Ralph Buttyan

Professor

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Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - Nov 2020)
Sox9 Mediated Reprogramming of Prostate Cancer Cells and Acquired Therapy Resistance (2019)

Treatment-induced neuroendocrine trans-differentiation (NEtD) complicates therapies for metastatic prostate cancer (PCa). Based on evidence that PCa cells can transdifferentiate to other neuroectodermally-derived cell lineages in vitro, we proposed that NEtD requires first an intermediary reprogramming to metastable cancer stem-like cells (CSCs) of a neural class and we demonstrate that several different AR⁺/PSA⁺ PCa cell lines were efficiently reprogrammed to, maintained and propagated as CSCs by growth in androgen-free neural/neural crest (N/NC) stem transition medium (STM). Such reprogrammed cells lost features of prostate differentiation; gained features of N/NC stem cells including tumour-initiating potential; were resistant to androgen signaling inhibition; and acquired an invasive phenotype in vitro and in vivo. These aggressive features were linked to overexpression of N/NC master regulator Sox9, with concomitant loss of Rb1 and p53 function. Functional analysis employing Sox9 constructs phenocopied invasive and expression profiles of STM-mediated development reprogramming. Meanwhile, targeting of Sox9 and it’s upstream activators reduced aggressive features and sensitized cells to androgen signalling inhibition. Acute androgen deprivation or anti-androgen treatment of PCa cells led to the transient appearance of a sub-population of cells with similar characteristics to the developmentally reprogrammed CSCs described above. Notably, a 132-gene signature derived from reprogrammed PCa cell lines distinguished tumours from PCa patients with adverse outcomes. When placed back into serum-containing mediums, reprogrammed cells could be re-differentiated to N-/NC-derived cell lineages or re-differentiate to an AR⁺ prostate-like state. Once returned, the AR⁺ cells were resistant to androgen signaling inhibition, and had retained aggressiveness markers like NRP1 and EZH2 associated with the CSC state. This model may explain neural manifestations of PCa associated with lethal disease. The metastable nature of the reprogrammed stem-like PCa cells suggests that cycles of PCa cell reprogramming followed by re-differentiation may support disease progression and therapeutic resistance. The ability of a gene signature from reprogrammed PCa cells to identify tumours from patients with metastasis or PCa-specific mortality implies that developmental reprogramming is linked to aggressive tumour behaviors. Finally, the signature of serum return cells functioned as a recurrence model in identifying genes associated with enhanced aggressiveness in therapy resistance.

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Master's Student Supervision (2010 - 2018)
Non-canonical activation of Hedgehog signaling in prostate cancer cells is mediated by the interaction of Gli proteins and transcriptionally active androgen receptor (2018)

The Hedgehog (Hh) pathway is an embryonic development pathway, driven by peptide ligands called hedgehogs. During progression of prostate cancer (PCa), Hh signaling is increased, with especially high activation in castration resistant disease (CRPC). Evidence is lacking for canonical Hh signaling in PCa, indicating the likelihood that non-canonical pathways are involved. Our work shows that transcriptionally active androgen receptor (AR) binding to Gli proteins drives non-canonical Hh signaling in PCa. Androgen-sensitive LNCaP and androgen-independent LNCaP-AI and LN95 cells were transfected with a Gli-promoter driven luciferase reporter and treated with R1881, enzalutamide, or both, and luciferase activity was measured. Androgen treatment (R1881) induced Gli transcriptional activity while enzalutamide reversed this effect. Similarly, siRNA knockdown of full-length AR (AR-FL) suppressed R1881-induced Gli transcription. Western blot and qPCR confirmed increased expression of endogenous Gli target genes Gli1 and Ptch1 with androgen treatment. Androgen treatment stabilized expression of full-length active Gli3 in a dose-dependent manner but this was reversed by AR knockdown using siRNA. AR binds to Gli3 at the protein processing domain (PPD), which, given the above data, suggests that AR binding to Gli stabilizes full-length Gli3 by preventing phosphorylation and ubiquitination of the PPD, thereby stopping proteolytic cleavage and proteosomal degradation from occurring. Finally, we found that an AR-binding decoy peptide derived from the Gli2 C-terminus can compete with Gli3 for binding to AR, suppressing Gli transcriptional activity in PCa cells. Our data supports the idea that transcriptionally active AR binding to Gli proteins provides a means for Hh signaling to occur. Not only does AR co-activate Gli transcriptional activity, but it also alters the proteolytic processing of Gli proteins by preventing phosphorylation and ubiquitination of the Gli PPD by competing with β-TrCP for binding to Gli. Collectively, our findings show the importance of AR-Gli interaction in PCa progression.

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