repair (assessed by gamma-H2AX), and suppressed radiation-induced cyclin B expression. HT29 xenografts exposed to five daily radiation fractions and to two daily AZD7762 doses exhibited significant radiation enhancement compared with radiation alone. AZD7762 effectively enhanced the radiosensitivity of mutated p53 tumor cell lines and HT29 xenografts and was without untoward toxicity when administered alone or in combination with radiation. The results of this study support combining AZD7762 with radiation in clinical trials.

Partial silencing of methyl cytosine protein binding 2 ( MECP2 ) in mesenchymal stem cells induces senescence with an increase in damaged DNA

DNA methylation is an epigenetic modification that occurs almost exclusively on CpG dinucleotides. MECP2 is a member of a family of proteins that preferentially bind to methylated CpGs. We analyzed the contribution of MECP2 to the physiology of mesenchymal stem cells (MSCs). Partial silencing of MECP2 in human MSCs induced a significant reduction of S-phase cells, along with an increase in G(1) cells. These changes were accompanied by a reduction of apoptosis, the triggering of senescence, a decrease in telomerase activity, and the down-regulation of genes involved in maintaining stem cell properties. Senescence appeared to rely on impairment of DNA damage repair and seemed to occur through RB- and P53-related pathways. The effects of MECP2 silencing could be related to the modification of the DNA methylation status. Our results indicate that the silencing of MECP2 induces an increase in methylated cytosines in the genome. Nevertheless, MECP2 partial silencing did not change the methylation of promoters, whose expression is affected by MECP2 down-regulation.

Targeting HER2 signaling pathway for radiosensitization: Alternative strategy for therapeutic resistance

Several studies have indicated the potential value of targeting HER-2 signaling to enhance the anti-tumor activity of ionizing radiation. However, therapeutic resistance resulting from several factors, including activation of the downstream pathway, represents a major obstacle to treatment. Here, we investigated whether inhibitors targeting downstream of HER-2 signaling would radiosensitize SKBR3 breast cancer cells that exhibit overamplification of HER2. Selective inhibition of MEK-ERK signaling using pharmacologic inhibitors (PD98059, UO126) did not increase the radiosensitivity of SKBR3 cells. Selective inhibition of the PI3K-AKT-mTOR pathway using pharmacologic inhibitors (LY294002, AKT inhibitor VIII, Rapamycin) significantly attenuated expression of p-AKT and p-70S6K, respectively and radiosensitized SKBR3 cells. MCF-7 cells those did not over-express HER-2, showed less radiosensitization compared to SKBR3 cells by inhibition of this pathway. Pre-treatment with these inhibitors also caused significant abrogation of typical G2 arrest following ionizing radiation and induced marked prolongation of γH2AX foci indicating impairment of DNA damage repair. A dual inhibitor of Class I PI3K and mTOR, PI103 effectively radiosensitized SKBR3 cells and showed significant prolongation of γH2AX foci. Inhibition of PI3K-AKT signaling was associated with down-regulation of DNA-PKs, respectively. While apoptosis was the major mode of cell death when the cells were pretreated with LY294002 or AKT inhibitor VIII, the cells were pretreated by rapamycin or PI103 showed mixed mode of cell death including autophagy. Our results suggest possible mechanisms to counteract the HER-2 prosurvival signaling implicated in radioresistance, and offer an alternative strategy to overcome resistance to HER-2 inhibitors combined with radiation.

Abstract B254: Extended target-coverage by selective Chk1 inhibitors enhances pharmacodynamic inhibition of Chk1 signaling and antitumor activity in vivo

Abstract Loss of coordination between cell cycle checkpoints and DNA damage repair is a fundamental feature tumor cells rely on for unregulated growth and developing chemotherapeutic resistance. The protein kinase Checkpoint kinase 1 (Chk1) is a sentinel molecule essential for cell cycle arrest at the S and G2M checkpoints, as well as regulating homologous recombination DNA repair. In tumor cells exposed to chemotherapy, Chk1 inhibition overrides cell cycle arrest and DNA repair functions, effectively driving tumor cells into a state of mitotic catastrophe and, ultimately, cell death. We have previously reported in schedule-dependence studies, using Chk1 inhibitors and irinotecan (CPT-11), that oral administration of Chk1 inhibitors allows for multi-day target-coverage, and thus continuous inhibition of Chk1 for a finite period of time, which maximizes anti-tumor efficacy. Here, we extend these studies and investigate the pharmacodynamic relationship to efficacy, as well as the specific biomarkers that are predictive of an anti-tumor effect, when Chk1 inhibitors are administered on a multi-day dosing schedule. Utilizing potent (IC50=24–27nM), selective, and orally bio-available small molecule Chk1 inhibitors of which Chk1-A and Chk1-C are representative, we find only modest inhibition of the functional biomarker phospho-cdc2, following a single dose of a Chk1 inhibitor. Alternatively, on multi-day Chk1 inhibitor dose schedules, we find dose-related pharmacodynamic inhibition of Chk1 signaling that is maximized at doses where we see significant tumor growth inhibition in efficacy experiments. Furthermore, multi-day dosing of Chk1 inhibitors induces marked inhibition of phospho-cdc2 and Rad51 protein levels, suggesting tumoricidal activity related to Chk1 inhibition is due to both checkpoint override and impairment of DNA damage repair. In human tumor xenografts administered combination therapy with gemcitabine, an orally-delivered Chk1 inhibitor dosed on a multi-day schedule shows superior efficacy over an IV administered compound. Taken together, our findings show a clear correlative relationship between pharmacodynamic target inhibition and anti-tumor activity that is exclusively achieved on multi-day dose schedules. These results demonstrate the need for prolonged Chk1 inhibition to provide robust pharmacodynamic inhibition and maximal anti-tumor efficacy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B254.

Suppressed expression of non-DSB repair genes inhibits gamma-radiation-induced cytogenetic repair and cell cycle arrest

Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in double-strand break (DSB) repair, and its impact on cytogenetic responses has not been well studied. The purpose of this study is to identify new roles of IR inducible genes in regulating DSB repair and cell cycle progression. In this study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by small interfering RNA in human fibroblast cells. Frequency of micronuclei (MN) formation and chromosome aberrations were measured to determine efficiency of cytogenetic repair, especially DSB repair. In response to IR, the formation of MN was significantly increased by suppressed expression of five genes: Ku70 (DSB repair pathway), XPA (nucleotide excision repair pathway), RPA1 (mismatch repair pathway), RAD17 and RBBP8 (cell cycle control). Knocked-down expression of four genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Moreover, decreased XPA, p21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Nine of these eleven genes, whose knock-down expression affected cytogenetic repair, were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate IR-induced biological consequences. Furthermore, eight non-DBS repair genes showed involvement in regulating DSB repair, indicating that successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. These results reveal that many genes play previously unrecognized roles in multiple DNA repair responses, all of which are required for successful repair of IR-induced damage.

Liver is a Target of Arsenic Carcinogenesis

Inorganic arsenic is clearly a human carcinogen causing tumors of the skin, lung, urinary bladder, and possibly liver (IARC, 2004). At the time of construction of this monograph, the evidence for arsenic as a hepatocarcinogen in humans was considered controversial and in rodents considered insufficient. However, recent data has accumulated indicating hepatocarcinogenicity of arsenic. This forum reevaluates epidemiology studies, rodent studies together with in vitro models, and focuses on the liver as a target organ of arsenic toxicity and carcinogenesis. Hepatocellular carcinoma and hepatic angiosarcoma, have been frequently associated with environmental or medicinal exposure to arsenicals. Preneoplastic lesions, including hepatomegaly, hepatoportal sclerosis, fibrosis, and cirrhosis often occur after chronic arsenic exposure. Recent work in mice clearly shows that exposure to inorganic arsenic during gestation induces tumors, including hepatocellular adenoma and carcinoma, in offspring when they reach adulthood. In rats, the methylated arsenicals, dimethylarsinic acid promotes diethylnitrosamine-initiated liver tumors, whereas trimethylarsine oxide induces liver adenomas. Chronic exposure of rat liver epithelial cells to low concentrations of inorganic arsenic induces malignant transformation, producing aggressive, undifferentiated epithelial tumors when inoculated into the Nude mice. There are a variety of potential mechanisms for arsenical-induced hepatocarcinogenesis, such as oxidative DNA damage, impaired DNA damage repair, acquired apoptotic tolerance, hyperproliferation, altered DNA methylation, and aberrant estrogen signaling. Some of these mechanisms may be liver specific/selective. Overall, accumulating evidence clearly indicates that the liver could be an important target of arsenic carcinogenesis.

Contributions of DNA interstrand cross-links to aging of cells and organisms.

Impaired DNA damage repair, especially deficient transcription-coupled nucleotide excision repair, leads to segmental progeroid syndromes in human patients as well as in rodent models. Furthermore, DNA double-strand break signalling has been pinpointed as a key inducer of cellular senescence. Several recent findings suggest that another DNA repair pathway, interstrand cross-link (ICL) repair, might also contribute to cell and organism aging. Therefore, we summarize and discuss here that (i) systemic administration of anti-cancer chemotherapeutics, in many cases DNA cross-linking drugs, induces premature progeroid frailty in long-term survivors; (ii) that ICL-inducing 8-methoxy-psoralen/UVA phototherapy leads to signs of premature skin aging as prominent long-term side effect and (iii) that mutated factors involved in ICL repair like ERCC1/XPF, the Fanconi anaemia proteins, WRN and SNEV lead to reduced replicative life span in vitro and segmental progeroid syndromes in vivo. However, since ICL-inducing drugs cause damage different from ICL and since all currently known ICL repair factors work in more than one pathway, further work will be needed to dissect the actual contribution of ICL damage to aging.

DNA damage repair in quiescent murine mammary carcinoma cells in culture

Murine mammary carcinoma cells (line 67) were grown in unfed cultures for up to 9 days. In cultures (day 2–3) in which cells were proliferatively active and in day 3–5 (transition) cells, a large fraction of nuclear DNA was retained on polycarbonate filters when assayed by the alkaline filter elution technique. In contrast, the fraction of DNA retained on filters was significantly reduced for nonproliferating (Q, quiescent) cells from unfed 7–9 day cultures. The increase in endogenous DNA breaks followed both the decrease in proliferative state and clonogenicity in these cells. When day 7 Q cells were refed these endogenous DNA breaks were removed with a half-time of about 2.5 h. When the cells were exposed to X-irradiation and the integrity of their nuclear DNA measured by the alkaline filter elution assay, as much as a 2-fold greater frequency of radiation-induced DNA breaks was produced in Q versus P cells. DNA breaks were also removed from irradiated Q cells at a rate which was 0.23 that observed in P cells. We suggest that the depressed capacity for DNA damage removal in Q cells is responsible for their greater radiosensitivity, and the impaired DNA damage repair is probably due to a reduced level of energy sources in these unfed Q cell cultures.