Breast cancer will cause roughly 40,000 deaths in the U.S. in 2015. The 10-20% of cases classified as triple- negative (ER, PR, and HER2 negative) breast cancers (TNBCs) account for an inordinate number of these deaths, in part owing to a lack of unifying molecular features and thus a lack of targeted therapies. As such, deeper knowledge of the underlying biology is desperately needed to define therapeutic vulnerabilities and provide better treatment outcomes for patients. A subset of TNBCs has been shown to respond to therapeutic inhibition of Ras and PI3K signaling pathways; however, it has historically been challenging to predict which individual tumors will respond to these treatments because of the mutational complexity of this disease. As such, an ideal approach would be to directly assess Ras and PI3K pathway dynamics in live cells in order to identify which tumors harbor functional pathway alterations and are therefore more likely to respond to pathway inhibitors. To accomplish this goal I will employ a multidisciplinary approach to profile Ras and PI3K signaling in TNBC cells using optogenetics, microscopy, bioinformatics, and drug cytotoxicity assays. This work is based on our lab's development of parallel optogenetic systems that harnesses a protein interaction from plants (the Phy/PIF binding pair) to control either Ras or PI3K activity with high resolution in single cells. These pathways will be placed under light control in 30 well-characterized human TNBC cell lines by fusing the Phy/PIF domains to proteins that selectively activate the Ras or PI3K modules. By varying light inputs and measuring live-cell fluorescent reporters of downstream pathway activity, I will measure detailed responses of signaling dynamics in both pathways at a single-cell level across a representative panel of TNBC mutational backgrounds. In parallel, I will also examine the cytotoxic response of these cells to a panel of targeted kinase inhibitors. I will then use publicl available genomic data from each cell line to identify the mutational signatures that underlie signaling abnormalities and drug sensitivities. The proposed study will produce several advances in scientific knowledge. First, it will develop a method for rapid, precise control of the Ras and PI3K pathways in a panel of tumor cells, highlighting how cell signaling and protein dynamics are dysregulated by mutation. Second, it will constitute the first functional study of temporal signaling dynamics in TNBC, expanding the understanding of how uncharacterized Ras and PI3K pathway alterations contribute to neoplastic growth. Third, the relevance of these findings to each cell lines' drug sensitivity and genomic alterations will be assessed, highlightin targetable molecular and temporal processes involved in oncogenic signaling defects. Successful completion of my research will broaden our understanding of TNBC pathogenesis and facilitate the design of rational treatment strategies with immediate clinical applicability.
Triple negative breast cancer (TNBC) is a poorly-understood, highly aggressive breast cancer subtype that lacks common molecular features, leaving patients without proven options for targeted therapy. Our lab recently discovered that altered temporal signaling dynamics of Ras and PI3K pathways can contribute to uncontrolled proliferation of cancer cells. Thus, the proposed study will investigate the relationship between functional signaling abnormalities in TNBC cells, genomic alterations, and sensitivity to targeted kinase inhibitors, potentially forming the basis for rational design of therapies targeting molecular and temporal vulnerabilities of TNBC.
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| Barrio-Real, Laura; Lopez-Haber, Cynthia; Casado-Medrano, Victoria et al. (2018) P-Rex1 is dispensable for Erk activation and mitogenesis in breast cancer. Oncotarget 9:28612-28624 |
| Goglia, Alexander G; Wilson, Maxwell Z; DiGiorno, Daniel B et al. (2017) Optogenetic Control of Ras/Erk Signaling Using the Phy-PIF System. Methods Mol Biol 1636:3-20 |
