Supplemental figure 1 ASC-J9® degraded androgen receptor expression in bladder cancer

PDF file - 471K, Figure. S5. AR suppresses the expression of Hif-2αand VEGF via could miR-145.

PDF file - 1477K, Figure. S3. RCC cell proliferative ability were determined by BrdU incorporation assay.

You have accessJournal of UrologyCME1 Apr 2023MP20-10 ENZALUTAMIDE INCREASES PROSTATE CANCER (PCa) CELL INVASION YET DECREASES BLADDER CANCER (BCa) CELL INVASION VIA DIFFERENTIALLY REGULATING THE AR/circRNA-ARC1/miR-125b-2-3p OR MiR-4736/PPARγ/MMP-9 SIGNALS Gang Deng, Yixi Hu, Yin Sun, Jean Joseph, Shuyuan Yeh, Edward M. Messing, and Chawnshang Chang Gang DengGang Deng More articles by this author , Yixi HuYixi Hu More articles by this author , Yin SunYin Sun More articles by this author , Jean JosephJean Joseph More articles by this author , Shuyuan YehShuyuan Yeh More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , and Chawnshang ChangChawnshang Chang More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003245.10AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Androgen-deprivation therapy (ADT) is a standard treatment to prevent and shrink PCa growing. However, some patients undergoing ADT may finally develop to hormone-insensitive PCa. The underlying mechanisms haven't been fully elucidated. Here we found ADT-Enz led to increased PCa cell invasion, yet decreased BCa cell invasion. Our goal is to 1.reveal mechanisms of Enz induced hormone-insensitive PCa and find a potential combination therapy. 2. reveal mechanisms of ADT role in BCa and provide an evidence of applying ADT in BCa. METHODS: The human PCa and BCa cells: C4-2, CWR22Rv1, T24 and TCC-SUP were used. Lentiviral plasmids were used to generate gene engineering tools. qPCR and Western blot were used to detect gene expressions. Invasion assay were applied to determine cancer cell invasion capability. Protein-DNA binding complexes were detected by Chromatin immunoprecipitation (ChIP) assays. PPARGC1B/PDK1 3′UTR regions were cloned into the psiCHECKTM-2 vector to assay the miRNA functions. Preclinical mouse PCa model was used to evaluate the anti-cancer efficacy of Enz and circRNA-ARC1 blockage. RESULTS: Mechanistic dissection revealed that suppressing androgens/AR signals could result in differential alterations of the selective circular RNAs (circRNAs) as a result of differential endogenous AR transcription. A negative autoregulation in PCa, yet a positive autoregulation in BCa, as a result of differential binding of AR to different androgen-response elements (AREs) and a discriminating histone H3K4 methylation. Further mechanistic studies indicated that AR-encoded circRNA-ARC1 might sponge the availability of the miR-125b-2-3p and/or miR-4736 to impact the metastasis-related PPARγ/MMP-9 signals. The preclinical mouse model confirms in vitro cell lines data, showing that Enz treatment could increase PCa metastasis, which can be suppressed after iknockdown of circRNA-ARC1. Together, these results demonstrate that antiandrogen therapy with Enz treatment may differentially lead to promoting PCa cell invasion, yet decreasing BCa cell invasion. CONCLUSIONS: Our study is the first to reveal ADT-Enz treatment can promote PCa Cell Invasion yet decrease BCa cell Invasion via differentially regulating the AR/circRNA-ARC1/miR-125b-2-3p or MiR-4736/PPARγ/MMP-9 Signals. Furthermore, our findings may help in development of novel therapeutic approaches to increase the Enz efficacy with less adverse effects in patients with PCa, and providing an evidence of applying ADT in BCa. Source of Funding: This work was partially supported by URMC Urology Department Research fund and George H. Whipple Professorship Endowment © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e279 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Gang Deng More articles by this author Yixi Hu More articles by this author Yin Sun More articles by this author Jean Joseph More articles by this author Shuyuan Yeh More articles by this author Edward M. Messing More articles by this author Chawnshang Chang More articles by this author Expand All Advertisement PDF downloadLoading ...

<p>Supplementary Material and Methods include information for performing/preparing (1) lentivirus particle production and transduction of RCC cells, (2) qPCR, (3) western blot analysis, (4) IHC staining and quantification, and (5) the shRNA#1 and #2 targeting sequences for circATP2B1 and ERβ.</p>

PDF file - 271K, Figure. S4. HIF2a promoter luciferase assay also demonstrated that AR could modulate the transcription of HIF-2α in RCC cells.

Supplemental figure 1 ASC-J9® degraded androgen receptor expression in bladder cancer cells.Supplemental figure 2 HF or ASC-J9® induced THP-1 cell differentiating to M1 macrophage

Quantitation for Western Blot data. A. Quantitation for Fig. 1C/D. B. Quantitation for Fig. 2A/D. C. Quantitation for Fig. 3D. D. Quantitation for Fig. 5A.

You have accessJournal of UrologyCME1 Apr 2023PD17-07 COMBINATION THERAPY FOR TKI RESISTANT KIDNEY CANCER VIA BLOCKING ERΒ/CIRCRNA-DGKD/MIR-125-5P/VE-CADHERIN SIGNAL INDUCED VASCULOGENIC MIMICRY Jie Ding, Yixi Hu, Chawnshang Chang, Edward M. Messing, Jean Joseph, and Shuyuan Yeh Jie DingJie Ding More articles by this author , Yixi HuYixi Hu More articles by this author , Chawnshang ChangChawnshang Chang More articles by this author , Edward M. MessingEdward M. Messing More articles by this author , Jean JosephJean Joseph More articles by this author , and Shuyuan YehShuyuan Yeh More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003272.07AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Tyrosine kinase inhibitors (TKIs), pazopanib and sunitinib, are widely used in renal cell carcinoma (RCC) therapy by inhibiting tumor angiogenesis. However, most patients become insensitive or develop resistance toward TKI therapy eventually, and the underlying mechanisms have not yet been fully elucidated. Our goal is to reveal the molecular mechanisms of TKI resistant RCC to find a potential combination therapy for TKI-resistant RCC. METHODS: The human RCC cell lines: 786-O, A498, Caki-1, and SW-839 were used. Lentiviral plasmids were used to generate gene engineering tools. The qPCR and Western blot were used to evaluate the gene expressions at RNA and protein levels, respectively. Vasculogenic mimicry (VM) assays were performed to examine vascular-like structure formation ability of cancer cells. Protein-DNA binding complexes were detected by Chromatin immunoprecipitation (ChIP) assays. The human DGKD promoter region was cloned into pGL3 vector for Luciferase Assays. Preclinical mouse RCC model was used to evaluate the anti-cancer effect of pazopanib and circRNA-DGKD blockage in TKI resistant RCC. RESULTS: TKI resistant RCC cells showed increased ability of forming VM to counteract the TKI-suppression function both in vivo and in vitro. Mechanism dissection revealed that pazopanib (or sunitinib)-resistant RCC cells had an increased ERβ, consequently enhancing the circRNA-DGKD expression. In turn, circRNA-DGKD could increase VE-cadherin expression via sponging the miR-125-5p. Supportively, targeting circRNA-DGKD intercepted the RCC VM formation, reduced metastases and improved survival in an orthotopic RCC mouse model. Collectively, the findings may aid in the development of novel therapies for metastatic or TKI-resistant RCC. CONCLUSIONS: Our study reveals that the resistance to TKIs (pazopanib or sunitinib) occurs by increasing vasculogenic mimicry (VM) ability to counteract the TKI angiogenesis suppression function. Furthermore, we find a potential therapy to more effectively treat TKI resistant RCC by blocking ERβ/circRNA-DGKD/miR-125-5p/VE-cadherin pathway together with pazopanib administration. Source of Funding: This work was partially supported by URMC Urology Department Research fund and George H. Whipple Professorship Endowment © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e498 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jie Ding More articles by this author Yixi Hu More articles by this author Chawnshang Chang More articles by this author Edward M. Messing More articles by this author Jean Joseph More articles by this author Shuyuan Yeh More articles by this author Expand All Advertisement PDF downloadLoading ...

PDF file - 365K, Figure. S6. VHL inhibit HIF-2a and VEGF expression independent AR.

Two different shERβs, shERβ#1 and shERβ#2, can independently and effectively knock down ERβ and regulate ERβ downstream signals in RCC cells.

PDF file - 3303K, Figure. S7. blocking AR signaling could inhibit AR positive cell migration, invasion and proliferation in a concentration-dependent in vivo and in vitro.

Supplemental figure 2 HF or ASC-J9® induced THP-1 cell differentiating to M1 macrophage

<p>Figure Legends for the Supplementary Figures</p>

Bladder cancer is the ninth most common cancer worldwide with a striking sex-based difference in incidence. Emerging evidence indicates that the androgen receptor (AR) might promote the development, progression and recurrence of bladder cancer, contributing to the observed sex differences. Targeting androgen–AR signalling has promise as potential therapy for bladder cancer and helps to suppress progression of this disease. In addition, the identification of a new membrane AR and AR-regulated non-coding RNAs has important implications for bladder cancer treatment. The success of human clinical trials of targeted-AR therapies will help in the development of improved treatments for patients with bladder cancer.

Supplemental Fig. S1 Endogenous AR expression in various HCC cell lines; Supplemental Fig. S2 Multiple E:T ratio for NK cells against HCC cells; Supplemental Fig. S3 Tunel assay for NK cells against SNU-423 cells; Supplemental Fig. S4 IL-12p35 release of HCC cells with various AR; Supplemental Fig. S5 MAGNET V2.0 calculated correlation between IL12A and AR; Supplemental Fig. S6 Distribution of NK cells in different region of HCC;Supplemental Fig. S7 IL-12p35 release induced by sorafenib treatment; Supplemental Fig. S8 Ectopic AR in HCC cells interrupted sorafenib induced IL-12A; Supplemental Fig. S9 Correlation analysis for animal tumor samples staining

30 metastasis-related genes used for screening.

PDF file - 1486K, Figure. S8. Treatment with ASC-J9 could inhibit RCC cells migration, invasion and proliferation.

PDF file - 267K, Fig. s1. HMECs induce the PCa cells invasion in vitro. Fig. s2. ECs down-regulated AR signaling in PCa cells. Fig. s3. Cytokines/chemokines level changes in ECs upon PCa cells co-culture. Fig. s4. EMT markers expressions changes in PCa cells upon ECs co-culture.