Androgen receptor variant-7 regulation by tenascin-c induced src activation.

BACKGROUND: Bone metastatic prostate cancer does not completely respond to androgen-targeted therapy and generally evolves into lethal castration resistant prostate cancer (CRPC). Expression of AR-V7- a constitutively active, ligand independent splice variant of AR is one of the critical resistant mechanisms regulating metastatic CRPC. TNC is an extracellular matrix glycoprotein, crucial for prostate cancer progression, and associated with prostate cancer bone metastases. In this study, we investigated the mechanisms that regulate AR-V7 expression in prostate cancer cells interacting with osteogenic microenvironment including TNC. METHODS: Prostate cancer/preosteoblast heterotypical organoids were evaluated via immunofluorescence imaging and gene expression analysis using RT-qPCR to assess cellular compartmentalization, TNC localization, and to investigate regulation of AR-V7 in prostate cancer cells by preosteoblasts and hormone or antiandrogen action. Prostate cancer cells cultured on TNC were assessed using RT-qPCR, Western blotting, cycloheximide chase assay, and immunofluorescence imaging to evaluate (1) regulation of AR-V7, and (2) signaling pathways activated by TNC. Identified signaling pathway induced by TNC was targeted using siRNA and a small molecular inhibitor to investigate the role of TNC-induced signaling activation in regulation of AR-V7. Both AR-V7- and TNC-induced signaling effectors were targeted using siRNA, and TNC expression assessed to evaluate potential feedback regulation. RESULTS: Utilizing heterotypical organoids, we show that TNC is an integral component of prostate cancer interaction with preosteoblasts. Interaction with preosteoblasts upregulated both TNC and AR-V7 expression in prostate cancer cells which was suppressed by testosterone but elevated by antiandrogen enzalutamide. Interestingly, the results demonstrate that TNC-induced Src activation regulated AR-V7 expression, post-translational stability, and nuclear localization in prostate cancer cells. Treatment with TNC neutralizing antibody, Src knockdown, and inhibition of Src kinase activity repressed AR-V7 transcript and protein. Reciprocally, both activated Src and AR-V7 were observed to upregulate autocrine TNC gene expression in prostate cancer cells. CONCLUSION: Overall, the findings reveal that prostate cancer cell interactions with the cellular and ECM components in the osteogenic microenvironment plays critical role in regulating AR-V7 associated with metastatic CRPC. Video Abstract.

Modulation of adenoviral transduction in vitro and in vivo by hyaluronan and its receptor CD44.

Adenovirus infection is a significant cause of ocular, respiratory, and gastrointestinal illness and can spread rapidly. Morbidity is considerable in immune-suppressed individuals and there is significant mortality. There are no effective therapies. During preclinical studies of adenoviral-mediated gene therapy for ocular disorders, we noticed a significant increase in transduction when the target cells were exposed to adenovirus in the presence of ocular vitreous. The vitreous is mainly comprised of water, collagen, and the large polysaccharide hyaluronan. In this paper, we report data that implicate hyaluronan in the adenoviral infectious process and show that interference with the interaction between hyaluronan and its cellular receptor CD44 can block adenovirus transduction in vitro and in vivo.

Efficient gene transfer into retinal cells using adenoviral vectors: dependence on receptor expression.

PURPOSE: A number of ocular diseases are potentially amenable to gene therapy interventions if appropriate vectors for the targeted administration of therapeutic genes can be identified. In vitro and in vivo transduction efficiency of a Group C serotype 5 adenoviral vector containing the fiber domain derived from a Group B serotype 35 adenovirus and the gene encoding green fluorescent protein (AdV5/F35-GFP) was compared to an AdV5-GFP vector for transgene delivery to human retinoblastoma and to human and murine retinas. METHODS: The distribution of the adenoviral receptors CAR and CD46 on normal and malignant retinal tissues was determined using immunohistochemistry. Human retinoblastoma cells were incubated with either AdV5-GFP or AdV5/F35-GFP, and the expression of the reporter protein was compared using quantitative fluorescence and fluorescent-activated cell sorting. Mice were given a single subretinal injection of either viral vector, and eyes were enucleated at specified times after injection for histopathologic examination. Human cadaver eyes were similarly examined ex vivo. RESULTS: CAR was expressed in retina except in photoreceptor outer segments. CD46 was expressed in photoreceptor inner and outer segments. Both vectors efficiently transduced the human retinoblastoma cells in vitro. However, the amount of the transgene expressed using AdV5/F35-GFP was more than sixfold greater than that when AdV5-GFP was used. In vivo, AdV5/F35-GFP at doses as low as 10(5) infectious units (IU) transduced cells in all layers of the retina especially photoreceptors and occasional neuronal cells, and Müller cells as well as retinal pigment epithelial cells, whereas AdV5-GFP transduced only retinal pigment epithelial cells and occasional photoreceptors and Müller cells. CONCLUSIONS: AdV5/F35 chimeric vectors may be superior to AdV5 for gene therapy applications targeting the photoreceptor.