My primary research interest has focused on the mechanistic underpinnings of how signal transduction pathways cross-talk and contribute to cancer progression, with specific emphasis on identifying and understanding the nodes within the cell signaling network that cause cancer progression and resistance to therapy. We are pursuing three major research projects. The first focuses on the androgen receptor (AR), an established signaling node in prostate cancer. We are studying the molecular mechanisms of AR activity with a specific emphasis on how AR phosphorylation regulates AR function. In our second research project, we are studying kinases that we recently identified through an RNAi screen to identify kinases that regulate prostate cancer cell growth with the goal of uncovering kinase cascades that can overcome the compensatory signaling mechanisms that limit the effectiveness of androgen ablation therapy. The third research project goal is to develop paradigms for the rational design of drug combinations.
AR phosphorylation in prostate cancer
The AR is essential for the growth and survival of prostate cancer. Most castration-resistant prostate tumors continue to express the AR as well as androgen responsive genes, despite the near absence of circulating androgen in these patients, and even late-stage prostate cancer is still dependent on the AR. Moreover, it is increasingly clear that the AR is regulated not only by its cognate steroid hormone, but also by interactions with a constellation of co-regulatory and signaling molecules, many of which are elevated as prostate cancer progresses. The ability of the AR to function in the absence of physiologic levels of androgen is clearly a consequence of these alternative regulatory events. We set out to unequivocally identify the in vivo sites of AR phosphorylation. This work was the first systematic exploration of regulated changes in AR phosphorylation as a possible mechanism for activation/sensitization of AR-dependent gene expression by cell surface receptors and associated downstream effectors. This led to the guiding hypothesis that AR phosphorylation plays a critical role in regulating AR function and that one or more of these phosphorylation sites play a role in prostate cancer, affecting gene expression, cell growth, and/or survival. Our research has determined that stress kinase signaling regulates AR Ser 650 phosphorylation and that this phosphorylation is required for optimal nuclear androgen export. Most recently, our work has shown that that the AR is phosphorylated on Ser 81 by CDK9 and that this phosphorylation regulates AR promoter selectivity and prostate cancer cell growth. Our current major focus is on determining the effect of the cell cycle on AR phosphorylation and transcriptional activity. Our data suggest that Ser 308 is phosphorylated by CDK1 in G2/M and that this phosphorylation event correlates with a change in AR transcriptional activity. Collectively, this work will provide important insight into the function of the AR, a major regulator of prostate cancer progression.
Kinase signaling in prostate cancer
Our research on growth factor signaling in prostate cancer began with a study in patient samples that demonstrated an increase in MAPK activity as prostate cancer progresses to a castration-resistant disease. This report was one of first to use a phosphospecific antibody to probe archival paraffin-embedded human patient material. This study, along with our work on the AR, led to the hypothesis that therapeutic strategies targeting kinase cascades can overcome the compensatory signaling mechanisms that limit the effectiveness of androgen ablation. Through screening a panel of shRNAs targeting 673 human kinases against prostate cancer cells grown in the presence and absence of hormone, gene targets have been identified that regulate prostate cancer cell growth. This research now focuses on the detailed evaluation of these kinases, specifically in regulating prostate cancer cell growth, survival, and androgen-AR signaling. For example, from the prioritized list, PSKH1 has been selected for a detailed evaluation as a regulator of androgen responsiveness and prostate cancer cell growth. In addition to RNAi knockdown of PSKH1 regulating prostate cancer cell growth, preliminary data suggest that PSKH1 may phosphorylate the AR. We are now looking at both the role of PSKH1 in regulating androgen-AR signaling, growth, and survival of prostate cancer cells as well as examining the critical intracellular proteins that mediate PSKH1 signaling and growth regulation of prostate cancer cells. These experiments are informing our understanding of the signaling networks important for regulating prostate cancer cells. Ultimately, the goal is to identify new kinase targets that, when inhibited or manipulated, can improve the effectiveness of androgen ablation therapy.
Paradigms for the rational design of drug combinations
Since inhibitors targeting a single signaling molecule that is overexpressed and activated in cancer have shown only modest clinical benefit when used as single agents, combinatorial therapies hold much promise. Therapeutic strategies targeting multiple pathways simultaneously can hypothetically overcome the inherent compensatory, feedback, and redundant signaling mechanisms that limit the effectiveness of single agent therapy. Our work uses two general paradigms to rationally develop effective therapeutic combinations. The first is to use global analysis to identify compensatory and redundant signaling pathways induced by specific targeted therapeutics. The second is to use synthetic lethal screening with small molecule inhibitors to search for combinatorial effects and functionally identify compensatory and redundant relationships.
Using the first paradigm, my laboratory identified compensatory signaling pathways in prostate cancer xenografts treated with an MEK inhibitor. Combining MEK inhibition of any one of the up-regulated pathways (NFkB, Hedgehog, and PI3K signaling) provided synergistic growth inhibition. This suggests that this paradigm can be used to successfully identify effective drug combinations for the treatment of cancer.
Using the second paradigm, we used a library of chemical inhibitors targeting signaling proteins to search for combinatorial effects on melanoma cell line growth in order to functionally identify compensatory and redundant relationships between different signaling components. We have been able to identify combinations of small molecule inhibitors that demonstrate synergistic inhibition of melanoma growth. These combinatorial therapies are now being evaluated both mechanistically and in preclinical xenograft models. This research has evolved into a team based project drawing on the capabilities of multiple research groups. Through collaborations, the signaling nodes predictive of drug sensitivity are being determined by mathematical modeling of proteomic, transcriptomic, and genomic analysis. The combination of both the functional and mathematical approaches will facilitate our understanding of the cell signaling network and how that network responds to therapeutic intervention. This information will then be used to both refine the selection of effective drug combinations and identify tumors that are sensitive to such combinations.
The goal of my research is to provide clear and sure stepping stones of knowledge for myself and others so that we can understand how crosstalk among signal transduction pathways contributes to cancer progression and how that information can be used to develop more effective cancer treatments.