Genotoxic Stress-induced Apoptosis Research Articles

TCTP regulates genotoxic stress and tumorigenicity via intercellular vesicular signaling

Oncogenic intercellular signaling is regulated by extracellular vesicles (EVs), but the underlying mechanisms remain mostly unclear. Since TCTP (translationally controlled tumor protein) is an EV component, we investigated whether it has a role in genotoxic stress signaling and malignant transformation. By generating a Tctp-inducible knockout mouse model (Tctp–/f–), we report that Tctp is required for genotoxic stress-induced apoptosis signaling via small EVs (sEVs). Human breast cancer cells knocked-down for TCTP show impaired spontaneous EV secretion, thereby reducing sEV-dependent malignant growth. Since Trp53–/– mice are prone to tumor formation, we derived tumor cells from Trp53–/–;Tctp–/f– double mutant mice and describe a drastic decrease in tumori-genicity with concomitant decrease in sEV secretion and content. Remarkably, Trp53–/–;Tctp–/f– mice show highly prolonged survival. Treatment of Trp53–/– mice with sertraline, which inhibits TCTP function, increases their survival. Mechanistically, TCTP binds DDX3, recruiting RNAs, including miRNAs, to sEVs. Our findings establish TCTP as an essential protagonist in the regulation of sEV-signaling in the context of apoptosis and tumorigenicity.

Chromatin-Associated SIN3B Protects Cancer Cells from Genotoxic Stress-Induced Apoptosis and Dictates DNA Damage Repair Pathway Choice.

Identifying SIN3B as a modulator of DNA damage repair choice provides novel potential therapeutic avenues to sensitize cancer cells to cytotoxic therapies.

Wip1 phosphatase inhibition enhances etoposide mediated apoptosis by increasing p53 phosphorylation in Rhabdoid tumor cells

Protein phosphatase magnesium-dependent 1 delta (PPM1D/Wip1) is a genotoxic stress-activated serine/threonine phosphatase playing a central role in the timely termination of DNA damage response (DDR). Wip1 is frequently mutated, amplified, and overexpressed in common human cancers and is recognized as an oncogene. Rhabdoid tumors (RT) are a type of soft tissue sarcoma being quite aggressive and resistant to chemotherapy. In this study, the role of Wip1 in the regulation of genotoxic stress-induced apoptosis was studied in RT cell line A204. First PPM1D mRNA levels were analyzed in RT compared to normal tissues by in silico analysis utilizing RNA-seq data generated by TARGET from TCGA Pan Cancer. Second, MCF-7, BJ, and A204 cells were used to test the gene copy number and mRNA levels of Wip1 by qRT-PCR. Small molecule inhibitor GSK2830371 and etoposide were used to target Wip1 and to trigger genotoxic stress, respectively. MTT and AnexinV/7AAD analyzes were used to determine the viability and apoptotic response of A204 cells. Inhibition of Wip1 and total and phosphorylated levels of p53 was demonstrated by WB analysis. Thus, here we showed that Wip1 is overexpressed and amplified in the A204 RT cell line. Wip1 overexpressing A204 cells display resistance to etoposide-induced apoptosis due to decreased phosphorylation of p53. Targeting of Wip1 by GSK2830371 increased the phosphorylation of p53 in response to etoposide treatment. Additionally combined use of GSK2830371 and etoposide enhances p53-mediated apoptosis in A204 cells.

The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions

Caspase-2 represents an evolutionary conserved caspase, which plays a role in genotoxic stress-induced apoptosis, ageing-related metabolic changes, and in deleting aneuploid cells in tumors. Genetic deletion of caspase-2 leads to increased tumor susceptibility in vivo. The exact downstream signaling mechanism by which caspase-2 accomplishes its specific tumor suppressor functions is not clear. Caspase-2, uniquely among caspases, resides in the nucleus and other cellular compartments. In this study, we identify a nuclear caspase-2 specific substrate, p54nrb, which is selectively cleaved by caspase-2 at D422, leading to disruption of the C-terminal site, the putative DNA binding region of the protein. P54nrb is an RNA and DNA binding protein, which plays a role in RNA editing, transport, and transcriptional regulation of genes. Overexpression of p54nrb is observed in several human tumor types, such as cervix adenocarcinoma, melanoma, and colon carcinoma. In contrast, the loss of p54nrb in tumor cell lines leads to increased cell death susceptibility and striking decrease in tumorigenic potential. By employing high resolution quantitative proteomics, we demonstrate that the loss/cleavage of p54nrb results in altered expression of oncogenic genes, among which the downregulation of the tumorigenic protease cathepsin-Z and the anti-apoptotic gelsolin can be detected universally across three tumor cell types, including adenocarcinoma, melanoma and colon carcinoma. Finally, we demonstrate that p54nrb interacts with cathepsin-Z and gelsolin DNA, but not RNA. Taken together, this study uncovers a so far not understood mechanism of caspase-2 tumor suppressor function in human tumor cells.

Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.

Involvement of Upregulated P53-Induced Death Domain Protein in Retinal Ganglion Cells Apoptosis After Optic Nerve Crush.

Retinal ganglion cells (RGCs) apoptosis is a common characteristic of optic neuropathies. p53-induced protein with a death domain (PIDD) is a well-known regulator of genotoxic stress-induced apoptosis, which is constitutively cleaved into three main fragments: PIDD-N, PIDD-C and PIDD-CC. Thus, we aim to determine the physiological relevance of PIDD in RGCs apoptosis in an optic nerve crush (ONC) model. All animals were evenly randomized into four groups: sham-control group, con-siRNA group, ONC group, and PIDD-siRNA group (ONC +PIDD-siRNA). Expressions of PIDD, caspase-2, Brn3a and tBid in ONC model were analyzed by Western blot and immunofluorescence. Mean densities of RGCs/mm2 were calculated with Fluoro-Gold (FG). Moreover, we tested the effect of PIDD-siRNA on ONC-induced RGCs apoptosis using TUNEL staining. The level of full-length PIDD was weakly present and showed no significant differences at any time points. PIDD-CC and PIDD-C were significantly up-regulated in the retina at 3 days after ONC. Meanwhile, the expression of PIDD was significantly increased in Brn3a (a marker of RGCs) positive cells, indicating that the localization of PIDD appeared to be confined to RGCs. Furthermore, inhibition of PIDD prevented RGCs apoptosis by inhibiting caspase-2 and tBid activation. Taken together, PIDD may play a crucial role in RGCs apoptosis after ONC, and this process may be relevant to caspase-2 and tBid.

ΔN-ASPP2, a novel isoform of the ASPP2 tumor suppressor, promotes cellular survival

ASPP2 is a tumor suppressor that works, at least in part, through enhancing p53-dependent apoptosis. We now describe a new ASPP2 isoform, ΔN-ASPP2, generated from an internal transcription start site that encodes an N-terminally truncated protein missing a predicted 254 amino acids. ΔN-ASPP2 suppresses p53 target gene transactivation, promoter occupancy, and endogenous p53 target gene expression in response to DNA damage. Moreover, ΔN-ASPP2 promotes progression through the cell cycle, as well as resistance to genotoxic stress-induced growth inhibition and apoptosis. Additionally, we found that ΔN-ASPP2 expression is increased in human breast tumors as compared to adjacent normal breast tissue; in contrast, ASPP2 is suppressed in the majority of these breast tumors. Together, our results provide insight into how this new ASPP2 isoform may play a role in regulating the ASPP2-p53 axis.

Single-Cell Gene Expression Analysis Identifies Alcam As a Novel Candidate Therapeutic Target in AML

Acute myeloid leukemia (AML) is the most common acute leukemia in adults, characterized by high rate of relapse even in recent days. AML is organized in a hierarchy of heterogeneous cell populations and relapse is generally attributable to chemoresistant leukemia stem cells (LSCs). Anthracyclins and cytarabine, which cannot eliminate LSCs, have remained the mainstay of AML therapy for almost 40 years and novel drugs targeting LSCs more efficiently are urgently needed. Toward that end, a potential strategy is to identify cell surface markers that can be differentially regulated in LSCs within the bulk of leukemic cells. Here in this study, focusing on heterogeneity of AML cells, we exploited single-cell gene expression analysis to find a novel cell surface antigen differentially expressed in LSCs, which might be missed by analyses as a whole.Firstly, we proposed "single-cell leukemia initiating cell (LIC) score", defined as sum of mRNA expression levels of 15 genes specifically expressed in functionally-determined LICs according to public transcriptome datasets (GSE30375), and graded primary AML samples on a single-cell basis. Single CD34+ CD38- cells (n = 66) and CD34+ CD38+ cells (n = 30) from bone marrow (BM) of two AML patients were analyzed. Single-cell gene expression analysis using Fluidigm technology revealed that within 50 candidate cell surface molecules highly expressed in LICs according to public datasets, expression of 19 genes were positively related to the LIC score. Among them, we focused on ALCAM (Activated Leucocyte Cell Adhesion, CD166), a cell adhesion molecule implicated in tumorigenesis, because survival analyses using public datasets showed that high expression of ALCAM was associated with shorter overall survival in AML.When we examined cell-surface expression of ALCAM in undifferentiated AML cells, ALCAM was highly expressed on CD34+ AML cells compared with CD34+ normal hematopoietic cells. We separated BM cells from AML patients into CD34+ ALCAM high fraction and CD34+ ALCAM low fraction. Colony-forming activities were enriched in CD34+ ALCAM high fraction in vitro (n = 5). And then, we did xenotransplantation model by intravenous injection of human AML BM cells to NSG mice. We show that LICs tended to be enriched within CD34+ ALCAM high fraction in some primary AML samples by using xenotransplantation model.To elucidate whether ALCAM exerts any biological functions and could be a novel therapeutic target in AML, we established human THP-1 AML cells in which expression of ALCAM was silenced by short hairpin RNA (shRNA). Knockdown of ALCAM reduced proliferation and enhanced chemosensitivity of THP-1 cells to cytarabine by curbing genotoxic stress-induced apoptosis in vitro. THP-1 cells expressing shRNA against ALCAM also showed decreased leukomogenic capacity in a xenotransplantation model. When we suppressed activities of ALCAM using a soluble isoform of ALCAM (soluble ALCAM), which binds to ALCAM and inhibits intercellular ALCAM-ALCAM hemophilic interactions, THP-1 cells expressing soluble ALCAM also showed decreased proliferation, enhanced chemosensitivity and decreased leukomogeneic capacity. Furthermore, we transplanted human primary AML cells transduced with a tetracycline-inducible lentiviral construct encoding soluble ALCAM into NSG mice. After engraftment of human cells, expression of soluble ALCAM was induced. BM engraftment of human primary AML cells expressing soluble ALCAM was reduced, which indicated that decreased BM engraftment was not due to impaired homing, but to leukemogenic ability.These findings implicate that LICs with high clonogenic capacity were enriched within CD34+ ALCAM high fraction and ALCAM could be a novel therapeutic target of AML cells. Furthermore, our results also suggests single-cell gene expression analysis and a scoring method such as "LIC score" could be applicable to other hematological malignancies and other cancers to identify and exploit a property of heterogenous cancer cells. DisclosuresNo relevant conflicts of interest to declare.

Speaker Presentations

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1−/− cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase–like disease in mice compared with less malignant CML-chronic phase–like disease from BCR-ABL1-Abl1+/+ cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

Sam68/KHDRBS1 is critical for colon tumorigenesis by regulating genotoxic stress-induced NF-κB activation.

Nuclear factor kappa B (NF-κB)-mediated transcription is an important mediator for cellular responses to DNA damage. Genotoxic agents trigger a 'nuclear-to-cytoplasmic' NF-κB activation signaling pathway; however, the early nuclear signaling cascade linking DNA damage and NF-κB activation is poorly understood. Here we report that Src-associated-substrate-during-mitosis-of-68kDa/KH domain containing, RNA binding, signal transduction associated 1 (Sam68/KHDRBS1) is a key NF-κB regulator in genotoxic stress-initiated signaling pathway. Sam68 deficiency abolishes DNA damage-stimulated polymers of ADP-ribose (PAR) production and the PAR-dependent NF-κB transactivation of anti-apoptotic genes. Sam68 deleted cells are hypersensitive to genotoxicity caused by DNA damaging agents. Upregulated Sam68 coincides with elevated PAR production and NF-κB-mediated anti-apoptotic transcription in human and mouse colon cancer. Knockdown of Sam68 sensitizes human colon cancer cells to genotoxic stress-induced apoptosis and genetic deletion of Sam68 dampens colon tumor burden in mice. Together our data reveal a novel function of Sam68 in the genotoxic stress-initiated nuclear signaling, which is crucial for colon tumorigenesis.

Abstract 3532: Compound Tc inhibits ATG4 to attenuate autophagic flux and sensitize cancer cells to chemotherapy

Abstract Autophagy is induced to protect cancer cells against stresses, including starvation, hypoxia and chemotherapy. Antimalarial drugs chloroquine and its derivative hydroxychloroquine are used to inhibit autophagy and being investigated with clinical trials for certain types of cancer. However, the tumor suppressive effects of the drugs might be autophagy independent, which require more specific inhibitors target autophagy components for clinical uses. Here, we screened FDA-approved drug library with both biochemical and cellular ATG4 reporter assays and found compound Tc blocked proteolysis activity of ATG4, essential proteases in autophagy machinery. Docking and MD simulation results appeared compound Tc was directly associated with active site of ATG4. Further, compound Tc obstructed the binding between ATG4 and MAP1LC3 and accumulated autophagosomes, which in turn reduce autophagic flux in cells. Moreover, compound Tc suppressed tumor viability and sensitized chemotherapy efficacy in two-dimensional and sphere culture model. Silencing ATG4 or overexpression of ATG4 catalytic mutant had no additional effects on tumor suppression compared to compound Tc alone, indicating compound Tc inhibited ATG4s activity to diminish tumor viability. In addition, compound Tc enhanced genotoxic stress-induced apoptosis to synergize chemotherapy efficacy in vitro and in vivo. Thus, we report that the clinical drug compound Tc is a potent ATG4 inhibitor, which could interrogate the role of autophagy in clinical setting and provide compound Tc as an anticancer drug or chemosensitizer. Citation Format: Chih-Wen Shu. Compound Tc inhibits ATG4 to attenuate autophagic flux and sensitize cancer cells to chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3532.

Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases

AbstractLeukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1−/− cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase–like disease in mice compared with less malignant CML-chronic phase–like disease from BCR-ABL1-Abl1+/+ cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin.

The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2−/− MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2−/− MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our study highlights the relevance of Sall2 in the apoptotic response to extended genotoxic stress, which is important for understanding its role in normal physiology and disease.

A central role of TRAX in the ATM-mediated DNA repair.

DNA repair is critical for the maintenance of genome stability. Upon genotoxic stress, dysregulated DNA repair may induce apoptosis. Translin-associated factor X (TRAX), which was initially identified as a binding partner of Translin, has been implicated in genome stability. However, the exact role of TRAX in DNA repair remains largely unknown. Here, we showed that TRAX participates in the ATM/H2AX-mediated DNA repair machinery by interacting with ATM and stabilizing the MRN complex at double-strand breaks. The exogenous expression of wild-type (WT) TRAX, but not a TRAX variant lacking the nuclear localization signal (NLS), rescued the vulnerability of TRAX-null mouse embryo fibroblasts (MEFs). This finding confirms the importance of the nuclear localization of TRAX in the repair of DNA damage. Compared with WT MEFs, TRAX-null MEFs exhibited impaired DNA repair (for example, reduced phosphorylation of ATM and H2AX) after treatment with ultra violet-C or γ-ray irradiation and a higher incidence of p53-mediated apoptosis. Our findings demonstrate that TRAX is required for MRN complex-ATM-H2AX signaling, which optimizes DNA repair by interacting with the activated ATM and protects cells from genotoxic stress-induced apoptosis.

Altered expression of long non-coding RNAs during genotoxic stress-induced cell death in human glioma cells.

Long non-coding RNAs (lncRNAs), a recently discovered class of non-coding genes, are transcribed throughout the genome. Emerging evidence suggests that lncRNAs may be involved in modulating various aspects of tumor biology, including regulating gene activity in response to external stimuli or DNA damage. No data are available regarding the expression of lncRNAs during genotoxic stress-induced apoptosis and/or necrosis in human glioma cells. In this study, we detected a change in the expression of specific candidate lncRNAs (neat1, GAS5, TUG1, BC200, Malat1, MEG3, MIR155HG, PAR5, and ST7OT1) during DNA damage-induced apoptosis in human glioma cell lines (U251 and U87) using doxorubicin (DOX) and resveratrol (RES). We also detected the expression pattern of these lncRNAs in human glioma cell lines under necrosis induced using an increased dose of DOX. Our results reveal that the lncRNA expression patterns are distinct between genotoxic stress-induced apoptosis and necrosis in human glioma cells. The sets of lncRNA expressed during genotoxic stress-induced apoptosis were DNA-damaging agent-specific. Generally, MEG3 and ST7OT1 are up-regulated in both cell lines under apoptosis induced using both agents. The induction of GAS5 is only clearly detected during DOX-induced apoptosis, whereas the up-regulation of neat1 and MIR155HG is only found during RES-induced apoptosis in both cell lines. However, TUG1, BC200 and MIR155HG are down regulated when necrosis is induced using a high dose of DOX in both cell lines. In conclusion, our findings suggest that the distinct regulation of lncRNAs may possibly involve in the process of cellular defense against genotoxic agents.

CCAR2 deficiency augments genotoxic stress-induced apoptosis in the presence of melatonin in non-small cell lung cancer cells.

Melatonin exhibits oncostatic activity in several cancers but does not lead to cytotoxicity in estrogen receptor (ER)-negative non-small cell lung cancers (NSCLCs). In an effort to overcome the melatonin resistance of these cancers, we investigated whether cell cycle and apoptosis regulator 2 (CCAR2) depletion would promote apoptosis following genotoxic stress in melatonin-resistant cancer cells. Ordinarily, the NSCLC cell lines A549 and A427 did not undergo cell death following melatonin treatment for short period. These cell lines were irradiated with UV, a source of genotoxic damage, to trigger apoptotic signaling. Treatment with melatonin prior to irradiation did not produce any significant change in apoptosis. By contrast, in CCAR2-deficient cells, melatonin treatment increased apoptosis induced by genotoxic stress; this effect was dependent on the dose of melatonin. The increase in apoptosis in CCAR2-deficient cells was not dependent on SIRT1. The results indicate that CCAR2 is critical for maintaining cell survival in the presence of melatonin under genotoxic stress. Furthermore, CCAR2 is overexpressed in NSCLC; therefore, melatonin could be used as a potential supplement to classical anticancer drugs in therapies against CCAR2-deficient cancers.

Acute dyskerin depletion triggers cellular senescence and renders osteosarcoma cells resistant to genotoxic stress-induced apoptosis

Dyskerin is a conserved, nucleolar RNA-binding protein implicated in an increasing array of fundamental cellular processes. Germline mutation in the dyskerin gene (DKC1) is the cause of X-linked dyskeratosis congenita (DC). Conversely, wild-type dyskerin is overexpressed in sporadic cancers, and high-levels may be associated with poor prognosis. It was previously reported that acute loss of dyskerin function via siRNA-mediated depletion slowed the proliferation of transformed cell lines. However, the mechanisms remained unclear. Using human U2OS osteosarcoma cells, we show that siRNA-mediated dyskerin depletion induced cellular senescence as evidenced by proliferative arrest, senescence-associated heterochromatinization and a senescence-associated molecular profile. Senescence can render cells resistant to apoptosis. Conversely, chromatin relaxation can reverse the repressive effects of senescence-associated heterochromatinization on apoptosis. To this end, genotoxic stress-induced apoptosis was suppressed in dyskerin-depleted cells. In contrast, agents that induce chromatin relaxation, including histone deacetylase inhibitors and the DNA intercalator chloroquine, sensitized dyskerin-depleted cells to apoptosis. Dyskerin is a core component of the telomerase complex and plays an important role in telomere homeostasis. Defective telomere maintenance resulting in premature senescence is thought to primarily underlie the pathogenesis of X-linked DC. Since U2OS cells are telomerase-negative, this leads us to conclude that loss of dyskerin function can also induce cellular senescence via mechanisms independent of telomere shortening.

Impaired PIASy–Tip60 signaling weakens activation of p53 in melanoma

The tumor suppressor p53 plays a central role in preventing tumor development by promoting transcription of genes that stall cell cycle and induce cell death. Although the majority of melanomas express wild-type p53, the molecular mechanisms that impede its activation remain unclear. We previously reported that the SUMO E3 ligase PIASy and the histone acetyltransferase Tip60 signaling cascade promote p53-dependent autophagy and apoptosis. We hypothesized that impairment in this signaling attenuates p53, thus disabling its apoptotic function in melanoma. Here, we show that human melanoma patient samples and cell lines maintain p53 expression but PIASy and/or Tip60 are frequently lost. We observed dysregulation of Tip60-mediated p53 transcription program in melanoma cell lines. Reconstitution of PIASy and Tip60 in melanoma cells increased genotoxic stress-induced apoptosis. Our study provides a clinical link of how sumoylation signaling may activate p53-mediated cell death in melanoma.

Abstract 1730: RET tyrosine kinase receptor represses Noxa transcription and prevents genotoxic or endoplasmic reticulum stress-induced apoptosis.

Abstract One of the hallmarks of human cancers is the intrinsic or acquired resistance to apoptosis. Apoptosis resistance contributes to carcinogenesis, tumor progression, and treatment resistance. Patients with medullary thyroid carcinoma (MTC) have been shown to be resistant to standard chemotherapy and conventional radiotherapy. Dominant activating mutations in the RET protooncogene are the cause of hereditary MTC and are found in more than 40% of sporadic cases. Despite an ever-growing wealth of information about RET's role in cell survival/proliferation, a detailed understanding of how this tyrosine kinase receptor acts to prevent cell death in tumorigenesis remains elusive. We hypothesized that activated RET prevents apoptosis by repressing the transcription of the proapoptotic gene Noxa in the nucleus in response to genotoxic or endoplasmic reticulum (ER) stress. Here we report that activated RET translocates to the nucleus in cultured MTC cells as well as MTC primary tumor tissues. RET is recruited to the Noxa promoter and is able to repress its transcription. The formation of the RET-Noxa promoter complex depends on RET expression and requires the tyrosine kinase activity of RET. Additionally, stable depletion of RET in MTC cells, using lentiviral shRNA, sensitizes MTC cells to genotoxic or endoplasmic reticulum stress. We specifically show that RET is recruited to the Activating Transcription Factor 4 (ATF4) binding site on the Noxa promoter. RET physically interacts with ATF4 and phosphorylates ATF4 leading to the blockage of its transcriptional activity. Moreover, RET knockdown is associated with increased ATF4-dependent expression of Noxa. Inhibition of RET kinase activity by sunitinib results in ATF4 activation, induction of Noxa expression and apoptosis. Conversely, silencing of ATF4 in MTC cells decreased sunitinib-mediated Noxa induction and apoptosis. ATF4 protein levels markedly decreased and/or localized in the cytoplasm of 60% of primary MTC (n=40) and associated with poor overall survival. Our data suggest that RET activating mutations confers resistance to chemotherapeutic agents by preventing apoptosis through inhibition of ATF4 activity and repression of proapoptotic gene Noxa. These findings reveal the importance of nuclear localization of RET, as newly discovered modulator of nuclear gene expression. This newly identified interaction between RET kinase and ATF4 has important implication for MTC and other RET/ATF4-mediated cancer types. Since ATF4 transcription is induced by endoplasmic reticulum stress, and activation of ATF4 induces cell cycle arrest and apoptosis, the concept of promoting endoplasmic reticulum stress in combination with tyrosine kinase inhibitors could be considered as a therapeutic strategy for cancer. Citation Format: Rozita Bagheri-Yarmand, Anupama E Gururaj, Michelle Williams, Zamal Ahmed, Jean E Ladbury, Oliver Bogler, Sue-chen Huang, Gilbert G Cote, Robert F Gagel. RET tyrosine kinase receptor represses Noxa transcription and prevents genotoxic or endoplasmic reticulum stress-induced apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1730. doi:10.1158/1538-7445.AM2013-1730

Enhanced levels of double-strand DNA break repair proteins protect ovarian cancer cells against genotoxic stress-induced apoptosis

BackgroundEarlier, proteomic profiling of a Serous Ovarian Carcinoma (SeOvCa) progression model in our lab had identified significantly enriched expression of three double-strand break (DSB) -repair proteins viz. RAD50, NPM1, and XRCC5 in transformed cells over pre-transformed, non-tumorigenic cells. Analysis of the functional relevance of enhanced levels of these proteins was explored in transformed ovarian cancer cells.MethodsExpression profiling, validation and quantitation of the DSB-repair proteins at the transcriptional and protein levels were carried out. Further analyses included identification of their localization, distribution and modulation on exposure to Estradiol (E2) and cisplatin. Effects on silencing of each of these under conditions of genomic-stress were studied with respect to apoptosis, alterations in nuclear morphology and DNA fragmentation; besides profiling known mitotic and spindle check-point markers in DSB-repair.ResultsWe identified that levels of these DSB-repair proteins were elevated not only in our model, but generally in cancer and are specifically triggered in response to genotoxic stress. Silencing of their expression led to aberrant DSB repair and consequently, p53/p21 mediated apoptosis. Further compromised functionality generated genomic instability.ConclusionsPresent study elucidates a functional relevance of NPM1, RAD50 and XRCC5 DSB-repair proteins towards ensuring survival and evasion of apoptosis during ovarian transformation, emphasizing their contribution and association with disease progression in high-grade SeOvCa.