Functional analysis of the protein tyrosine phosphatase family in mammary epithelial cell models of breast cancer

Abstract

The protein tyrosine phosphatase (PTP) superfamily of enzymes is encoded by ~100 genes. In coordinating with protein tyrosine kinases (PTK), they regulate signaling pathways that control a broad spectrum of fundamental physiological processes and their disruption underlie human diseases. To date, however, the functional analysis of PTPs is not as advanced as for PTKs. Therefore, the ultimate goal of this dissertation is to develop the tools for global analysis of the function of PTPs through RNAi-mediated loss-of-function screens. I constructed a PTP short-hairpin RNA (shRNA) library, which contains ~5 shRNAs specifically designed to target each of the PTPs. In collaboration with Min Yu, I studied the function of PTPRO in mammary epithelial cell morphogenesis by using these shRNAs. We found that suppression of PTPRO promoted cell proliferation and by recognizing the oncoprotein tyrosine kinase ErbB2 as a direct substrate, PTPRO augmented the ability of ErbB2 to induce multi-acinar structure in 3D culture. Preliminary analysis of other PTPs revealed different phenotypes consistent with distinct mechanisms of the enzymes. I also employed an RNAi mediated loss-of-function screen to study systematically the role of the PTP family in mammary epithelial cell motility, in the absence or presence of ErbB2. I identified PTPs that either promoted or inhibited mammary epithelial cell motility. I further characterized 3 PTPs, PRPN23, PTPRG and PTPRR, which function to inhibit cell motility. I found that suppression of PTPN23, but not PTPRG or PTPRR, induced cell invasion. Suppression of PTPN23 increased E-cadherin internalization, impaired early endosome forward trafficking of E-cadherin and induced the expression of mesenchymal proteins. Moreover, I identified Src as a direct substrate of PTPN23, and demonstrated that Src mediates the effects of suppressing PTPN23 on control cell motility and invasion. In this dissertation, I have revealed novel functions for members of the PTP family. In addition, I illustrate how RNAi-mediated loss-of-function screen reveals new roles for PTPs in mammary epithelial cell motility and invasion. These findings highlight the specificity of PTPs in breast cancer, suggesting PTPs or the signaling controlled by them could be biomarkers or therapeutic targets for the treatment of breast cancer.