Project description:Although androgen deprivation therapy triggers a rapid response in men with metastatic prostate cancer, most patients eventually progress to lethal castration resistant prostate cancer (CRPC) characterized by androgen receptor (AR)-dependent tumor growth despite castrate levels of circulating androgens. Current treatment options for CRPC remain limited and non-curative, rendering improved outcomes dependent on deeper mechanistic insight and new therapeutic targets. In this RNA sequencing study we profiled the transcriptomes of SPOP WT and mutant LNCaP cells before and after androgen deprivation by RNA-sequencing. Using standard criteria (minimum 2-fold change), we identified 981 genes that were differentially expressed in SPOP mutant vs SPOP WT LNCaP cells grown in the absence of androgens . Strikingly, a significant fraction (595 or ~60%) of these genes overlapped with genes (3510 in number) differentially expressed as a function of androgen in WT LNCaP cells, indicating partial restoration of AR signaling in SPOP mutant cells following androgen withdrawal. To determine the contribution of GLI3 to this process, we performed RNA-sequencing following GLI3 knockdown in androgen-deprived SPOP mutant LNCaP cells. Among the 595 androgen-responsive genes in LNCaP cells whose hormone-independent expression is regulated by SPOP 187 (or ~31%) are also regulated by GLI3, thus revealing a prominent role for GLI3 in restoration of AR signaling in SPOP mutant cells following androgen withdrawal. Enrichment analysis using Gene Ontology Biological Processes revealed these 187 GLI3-regulated genes to be prominently linked with “chromosome segregation”, “DNA replication”, “transcriptional regulation of in G1/S transition”, “regulation of mitotic cell cycle”, and “chromatin silencing” among others, suggesting possible molecular genetic bases for androgen independent growth of SPOP mutant cells. Taken together, these findings indicate that SPOP mutant prostate cancer cells acquire robust androgen-independent growth through reestablishment of an AR signaling axis that involves functional crosstalk with the SHH/GLI3 pathway.

Project description:Following androgen ablation therapy (AAT), the vast majority of prostate cancer patients develop treatment resistance with a median time of 18-24 months to disease progression. To identify molecular targets that aid in prostate cancer cell survival and contribute to the androgen independent phenotype, we evaluated changes in LNCaP cell gene expression during 12 months of androgen deprivation. At time points reflecting critical growth and phenotypic changes, we performed Affymetrix expression array analysis to examine the effects of androgen deprivation during the acute response, during the period of apparent quiescence, and during the emergence of highly proliferative, androgen-independent prostate cancer cells (LNCaP-AI). We discovered alterations in gene expression for a host of molecules associated with promoting prostate cancer cell growth and survival, regulating cell cycle progression, apoptosis and adrenal androgen metabolism, in addition to AR co-regulators and markers of neuroendocrine disease. These findings illustrate the complexity and unpredictable nature of cancer cell biology and contribute greatly to our understanding of how prostate cancer cells likely survive AAT. The value of this longitudinal approach lies in the ability to examine gene expression changes throughout the cellular response to androgen deprivation; it provides a more dynamic illustration of those genes which contribute to disease progression in addition to specific genes which constitute a malignant androgen-independent phenotype. In conclusion, it is of great importance that we employ new approaches, such as the one proposed here, to continue exploring the cellular mechanisms of therapy resistance and identify promising targets to improve cancer therapeutics. Experiment Overall Design: To identify molecular targets that aid in prostate cancer cell survival and contribute to the androgen independent phenotype, we evaluated changes in LNCaP cell gene expression during 12 months of androgen deprivation. At time points reflecting critical growth and phenotypic changes, we performed Affymetrix expression array analysis to examine the effects of androgen deprivation during the acute response, during the period of apparent quiescence, and during the emergence of highly proliferative, androgen-independent prostate cancer cells (LNCaP-AI).

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Homo sapiens

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