ZBTB7A interferes with the RPL5-P53 feedback loop and reduces endoplasmic reticulum stress-induced apoptosis of pancreatic cancer cells.

Endoplasmic reticulum (ER) stress is a primary mechanism leading to cell apoptosis, making it of great research interests in cancer management. This study delves into the function of ribosomal protein L5 (RPL5) in ER stress within pancreatic cancer (PCa) cells and investigates its regulatory mechanisms. Bioinformatics predictions pinpointed RPL5 as an ER stress-related gene exhibiting diminished expression in PCa. Indeed, RPL5 was found to be poorly expressed in PCa tissues and cells, with this reduced expression correlating with an unfavorable prognosis. Moreover, RPL5 overexpression led to heightened levels of p-PERK, p-eIF2α, and CHOP, bolstering the proapoptotic effect of Tunicamycin, an ER stress activator, on PCa cells. Additionally, the RPL5 overexpression curbed cell proliferation, migration, and invasion. Tunicamycin enhanced the binding between RPL5 and murine double minute 2 (MDM2), thus suppressing MDM2-mediated ubiquitination and degradation of P53. Consequently, P53 augmentation intensified ER stress, which further enhanced the binding between RPL5 and MDM2 through PERK-dependent eIF2α phosphorylation, thereby establishing a positive feedback loop. Zinc finger and BTB domain containing 7A (ZBTB7A), conspicuously overexpressed in PCa samples, repressed RPL5 transcription, thereby reducing P53 expression. Silencing of ZBTB7A heightened ER stress and subdued the malignant attributes of PCa cells, effects counteracted upon RPL5 silencing. Analogous outcomes were recapitulated in vivo employing a xenograft tumor mouse model, where ZBTB7A silencing dampened the tumorigenic potential of PCa cells, an effect reversed by additional RPL5 silencing. In conclusion, this study suggests that ZBTB7A represses RPL5 transcription, thus impeding the RPL5-P53 feedback loop and mitigating ER-induced apoptosis in PCa cells.

Mechanism of miR-7 mediating TLR4/TRAF6/NF-κB inflammatory pathway in colorectal cancer.

This study is aimed at investigating the roles of Toll-like receptor 4 (TLR4) and microRNA-7 (miR-7) in colorectal cancer (CRC) development and progression. We assessed TLR4 and miR-7 expression in CRC cells and tissues using reverse transcription-quantitative polymerase chain reaction. The relationship between miR-7 and TLR4 was analyzed through dual luciferase reporter assays. MTT, wound healing, and cell invasion assays were conducted to examine the effects of TLR4 and miR-7 on CRC cell proliferation, migration, and invasion. Western blotting was used to explore the involvement of the TRAF6/NF-κB signaling pathway. miR-7 was underexpressed in CRC, while TLR4 levels were increased. miR-7 negatively regulated TLR4 expression and its knockdown enhanced CRC cell proliferation, migration, and invasion. TLR4 knockdown had the opposite effects. The TRAF6/NF-κB pathway was linked to TLR4's role in tumor progression. miR-7 might inhibit TRAF6/NF-κB target a signaling pathway of TLR4 and promote CRC occurrence. miR-7 may therefore be used as a sensitive biomarker in CRC patients.

USP7-mediated JUND suppresses RCAN2 transcription and elevates NFATC1 to enhance stem cell property in colorectal cancer.

Cancer stem cells (CSCs) encompass a subset of highly aggressive tumor cells that are involved in tumor initiation and progression. This study investigates the function of regulator of calcineurin 2 (RCAN2) in the stem cell property in colorectal cancer (CRC). By analyzing four GEO datasets, we obtained RCAN2 as a stemness-related gene in CRC. RCAN2 was poorly expressed in CRC tissues and cells, especially in CSCs. RCAN2 restoration reduced calcineurin activity and promoted phosphorylation and degradation of nuclear factor of activated T cells 1 (NFATC1) protein, leading to reduced stemness of CSCs. JunD proto-oncogene (JUND), whose protein level was increased in CRC samples and CRC stem cells, bound to RCAN2 and suppressed its transcription. The abundant ubiquitin specific peptidase 7 (USP7) in CSCs enhanced JUND protein stability through deubiquitination modification. Lentivirus-mediated knockdown of USP7 or JUND also blocked the calcineurin-NFATC1 signaling and reduced the protein levels of stemness-related proteins. Moreover, the USP7 knockdown weakened the colony/sphere formation ability as well as the tumorigenicity of CSCs, and it reduced the CSC content in xenograft tumors. However, further restoration of JUND rescued the stemness of the CSCs. Overall, this study demonstrates that USP7-mediated JUND suppresses RCAN2 transcription and activates NFATC1 to enhance stem cell property in CRC. 1. RCAN2 is poorly expressed in CRC tissues and cells and especially in CSCs. 2. RCAN2 reduces stemness of CSCs by blocking calcineurin-NFATC1 signal transduction. 3. JUND binds to RCAN2 promoter to suppresses RCAN2 transcription. 4. USP7 enhances JUND protein stability via deubiquitination modification. 5. Downregulation of USP7 or JUND restores RCAN2 level and suppresses stemness of CSCs.

ETS-1 facilitates Th1 cell-mediated mucosal inflammation in inflammatory bowel diseases through upregulating CIRBP.

BACKGROUND & AIMS: As a susceptibility gene for human inflammatory bowel diseases (IBD), how avian erythroblastosis virus E26 oncogene homolog-1 (ETS-1) modulates intestinal mucosal immune response remains unclear. Here we studied the potential roles of ETS-1 in the pathogenesis of IBD. METHODS: ETS-1 expression was examined in IBD patients. CD45RBhighCD4+ T cell-transfer colitis, dextran sulfate sodium (DSS)-induced colitis, and azomethane (AOM)/DSS-induced colitis-associated cancer (CAC) models were constructed to probe the function of ETS-1 in vivo. RNA-sequencing of CD4+ T cells from Ets-1 transgenic (Tg) mice was performed to decipher the key differentially expressed genes. Adenovirus transduction was conducted to verify the therapeutic potentials of ETS-1 in vivo. RESULTS: ETS-1 expression was significantly increased in CD4+ T cells from active IBD patients compared with healthy controls, which was upregulated by TNF-α but markedly suppressed by anti-TNF-α mAb therapy. More severe colitis was observed in Rag1-/- mice reconstituted with Ets-1TgCD45RBhighCD4+ T cells or in Ets-1 Tg mice after DSS exposure compared with controls, characterized by higher TNF-α and IFN-γ expression in inflamed colon. Ets-1 Tg mice were more prone to develop AOM/DSS-induced CAC, and bone marrow chimeras further proved that lamina propria immune cells but not intestinal epithelial cells contributed to the development of colitis. RNA-sequencing and luciferase analysis revealed cold-inducible RNA-binding protein (CIRBP) as a functional target of ETS-1 to promote Th1 cell-driven immune response. Consistently, intraperitoneal administration of adenovirus-m-cirbp-shRNA ameliorated trinitrobenzene sulfonic acid (TNBS)-induced colitis of Ets-1 Tg mice. CONCLUSIONS: Our data identify that ETS-1 is highly expressed in IBD patients and promotes Th1-driven mucosal inflammation through CIRBP. CIRBP may serve as a novel therapeutic target for treatment of human IBD.

BRCA2 loss-of-function germline mutations are associated with esophageal squamous cell carcinoma risk in Chinese.

Esophageal squamous cell carcinoma (ESCC) occurs with highest frequency in China with over 90% mortality, highlighting the need for early detection and improved treatment strategies. We aimed to identify ESCC cancer predisposition gene(s). Our study included 4,517 individuals. The discovery phase using whole-exome sequencing (WES) included 186 familial ESCC patients from high-risk China. Targeted gene sequencing validation of 598 genes included 3,289 Henan and 1,228 moderate-risk Hong Kong Chinese. A WES approach identified BRCA2 loss-of-function (LOF) mutations in 3.23% (6/186) familial ESCC patients compared to 0.21% (9/4300) in the ExAC East Asians (odds ratio [OR] = 15.89, p = 2.48 × 10-10 ). BRCA2 LOF mutation frequency in the combined Henan cohort has significantly higher prevalence (OR = 10.55, p = 0.0035). Results were independently validated in an ESCC Hong Kong cohort (OR = 10.64, p = 0.022). One Hong Kong pedigree was identified to carry a BRCA2 LOF mutation. BRCA2 inactivation in ESCC was via germline LOF mutations and wild-type somatic allelic loss via loss of heterozygosity. Gene-based association analysis, including LOF mutations and rare deleterious missense variants defined with combined annotation dependent depletion score ≥30, confirmed the genetic predisposition role of BRCA2 (OR = 9.50, p = 3.44 × 10-5 ), and provided new evidence for potential association of ESCC risk with DNA repair genes (POLQ and MSH2), inflammation (TTC39B) and angiogenesis (KDR). Our findings are the first to provide compelling evidence of the role of BRCA2 in ESCC genetic susceptibility in Chinese, suggesting defective homologous recombination is an underlying cause in ESCC pathogenesis, which is amenable to therapeutic options based on synthetic lethality approaches such as targeting BRCA2 with PARP1 inhibitors in ESCC.

Imbalance of a KLF4-miR-7 auto-regulatory feedback loop promotes prostate cancer cell growth by impairing microRNA processing.

The microRNA-transcription factor auto-regulatory feedback loop is a pivotal mechanism for homeostatic regulation of gene expression, and dysregulation of the feedback loop is tightly associated with tumorigenesis and progression. However, the mechanism underlying such dysregulation is still not well-understood. Here we reported that Krüppel-like factor 4 (KLF4), a stemness-associated transcription factor, promotes the transcription of miR-7 to repress its own translation so that a KLF4-miR-7 auto-regulatory feedback loop is established for mutual regulation of their expression. Interestingly, this feedback loop is unbalanced in prostate cancer (PCa) cell lines and patient samples due to an impaired miR-7-processing, leading to decreased mature miR-7 production and attenuated inhibition of KLF4 translation. Mechanistically, enhanced oncogenic Yes associated protein (YAP) nuclear translocation mediates sequestration of p72, a co-factor of the Drosha/DGCR8 complex for pri-miR-7s processing, leading to attenuation of microprocessors' efficiency. Knockdown of YAP or transfection with a mature miR-7 mimic can significantly recover miR-7 expression to restore this feedback loop, and in turn to inhibit cancer cell growth by repressing KLF4 expression in vitro. Thus, our findings indicate that targeting the KLF4-miR-7 feedback loop might be a potential strategy for PCa therapy.

TIM4-TIM1 interaction modulates Th2 pattern inflammation through enhancing SIRT1 expression.

Skewed T helper 2 (Th2)­cell polarization plays a critical role in the pathogenesis of allergic inflammations; however, the underlying mechanisms require further elucidation. The aim of the present study was to investigate the mechanisms through which the interaction between T­cell immunoglobulin and mucin domain (TIM)4 and TIM1 regulates the expression of silent information regulator 1 (SIRT1) in Th2 cells, and the role of SIRT1 in Th2­cell polarization during nasal allergic inflammation. The results demonstrated that TIM4 expression by splenic dendritic cells was increased in mice with allergic rhinitis, and the TIM4̸TIM1 interaction promoted CD4+ T cells to express SIRT1 during allergic inflammation via enhancing phosphoinositide 3­kinase/Akt phosphorylation. SIRT1 then facilitated CD4+ T­cell proliferation through downregulating the expression of Fas ligand, caspase-3 and p53 in mice with nasal allergic inflammation. In conclusion, the interaction of TIM4̸TIM1 was found to promote Th2­cell proliferation through enhancing SIRT1 expression in mice with nasal allergic rhinitis.

MicroRNA-7 inhibits the stemness of prostate cancer stem-like cells and tumorigenesis by repressing KLF4/PI3K/Akt/p21 pathway.

Up to now, the molecular mechanisms underlying the stemness of prostate cancer stem cells (PCSCs) are still poorly understood. In this study, we demonstrated that microRNA-7 (miR-7) appears to be a novel tumor-suppressor miRNA, which abrogates the stemness of PCSCs and inhibits prostate tumorigenesis by suppressing a key stemness factor KLF4. MicroRNA-7 is down-regulated in prostate cancer cells compared to non-tumorigenic prostate epithelial cells. Restoration of miR-7 suppresses the expression of the stemness factor KLF4 in PCSCs and inhibits prostate tumorigenesis both in vitro and in vivo. Interestingly, the suppression of the stemness of PCSCs by miR-7 is sustained for generations in xenografts. Analysis of clinical samples also revealed a negative correlation between miR-7 expression and prostate tumor progression. Mechanistically, overexpression of miR-7 may lead to a cell cycle arrest but not apoptosis, which seems achieved via suppressing the KLF4/PI3K/Akt/p21 pathway. This study identifies miR-7 as a suppressor of PCSCs' stemness and implicates its potential application for PCa therapy.

MicroRNAs targeting prostate cancer stem cells.

Prostate cancer is a frequently diagnosed cancer in males with high mortality in the world. As a heterogeneous tissue, the tumor mass contains a subpopulation that is called as cancer stem cells and displays stem-like properties such as self-renewal, epithelial-mesenchymal transition, metastasis, and drug resistance. Cancer stem cells have been identified in variant tumors and shown to be regulated by various molecules including microRNAs. MicroRNAs are a class of small non-coding RNAs, which can influence tumorigenesis via different mechanisms. In this review, we focus on the functions of microRNAs on regulating the stemness of prostate cancer stem cells with different mechanisms and propose the potential roles of microRNAs in prostate cancer therapy.

Regulation and methylation of tumor suppressor miR-124 by androgen receptor in prostate cancer cells.

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.

Hypermethylation-mediated transcriptional repression of TMPRSS2 in androgen receptor-negative prostate cancer cells.

Prostate cancer is the most common type of cancer for men in the developed world. Androgen receptor (AR) is very important in prostate cancer progression. TMPRSS2 is an AR signaling downstream gene and closely related to prostate carcinogenesis. DNA methylation is a key mechanism to influence gene expression. Though previous reports have shown that AR signaling plays a critical role in the regulation of TMPRSS2 in prostate cancer, hardly any studies have examined whether the DNA methylation has been involved in the regulation of TMPRSS2. In the present study, we demonstrated that AR-negative prostate cancer (PCa) cells showed low expression levels and hypermethylation of TMPRSS2. In contrast, AR-positive PCa cells displayed high levels and hypomethylation of TMPRSS2. Treatment with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine reversed the low expression levels of TMPRSS2 in the AR-negative PCa cells. Additionally, we found that the level of DNA methyltransferases 1 (DNMT1) was high in AR-negative PCa cells, in which hypermethylation of TMPRSS2 and low expression level of TMPRSS2 were observed. Collectively, these data suggest that the high level of DNMT1 might be the mechanism for the hypermethylation-mediated transcriptional repression of TMPRSS2 in AR-negative PCa cells.

Androgen receptor is negatively correlated with the methylation-mediated transcriptional repression of miR-375 in human prostate cancer cells.

Androgen receptor (AR) plays a critical role during the development and progression of prostate cancer in which microRNA miR-375 is overexpressed and correlated with tumor progression. Although DNA methylation is a key mechanism for the repression of gene expression, the relationship between AR and the expression or the hypermethylation of miR-375 is unknown. In this study, we found that AR-positive prostate cancer (PCa) cells showed high expression levels and hypomethylation of the miR-375. In contrast, AR-negative PCa cells displayed low levels and hypermethylation of the miR-375. Addition of 5-Aza-2'-deoxycytidine, a specific inhibitor of DNA methylation, into the culture medium reversed the low expression levels of miR-375 in the AR negative PCa cells. In addition, the total activity levels of DNA methyltransferases (DNMTs) were high in AR-negative PCa cells, in which hypermethylation of miR-375 promoter and low expression levels of miR-375 were observed. Taken together, these findings indicate that the negative correlation between AR and total DNMT activity is one of mechanisms to influence the methylation status of miR-375 promoter, which in turn regulates the expression of miR-375.