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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

This study was performed to demonstrate the transportation of an engineered MSC-produced intracellular anticancer gene product between mesenchymal stem cell (MSC) and cancer cells. MSC-mediated anticancer strategy has held great promise owing to MSCs' capacity of tumor-directed migration and the availability of specific anticancer genes. All anticancer genes that have been used in previous MSC-mediated anticancer studies were limited in functioning via extracellular mechanisms, mainly because of the restriction by cell membrane to macromolecules including proteins. In order to apply the majority of potent anticancer genes to the MSCmediated anticancer system, a specifically designed expression vector which bears an intracellular anticancer gene, PTEN, is utilized to demonstrate the feasibility of the system in cancer therapies. A transacting activator of transcription (TAT) was introduced into an expression vector followed by a segment for PTEN-RFP fusion protein. A direct demonstration of PTEN-RFP transportation between MSC and cancer cells was obtained from direct co-cultures. A marked cancer cell death was observed in indirect co-cultures with conditioned media from PTEN-transfected MSCs. The demonstration of PTEN-engineered MSC-produced PTEN transportation indicates the feasibility of applying intracellular anticancer gene expression system in MSC-mediated strategies for cancer therapy.

Despite the development of several successful targeted therapies, drug resistance and metastasis remain a significant challenge in the treatment of both breast (BCa) and prostate (PCa) cancers. For these reasons, there is an immediate need to identify a novel class of therapeutics targeting alternative factors, such as those that promote the metastatic capacity of tumour cells. ETS translocation variant 4 (ETV4) is a member of the ETS transcription factor family and is a significant mediator of tumorigenesis through its activation of several downstream pathways that are associated with migration and invasion. ETV4 is overexpressed in breast tumours and is associated with distant metastasis and poor prognosis particularly in triple negative BCa, which still lacks an approved targeted therapy. Similarly in PCa, overexpression of ETV4 is associated with the deregulation of the PI3K and Ras signalling pathways that are commonly implicated in metastatic disease. Like other ETS factors such as ERG and ETV1, fusion of the ETV4 gene can be found in a subtype of PCa cases and is associated with the disease progression. Thus, ETV4 is an important therapeutic candidate with potential applications in both advanced BCa and PCa. Drug development against ETV4 is made even more significant due to the lack of any approved therapy that directly targets it or any other members of the ETS family. Using a combination of in silico screening and in vitro assays, we have identified several small molecules with strong binding affinities and selectivity toward the DNA binding domain of ETV4. The prostate cell line, PC3, and the triple negative breast cancer cell line, MDA-MB-231, were used for in vitro studies as they endogenously express moderate to high levels of ETV4. Over 100 candidate compounds, which were selected from virtual screening against millions of small-molecular structures, were tested across the two cell lines using a luciferase-based transcriptional reporter assay. From this assay approximately 30 compounds were identified with significant inhibition on the transcriptional activity of ETV4 without a cytotoxic effect. The most potent of these compounds was shown by protein nuclear magnetic resonance (NMR) to directly interact with specific residues within the DNA binding domain. These compounds were also able to inhibit the migratory capacity of cancer cells. This data provides evidence for the direct targeting of ETV4 by small molecules, and future work will aim to further characterize their mechanism of action and effects on downstream targets in an effort to create a novel therapeutic strategy to treat metastatic breast and prostate cancers. (Supported by grants from the Canadian Cancer Society Research Initiative and Prostate Cancer Canada) Citation Format: Miriam S. Butler, Michael Hsing, Mani Roshan Moniri, Desmond Lau, Paul Yen, Ari Kim, Scott Lien, Marta Mroczek, Fariba Ghaidi, Eric LeBlanc, Lawrence McIntosh, Michael Cox, Artem Cherkasov, Paul S. Rennie. Targeting ETS factor ETV4 as a novel therapeutic for the management of breast and prostate cancer. [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 1648. doi:10.1158/1538-7445.AM2015-1648