TRIM8 regulated autophagy modulates the level of cleaved Caspase-3 subunit to inhibit genotoxic stress induced cell death

In cancer patients, treatment modalities like chemotherapy and radiation exert their anticancer effects by inducing DNA damage. The cancer cells can survive under genotoxic stress by inducing DNA damage response (DDR) or can undergo cell death. The process of autophagy is emerging as crucial regulator of cell survival during different stress conditions. Post translational modification through ubiquitin plays an essential role in DDR during genotoxic stress conditions. Ubiquitin ligases regulate autophagy and cell death pathways however their role during genotoxic stress conditions is not understood. In the current study we identified TRIM8, RING E3 Ligase, as a novel regulator of autophagy during DDR. TRIM8 regulates lysosomal biogenesis and autophagy flux. The turnover of TRIM8 is high and is stabilized during genotoxic stress conditions. TRIM8 regulated autophagy is essential for its cytoprotective role during genotoxic stress induced cell death. TRIM8 stabilizes the turnover of XIAP during genotoxic stress and forms complex with XIAP and caspase-3 to inhibit its activation in presence of etoposide. TRIM8 mediated autophagy promotes degradation of cleaved caspase-3 subunits. This study described TRIM8, as a novel regulator of DDR-autophagy crosstalk, which may play role in survival of cancer cells in presence of genotoxic agents.     

hMSH5 Facilitates the Repair of Camptothecin-induced Double-strand Breaks through an Interaction with FANCJ

Replication stress from stalled or collapsed replication forks is a major challenge to genomic integrity. The anticancer agent camptothecin (CPT) is a DNA topoisomerase I inhibitor that causes fork collapse and double-strand breaks amid DNA replication. Here we report that hMSH5 promotes cell survival in response to CPT-induced DNA damage. Cells deficient in hMSH5 show elevated CPT-induced γ-H2AX and RPA2 foci with concomitant reduction of Rad51 foci, indicative of impaired homologous recombination. In addition, CPT-treated hMSH5-deficient cells exhibit aberrant activation of Chk1 and Chk2 kinases and therefore abnormal cell cycle progression. Furthermore, the hMSH5-FANCJ chromatin recruitment underlies the effects of hMSH5 on homologous recombination and Chk1 activation. Intriguingly, FANCJ depletion desensitizes hMSH5-deficient cells to CPT-elicited cell killing. Collectively, our data point to the existence of a functional interplay between hMSH5 and FANCJ in double-strand break repair induced by replication stress.     

Pro-tumorigenic function of autophagy in mammary oncogenesis

Autophagy is a highly conserved catabolic cellular process by which cells degrade intracellular constituents in lysosomes, and its dysfunctions have been associated with a variety of human diseases including cancer. Previous studies have linked autophagy to both tumor-suppressive and promoting functions in different contexts, although the pro-tumorigenic function of autophagy has not been examined directly in breast or other cancers in animal models with intact immune functions in vivo. FIP200 (focal adhesion kinase family interacting protein of 200 kD) is a component of the ULK1-Atg13-FIP200-Atg101 complex that is essential for the induction of mammalian autophagy. In our recent study, we show that conditional knockout (KO) of FIP200 in the well-characterized MMTV-PyMT mouse model of human breast cancer significantly suppresses mammary tumorigenesis and progression. Similar to a number of recent studies in Ras-transformed cells, our studies revealed the importance of autophagy in promoting tumorigenesis through regulation of tumor cell glycolysis and proliferation. In addition to the intrinsic defects in proliferation of FIP200-null tumor cells, we also showed that FIP200 deletion in mammary tumor cells triggers increased host anti-tumor immune surveillance, which also contributes to the decreased mammary tumorigenesis and progression. Our study provides the first direct demonstration of a pro-tumorigenic role of autophagy in oncogene-driven tumor models with intact immune functions in vivo. They also suggest FIP200 and other autophagy proteins as potential therapeutic targets for cancer treatment, and raise a number of questions for future studies on the potentially dual functions of autophagy in promoting and suppressing tumorigenesis under different conditions in vivo.     

Role of FIP200 in cardiac and liver development and its regulation of TNFalpha and TSC-mTOR signaling pathways

Focal adhesion kinase family interacting protein of 200 kD (FIP200) has been shown to regulate diverse cellular functions such as cell size, proliferation, and migration in vitro. However, the function of FIP200 in vivo has not been investigated. We show that targeted deletion of FIP200 in the mouse led to embryonic death at mid/late gestation associated with heart failure and liver degeneration. We found that FIP200 knockout (KO) embryos show reduced S6 kinase activation and cell size as a result of increased tuberous sclerosis complex function. Furthermore, FIP200 KO embryos exhibited significant apoptosis in heart and liver. Consistent with this, FIP200 KO mouse embryo fibroblasts and liver cells showed increased apoptosis and reduced c-Jun N-terminal kinase phosphorylation in response to tumor necrosis factor (TNF) alpha stimulation, which might be mediated by FIP200 interaction with apoptosis signal-regulating kinase 1 (ASK1) and TNF receptor-associated factor 2 (TRAF2), regulation of TRAF2-ASK1 interaction, and ASK1 phosphorylation. Together, our results reveal that FIP200 functions as a regulatory node to couple two important signaling pathways to regulate cell growth and survival during mouse embryogenesis.     

Targeting of cancer cell death mechanisms by resveratrol: a review

Cancer cell death is the utmost aim in cancer therapy. Anti-cancer agents can induce apoptosis, mitotic catastrophe, senescence, or autophagy through the production of free radicals and induction of DNA damage. However, cancer cells can acquire some new properties to adapt to anti-cancer agents. An increase in the incidence of apoptosis, mitotic catastrophe, senescence, and necrosis is in favor of overcoming tumor resistance to therapy. Although an increase in the autophagy process may help the survival of cancer cells, some studies indicated that stimulation of autophagy cell death may be useful for cancer therapy. Using some low toxic agents to amplify cancer cell death is interesting for the eradication of clonogenic cancer cells. Resveratrol (a polyphenol agent) may affect various signaling pathways related to cell death. It can induce death signals and also downregulate the expression of anti-apoptotic genes. Resveratrol has also been shown to modulate autophagy and induce mitotic catastrophe and senescence in some cancer cells. This review focuses on the important targets and mechanisms for the modulation of cancer cell death by resveratrol.

     

The human DEK oncogene regulates DNA damage response signaling and repair

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.     

KDM6B promotes PARthanatos via suppression of O6-methylguanine DNA methyltransferase repair and sustained checkpoint response

Poly(ADP-ribose) polymerase-1 (PARP-1) is a DNA damage sensor and contributes to both DNA repair and cell death processes. However, how PARP-1 signaling is regulated to switch its function from DNA repair to cell death remains largely unknown. Here, we found that PARP-1 plays a central role in alkylating agent-induced PARthanatic cancer cell death. Lysine demethylase 6B (KDM6B) was identified as a key regulator of PARthanatos. Loss of KDM6B protein or its demethylase activity conferred cancer cell resistance to PARthanatic cell death in response to alkylating agents. Mechanistically, KDM6B knockout suppressed methylation at the promoter of O⁶-methylguanine-DNA methyltransferase (MGMT) to enhance MGMT expression and its direct DNA repair function, thereby inhibiting DNA damage-evoked PARP-1 hyperactivation and subsequent cell death. Moreover, KDM6B knockout triggered sustained Chk1 phosphorylation and activated a second XRCC1-dependent repair machinery to fix DNA damage evading from MGMT repair. Inhibition of MGMT or checkpoint response re-sensitized KDM6B deficient cells to PARthanatos induced by alkylating agents. These findings provide new molecular insights into epigenetic regulation of PARP-1 signaling mediating DNA repair or cell death and identify KDM6B as a biomarker for prediction of cancer cell vulnerability to alkylating agent treatment.     

TDP1 deficiency sensitizes human cells to base damage via distinct topoisomerase I and PARP mechanisms with potential applications for cancer therapy

Base damage and topoisomerase I (Top1)-linked DNA breaks are abundant forms of endogenous DNA breakage, contributing to hereditary ataxia and underlying the cytotoxicity of a wide range of anti-cancer agents. Despite their frequency, the overlapping mechanisms that repair these forms of DNA breakage are largely unknown. Here, we report that depletion of Tyrosyl DNA phosphodiesterase 1 (TDP1) sensitizes human cells to alkylation damage and the additional depletion of apurinic/apyrimidinic endonuclease I (APE1) confers hypersensitivity above that observed for TDP1 or APE1 depletion alone. Quantification of DNA breaks and clonogenic survival assays confirm a role for TDP1 in response to base damage, independently of APE1. The hypersensitivity to alkylation damage is partly restored by depletion of Top1, illustrating that alkylating agents can trigger cytotoxic Top1-breaks. Although inhibition of PARP activity does not sensitize TDP1-deficient cells to Top1 poisons, it confers increased sensitivity to alkylation damage, highlighting partially overlapping roles for PARP and TDP1 in response to genotoxic challenge. Finally, we demonstrate that cancer cells in which TDP1 is inherently deficient are hypersensitive to alkylation damage and that TDP1 depletion sensitizes glioblastoma-resistant cancer cells to the alkylating agent temozolomide.     

WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and S-phase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was observed for DNA single- and double-strand break formation in response to topotecan. Topotecan induced DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA double-strand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA single-strand breaks into DSBs during the resolution of stalled replication forks at topo I–DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.     

Enhanced killing of cancer cells by poly(ADP-ribose) polymerase inhibitors and topoisomerase I inhibitors reflects poisoning of both enzymes

Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1(-/-) MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1(-/-) MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair.     

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint

Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47^PHOX (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.     

DAPK plays an important role in panobinostat-induced autophagy and commits cells to apoptosis under autophagy deficient conditions

The histone deacetylase inhibitor (HDACi) LBH589 has been verified as an effective anticancer agent. The identification and characterization of new targets for LBH589 action would further enhance our understanding of the molecular mechanisms involved in HDACi therapy. The role of the tumor suppressor death-associated protein kinase (DAPK) in LBH589-induced cytotoxicity has not been investigated to date. Stable DAPK knockdown (shRNA) and DAPK overexpressing (DAPK+++) cell lines were generated from HCT116 wildtype colon cancer cells. LBH589 inhibited cell proliferation, reduced the long-term survival, and up-regulated and activated DAPK in colorectal cancer cells. Moreover, LBH589 significantly suppressed the growth of colon tumor xenografts and in accordance with the in vitro studies, increased DAPK levels were detected immunohistochemically. LBH589 induced a DAPK-dependent autophagy as assessed by punctuate accumulation of LC3-II, the formation of acidic vesicular organelles, and degradation of p62 protein. LBH589-induced autophagy seems to be predominantly caused by DAPK protein interactions than by its kinase activity. Caspase inhibitor zVAD increased autophagosome formation, decreased the cleavage of caspase 3 and PARP but didn’t rescue the cells from LBH589-induced cell death in crystal violet staining suggesting both caspase-dependent as well as caspase-independent apoptosis pathways. Pre-treatment with the autophagy inhibitor Bafilomycin A1 caused caspase 3-mediated apoptosis in a DAPK-dependent manner. Altogether our data suggest that DAPK induces autophagy in response to HDACi-treatment. In autophagy deficient cells, DAPK plays an essential role in committing cells to HDACi-induced apoptosis.     

DNA damage and autophagy

Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.     

Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

The topoisomerase IIα inhibitor etoposide is a ‘broad spectrum’ anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK_cs as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G₂/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G₂/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G₂/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.     

Activity of OGG1 variants in the repair of pro-oxidant-induced 8-oxo-2'-deoxyguanosine

Cells are continuously exposed to damaging reactive oxygen species (ROS), which are produced from both endogenous and exogenous sources. 8-Oxodeoxyguanosine (8-oxodG) is an abundant base lesion formed during oxidative stress which, if not repaired, can give rise to G:C-->T:A transversions in DNA. The 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated base excision repair (BER) pathway operates to remove 8-oxodG lesions. Ogg1 deletion and polymorphism may result in a hypermutator phenotype and susceptibility to oxidative pathologies including cancer. Limited and conflicting evidence exists regarding the repair capacity of a prevalent human OGG1 (hOGG1) polymorphism, the Cys326-hOGG1 variant. The formamidopyrimidine DNA glycosylase (FPG)-modified comet assay was used to investigate the ability of sodium dichromate, potassium bromate and Ro19-8022 (+light) to induce DNA damage in mogg1(-/-) null (KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs) and to assess hOGG1 variant-initiated BER capacities under conditions of oxidative stress. Treatment of WT MEFs with these pro-oxidant agents induced direct DNA strand breaks in a concentration-dependent manner, whereas, identical treatment of KO MEFs produced no effect. In contrast, KO MEFs accumulated significantly more FPG-sensitive sites than WT MEFs. Expression of hOGG1 in KO MEFs restored the WT phenotype in response to all pro-oxidants tested. The results suggest OGG1-initiated BER generates direct DNA strand breaks detected by the conventional comet assay, thus it is important that researchers do not interpret these as direct damage per se but rather a reflection of the repair process. The data also indicate Cys326-hOGG1-initiated BER is transiently impaired with respect to Ser326-hOGG1 (wild-type)- and Gly326-hOGG1 (artificial)-initiated BER following pro-oxidant treatment, possibly via hOGG1 cysteine 326 oxidation. This finding suggests the homozygous cys326/cys326 genotype may be classified as a biomarker of disease susceptibility, which is in support of a growing body of epidemiological evidence.     

GADD34 suppresses wound healing by upregulating expression of myosin IIA

Wound healing consists of sequential steps of tissue repair, and cell migration is particularly important. In order to analyze the potential function of growth arrest and DNA damage inducible protein 34 (GADD34) in tissue repair, we performed in vitro and in vivo wound healing experiments. In an in vitro scratch assay, GADD34 knockout (KO) mouse embryonic fibroblasts (MEFs) had higher migration rates than did wild type (WT) MEFs. Furthermore, the rate of wound closure was faster in GADD34 KO MEFs than in WT MEFs. Using in vivo punch biopsy assays, GADD34 KO mice had accelerated wound healing compared to WT mice. WT mice expressed higher amounts of myosin IIA in migrating macrophages and myofibroblasts than did GADD34 KO mice. These results indicate that GADD34 negatively regulates cell migration in wound healing via expression of myosin IIA.     

Protein Expression of DNA Damage Repair Proteins Dictates Response to Topoisomerase and PARP Inhibitors in Triple-Negative Breast Cancer

Patients with metastatic triple-negative breast cancer (TNBC) have a poor prognosis. New approaches for the treatment of TNBC are needed to improve patient survival. The concept of synthetic lethality, brought about by inactivating complementary DNA repair pathways, has been proposed as a promising therapeutic option for these tumors. The TNBC tumor type has been associated with BRCA mutations, and inhibitors of Poly (ADP-ribose) polymerase (PARP), a family of proteins that facilitates DNA repair, have been shown to effectively kill BRCA defective tumors by preventing cells from repairing DNA damage, leading to a loss of cell viability and clonogenic survival. Here we present preclinical efficacy results of combining the PARP inhibitor, ABT-888, with CPT-11, a topoisomerase I inhibitor. CPT-11 binds to topoisomerase I at the replication fork, creating a bulky adduct that is recognized as damaged DNA. When DNA damage was stimulated with CPT-11, protein expression of the nucleotide excision repair enzyme ERCC1 inversely correlated with cell viability, but not clonogenic survival. However, 4 out of the 6 TNBC cells were synergistically responsive by cell viability and 5 out of the 6 TNBC cells were synergistically responsive by clonogenic survival to the combination of ABT-888 and CPT-11. In vivo, the BRCA mutant cell line MX-1 treated with CPT-11 alone demonstrated significant decreased tumor growth; this decrease was enhanced further with the addition of ABT-888. Decrease in tumor growth correlated with an increase in double strand DNA breaks as measured by γ-H2AX phosphorylation. In summary, inhibiting two arms of the DNA repair pathway simultaneously in TNBC cell lines, independent of BRCA mutation status, resulted in un-repairable DNA damage and subsequent cell death.