Dual role of CASP8AP2/FLASH in regulating epithelial-to-mesenchymal transition plasticity (EMP).

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a developmental program that consists of the loss of epithelial features concomitant with the acquisition of mesenchymal features. Activation of EMT in cancer facilitates the acquisition of aggressive traits and cancer invasion. EMT plasticity (EMP), the dynamic transition between multiple hybrid states in which cancer cells display both epithelial and mesenchymal markers, confers survival advantages for cancer cells in constantly changing environments during metastasis. METHODS: RNAseq analysis was performed to assess genome-wide transcriptional changes in cancer cells depleted for histone regulators FLASH, NPAT, and SLBP. Quantitative PCR and Western blot were used for the detection of mRNA and protein levels. Computational analysis was performed on distinct sets of genes to determine the epithelial and mesenchymal score in cancer cells and to correlate FLASH expression with EMT markers in the CCLE collection. RESULTS: We demonstrate that loss of FLASH in cancer cells gives rise to a hybrid E/M phenotype with high epithelial scores even in the presence of TGFß, as determined by computational methods using expression of predetermined sets of epithelial and mesenchymal genes. Multiple genes involved in cell-cell junction formation are similarly specifically upregulated in FLASH-depleted cells, suggesting that FLASH acts as a repressor of the epithelial phenotype. Further, FLASH expression in cancer lines is inversely correlated with the epithelial score. Nonetheless, subsets of mesenchymal markers were distinctly up-regulated in FLASH, NPAT, or SLBP-depleted cells. CONCLUSIONS: The ZEB1low/SNAILhigh/E-cadherinhigh phenotype described in FLASH-depleted cancer cells is driving a hybrid E/M phenotype in which epithelial and mesenchymal markers coexist.

Interaction between CASP8AP2 and ZEB2-CtBP2 Regulates the Expression of LEF1.

Low expression of CTBP2 and CASP8AP2 correlated with poor outcome and predicted risk of relapse in pediatric B-cell acute lymphoblastic leukemia (B-ALL). This study aimed to investigate the molecular mechanism by which CASP8AP2 regulates LEF1 expression by interacting with CtBP2 and ZEB2 in Acute lymphoblastic lymphoma (ALL). There was an interaction between CASP8AP2, ZEB2, and CtBP2, and then the interaction between CtBP2 and ZEB2 was observed after downregulating the expression of CASP8AP2. The wild type (containing the ZEB2 binding site) or mutant (containing a mutant binding site) LEF1 gene promoter sequence was inserted into the pGL3-basic plasmid, and a dual-luciferase reporter gene detection system was used to observe how CASP8AP2, ZEB2, and CtBP2 regulate the transcription of the LEF1 gene. We conclude that CASP8AP2, CtBP2, and ZEB2 can all bind to the LEF1 gene promoter region and reduce the luciferase activity of the LEF1 promoter. Meanwhile, the interaction of ZEB2 and the LEF1 promoter was significantly weakened after downregulation of CASP8AP2. Knockdown of CASP8AP2 in the 697 cell lines resulted in the significant upregulation of the mRNA expression levels of the stemness-related genes CD44, JAG1, and SALL4. In conclusion, CASP8AP2 is vital for the interaction between CtBP2 and ZEB2, inhibiting LEF1 and stemness-related genes expression ALL.Supplemental data for this article is available online at https://doi.org/10.1080/08880018.2022.2033369 .

The microRNA-210/Casp8ap2 Axis Alleviates Hypoxia-Induced Myocardial Injury by Regulating Apoptosis and Autophagy.

Coronary heart disease (CHD) is a serious condition comprising atherosclerosis-mediated ischaemic and hypoxic myocardial injury. This study aimed to investigate the mechanism of the miR-210/Casp8ap2 signalling pathway in hypoxic myocardial cells. mRNA and protein expression levels were determined by quantitative real-time PCR and western blotting, respectively. MTT was used to evaluate cell survival, and flow cytometry was used to assess apoptosis and the cell cycle distribution. The interaction between miR-210 and -Casp8ap2 was detected by dual-luciferase reporter assay. As a result, overexpression of miR-210 significantly inhibited apoptosis and reduced the proportion of cells in G1 phase. Moreover, miR-210 suppressed autophagy by upregulating p62 levels and reducing the LC3-II/I ratio in hypoxic cardiomyocytes. miR-210 regulated apoptosis and autophagy by directly targeting Casp8ap2. Furthermore, the expression levels of Casp8ap2, Cleaved caspase 8, Cleaved caspase 3and Beclin-1 were all decreased in response to miR-210. In short, our results suggest that miR-210 exerts anti-apoptotic and anti-autophagic effects in hypoxic cardiomyocytes, which alleviates myocardial injury in response to hypoxia.

Omega-3 fatty acid protects cardiomyocytes against hypoxia-induced injury through targeting MiR-210-3p/CASP8AP2 axis.

MicroRNAs (miRs) regulate diverse biological functions in both normal and pathological cellular conditions by post-transcriptional regulation of various genes expression. Nevertheless, the role of miRs in regulating the protective functions of omega-3 fatty acid in relation to hypoxia in cardiomyocytes remains unknown. The aim of this study was to investigate the effects of omega-3 fatty acid supplementation on cardiomyocyte apoptosis and further delineate the mechanisms underlying microRNA-210 (miRNA-210)-induced cardiomyocyte apoptosis in vitro. H9C2 cultured cells were first subjected to hypoxia followed by a subsequent treatment with main component of the Omega-3 fatty acid, Docosahexaenoic Acid (DHA). Cell apoptosis were detected by flow cytometry and the expression of miR-210-3p were detected by RT-qPCR and caspase-8-associated protein 2 (CASP8AP2) at protein levels by immunoblotting. Dual luciferase assay was used to verify the mutual effect between miR-210-3p and the 3'-untranslated region (UTR) of CASP8AP2 gene. DHA was shown to reduce apoptosis in H9C2 cells subjected to hypoxia. While DHA caused a significant increase in the expression of miR-210-3p, there was a marked reduction in the protein expression of CASP8AP2. MiR-210-3p and CASP8AP2 were significantly increased in H9C2 cardiomyocyte subjected to hypoxia. Overexpression of miR-210-3p could ameliorate hypoxia-induced apoptosis in H9C2 cells. MiR-210-3p negatively regulated CASP8AP2 expression at the transcriptional level. Both miR-210-3p mimic and CASP8AP2 siRNA could efficiently inhibit apoptosis in H9C2 cardiomyocyte subjected to hypoxia. We provide strong evidence showing that Omega-3 fatty acids can attenuate apoptosis in cardiomyocyte under hypoxic conditions via the up-regulation of miR-210-3p and targeting CASP8AP2 signaling pathway.

Low expression of CTBP2 and CASP8AP2 predicts risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia: a retrospective cohort study.

CtBP is a known corepressor abundantly expressed in cancer and regulates genes involved in cancer initiation, progression, and metastasis. This study aimed to investigate the prognostic significance of CTBP2 expression in a cohort of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL). It further evaluated the role of combined CTBP2 and CASP8AP2 expression in risk of relapse of BCP-ALL. The expression of CTBP2 mRNA was retrospectively detected by a qRT-PCR approach in bone marrow samples from 104 children with newly diagnosed BCP-ALL. CASP8AP2 was assessed simultaneously in the 100 patients included in this study. The receiver operating characteristic (ROC) curve analysis determined the cut off levels for CTBP2 and CASP8AP2 expression with good predictive significance for relapse of BCP-ALL. Patients with low CTBP2 expression had inferior relapse-free survival (RFS) and event-free survival (EFS) when compared to patients with high-CTBP2 expression. The expression level of CTBP2 was significantly associated with CASP8AP2 expression (r = 0.449, P < 0.001). Patients were stratified into three groups according to the combined evaluation of the two gene expression, and patients with simultaneous low-expression had the worst outcome (6-year RFS: 64.6%±12.8%, P < 0.001). Multivariate analysis demonstrated the expression of CTBP2 and CASP8AP2, minimal residual disease (MRD) at day 33 remained as independent prognostic factors for RFS. Based on the final Cox hazards model, we proposed an algorithm to calculate the risk index, which was more precise for predicting relapse. In conclusion, low expression of CTBP2 and CASP8AP2 correlated with poor outcome and predicted risk of relapse in pediatric BCP-ALL.

Transcription factor E2F3a regulates CASP8AP2 transcription and enhances sensitivity to chemotherapeutic drugs in acute lymphoblastic leukemia.

BACKGROUND: Low expression of E2F3a and caspase 8 associated protein 2 (CASP8AP2) are associated with poor prognosis of childhood acute lymphoblastic leukemia (ALL). METHODS: Dual-luciferase reporter assay and wild type as well as four mutated types of reporter plasmids were used to demonstrate the activation of E2F3a on CASP8AP2 transcription. The direct binding of E2F3a with the promoter of CASP8AP2 was shown by Chromatin Immunoprecipitation (ChIP). Cell proliferation activity and cell cycle were determined by MTS and flow cytometry in leukemic cells after treating with common chemotherapeutic drugs vincristine and daunorubicin. RESULTS: In this study, we found that up-regulation of E2F3a in leukemic cells led to increased fraction of cells in S and G2/M phase, accelerated proliferation, and enhanced sensitivity to vincristine and daunorubicin. ChIP and luciferase assay indicated that E2F3a could directly bind to two fragments in the wild type of CASP8AP2 promotor (- 206 to - 69 and - 677 to - 507), and activate its transcription activity which was reduced in mutated promotors. The effect of E2F3a on chemotherapeutic sensitivity of leukemic cells could be reversed by down-regulating CASP8AP2. CONCLUSIONS: E2F3a could promote transcription and expression of CASP8AP2. The effect of E2F3a on chemotherapeutic sensitivity of ALL cells was implemented by regulating CASP8AP2 expression to a great extent.

Low expressions of ARS2 and CASP8AP2 predict relapse and poor prognosis in pediatric acute lymphoblastic leukemia patients treated on China CCLG-ALL 2008 protocol.

ARS2 protein is important to early development and cell proliferation, in which ARS2-CASP8AP2 interaction is implicated. However, the predictive significance of ARS2 in childhood acute lymphoblastic leukemia (ALL) is unknown. Here we evaluate the predictive values of ARS2 expression and combined ARS2 and CASP8AP2 expression in relapse. We showed that ARS2 expression in ALL bone marrow samples at initial diagnosis was markedly lower than that in complete remission (CR). Likewise, the levels of ARS2 expression in the patients suffering from relapse were significantly lower than that of patients in continuous CR. Furthermore, low expression of ARS2 was closely correlated to poor treatment response including poor prednisone response and high minimal residual disease (MRD), and the patients with high MRD (≥10(-4)) and low ARS2 were more subject to relapse. The multivariate analyses for relapse free survival and event free survival revealed that ARS2 expression remained an independent prognostic factor after adjusting other risk factors. In addition, combined assessment of ARS2 and CASP8AP2 expression was more accurate to predict relapse, based on which an algorithm composed of ARS2 and CASP8AP2 expression, prednisone response and MRD (day 78) was proposed. Together, ARS2 and CASP8AP2 expressions can precisely predict high-risk of relapse and ALL prognosis.

Significance of CASP8AP2 and H2AFZ expression in survival and risk of relapse in children with acute lymphoblastic leukemia.

Novel biomarkers for risk refinement and stratification in childhood acute lymphoblastic leukemia (ALL) are needed to optimize treatment results. We studied the expression of CASP8AP2 and H2AFZ associated with relapse and survival in bone marrow samples from newly diagnosed children with ALL. We found: (a) an increased risk for early relapse in those patients with low expression of CASP8AP2 (odds ratio [OR] 3.93, 95% confidence interval [CI] 1.40-11.02, p < 0.05) confirming its usefulness as a predictive risk marker, although H2AFZ did not present the same effect; (b) patients with low expressions of CASP8AP2 and H2AFZ had inferior survival rates (p < 0.001); (c) the predictive values regarding low expressions of H2AFZ and CASP8AP2 and high white blood cell count suggest that these features could help to identify more accurately patients at greater risk of relapse.

Hypermethylation of two CpG sites upstream of CASP8AP2 promoter influences gene expression and treatment outcome in childhood acute lymphoblastic leukemia.

DNA hypermethylation of Caspase 8 associated protein 2 (CASP8AP2) and its role in childhood acute lymphoblastic leukemia (ALL) is unclear. We analyzed methylation status of CpG sites upstream of CASP8AP2 gene in 86 children with ALL by bisulfite sequencing and quantitative PCR. Methylation percentage of two CpG sites at positions of -1189 and -1176 was inversely correlated with mRNA expression (Spearman correlation: -0.333, P=0.002). High methylation was associated with the existence of minimal residual disease (MRD) at day 78 (P=0.035), The patients in high methylation group had a poor treatment outcome. The combination of methylation level and MRD at day 33 might improve current risk stratification.

SUMO regulates proteasome-dependent degradation of FLASH/Casp8AP2.

FLASH/Casp8AP2 is a huge multifunctional protein involved in multiple cellular processes, reaching from death receptor signaling to regulation of histone gene transcription and histone mRNA processing. Previous work has shown that FLASH localizes to Cajal bodies and promyelocytic leukemia (PML) bodies. However, the function of its nuclear body association remains unclear. Here we demonstrate that murine FLASH is covalently modified by SUMO at Lys residue 1792. Interestingly, ectopic expression of SUMO results in proteasome-dependent degradation of FLASH. A point mutant of FLASH with a mutated SUMO acceptor lysine residue, FLASH(K1792R), is resistant to SUMO-induced degradation. Finally, we show that arsenic trioxide, a drug known to potentiate SUMO modification and degradation of PML, triggers recruitment of FLASH to PML bodies and concomitant loss of FLASH protein. Our data suggest that SUMO targets FLASH for proteasome-dependent degradation, which is associated with recruitment of FLASH to PML bodies.