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Cell viability and cell migration capacities are critical parameters for cell culture-related studies. It is essential to monitor the dynamic changes of cell properties under various co-culture conditions to our better understanding of their behaviours and characteristics. The real time cell analyzer (RTCA, xCELLigence, Roche) is an impedance-based technology that can be used for label-free and real-time monitoring of cell properties, such as cell adherence, proliferation, migration and cytotoxicity. The practicality of this system has been proven in our recent cancer studies. In the present method, we intend to use co-cultures of pancreatic cancer cells (HP62) and mesenchymal stem cells to describe in detail, the procedures and benefits of RTCA.

The Androgen Receptor (AR) is a ligand inducible transcription factor that drives expression of genes contributing to the growth, recurrence and metastasis of prostate cancer (PCa). The AR is activated by steroid hormones (androgens) that bind to a C-terminal ligand-binding domain (LBD) and cause the transcription factor to enter the nucleus, dimerize and interact with DNA via its conserved DNA-binding domain (DBD). Accordingly, treatment for PCa involves reducing DHT production or using small molecules (i.e. antiandrogens) to compete with the specific interaction between hormones and the AR-LBD, preventing AR activation. This treatment will eventually fail as recurrent PCa changes into a castration resistant form (CRPC) where AR signalling continues even in the absence of androgens. The occurrence of CRPC is driven, in part, by mutations in the AR-LBD that alter the binding of antiandrogens, or by the presence of constitutively active AR splice variants that lack the LBD, effectively removing the site of action of conventional antiandrogens. Here, we describe a surface exposed pocket on the AR-DBD, based on its 3D-crystal structure, which may qualify as an alternative target for small molecule inhibitors of AR transcriptional activity. Following virtual screening of many compounds, we characterize the interaction of the AR-DBD with various potential inhibitors and measure the efficacy of these agents to block the transcriptional activity of both full-length and splice variant AR in cell culture. Drug-inhibition was reduced upon site-directed mutagenesis of amino-acid residues in the DBD that are suspected of interacting with the compounds. Biochemical approaches are used to further examine the physical interaction of the purified AR-DBD protein with small molecules and to investigate their effect on protein-DNA interactions in vitro. Lastly, we use confocal microscopy of live PCa cells to investigate the effect of our compounds on the spatial localization and dimerization of fluorescently tagged AR proteins. These results illustrate the feasibility of targeting the AR-DBD to potentially inhibit the reactivation of AR transcriptional activity that is observed in CRPC. Citation Format: Kush Dalal, Huifang Li, Mani R. Moniri, Fuqiang Ban, Aishwariya Sharma, Artem Cherkasov, Paul S. Rennie. Targeting the DNA binding domain of the androgen receptor as a potential therapy for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2014-1235

No abstract

Androgen receptor- (AR-) expressing prostate cancer (PCa) cell lines can transdifferentiate to neuro-ectodermal cell lineages under androgen deprivation. We have proposed that this transdifferentiation is mediated by initial regression to an unstable cancer stem cell-like (CSCL) intermediate state and we have developed a serum- and androgen-free custom cell culture medium that now allows us to efficiently reprogram PCa cells to this CSCL state and expand them for characterization. Exposure of adherent cultures of parental LNCaP, VCaP, LAPC4 or CWR22rv1 cells to the reprogramming medium induced a rapid and efficient (>67%) morphological change of plated cells to smaller, rounded cells with large nuclei and prominent nucleoli that grow in 3-dimensions. Following transfer of the reprogrammed cells they, thereafter, grow as cell rosettes that will form larger spheroids. Analysis of changes in gene expression by microarray gene expression profiling or immunohistochemistry (IHC) showed a remarkable overexpression of stem cell genes in the reprogrammed cells and a change in cell surface CD protein expression that resembles neural stem cells. Reprogramming drastically reduced expression of AR RNA and protein in all cell lines. Once reprogrammed, the CSCLs have significantly higher tumor-initiating capacity based upon tumors formed from cell serial dilutions xenografted into intact or castrated male nude mice. In vitro, reprogrammed cells were significantly more resistant to the anti-androgen enzalutamide and to hypoxia compared to parentals. By placing CSCLs into specific differentiation mediums, we could differentiate them back into neural-, oligodendrocyte-, glia- or osteoblast-like cells based upon induced expression of lineage-specific RNAs/proteins or return them to prostate-like cells (that re-express AR) by supplementing them with serum and androgen. Reprogrammed cells have an EMT phenotype based on increased expression of EMT-related genes and significantly increased invasiveness in in vitro and in vivo models. Comparison of gene expression changes acquired by reprogrammed CSCLs amongst the different cell models revealed a shared set of 65 over-/under-expressed genes (≥1.8-fold) between them. Finally, we have found that growth of parental LNCaP cells in androgen-deprived or enzalutamide-supplemented (serum-containing) growth medium increased the population of cells bearing CSCL surface CD stem markers. In summary, our studies show that commonly used PCa cell lines have the ability to efficiently transition between a differentiated and a CSCL state in an appropriate microenvironment that is depleted of androgens. While in the CSCL state, these cells show increased invasiveness, increased resistance to hormonal therapeutics and a propensity to transdifferentiate to alternate cell lineages under appropriate stimulation. Citation Format: Josselin Caradec, Amy A. Lubik, Mannan Nouri, Na Li, Manuel Altimarano-Dimas, Jennifer Bishop, Mani Moniri, Down Cochrane, Martin Gleave, Ralph Buttyan. Efficient conditional reprogramming of differentiated prostate cancer cells back to a stem cell-like state with increased aggressive properties. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4073. doi:10.1158/1538-7445.AM2015-4073