Identification of Kinases Regulating Prostate Cancer Cell Growth Using an RNAi Phenotypic Screen

As prostate cancer progresses to castration-resistant disease, there is an increase in signal transduction activity. Most castration-resistant prostate tumors continue to express the androgen receptor (AR) as well as androgen-responsive genes, despite the near absence of circulating androgen in these patients. The AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules. Thus, the elevated signaling activity that occurs during progression to castration resistance can affect prostate cancer cell growth either through the AR or independent of the AR. In order to identify signaling pathways that regulate prostate cancer cell growth, we screened a panel of shRNAs targeting 673 human kinases against LNCaP prostate cancer cells grown in the presence and absence of hormone. The screen identified multiple shRNA clones against known and novel gene targets that regulate prostate cancer cell growth. Based on the magnitude of effect on growth, we selected six kinases for further study: MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1. Knockdown of these kinases decreased cell growth in both androgen-dependent and castration-resistant prostate cancer cells. However, these kinases had different effects on basal or androgen-induced transcriptional activity of AR target genes. MAP3K11 knockdown most consistently altered transcription of AR target genes, suggesting that MAP3K11 affected its growth inhibitory effect by modulating the AR transcriptional program. Consistent with MAP3K11 acting on the AR, knockdown of MAP3K11 inhibited AR Ser 650 phosphorylation, further supporting stress kinase regulation of AR phosphorylation. This study demonstrates the applicability of lentiviral-based shRNA for conducting phenotypic screens and identifies MAP3K11, DGKD, ICK, CIT, GALK2, and PSKH1 as regulators of prostate cancer cell growth. The thorough evaluation of these kinase targets will pave the way for developing more effective treatments for castration-resistant prostate cancer

Insights from AR gene mutations

The accumulation of somatic mutations on the background of natural germline variation is one of the fundamental mechanisms underpinning both disease and the development and progression of perhaps all tumors. For example, germline inactivating mutations in one allele of a tumor suppressor gene (TSG) (e.g., p53 or BRCA1) predisposes an individual to a lifetime of increased risk of cancer in particular tissues. In those cases, tumors commonly arise after somatic inactivation of the second TSG allele, and are often characterized by a younger age of onset and a more aggressive phenotype than tumors arising in the same tissues without a common or dominant genetic predisposition. A small proportion of prostate cancers do indeed occur in a hereditary manner, but it has been difficult to attribute inheritance and risk to a single gene. Even for the monoallelic androgen receptor (AR) gene on the X chromosome, which exhibits variously traits of an at-risk allele, oncogene, and mediator of prostate cancer progression and therapy resistance, inactivating germline variation in the AR is essentially nonexistent in this disease. Instead, germline AR variants often underpin the relatively common inherited syndrome of androgen insensitivity (AIS). In contrast, however, somatic variation of the AR is potentially a frequent event during tumor progression. As a consequence of these complexities, unraveling the precise role of the AR at each stage of prostate cancer progression, and indeed in different prostatic compartments or populations of tumor cells is challenging. In this chapter, we detail how the identification and characterization of somatic AR variants arising in prostate cancer has provided crucial information on (1) the role of the receptor throughout disease etiology and the emergence of castrate-recurrent disease, (2) the fine functional subdomain structure of the AR, (3) the oncogenic potential of aberrant AR signaling, and (4) new approaches to targeting AR function in disease management.Grant Buchanan, Eleanor F. Need, Tina Bianco-Miotto, Norman M. Greenberg, Howard I. Scher, Margaret M. Centenera, Lisa M. Butler, Diane M. Robins, and Wayne D. Tille