Guosong Jiang

Isorhapontigenin (ISO) Inhibits Invasive Bladder Cancer Formation In Vivo and Human Bladder Cancer Invasion In Vitro by Targeting STAT1/FOXO1 Axis

Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo. We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro. Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancers propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1–FOXO1–MMP-2 axis. Cancer Prev Res; 9(7); 567–80. ©2016 AACR.

SESN2/sestrin 2 induction-mediated autophagy and inhibitory effect of isorhapontigenin (ISO) on human bladder cancers.

ABSTRACT Isorhapontigenin (ISO) is a new derivative of stilbene isolated from the Chinese herb Gnetum cleistostachyum. Our recent studies have revealed that ISO treatment at doses ranging from 20 to 80 μM triggers apoptosis in multiple human cancer cell lines. In the present study, we evaluated the potential effect of ISO on autophagy induction. We found that ISO treatment at sublethal doses induced autophagy effectively in human bladder cancer cells, which contributed to the inhibition of anchorage-independent growth of cancer cells. In addition, our studies revealed that ISO-mediated autophagy induction occurred in a SESN2 (sestrin 2)-dependent and BECN1 (Beclin 1, autophagy related)-independent manner. Furthermore, we identified that ISO treatment induced SESN2 expression via a MAPK8/JNK1 (mitogen-activated protein kinase 8)/JUN-dependent mechanism, in which ISO triggered MAPK8-dependent JUN activation and facilitated the binding of JUN to a consensus AP-1 binding site in the SESN2 promoter region, thereby led to a significant transcriptional induction of SESN2. Importantly, we found that SESN2 expression was dramatically downregulated or even lost in human bladder cancer tissues as compared to their paired adjacent normal tissues. Collectively, our results demonstrate that ISO treatment induces autophagy and inhibits bladder cancer growth through MAPK8-JUN-dependent transcriptional induction of SESN2, which provides a novel mechanistic insight into understanding the inhibitory effect of ISO on bladder cancers and suggests that ISO might act as a promising preventive and/or therapeutic drug against human bladder cancer.

Induction of miR-137 by Isorhapontigenin (ISO) Directly Targets Sp1 Protein Translation and Mediates Its Anticancer Activity Both In Vitro and In Vivo

Our recent studies found that isorhapontigenin (ISO) showed a significant inhibitory effect on human bladder cancer cell growth, accompanied with cell-cycle G0–G1 arrest as well as downregulation of Cyclin D1 expression at transcriptional level via inhibition of Sp1 transactivation in bladder cancer cells. In the current study, the potential ISO inhibition of bladder tumor formation has been explored in a xenograft nude mouse model, and the molecular mechanisms underlying ISO inhibition of Sp1 expression and anticancer activities have been elucidated both in vitro and in vivo. Moreover, the studies demonstrated that ISO treatment induced the expression of miR-137, which in turn suppressed Sp1 protein translation by directly targeting Sp1 mRNA 3′-untranslated region (UTR). Similar to ISO treatment, ectopic expression of miR-137 alone led to G0–G1 cell growth arrest and inhibition of anchorage-independent growth in human bladder cancer cells, which could be completely reversed by overexpression of GFP-Sp1. The inhibition of miR-137 expression attenuated ISO-induced inhibition of Sp1/Cyclin D1 expression, induction of G0–G1 cell growth arrest, and suppression of cell anchorage-independent growth. Taken together, our studies have demonstrated that miR-137 induction by ISO targets Sp1 mRNA 3′-UTR and inhibits Sp1 protein translation, which consequently results in reduction of Cyclin D1 expression, induction of G0–G1 growth arrest, and inhibition of anchorage-independent growth in vitro and in vivo. Our results have provided novel insights into understanding the anticancer activity of ISO in the therapy of human bladder cancer. Mol Cancer Ther; 15(3); 512–22. ©2016 AACR.

Role of STAT3 and FOXO1 in the divergent therapeutic responses of non-metastatic and metastatic bladder cancer cells to miR-145

Although miR-145 is the most frequently downregulated miRNA in bladder cancer, its exact stage association and downstream effector have not been defined. Here, we found that miR-145 was upregulated in human patients with bladder cancer with lymph node metastasis and in metastatic T24T cell line. Forced expression of miR-145 promoted anchorage-independent growth of T24T cells accompanied by the downregulation of forkhead box class O1 (FOXO1). In contrast, in non-metastatic T24 cells, miR-145 overexpression inhibited cell growth with upregulation of FOXO1, and the knockdown of FOXO1 abolished the miR-145–mediated inhibition of cell growth. Mechanistic studies revealed that miR-145 directly bound to and attenuated 3′-untranslated region (UTR) activity of foxo1 mRNA in both T24 and T24T cells. Interestingly, miR-145 suppressed STAT3 phosphorylation at Tyr705 and increased foxo1 promoter transcriptional activity in T24 cells, but not in T24T cells, suggesting a role of STAT3 in the divergent responses to miR-145. Supporting this was our finding that STAT3 knockdown mimicked miR-145–mediated upregulation of FOXO1 in T24T cells and inhibition of anchorage-independent growth. Consistently, ectopic expression of miR-145 promoted tumor formation of xenograft T24T cells, whereas such promoting effect became inhibitory due to specific knockdown of STAT3. Together, our findings demonstrate the stage-specific association and function of miR-145 in bladder cancers and provide novel insights into the therapeutic targeting of miR-145. Mol Cancer Ther; 16(5); 924–35. ©2017 AACR.

XIAP overexpression promotes bladder cancer invasion in vitro and lung metastasis in vivo via enhancing nucleolin-mediated Rho-GDIβ mRNA stability: XIAP mediates bladder cancer invasion

Our recent studies demonstrate that X‐linked inhibitor of apoptosis protein (XIAP) is essential for regulating colorectal cancer invasion. Here, we discovered that RhoGDIβ was a key XIAP downstream effector mediating bladder cancer (BC) invasion in vitro and in vivo. We found that both XIAP and RhoGDIβ expressions were consistently elevated in BCs of N‐butyl‐N‐(4‐hydroxybutyl)‐nitrosamine (BBN)‐treated mice in comparison to bladder tissues from vehicle‐treated mice and human BCs in comparison to the paired adjacent normal bladder tissues. Knockdown of XIAP attenuated RhoGDIβ expression and reduced cancer cell invasion, whereas RhoGDIβ expression was attenuated in BBN‐treated urothelium of RING‐deletion knockin mice. Mechanistically, XIAP stabilized RhoGDIβ mRNA by its positively regulating nucleolin mRNA stability via Erks‐dependent manner. Moreover, ectopic expression of GFP‐RhoGDIβ in T24T(shXIAP) cells restored its lung metastasis in nude mice. Our results demonstrate that XIAP‐regulated Erks/nucleolin/RhoGDIβ axis promoted BC invasion and lung metastasis.

ATG7 Overexpression Is Crucial for Tumorigenic Growth of Bladder Cancer In Vitro and In Vivo by Targeting the ETS2/miRNA196b/FOXO1/p27 Axis

Human bladder cancer (BC) is the fourth most common cancer in the United States. Investigation of the strategies aiming to elucidate the tumor growth and metastatic pathways in BC is critical for the management of this disease. Here we found that ATG7 expression was remarkably elevated in human bladder urothelial carcinoma and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced mouse invasive BC. Knockdown of ATG7 resulted in a significant inhibitory effect on tumorigenic growth of human BC cells both in vitro and in vivo by promoting p27 expression and inducing cell cycle arrest at G2/M phase. We further demonstrated that knockdown of ATG7 upregulated FOXO1 (forkhead box protein O 1) expression, which specifically promoted p27 transcription. Moreover, mechanistic studies revealed that inhibition of ATG7 stabilized ETS2 mRNA and, in turn, reduced miR-196b transcription and expression of miR-196b, which was able to bind to the 3′ UTR of FOXO1 mRNA, consequently stabilizing FOXO1 mRNA and finally promoting p27 transcription and attenuating BC tumorigenic growth. The identification of the ATG7/FOXO1/p27 mechanism for promoting BC cell growth provides significant insights into understanding the nature of BC tumorigenesis. Together with our most recent discovery of the crucial role of ATG7 in promoting BC invasion, it raises the potential for developing an ATG7-based specific therapeutic strategy for treatment of human BC patients.

XIAP BIR domain suppresses miR-200a expression and subsequently promotes EGFR protein translation and anchorage-independent growth of bladder cancer cell.

BackgroundThe X-linked inhibitor of apoptosis protein (XIAP) is a well-known potent apoptosis suppressor and also participates in cancer cell biological behaviors, therefore attracting great attentions as a potential antineoplastic therapeutic target for past years. Anti-IAP therapy is reported to be closely related to epidermal growth factor receptor (EGFR) expression level. However, whether and how XIAP modulates EGFR expression remains largely unknown.MethodsHuman XIAP was knockdown with short-hairpin RNA in two different bladder cancer cell lines, T24T and UMUC3. Two XIAP mutants, XIAP ∆BIR (deletion of N-terminal three BIR domains) and XIAP ∆RING (deletion of C-terminal RING domain and keeping the function of BIR domains), were generated to determine which domain is involved in regulating EGFR.ResultsWe found here that lacking of XIAP expression resulted in a remarkable suppression of EGFR expression, consequently leading to the deficiency of anchorage-independent cell growth. Further study demonstrated that BIR domain of XIAP was crucial for regulating the EGFR translation by suppressing the transcription and expression of miR-200a. Mechanistic studies indicated that BIR domain activated the protein phosphatase 2 (PP2A) activity by decreasing the phosphorylation of PP2A at Tyr307 in its catalytic subunit, PP2A-C. Such activated PP2A prevented the deviant phosphorylation and activation of MAPK kinases/MAPKs, their downstream effector c-Jun, and in turn inhibiting transcription of c-Jun-regulated the miR-200a.ConclusionsOur study uncovered a novel function of BIR domain of XIAP in regulating the EGFR translation, providing significant insight into the understanding of the XIAP overexpression in the cancer development and progression, further offering a new theoretical support for using XIAP BIR domain and EGFR as targets for cancer therapy.

MP49-06 STAT3 ACTIVATION STATUS CONTRIBUTES TO THE DIFFERENTIAL RESPONSES OF METASTATIC AND NO-METASTATIC HUMAN BLADDER CANCER CELLS TO MIR-145 INHIBITION OF ANCHORAGE-INDEPENDENT GROWTH THROUGH REGULATION OF FOXO1 EXPRESSION

INTRODUCTION AND OBJECTIVES: miR-145 was reported to be the most frequently downregulated miRNA in bladder cancer. However, the accurately determining of the networks regulated by miRNA in a specific condition is of great important. In this study, we address the stage-specific effects of miR-145 on bladder cancer. METHODS: TheexpressionofmiR-145wasassessedby real-time RT-PCR. Soft agar assay was used to determine anchorage-independent growth of the cancer cells. RT-PCR was used to evaluate mRNA expression, and western blot was used to detect the expression of protein level. Luciferase activity was evaluated by the Dual-Luciferase Reporter Assay. RESULTS: miR-145 was upregulated in human bladder cancer metastatic tissues in comparison to non-metastatic tissues and in metastatic T24T cells compared to parental non-metastatic T24 cells. Forced expression of miR-145 inhibited anchorage-independent growth of T24 cells, whereas it promoted growth of T24T cells. Overexpression of miR-145 resulted in the upregulation of both mRNA and protein levels of forkhead box class O1 (FOXO1) and its downstream effector, Cyclindependent kinase inhibitor 1B, in T24 cells, while they were downregulated by miR-145 in T24T cells. Moreover, knockdown of FOXO1 abolished the growth inhibition by miR-145 in T24 cells. Further studies showed that miR-145 could block FOXO1 mRNA 3’UTR activation in both T24 and T24T cells by targeting miR-145 binding site. On the other hand, miR-145 was demonstrated to suppress STAT3 activation and increase FOXO1 promoter transcription activation in T24 cells, and the mutation of STAT3 binding site at FOXO1 promoter effectively diminished the effects. In contrast, miR-145 overexpression did not show observable induction of FOXO1 promoter transcription activation in T24T cells that STAT3 was constitutively activated, whereas knockdown of STAT3 could restore the response of miR-145 induction of FOXO1 promoter transactivation, upregulation of FOXO1 protein expression and inhibition of growth in T24T cells. CONCLUSIONS: STAT3 activation status contributes to the differential responses of metastatic and no-metastatic bladder cancer cells to miR-145 inhibition of growth through regulation of FOXO1 expression. Our findings reveal the stage-specific function of miR-145 in bladder cancer, and provide novel useful information of the future potential utilization of miR-145 for therapy of bladder cancer.