Up-regulation of the proapoptotic caspase 2 splicing isoform by a candidate tumor suppressor, RBM5.
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ABSTRACT:
SUBMITTER: Fushimi K
PROVIDER: S-EPMC2572934 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The retinoblastoma protein (pRB) tumor suppressor blocks cell proliferation by repressing the E2F transcription factors. This inhibition is relieved through mitogen-induced phosphorylation of pRB, triggering E2F release and activation of cell-cycle genes. E2F1 can also activate proapoptotic genes in response to genotoxic or oncogenic stress. However, pRB's role in this context has not been established. Here we show that DNA damage and E1A-induced oncogenic stress promote formation of a pRB-E2F1 complex even in proliferating cells. Moreover, pRB is bound to proapoptotic promoters that are transcriptionally active, and pRB is required for maximal apoptotic response in vitro and in vivo. Together, these data reveal a direct role for pRB in the induction of apoptosis in response to genotoxic or oncogenic stress.
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Project description:Apoptosis is mediated by the caspase family of proteases that act as effectors of cell death by cleaving many cellular substrates. Caspase-2 is one of the most evolutionarily conserved caspases, yet its physiological function has remained enigmatic because caspase-2-deficient mice develop normally and are viable. We report here that the caspase-2(-/-) mouse embryonic fibroblasts (MEFs) show increased proliferation. When transformed with E1A and Ras oncogenes, caspase-2(-/-) MEFs grew significantly faster than caspase-2(+/+) MEFs and formed more aggressive and accelerated tumors in nude mice. To assess whether the loss of caspase-2 predisposes animals to tumor development, we used the mouse Emu-Myc lymphoma model. Our findings suggest that loss of even a single allele of caspase-2 resulted in accelerated tumorigenesis, and this was further enhanced in caspase-2(-/-) mice. The caspase-2(-/-) cells showed resistance to apoptosis induced by chemotherapeutic drugs and DNA damage. Furthermore, caspase-2(-/-) MEFs had a defective apoptotic response to cell-cycle checkpoint regulation and showed abnormal cycling following gamma-irradiation. These data show that loss of caspase-2 results in an increased ability of cells to acquire a transformed phenotype and become malignant, indicating that caspase-2 is a tumor suppressor protein.
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Project description:INTRODUCTION:ECRG4/C2ORF40 is a potential tumor suppressor gene (TSG) recently identified in esophageal carcinoma. Its expression, gene copy number and prognostic value have never been explored in breast cancer. METHODS:Using DNA microarray and array-based comparative genomic hybridization (aCGH), we examined ECRG4 mRNA expression and copy number alterations in 353 invasive breast cancer samples and normal breast (NB) samples. A meta-analysis was done on a large public retrospective gene expression dataset (n?=?1,387) in search of correlations between ECRG4 expression and histo-clinical features including survival. RESULTS:ECRG4 was underexpressed in 94.3% of cancers when compared to NB. aCGH data revealed ECRG4 loss in 18% of tumors, suggesting that DNA loss is not the main mechanism of underexpression. Meta-analysis showed that ECRG4 expression was significantly higher in tumors displaying earlier stage, smaller size, negative axillary lymph node status, lower grade, and normal-like subtype. Higher expression was also associated with disease-free survival (DFS; HR?=?0.84 [0.76-0.92], p?=?0.0002) and overall survival (OS; HR?=?0.72 [0.63-0.83], p?=?5.0E-06). In multivariate analysis including the other histo-clinical prognostic features, ECRG4 expression remained the only prognostic factor for DFS and OS. CONCLUSIONS:Our data suggest that ECRG4 is a candidate TSG in breast cancer, the expression of which may help improve the prognostication. If functional analyses confirm this TSG role, restoring ECRG4 expression in the tumor may represent a promising therapeutic approach.
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Project description:BackgroundApoptosis-stimulating Protein of TP53-2 (ASPP2) is a tumor suppressor enhancing TP53-mediated apoptosis via binding to the TP53 core domain. TP53 mutations found in cancers disrupt ASPP2 binding, arguing for an important role of ASPP2 in TP53-mediated tumor suppression. We now identify an oncogenic splicing variant, ASPP2κ, with high prevalence in acute leukemia.MethodsAn mRNA screen to detect ASPP2 splicing variants was performed and ASPP2κ was validated using isoform-specific PCR approaches. Translation into a genuine protein isoform was evaluated after establishing epitope-specific antibodies. For functional studies cell models with forced expression of ASPP2κ or isoform-specific ASPP2κ-interference were created to evaluate proliferative, apoptotic and oncogenic characteristics of ASPP2κ.FindingsExon skipping generates a premature stop codon, leading to a truncated C-terminus, omitting the TP53-binding sites. ASPP2κ translates into a dominant-negative protein variant impairing TP53-dependent induction of apoptosis. ASPP2κ is expressed in CD34+ leukemic progenitor cells and functional studies argue for a role in early oncogenesis, resulting in perturbed proliferation and impaired induction of apoptosis, mitotic failure and chromosomal instability (CIN) - similar to TP53 mutations. Importantly, as expression of ASPP2κ is stress-inducible it defines a novel class of dynamic oncogenes not represented by genomic mutations.InterpretationOur data demonstrates that ASPP2κ plays a distinctive role as an antiapoptotic regulator of the TP53 checkpoint, rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and ASPP2κ expression results in acquisition of genomic mutations, a first initiating step in leukemogenesis. We provide proof-of-concept to establish ASPP2κ as a clinically relevant biomarker and a target for molecule-defined therapy. FUND: Unrestricted grant support from the Wilhelm Sander Foundation for Cancer Research, the IZKF Program of the Medical Faculty Tübingen, the Brigitte Schlieben-Lange Program and the Margarete von Wrangell Program of the State Ministry Baden-Wuerttemberg for Science, Research and Arts and the Athene Program of the excellence initiative of the Eberhard-Karls University, Tübingen.
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Project description:Mutations in the spliceosomal RNA binding protein RBM10 cause TARP syndrome and are frequently observed in lung adenocarcinoma (LUAD). We have previously shown that RBM10 enhances exon skipping of its target genes, including its paralog RBM5. Here, we report that RBM10 negatively regulates its own mRNA and protein expression and that of RBM5 by promoting alternative splicing-coupled nonsense-mediated mRNA decay (AS-NMD). Through computational analysis and experimental validation, we identified RBM10-promoted skipping of exon 6 or 12 in RBM10 and exon 6 or 16 in RBM5 as the underlying AS-NMD events. Importantly, we showed that LUAD-associated mutations affecting splice sites of RBM10 exons 6 or 12 abolished exon inclusion and correlated with reduced expression of RBM10 RNA. Together, our investigations have revealed novel molecular mechanisms underlying RBM10 autoregulation and cross-regulation of RBM5, thereby providing insights concerning the functions of RBM10 under various physiological and pathological conditions. Our combined computational and experimental approach should be useful for elucidating the role of AS-NMD in auto- and cross-regulation by other splicing regulators.
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Project description:L. monocytogenes causes listeriosis, a foodborne disease that is particularly dangerous for immunocompromised individuals and fetuses. Several virulence factors of this bacterial pathogen belong to a family of leucine-rich repeat (LRR)-containing proteins called internalins. Among these, InlP is known for its role in placental infection. We report here a function of InlP in mammalian cell nucleus organization. We demonstrate that bacteria do not produce InlP under in vitro culture conditions. When ectopically expressed in human cells, InlP translocates into the nucleus and changes the morphology of nuclear speckles, which are membrane-less organelles storing splicing factors. Using yeast two-hybrid screen, immunoprecipitation and pull-down experiments, we identify the tumor suppressor and splicing factor RBM5 as a major nuclear target of InlP. InlP inhibits RBM5-induced cell death and stimulate the formation of RBM5-induced nuclear granules, where the SC35 speckle protein redistributes. Taken together, these results suggest that InlP acts as a nucleomodulin controlling compartmentalization and function of RBM5 in the nucleus and that L. monocytogenes has developed a mechanism to target the host cell splicing machinery.
| S-EPMC9763339 | biostudies-literature
Project description:RBM5, a regulator of alternative splicing of apoptotic genes, and its close homologues, RBM6 and RBM10, are RNA binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation and its modulation recapitulates or antagonizes the effects of RBM5, 6 and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation. RNA from 3 biological replicates of knockdowns of RBM5, 6 and 10 and a control set were used. Changes between the control and knockdowns were measured based on using a splice-junction array (Affymetrix HJAY).
| S-ECPF-GEOD-47431 | biostudies-other
Project description:PurposeCell adhesion molecules (CADM) comprise a newly identified protein family whose functions include cell polarity maintenance and tumor suppression. CADM-1, CADM-3, and CADM-4 have been shown to act as tumor suppressor genes in multiple cancers including prostate cancer. However, CADM-2 expression has not been determined in prostate cancer.Experimental designThe CADM-2 gene was cloned and characterized and its expression in human prostatic cell lines and cancer specimens was analyzed by reverse transcription-PCR and an immunohistochemical tissue array, respectively. The effects of adenovirus-mediated CADM-2 expression on prostate cancer cells were also investigated. CADM-2 promoter methylation was evaluated by bisulfite sequencing and methylation-specific PCR.ResultsWe report the initial characterization of CADM-2 isoforms: CADM-2a and CADM-2b, each with separate promoters, in human chromosome 3p12.1. Prostate cancer cell lines, LNCaP and DU145, expressed negligible CADM-2a relative to primary prostate tissue and cell lines, RWPE-1 and PPC-1, whereas expression of CADM-2b was maintained. Using immunohistochemistry, tissue array results from clinical specimens showed statistically significant decreased expression in prostate carcinoma compared with normal donor prostate, benign prostatic hyperplasia, prostatic intraepithelial neoplasia, and normal tissue adjacent to tumor (P < 0.001). Adenovirus-mediated CADM-2a expression suppressed DU145 cell proliferation in vitro and colony formation in soft agar. The decrease in CADM-2a mRNA in cancer cell lines correlated with promoter region hypermethylation as determined by bisulfite sequencing and methylation-specific PCR. Accordingly, treatment of cells with the demethylating agent 5-aza-2'-deoxycytidine alone or in combination with the histone deacetylase inhibitor trichostatin A resulted in the reactivation of CADM-2a expression.ConclusionsCADM-2a protein expression is significantly reduced in prostate cancer. Its expression is regulated in part by promoter methylation and implicates CADM-2 as a previously unrecognized tumor suppressor gene in a proportion of human prostate cancers.
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Project description:NORE1A (RASSF5) is a proapoptotic Ras effector that is frequently inactivated by promoter methylation in human tumors. It is structurally related to the RASSF1A tumor suppressor and is itself implicated as a tumor suppressor. In the presence of activated Ras, NORE1A is a potent inducer of apoptosis. However, when expressed at lower levels in the absence of activated Ras, NORE1A seems to promote cell cycle arrest rather than apoptosis. The mechanisms underlying NORE1A action are poorly understood. We have used microarray analysis of an inducible NORE1A system to screen for physiologic signaling targets of NORE1A action. Using this approach, we have identified several potential signaling pathways modulated by NORE1A. In particular, we identify the cyclin-dependent kinase inhibitor p21(CIP1) as a target for NORE1A activation and show that it is a vital component of NORE1A-mediated growth inhibition. In primary human hepatocellular carcinomas (HCC), loss of NORE1A expression is frequent and correlates tightly with loss of p21(CIP1) expression. NORE1A down-regulation in HCC also correlates with poor prognosis, enhanced proliferation, survival, and angiogenic tumor characteristics. Experimental inactivation of NORE1A results in the loss of p21(CIP1) expression and promotes proliferation. The best characterized activator of p21(CIP1) is the p53 master tumor suppressor. Further experiments showed that NORE1A activates p21(CIP1) via promoting p53 nuclear localization. Thus, we define the molecular basis of NORE1A-mediated growth inhibition and implicate NORE1A as a potential component of the ill-defined connection between Ras and p53.
| S-EPMC6957251 | biostudies-literature
Project description:Alternative splicing occurs in the C-terminal region of the p53 tumor suppressor gene between two alternative 3' splice sites in intron 10. This alternative splicing event has been detected in murine cells, but not in rat or human tissues. In this paper, we have characterized the pattern of p53 alternative splicing in cell lines from five different species. Our results confirm that p53 alternative splicing is species-specific, being detected only in cell lines of rodent origin. Using transient transfection assays, we have established that the rat p53 gene undergoes efficient alternative splicing in both mouse and rat cell lines, thus demonstrating that it has all the necessary cis -acting sequences to be alternatively spliced. In contrast, we were unable to detect any usage of the human alternative 3' splice site under the same experimental conditions. Thus, the low levels or absence of alternatively spliced p53 mRNA in rat and human cell lines seems to be the result of different mechanisms. Our results support the hypothesis that there are species-specific mechanisms implicated in the regulation of p53 activity.
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