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

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

Recent studies showed the potential linkage of estrogen/estrogen receptor signaling with bladder tumorigenesis, yet detailed mechanisms remain elusive. Here we found a new potential therapy with the combination of Bacillus Calmette-Guerin (BCG) and the anti-estrogen ICI 182,780 led to better suppression of bladder cancer (BCa) than BCG alone. Mechanism dissection found ICI 182,780 could promote BCG attachment/internalization to the BCa cells through increased integrin-α5β1 expression and IL-6 release, which may enhance BCG-induced suppression of BCa cell growth via recruiting more monocytes/macrophages to BCa cells and increased TNF-α release. Consistently, in vivo studies found ICI 182,780 could potentiate the anti-BCa effects of BCG in the carcinogen-induced mouse BCa models. Together, these in vitro and in vivo results suggest that combining BCG with anti-estrogen may become a new therapeutic approach with better efficacy to suppress BCa progression and recurrence.

Fatty acid synthase (FASN) is the key enzyme for the control of fatty acid synthesis that contributes significantly to the prostate cancer (PCa) progression. It was reported that androgens were able to induce FASN expression in PCa, and addition of the anti‐androgen Casodex might suppress the androgen‐induced FASN expression. However, here we found androgen‐deprivation‐therapy (ADT) with anti‐androgens Bicalutamide (Casodex) or Enzalutamide (MDV3100) had little effect to suppress FASN expression and FASN‐mediated cell growth and invasion during the castration resistant stage when the androgen concentration is 1 nM DHT (dihydrotestosterone). In contrast, the newly developed androgen receptor (AR) degradation enhancer ASC‐J9® suppressed FASN expression and FASN‐mediated cell growth and invasion in various PCa cell lines at 1 nM DHT. Mechanism dissection found ASC‐J9® could suppress significantly the FASN expression and FASN‐mediated PCa progression via the AR‐dependent pathway involving AR→SREBP‐1→FASN signaling in AR‐positive C4‐2 and LNCaP cells and via the AR‐independent pathway involving the modulation of PI3K/AKT→SREBP‐1→FASN signaling in AR‐negative PC‐3 and DU145 cells. Together, these results suggest that FASN is one of the important mechanism why the current ADT eventually fails. ASC‐J9® might represent a new potential therapeutic approach to suppress FASN‐mediated PCa progression via both AR‐dependent and AR‐independent pathways during the castration resistant stage of PCa. © 2016 Wiley Periodicals, Inc.

Circulating tumor cells (CTCs) are a group of rare cancer cells that have detached from a primary tumor and circulate in the bloodstream. Herein, light addressable potentiometric sensor (LAPS) was exploited in the label-free detection of CTCs in the prostate cancer. To this end, the mouse anti-human epithelial cell adhesion molecule (anti-EpCAM) monoclonal antibody was selected as the probe to capture CTCs according to our western blot experiments, and therefore the anti-EpCAM was immobilized on the surface of carboxylated graphene oxide (GO-COOH) modified LAPS. Spiking experiments confirmed that LAPS' voltage decreased with the increasing of CTCs' concentration both in phosphate buffer (PBS) and blood, and as few as 10 CTCs in 1 ml of blood could be detected, illustrating the high sensitivity of the proposed strategy. The analysis of healthy blood samples revealed no change in electrical signal, confirming the specificity of the system. Ultraviolet–visible (UV–vis) spectroscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and immunofluorescent assay (IFA) were conducted to characterize GO-COOH, testify its existence on LAPS and validate CTCs' capturing by anti-EpCAM grafted on GO-COOH modified substrates. It is indicated that LAPS could be a potential platform for CTCs detection and may provide a powerful tool for downstream analysis.

Abstract Recent studies suggest that the androgen receptor (AR) might play important roles in influencing bladder cancer progression, yet its clinical application remains unclear. Here, we developed a new combined therapy with Bacillus Calmette–Guérin (BCG) and the AR degradation enhancer ASC-J9 or antiandrogen hydroxyflutamide (HF) to better suppress bladder cancer progression. Mechanism dissection revealed that ASC-J9 treatment enhanced BCG efficacy to suppress bladder cancer cell proliferation via increasing the recruitment of monocytes/macrophages that involved the promotion of BCG attachment/internalization to the bladder cancer cells through increased integrin-α5β1 expression and IL6 release. Such consequences might then enhance BCG-induced bladder cancer cell death via increased TNFα release. Interestingly, we also found that ASC-J9 treatment could directly promote BCG-induced HMGB1 release to enhance the BCG cytotoxic effects for suppression of bladder cancer cell growth. In vivo approaches also concluded that ASC-J9 could enhance the efficacy of BCG to better suppress bladder cancer progression in BBN-induced bladder cancer mouse models. Together, these results suggest that the newly developed therapy combining BCG plus ASC-J9 may become a novel therapy to better suppress bladder cancer progress. Mol Cancer Ther; 14(11); 2586–94. ©2015 AACR.