Tumorigenesis of basal muscle invasive bladder cancer was mediated by PTEN protein degradation resulting from SNHG1 upregulation.

BACKGROUND: Phosphatase and tensin homolog deleted on chromosome ten (PTEN) serves as a powerful tumor suppressor, and has been found to be downregulated in human bladder cancer (BC) tissues. Despite this observation, the mechanisms contributing to PTEN's downregulation have remained elusive. METHODS: We established targeted genes' knockdown or overexpressed cell lines to explore the mechanism how it drove the malignant transformation of urothelial cells or promoted anchorageindependent growth of human basal muscle invasive BC (BMIBC) cells. The mice model was used to validate the conclusion in vivo. The important findings were also extended to human studies. RESULTS: In this study, we discovered that mice exposed to N-butyl-N-(4-hydroxybu-tyl)nitrosamine (BBN), a specific bladder chemical carcinogen, exhibited primary BMIBC accompanied by a pronounced reduction in PTEN protein expression in vivo. Utilizing a lncRNA deep sequencing high-throughput platform, along with gain- and loss-of-function analyses, we identified small nucleolar RNA host gene 1 (SNHG1) as a critical lncRNA that might drive the formation of primary BMIBCs in BBN-treated mice. Cell culture results further demonstrated that BBN exposure significantly induced SNHG1 in normal human bladder urothelial cell UROtsa. Notably, the ectopic expression of SNHG1 alone was sufficient to induce malignant transformation in human urothelial cells, while SNHG1 knockdown effectively inhibited anchorage-independent growth of human BMIBCs. Our detailed investigation revealed that SNHG1 overexpression led to PTEN protein degradation through its direct interaction with HUR. This interaction reduced HUR binding to ubiquitin-specific peptidase 8 (USP8) mRNA, causing degradation of USP8 mRNA and a subsequent decrease in USP8 protein expression. The downregulation of USP8, in turn, increased PTEN polyubiquitination and degradation, culminating in cell malignant transformation and BMIBC anchorageindependent growth. In vivo studies confirmed the downregulation of PTEN and USP8, as well as their positive correlations in both BBN-treated mouse bladder urothelium and tumor tissues of bladder cancer in nude mice. CONCLUSIONS: Our findings, for the first time, demonstrate that overexpressed SNHG1 competes with USP8 for binding to HUR. This competition attenuates USP8 mRNA stability and protein expression, leading to PTEN protein degradation, consequently, this process drives urothelial cell malignant transformation and fosters BMIBC growth and primary BMIBC formation.

Induction of p27 contributes to inhibitory effect of isorhapontigenin (ISO) on malignant transformation of human urothelial cells.

Bladder cancer (BC) is the most expensive cancer to manage on a per-patient basis, costing about $4 billion in total healthcare expenditure per annum in America alone. Therefore, identifying a natural compound for prevention of BC is of tremendous importance for managing this disease. Previous studies have identified isorhapontigenin (ISO) as having an 85% preventive effect against invasive BC formation induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The results showed here that ISO treatment inhibited EGF-induced cell transformation of human urothelial cells through induction of tumor suppressor p27 transcription secondary to activation of an E2F1-dependentpathway.ISOtreatmentrenderedcellsresistanttoEGF-induced anchorage-independent growth concurrent with p27 protein induction in both UROtsa and SV-HUC-1 cells. ISO inhibition of EGF-induced cell transformation could be completely reversed by knockdown of p27, indicating that this protein was essential for the noted ISO inhibitory action. Mechanistic studies revealed that ISO treatment resulted in increased expression of E2F1, which in turn bound to its binding site in p27 promoter and initiated p27 transcription. The E2F1 induction was due to the elevation of its translation caused by ISO-induced miR-205 downregulation. Consistently, miR-205 was found to be overexpressed in human BCs, and ectopic expression of miR-205 mitigated ISO inhibitory effects against EGF-induced outcomes. Collectively, the results here demonstrate that ISO exhibits its preventive effect on EGF-induced human urothelial cell transformation by induction of p27 through a miR-205/E2F1 axis. This is distinct from what has been described for the therapeutic effects of ISO on human BC cells.

lncRNA SNHG1 Promotes Basal Bladder Cancer Invasion via Interaction with PP2A Catalytic Subunit and Induction of Autophagy.

Although basal muscle-invasive bladder cancers (MIBCs) are predominant, are more aggressive, and have bad prognoses, molecular mechanisms underlying how basal MIBC formation/progression have been barely explored. In the present study, SNHG1, a long non-coding RNA, was shown to be expressed at higher levels in basal MIBC cells than in other types of bladder BC cells, and its presence could promote basal MIBC cell invasion. The results revealed that SNHG1 specifically induced MMP2 expression via increasing its transcription and mRNA stability. In one mechanism, SNHG1 directly bound with PP2A catalytic subunit (PP2A-c) to inhibit interactions of PP2A-c with c-Jun and then promoted c-Jun phosphorylation that, in turn, mediated MMP2 transcription. In another mechanism, SNHG1 markedly induced autophagy in the cells via induction of increases in the abundance of autophagy-related proteins. The latter initiated autophagy and further abolished miR-34a stability, which reduced overall miR-34a binding directly to the 3' UTR of MMP2 mRNA, thereby promoting MMP2 mRNA stabilization. These results provided novel insight into understanding the specific functions of SNHG1 in basal MIBC. Such findings may ultimately prove highly significant for the design/synthesis of new SNHG1-based compounds for the treatment of basal MIBC patients.

Isorhapontigenin (ISO) inhibits stem cell-like properties and invasion of bladder cancer cell by attenuating CD44 expression.

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.

The inhibitory effect of compound ChlA-F on human bladder cancer cell invasion can be attributed to its blockage of SOX2 protein.

Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that ChlA-F downregulates SOX2 at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8 mRNA, resulting in elevation of USP8 mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3'-UTR of SOX2 mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients.

XIAP Interaction with E2F1 and Sp1 via its BIR2 and BIR3 domains specific activated MMP2 to promote bladder cancer invasion.

XIAP has generally been thought to function in bladder cancer. However, the potential function of structure-based function of XIAP in human BC invasion has not been well explored before. We show here that ectopic expression of the BIR domains of XIAP specifically resulted in MMP2 activation and cell invasion in XIAP-deleted BC cells, while Src was further defined as an XIAP downstream negative regulator for MMP2 activation and BC cell invasion. The inhibition of Src expression by the BIR domains was caused by attenuation of Src protein translation upon miR-203 upregulation; which was resulted from direct interaction of BIR2 and BIR3 with E2F1 and Sp1, respectively. The interaction of BIR2/BIR3 with E2F1/Sp1 unexpectedly occurred, which could be blocked by serum-induced XIAP translocation. Taken together, our studies, for the first time revealed that: (1) BIR2 and BIR3 domains of XIAP play their role in cancer cell invasion without affecting cell migration by specific activation of MMP2 in human BC cells; (2) by BIR2 interacting with E2F1 and BIR3 interacting with Sp1, XIAP initiates E2F1/Sp1 positive feedback loop-dependent transcription of miR-203, which in turn inhibits Src protein translation, further leading to MMP2-cleaved activation; (3) XIAP interaction with E2F1 and Sp1 is observed in the nucleus. Our findings provide novel insights into understanding the specific function of BIR2 and BIR3 of XIAP in BC invasion, which will be highly significant for the design/synthesis of new BIR2/BIR3-based compounds for invasive BC treatment.