The p21CIP1-CDK4-DREAM axis is a master regulator of genotoxic stress-induced cellular senescence.

Cellular senescence, a major driver of aging, can be stimulated by DNA damage, and is counteracted by the DNA repair machinery. Here we show that in p16INK4a-deficient cells, senescence induction by the environmental genotoxin B[a]P or ionizing radiation (IR) completely depends on p21CIP1. Immunoprecipitation-based mass spectrometry interactomics data revealed that during senescence induction and maintenance, p21CIP1 specifically inhibits CDK4 and thereby activates the DREAM complex. Genome-wide transcriptomics revealed striking similarities in the response induced by B[a]P and IR. Among the top 100 repressed genes 78 were identical between B[a]P and IR and 76 were DREAM targets. The DREAM complex transcriptionally silences the main proliferation-associated transcription factors E2F1, FOXM1 and B-Myb as well as multiple DNA repair factors. Knockdown of p21CIP1, E2F4 or E2F5 diminished both, repression of these factors and senescence. The transcriptional profiles evoked by B[a]P and IR largely overlapped with the profile induced by pharmacological CDK4 inhibition, further illustrating the role of CDK4 inhibition in genotoxic stress-induced senescence. Moreover, data obtained by live-cell time-lapse microscopy suggest the inhibition of CDK4 by p21CIP1 is especially important for arresting cells which slip through mitosis. Overall, we identified the p21CIP1/CDK4/DREAM axis as a master regulator of genotoxic stress-induced senescence.

Genotoxic and Cytotoxic Activity of Fisetin on Glioblastoma Cells.

BACKGROUND/AIM: Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells. MATERIALS AND METHODS: We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis. RESULTS: Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 µM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells. CONCLUSION: Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.

DNA Alkylation Damage by Nitrosamines and Relevant DNA Repair Pathways.

Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as impurities in various drugs. This is of particular concern as nitrosamines are alkylating agents that are genotoxic and carcinogenic. We first summarize the current knowledge on the different sources and chemical nature of alkylating agents with a focus on relevant nitrosamines. Subsequently, we present the major DNA alkylation adducts induced by nitrosamines upon their metabolic activation by CYP450 monooxygenases. We then describe the DNA repair pathways engaged by the various DNA alkylation adducts, which include base excision repair, direct damage reversal by MGMT and ALKBH, as well as nucleotide excision repair. Their roles in the protection against the genotoxic and carcinogenic effects of nitrosamines are highlighted. Finally, we address DNA translesion synthesis as a DNA damage tolerance mechanism relevant to DNA alkylation adducts.

Expression of DNA repair genes and its relevance for DNA repair in peripheral immune cells of patients with posttraumatic stress disorder.

Posttraumatic stress disorder (PTSD) involves elevated levels of cellular oxidative stress which jeopardizes the integrity of essential cell compartments. Previously, we demonstrated higher levels of DNA lesions in peripheral blood mononuclear cells (PBMCs) in PTSD. Retaining vital levels of DNA integrity requires cells to mobilize compensatory efforts in elevating their DNA-repair capacity. Accordingly, we hypothesized to find increased expression rates of the DNA-repair genes X-ray repair cross complementing 1 (XRCC1), poly (ADP-ribose) polymerase 1 (PARP1), and polymerase ß (Polß) in PBMCs of PTSD patients as compared to controls, leading to functionally relevant changes in DNA-repair kinetics. In a cohort of 14 refugees with PTSD and 15 without PTSD, we found significantly higher XRCC1 expression in PTSD patients than controls (U = 161.0, p = 0.009, Cohen's r = 0.49), and positive correlations between the severity of PTSD symptoms and the expression of XRCC1 (rS = 0.57, p = 0.002) and PARP1 (rS = 0.43, p = 0.022). Higher XRCC1 (F = 2.39, p = 0.010, η2p = 0.10) and PARP1 (F = 2.15, p = 0.022, η2p = 0.09) expression accounted for slower repair of experimentally X-ray irradiation-induced DNA damage, highlighting the possible physiological relevance of altered DNA-repair gene expression in PTSD. Our study provides first evidence for a compensatory regulation of DNA-repair mechanisms in PTSD. We discuss the implications of increased DNA damage and altered DNA-repair mechanisms in immune senescence, premature aging, and increased physical morbidity in PTSD.

p53 triggers mitochondrial apoptosis following DNA damage-dependent replication stress by the hepatotoxin methyleugenol.

Liver cancer is one of the most frequent tumor entities worldwide, which is causally linked to viral infection, fatty liver disease, life-style factors and food-borne carcinogens, particularly aflatoxins. Moreover, genotoxic plant toxins including phenylpropenes are suspected human liver carcinogens. The phenylpropene methyleugenol (ME) is a constituent of essential oils in many plants and occurs in herbal medicines, food, and cosmetics. Following its uptake, ME undergoes Cytochrome P450 (CYP) and sulfotransferase 1A1 (SULT1A1)-dependent metabolic activation, giving rise to DNA damage. However, little is known about the cellular response to the induced DNA adducts. Here, we made use of different SULT1A1-proficient cell models including primary hepatocytes that were treated with 1'-hydroxymethyleugenol (OH-ME) as main phase I metabolite. Firstly, mass spectrometry showed a concentration-dependent formation of N2-MIE-dG as major DNA adduct, strongly correlating with SULT1A1 expression as attested in cells with and without human SULT1A1. ME-derived DNA damage activated mainly the ATR-mediated DNA damage response as shown by phosphorylation of CHK1 and histone 2AX, followed by p53 accumulation and CHK2 phosphorylation. Consistent with these findings, the DNA adducts decreased replication speed and caused replication fork stalling. OH-ME treatment reduced viability particularly in cell lines with wild-type p53 and triggered apoptotic cell death, which was rescued by pan-caspase-inhibition. Further experiments demonstrated mitochondrial apoptosis as major cell death pathway. ME-derived DNA damage caused upregulation of the p53-responsive genes NOXA and PUMA, Bax activation, and cytochrome c release followed by caspase-9 and caspase-3 cleavage. We finally demonstrated the crucial role of p53 for OH-ME triggered cell death as evidenced by reduced pro-apoptotic gene expression, strongly attenuated Bax activation and cell death inhibition upon genetic knockdown or pharmacological inhibition of p53. Taken together, our study demonstrates for the first time that ME-derived DNA damage causes replication stress and triggers mitochondrial apoptosis via the p53-Bax pathway.

Abrogation of Cellular Senescence Induced by Temozolomide in Glioblastoma Cells: Search for Senolytics.

A first-line therapeutic for high-grade glioma, notably glioblastoma (GBM), is the DNA methylating drug temozolomide (TMZ). Previously, we showed that TMZ induces not only apoptosis and autophagy, but also cellular senescence (CSEN). We presented the hypothesis that GBM cells may escape from CSEN, giving rise to recurrent tumors. Furthermore, the inflammatory phenotype associated with CSEN may attenuate chemotherapy and drive tumor progression. Therefore, treatments that specifically target senescent cells, i.e., senolytic drugs, may lead to a better outcome of GBM therapy by preventing recurrences and tumor inflammation. Here, we tested Bcl-2 targeting drugs including ABT-737, ABT-263 (navitoclax), several natural substances such as artesunate, fisetin and curcumin as well as lomustine (CCNU) and ionizing radiation (IR) for their senolytic capacity in GBM cells. Additionally, several proteins involved in the DNA damage response (DDR), ATM, ATR, Chk1/2, p53, p21, NF-kB, Rad51, PARP, IAPs and autophagy, a pathway involved in CSEN induction, were tested for their impact in maintaining CSEN. Treatment of GBM cells with a low dose of TMZ for 8-10 days resulted in >80% CSEN, confirming CSEN to be the major trait induced by TMZ. To identify senolytics, we treated the senescent population with the compounds of interest and found that ABT-737, navitoclax, chloroquine, ATMi, ATRi, BV-6, PX-866 and the natural compounds fisetin and artesunate exhibit senolytic activity, inducing death in senescent cells more efficiently than in proliferating cells. Curcumin showed the opposite effect. No specific effect on CSEN cells was observed by inhibition of Chk1/Chk2, p21, NF-kB, Rad51 and PARP. We conclude that these factors neither play a critical role in maintaining TMZ-induced CSEN nor can their inhibitors be considered as senolytics. Since IR and CCNU did not exhibit senolytic activity, radio- and chemotherapy with alkylating drugs is not designed to eliminate TMZ-induced senescent cancer cells.

Senescence Is the Main Trait Induced by Temozolomide in Glioblastoma Cells.

First-line drug in the treatment of glioblastoma, the most severe brain cancer, is temozolomide (TMZ), a DNA-methylating agent that induces the critical damage O6-methylguanine (O6MeG). This lesion is cytotoxic through the generation of mismatch repair-mediated DNA double-strand breaks (DSBs), which trigger apoptotic pathways. Previously, we showed that O6MeG also induces cellular senescence (CSEN). Here, we show that TMZ-induced CSEN is a late response which has similar kinetics to apoptosis, but at a fourfold higher level. CSEN cells show a high amount of DSBs, which are located outside of telomeres, a high level of ROS and oxidized DNA damage (8-oxo-guanine), and sustained activation of the DNA damage response and histone methylation. Despite the presence of DSBs, CSEN cells are capable of repairing radiation-induced DSBs. Glioblastoma cells that acquired resistance to TMZ became simultaneously resistant to TMZ-induced CSEN. Using a Tet-On glioblastoma cell system, we show that upregulation of MGMT immediately after TMZ completely abrogated apoptosis and CSEN, while induction of MGMT long-term (>72 h) after TMZ did not reduce apoptosis and CSEN. Furthermore, upregulation of MGMT in the senescent cell population had no impact on the survival of senescent cells, indicating that O6MeG is required for induction, but not for maintenance of the senescent state. We further show that, in recurrent GBM specimens, a significantly higher level of DSBs and CSEN-associated histone H3K27me3 was observed than in the corresponding primary tumors. Overall, the data indicate that CSEN is a key node induced in GBM following chemotherapy.

Alterations in Molecular Profiles Affecting Glioblastoma Resistance to Radiochemotherapy: Where Does the Good Go?

Glioblastoma multiforme (GBM) is a brain tumor characterized by high heterogeneity, diffuse infiltration, aggressiveness, and formation of recurrences. Patients with this kind of tumor suffer from cognitive, emotional, and behavioral problems, beyond exhibiting dismal survival rates. Current treatment comprises surgery, radiotherapy, and chemotherapy with the methylating agent, temozolomide (TMZ). GBMs harbor intrinsic mutations involving major pathways that elicit the cells to evade cell death, adapt to the genotoxic stress, and regrow. Ionizing radiation and TMZ induce, for the most part, DNA damage repair, autophagy, stemness, and senescence, whereas only a small fraction of GBM cells undergoes treatment-induced apoptosis. Particularly upon TMZ exposure, most of the GBM cells undergo cellular senescence. Increased DNA repair attenuates the agent-induced cytotoxicity; autophagy functions as a pro-survival mechanism, protecting the cells from damage and facilitating the cells to have energy to grow. Stemness grants the cells capacity to repopulate the tumor, and senescence triggers an inflammatory microenvironment favorable to transformation. Here, we highlight this mutational background and its interference with the response to the standard radiochemotherapy. We discuss the most relevant and recent evidence obtained from the studies revealing the molecular mechanisms that lead these cells to be resistant and indicate some future perspectives on combating this incurable tumor.

The Mitochondrial Disruptor Devimistat (CPI-613) Synergizes with Genotoxic Anticancer Drugs in Colorectal Cancer Therapy in a Bim-Dependent Manner.

Colorectal cancer is one of the most frequent tumor entities, with an increasing incidence and mortality in younger adults in Europe and the United States. Five-year survival rates for advanced colorectal cancer are still low, highlighting the need for novel targets in colorectal cancer therapy. Here, we investigated the therapeutic potential of the compound devimistat (CPI-613) that targets altered mitochondrial cancer cell metabolism and its synergism with the antineoplastic drugs 5-fluorouracil (5-FU) and irinotecan (IT) in colorectal cancer. Devimistat exerted a comparable cytotoxicity in a panel of established colorectal cancer cell lines and patient-derived short-term cultures independent of their genetic and epigenetic status, whereas human colonic epithelial cells were more resistant, indicating tumor selectivity. These findings were corroborated in intestinal organoid and tumoroid models. Mechanistically, devimistat disrupted mitochondrial membrane potential and severely impaired mitochondrial respiration, resulting in colorectal cancer cell death induction independent of p53. Combination treatment of devimistat with 5-FU or IT demonstrated synergistic cell killing in colorectal cancer cells as shown by Combenefit modeling and Chou-Talalay analysis. Increased cell death induction was revealed as a major mechanism involving downregulation of antiapoptotic genes and accumulation of proapoptotic Bim, which was confirmed by its genetic knockdown. In human colorectal cancer xenograft mouse models, devimistat showed antitumor activity and synergized with IT, resulting in prolonged survival and enhanced therapeutic efficacy. In human tumor xenografts, devimistat prevented IT-triggered p53 stabilization and caused synergistic Bim induction. Taken together, our study revealed devimistat as a promising candidate in colorectal cancer therapy by synergizing with established antineoplastic drugs in vitro and in vivo.

Identification of a highly efficient dual type I/II FMS-like tyrosine kinase inhibitor that disrupts the growth of leukemic cells.

Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.

Natural Merosesquiterpenes Activate the DNA Damage Response via DNA Strand Break Formation and Trigger Apoptotic Cell Death in p53-Wild-type and Mutant Colorectal Cancer.

Colorectal cancer (CRC) is a frequently occurring malignant disease with still low survival rates, highlighting the need for novel therapeutics. Merosesquiterpenes are secondary metabolites from marine sponges, which might be useful as antitumor agents. To address this issue, we made use of a compound library comprising 11 isolated merosesquiterpenes. The most cytotoxic compounds were smenospongine > ilimaquinone ≈ dactylospontriol, as shown in different human CRC cell lines. Alkaline Comet assays and γH2AX immunofluorescence microscopy demonstrated DNA strand break formation in CRC cells. Western blot analysis revealed an activation of the DNA damage response with CHK1 phosphorylation, stabilization of p53 and p21, which occurred both in CRC cells with p53 knockout and in p53-mutated CRC cells. This resulted in cell cycle arrest followed by a strong increase in the subG1 population, indicative of apoptosis, and typical morphological alterations. In consistency, cell death measurements showed apoptosis following exposure to merosesquiterpenes. Gene expression studies and analysis of caspase cleavage revealed mitochondrial apoptosis via BAX, BIM, and caspase-9 as the main cell death pathway. Interestingly, the compounds were equally effective in p53-wild-type and p53-mutant CRC cells. Finally, the cytotoxic activity of the merosesquiterpenes was corroborated in intestinal tumor organoids, emphasizing their potential for CRC chemotherapy.

Targeting c-IAP1, c-IAP2, and Bcl-2 Eliminates Senescent Glioblastoma Cells Following Temozolomide Treatment.

Therapy of malignant glioma depends on the induction of O6-methylguanine by the methylating agent temozolomide (TMZ). However, following TMZ exposure, most glioma cells evade apoptosis and become senescent and are thereby protected against further anticancer therapy. This protection is thought to be dependent on the senescent cell anti-apoptotic pathway (SCAP). Here we analyzed the factors involved in the SCAP upon exposure to TMZ in glioblastoma cell lines (LN-229, A172, U87MG) and examined whether inhibition of these factors could enhance TMZ-based toxicity by targeting senescent cells. We observed that following TMZ treatment, c-IAP2 and Bcl-2 were upregulated. Inhibition of these SCAP factors using non-toxic concentrations of the small molecule inhibitors, BV6 and venetoclax, significantly increased cell death, as measured 144 h after TMZ exposure. Most importantly, BV6 and venetoclax treatment of senescent cells strongly increased cell death after an additional 120 h. Moreover, Combenefit analyses revealed a significant synergy combining BV6 and venetoclax. In contrast to BV6 and venetoclax, AT406, embelin, and TMZ itself, teniposide and the PARP inhibitor pamiparib did not increase cell death in senescent cells. Based on these data, we suggest that BV6 and venetoclax act as senolytic agents in glioblastoma cells upon TMZ exposure.

Repair of O6-carboxymethylguanine adducts by O6-methylguanine-DNA methyltransferase in human colon epithelial cells.

The protein O6-methylguanine-DNA methyltransferase (MGMT) is able to repair the mutagenic O6-methylguanine (O6-MeG) adduct back to guanine. In this context, it may protect against colorectal cancer formation associated with N-nitroso compounds. Such compounds may be endogenously formed by nitrosylation of amino acids, which can give rise to mutagenic O6-MeG and O6-carboxymethylguanine (O6-CMG) adducts. It is well established that O6-MeG is repaired by MGMT. However, up to now, whether O6-CMG is repaired by this enzyme remains unresolved. Therefore, the aim of the present study was to analyze the fate of both types of O6-guanine adducts in the presence and absence of MGMT activity. To this end, MGMT activity was efficiently blocked by its chemical inhibitor O6-benzylguanine in human colon epithelial cells (HCECs). Exposure of cells to azaserine (AZA) caused significantly higher levels of both O6-MeG and O6-CMG adducts in MGMT-inhibited cells, with O6-CMG as the more abundant DNA lesion. Interestingly, MGMT inhibition did not result in higher levels of AZA-induced DNA strand breaks in spite of elevated DNA adduct levels. In contrast, MGMT inhibition significantly increased DNA strand break formation after exposure to temozolomide (TMZ), a drug that exclusively generates O6-MeG adducts. In line with this finding, the viability of the cells was moderately reduced by TMZ upon MGMT inhibition, whereas no clear effect was observed in cells treated with AZA. In conclusion, our study clearly shows that O6-CMG is repaired by MGMT in HCEC, thereby suggesting that MGMT might play an important role as a tumor suppressor in diet-mediated colorectal cancer.

Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination.

To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

Oxaliplatin-Induced Senescence in Colorectal Cancer Cells Depends on p14ARF-Mediated Sustained p53 Activation.

Senescence is an important consequence of cytostatic drug-based tumor therapy. Here we analyzed to which degree the anticancer drug oxaliplatin induces cell death, cell cycle arrest, and senescence in colorectal cancer (CRC) cells and elucidated the role of p53. Oxaliplatin treatment resulted in the G2-phase arrest in all CRC lines tested (HCT116p53+/+, HCT116p53-/-, LoVo, SW48 and SW480). Immunoblot analysis showed that within the p53-competent lines p53 and p21CIP1 are activated at early times upon oxaliplatin treatment. However, at later times, only LoVo cells showed sustained activation of the p53/p21CIP1 pathway, accompanied by a strong induction of senescence as measured by senescence-associated ß-Gal staining and induction of senescence-associated secretory phenotype (SASP) factors. Opposite to LoVo, the p53/p21CIP1 response and senescence induction was much weaker in the p53-proficient SW48 and SW480 cells, which was due to deficiency for p14ARF. Thus, among lines studied only LoVo express p14ARF protein and siRNA-mediated knockdown of p14ARF significantly reduced sustained p53/p21CIP1 activation and senescence. Vice versa, ectopic p14ARF expression enhanced oxaliplatin-induced senescence in SW48 and SW480 cells. Our data show that oxaliplatin-induced senescence in CRC cells is dependent on p53 proficiency; however, a significant induction can only be observed upon p14ARF-mediated p53 stabilization.

Benzo[a]pyrene represses DNA repair through altered E2F1/E2F4 function marking an early event in DNA damage-induced cellular senescence.

Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.

Lipoic Acid Synergizes with Antineoplastic Drugs in Colorectal Cancer by Targeting p53 for Proteasomal Degradation.

Lipoic acid (LA) is a redox-active disulphide compound, which functions as a pivotal co-factor for mitochondrial oxidative decarboxylation. LA and chemical derivatives were shown to target mitochondria in cancer cells with altered energy metabolism, thereby inducing cell death. In this study, the impact of LA on the tumor suppressor protein p53 was analyzed in various colorectal cancer (CRC) cell lines, with a focus on the mechanisms driving p53 degradation. First, LA was demonstrated to trigger the depletion of both wildtype and mutant p53 protein in all CRC cells tested without influencing its gene expression and preceded LA-triggered cytotoxicity. Depletion of p53 coincided with a moderate, LA-dependent ROS production, but was not rescued by antioxidant treatment. LA induced the autophagy receptor p62 and differentially modulated autophagosome formation in CRC cells. However, p53 degradation was not mediated via autophagy as shown by chemical inhibition and genetic abrogation of autophagy. LA treatment also stabilized and activated the transcription factor Nrf2 in CRC cells, which was however dispensable for p53 degradation. Mechanistically, p53 was found to be readily ubiquitinylated and degraded by the proteasomal machinery following LA treatment, which did not involve the E3 ubiquitin ligase MDM2. Intriguingly, the combination of LA and anticancer drugs (doxorubicin, 5-fluorouracil) attenuated p53-mediated stabilization of p21 and resulted in synergistic killing in CRC cells in a p53-dependant manner.

Epigenetic regulation of DNA repair genes and implications for tumor therapy.

DNA repair represents the first barrier against genotoxic stress causing metabolic changes, inflammation and cancer. Besides its role in preventing cancer, DNA repair needs also to be considered during cancer treatment with radiation and DNA damaging drugs as it impacts therapy outcome. The DNA repair capacity is mainly governed by the expression level of repair genes. Alterations in the expression of repair genes can occur due to mutations in their coding or promoter region, changes in the expression of transcription factors activating or repressing these genes, and/or epigenetic factors changing histone modifications and CpG promoter methylation or demethylation levels. In this review we provide an overview on the epigenetic regulation of DNA repair genes. We summarize the mechanisms underlying CpG methylation and demethylation, with de novo methyltransferases and DNA repair involved in gain and loss of CpG methylation, respectively. We discuss the role of components of the DNA damage response, p53, PARP-1 and GADD45a on the regulation of the DNA (cytosine-5)-methyltransferase DNMT1, the key enzyme responsible for gene silencing. We stress the relevance of epigenetic silencing of DNA repair genes for tumor formation and tumor therapy. A paradigmatic example is provided by the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT), which is silenced in up to 40% of various cancers through CpG promoter methylation. The CpG methylation status of the MGMT promoter strongly correlates with clinical outcome and, therefore, is used as prognostic marker during glioblastoma therapy. Mismatch repair genes are also subject of epigenetic silencing, which was shown to correlate with colorectal cancer formation. For many other repair genes shown to be epigenetically regulated the clinical outcome is not yet clear. We also address the question of whether genotoxic stress itself can lead to epigenetic alterations of genes encoding proteins involved in the defense against genotoxic stress.

Functional mismatch repair and inactive p53 drive sensitization of colorectal cancer cells to irinotecan via the IAP antagonist BV6.

A common strategy to overcome acquired chemotherapy resistance is the combination of a specific anticancer drug (e.g., topoisomerase I inhibitor irinotecan) together with a putative sensitizer. The purpose of this study was to analyze the cytostatic/cytotoxic response of colorectal carcinoma (CRC) cells to irinotecan, depending on the mismatch repair (MMR) and p53 status and to examine the impact of BV6, a bivalent antagonist of inhibitors of apoptosis c-IAP1/c-IAP2, alone or combined with irinotecan. Therefore, several MSH2- or MSH6-deficient cell lines were complemented for MMR deficiency, or MSH6 was knocked out/down in MMR-proficient cells. Upon irinotecan, MMR-deficient/p53-mutated lines repaired DNA double-strand breaks by homologous recombination less efficiently than MMR-proficient/p53-mutated lines and underwent elevated caspase-9-dependent apoptosis. Opposite, BV6-mediated sensitization was achieved only in MMR-proficient/p53-mutated cells. In those cells, c-IAP1 and c-IAP2 were effectively degraded by BV6, caspase-8 was fully activated, and both canonical and non-canonical NF-κB signaling were triggered. The results were confirmed ex vivo in tumor organoids from CRC patients. Therefore, the particular MMR+/p53mt signature, often found in non-metastasizing (stage II) CRC might be used as a prognostic factor for an adjuvant therapy using low-dose irinotecan combined with a bivalent IAP antagonist.