FUNCTIONAL IDENTIFICATION OF MASTER REGULATORS OF PROSTATE CANCER METASTASIS THROUGH CRISPR/CAS9 SCREENING

Training Plan: It is my strong desire to pursue a career in prostate cancer research devoted to understanding its molecular underpinnings and how this knowledge may be effectively translated to harness new and better therapeutic strategies that improve patient care. Since metastasis accounts for the overwhelming majority of cancer deaths, I want to focus on this aspect of tumor progression and, through the use of models, understand its most basic molecular features so as to ultimately be able to develop targeted therapies. I have recently arrived at the Abate-Shen laboratory, attracted by its unique mouse models and its bioinformatics approaches to directly translate their results to human cancer. With more than 20 years in prostate cancer research, Dr. Abate-Shen has ample knowledge of this disease and great experience as a mentor. The laboratory is located at Columbia University Medical Center, one of the top universities in the country, has strong collaborations with other research groups, and is an ideal place for me to get the necessary training to launch my prostate cancer research career. This research plan is thought for me to understand the molecular drivers of metastasis and to evaluate which of these may be targeted to achieve inhibition of metastatic growth. My training will therefore pave the way for future research on how to translate these discoveries into new therapeutic strategies for advanced prostate cancer, which are urgently needed.Research Plan: Despite its clinical relevance, strikingly little is known about the molecular underpinnings, let alone the key driver genes, that are responsible for metastasis. This dearth of knowledge is spurred by the lack of suitable models with which to study metastasis, a process that can only be faithfully modeled in an in vivo system such as the mouse. In order to overcome these shortcomings, the Abate-Shen laboratory has recently developed a unique mouse model of prostate cancer that generates metastases with 100% penetrance, based on the overactivation of the signaling pathways altered in advanced stages of human cancer. Furthermore, through state-of-the-art bioinformatic analyses, the laboratory has also generated the first and only prostate cancer interspecies gene regulatory networks, known as interactomes, which allow identification of conserved driver genes of complex phenotypes such as metastasis. We will take advantage of these two invaluable assets of our laboratory to uncover the driver genes responsible for metastasis, which we will call 'master regulators of metastasis' (MRMs), and further aim to identify which of these can actually be therapeutically targeted to inhibit metastatic growth. We will do so by inhibiting the expression of these genes in NPK cell lines using the modern CRISPR-CAS9 genomic-editing technology and screening for those that result in inhibition of metastatic growth of tumor allografts in nude mice. In essence, after lentiviral infection with pooled sgRNAs, tumor inoculation and metastasis formation, high-throughput analysis of individual sgRNA abundance in the resulting metastases compared to 'primary' tumors will allow us to identify MRM-targeted sgRNAs as being under-represented in the metastatic pool. Selected genes will be re-tested in a second phenotypic screen, this time by direct injection of sgRNAs into prostates of intact NPK mice, and further evaluation of metastatic sgRNA abundance as before. To confirm their role as drivers of human metastasis, we will use human cell line xenografts in nude mice to confirm that their inhibition impairs metastatic growth. The expression of selected MRMs will also be investigated on human metastatic tissues samples, to be further evaluated as candidate biomarkers for prediction of metastatic relapse.Impact: In this way, these studies will provide comprehensive insights into the molecular mechanisms of prostate cancer metastasis and will test which of the key genes may be effective targets for therapeutic intervention. Although we will be mainly using mouse models, through state-of-the-art bioinformatic analyses and validation screenings using human cells, we aim for the insights gained on mouse models to be translatable to human cancer. We expect that specifically targeting the metastatic cascade will provide a fundamentally different therapeutic strategy than that of present-day therapies and therefore provide new tools to combat this deadly disease.