Faculty Bibliography

Androgen receptor (AR) signaling in stromal cells is important in prostate cancer, yet the mechanisms underpinning stromal AR contribution to disease development and progression remain unclear. Using patient-matched benign and malignant prostate samples, we show a significant association between low AR levels in cancer associated stroma and increased prostate cancer-related death at one, three and five years post-diganosis, and in tissue recombination models with primary prostate cancer cells that low stromal AR decreases castration-induced apoptosis. AR-regulation was found to be different in primary human fibroblasts isolated from adjacent to cancerous and non-cancerous prostate epithelia, and to represent altered activation of myofibroblast pathways involved in cell cycle, adhesion, migration, and the extracellular matrix (ECM). Without AR signaling, the fibroblast-derived ECM loses the capacity to promote attachment of both myofibroblasts and cancer cells, is less able to prevent cell-matrix disruption, and is less likely to impede cancer cell invasion. AR signaling in prostate cancer stroma appears therefore to alter patient outcome by maintaining an ECM microenvironment inhibitory to cancer cell invasion. This paper provides comprehensive insight into AR signaling in the non-epithelial prostate microenvironment, and a resource from which the prognostic and therapeutic implications of stromal AR levels can be further explored.

Although the antitumor role of metformin has been widely reported, the molecular mechanism of this biguanide agent in the inhibition of tumor progression remains unclear. Here, we identified miR-708-5p as a novel target of metformin in prostate cancer cells. Metformin promotes increased expression of miR-708-5p, leading to suppression of endoplasmic reticulum (ER) membrane protein neuronatin (NNAT) expression and subsequently induces apoptosis of prostate cancer cells through the ER stress pathway. Further, miR-708-5p-induced knockdown of NNAT is associated with downregulated intracellular calcium levels and induced malformation of ER-ribosome structure revealed by electronic microscopy. Meanwhile, the unfolded protein response regulator CHOP, p-eIF2alpha, calreticulin, GRP78 and ATP2A1, all of which are also considered as ER stress markers, are upregulated by metformin and miR-708-5p. Taken together, our findings clearly demonstrate that metformin stimulates increased expression of miR-708-5p to target the NNAT-mediated response to ER stress and apoptosis. This novel regulatory mechanism of metformin in prostate cancer cells not only advances our knowledge on the molecular mechanism of metformin but also provides a promising therapeutic strategy by targeting miR-708-5p and NNAT for prostate cancer treatment.

The microRNA-34a (miR-34a), a tumor suppressive microRNA (miRNA), is implicated in epithelial-mesenchymal transition (EMT) and cancer stem cells. Lymphoid enhancer-binding factor-1 (LEF1) is a key transcription factor in the Wnt signaling pathway, and has been suggested to be involved in regulation of cell proliferation and invasion. Here, the molecular mechanism of miR-34a and LEF1 in cooperatively regulating prostate cancer (PCa) cell invasion is described. Molecular profiling analysis of miRNA levels in PCa cells revealed a negative correlation between miR-34a and LEF1 expression, and the downregulation of LEF1 by miR-34a was confirmed by luciferase assays. Further, miR-34a specifically repressed LEF1 expression through direct binding to its 3'-untranslated (3'-UTR) regions. miR-34a modulated the levels of LEF1 to regulate EMT in PCa cells. Functionally, miR-34a negatively correlated with the migration and invasion of PCa cells through LEF1. An analysis of miR-34a expression levels in matched human tumor and benign tissues demonstrated consistent and statistically significant downregulation of miR-34a in primary prostate cancer specimens. These data strongly suggest that miR-34a/LEF1 regulation of EMT plays an important role in PCa migration and invasion. Implications: The miR-34a/LEF1 axis represents a potential molecular target for novel therapeutic strategies in PCa.

IL-6 plays an important role in various inflammatory ocular diseases, including diabetic retinopathy. Muller cells are the major source of inflammatory mediators, including IL-6, in the retina. However, the mechanism of regulating IL-6 production in these cells remains unclear. Examination of signaling pathways in human retinal Muller cells (MIO-M1 cell line) cultured with IL-1beta, TNF-alpha, IL-6, IL-8, VEGF, IFN-gamma, glucose or mannitol showed that IL-1beta was the most potent stimulator of IL-6 production. In addition, IL-1 beta also increased NF-kappaB p50 protein level and phosphorylation of p38 MAPK, ERK1/2 and c-Jun. Induction of IL-6 production by IL-1beta was significantly reduced by addition of p38 MAPK (SB203580), MEK1/2 (U0126) or NF-kappaB (BAY11-7082) inhibitors, with the highest effect being observed with SB203580. To explore the specific elements in IL-6 promoter responsible for IL-1beta-induction of IL-6 expression, a series of plasmids bearing various IL-6 promoter mutations were transiently expressed in MIO-MI cells cultured in the presence or absence of IL-1beta (10ng/ml) and/or SB203580 (10microM). Results showed that IL-6 promoter activity of the parent pIL-6-Luc651 was significantly enhanced by IL-1beta, but the level was significantly attenuated by SB203580. Furthermore, the IL-6 promoter activity was also reduced upon deletion of NF-kappaB, AP-1 or C/EBP binding sites, with NF-kappaB deletion being the greatest. These results are the first demonstration that IL-1beta induces IL-6 production in Muller cells by activation of IL-6 promoter activity predominantly through the p38 MAPK/NF-kappaB pathway.

Post-translational modifications have been identified to be of great importance in cancers and lysine acetylation, which can attract the multifunctional transcription factor BRD4, has been identified as a potential therapeutic target. In this paper, we identify that BRD4 has an important role in colorectal cancer; and that its inhibition substantially wipes out tumor cells. Treatment with inhibitor MS417 potently affects cancer cells, although such effects were not always outright necrosis or apoptosis. We report that BRD4 inhibition also limits distal metastasis by regulating several key proteins in the progression of epithelial-to-mesenchymal transition (EMT). This effect of BRD4 inhibitor is demonstrated via liver metastasis in animal model as well as migration and invasion experiments in vitro. Together, our results demonstrate a new application of BRD4 inhibitor that may be of clinical use by virtue of its ability to limit metastasis while also being tumorcidal.

The inhibitory effects of metformin have been observed in many types of cancer. However, its effect on human salivary gland carcinoma is unknown. The effect of metformin alone or in combination with pp242 (an mTOR inhibitor) on salivary adenocarcinoma cells growth were determined in vitro and in vivo. We found that metformin suppressed HSY cell growth in vitro in a time and dose dependent manner associated with a reduced expression of MYC onco-protein, and the same inhibitory effect of metformin was also confirmed in HSG cells. In association with the reduction of MYC onco-protein, metformin significantly restored p53 tumor suppressor gene expression. The distinctive effects of metformin and PP242 on MYC reduction and P53 restoration suggested that metformin inhibited cell growth through a different pathway from PP242 in salivary carcinoma cells. Furthermore, the anti-tumor efficacy of metformin was confirmed in vivo as indicated by the increases of tumor necrosis and reduced proliferation in xenograft tumors from metformin treated group. For the first time, the inhibitory effect of metformin on human salivary gland tumor cells was documented. Moreover, metformin inhibitory effects were enhanced by mTOR inhibitor suggesting that metformin and mTOR inhibitor utilize distinctive signaling pathways to suppress salivary tumor growth.

Lymphoid enhancer-binding factor-1 (LEF1) is a key transcription factor mediating Wnt signaling pathway. Our previous studies indicate that LEF1 is highly expressed in androgen-independent prostate cancer (PCa) and enhances invasion ability in androgen-independent PCa cells. However, the molecular mechanism of LEF1 effect on invasion remains largely unknown. Using microRNA profiling analysis comparing androgen-independent LNCaP-AI PCa cells with high levels of endogenous LEF1 to LNCaP-AI cells with LEF1 knockdown by LEF1shRNA, we found miR-181a to be increased 12.3-fold in LNCaP-AI cells. We confirmed a positive correlation between LEF1 and miR-181a expression across multiple PCa cell lines. Additionally, we showed that in PCa cells, overexpression of LEF1 increased miR-181a expression and subsequently induced EMT associated migration and invasion, whereas LEF1 knockdown decreased miR-181a expression and subsequently resulted in inhibition of EMT, migration and invasion. Mechanistically, we demonstrated by chromatin immunoprecipitation assays that LEF1 could enhance miR-181a expression via its binding to the promoter regions of hsa-miR-181a. Overall, this study identified a novel LEF1-miR-181a-EMT axis in regulation of PCa migration and invasion.

The androgen receptor (AR) in stromal cells contributes significantly to the development and growth of prostate during fetal stages as well as during prostate carcinogenesis and cancer progression. During prostate development, stromal AR induces and promotes epithelial cell growth, as observed from tissue recombinant and mouse knockout studies. During prostate carcinogenesis and progression, the stromal cells begin to lose AR expression as early as at the stage of high-grade prostatic intraepithelial neoplasia. The extent of loss of stromal AR is directly proportional to the degree of differentiation (Gleason grade) and progression of prostate cancer (PCa). Co-culture studies suggested that stromal AR inhibits the growth of malignant epithelial cells, possibly through expression of certain paracrine factors in the presence of androgens. This functional reversal of stromal AR, from growth promotion during fetal prostate development to mediating certain growth-inhibiting effects in cancer, explains to some extent the reason that loss of AR expression in stromal cells may be crucial for development of resistance to androgen ablation therapy for PCa. From a translational perspective, it generates the need to re-examine the current therapeutic options and opens a fundamental new direction for therapeutic interventions, especially in advanced PCa.

The microbiome is a new center of attention for studies on the pathogenesis of human disease by focusing on the alterations of all microorganisms living in a particular site or system of human body, referred as microbiota. Evidence suggests that microbiota could contribute to the pathogenesis of a number of chronic diseases, including cancers, both locally and remotely. Multiple mechanisms have been proposed and/or proven for the microbiota's role in tumorigenesis, such as via induction of chronic inflammation, genotoxicity, bacterium-mediated cell proliferation, and activation of procarcinogens. Emerging data suggest that indigenous microbiota in the urinary tract may play an important role in the tumorigenesis of urothelial carcinoma, similar to other tumors. Future studies are needed to adequately define the microbiota composition and correlate its change with urothelial carcinoma.