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Functional Analysis of the Protein Tyrosine Phosphatase Family in Mammary Epithelial Cell Models of Breast Cancer

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Functional Analysis of the Protein Tyrosine Phosphatase Family in Mammary Epithelial Cell Models of Breast Cancer SSStttooonnnyyy BBBrrrooooookkk UUUnnniiivvveeerrrsssiiitttyyy The official electronic file of this thesis or dissertation is maintained by the University Libraries on behalf of The Graduate School at Stony Brook University. ©©© AAAllllll RRRiiiggghhhtttsss RRReeessseeerrrvvveeeddd bbbyyy AAAuuuttthhhooorrr... Functional analysis of the protein tyrosine phosphatase family in mammary epithelial cell models of breast cancer A Dissertation Presented by Guang Lin to The Graduate School in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Molecular and Cellular Biology Stony Brook University December 2010 Stony Brook University The Graduate School Guang Lin We, the dissertation committee for the above candidate for the Doctor of Philosophy degree, hereby recommend acceptance of this dissertation. Nicholas K. Tonks – Dissertation Advisor Professor, Cold Spring Harbor Laboratory Scott Powers – Chairperson of Defense Associate Professor, Cold Spring Harbor Laboratory Gregory J. Hannon Professor, Cold Spring Harbor Laboratory Senthil K. Muthuswamy Associate Professor, Cold Spring Harbor Laboratory Mark M. Moasser – Outside Member Professor in Residence, Dept. of Medicine, Division of Hematology/Oncology, University of California, San Francisco This dissertation is accepted by the Graduate School Lawrence Martin Dean of the Graduate School ii Abstract of the Dissertation Functional analysis of the protein tyrosine phosphatase family in mammary epithelial cell models of breast cancer by Guang Lin Doctor of Philosophy in Molecular and Cellular Biology Stony Brook University 2010 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 E-cadherin, β-catenin and Src as iii direct substrates 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. vi Table of Contents List of Figures…………………………………………………………………………....x Acknowledgments……………………………………………………………………xiii Chapter 1: General Introduction……………………………………………………….1 Preface…………………………………………………………………………..1 1.1 Protein Phosphorylation…………………………………………………2 1.1.1 Protein phosphorylation is reversible……………………………2 1.1.2 Protein tyrosine phosphorylation…………………………………3 1.1.3 PTKs as therapeutic targets of human cancers………………4 1.2 Protein Tyrosine Phosphatases…………………………………………5 1.2.1 The Classical PTP…………………………………………………6 1.2.2 The Dual Specificity Phosphatases…………………………...…8 1.2.3 Pseudophosphatases……………………………………………11 1.3 PTPs and cancer…………………………………………………………12 1.3.1 PTEN, a well known tumor suppressor…………………..……13 1.3.2 SHP2, an oncogenic PTP………………………………………13 1.3.3 CDC25s and cancer……………………………………………..15 1.3.4 PTPs and breast cancer…………………………………………15 1.4 PTPs characterized in this dissertation………………………………...16 1.4.1 PTPRO…………………………………………………………….16 1.4.2 DEP1………………………………………………………………17 1.4.3 PTPN23…………………………………………………………...18 1.4.4 PTPRG…………………………………………………………….20 v 1.4.5 PTPRR……………………………………………………………21 1.4.6 PTP1B…………………………………………………………….22 1.5 Study the function of PTP in an in vitro model of breast cancer…….23 1.5.1 The MCF10A 3D culture system…………………………….…24 1.5.2 MCF10A 3D culture system for the study of cell signaling and breast tumorigenesis………………………………………….…25 1.6 ErbB2 signaling and breast cancer……………………………………26 1.6.1 ErbB2 signaling………………………………………………..…26 1.6.2 ErbB2 and breast cancer……………………………………..…27 1.6.3 ErbB2 as therapeutic target…………………………………..…28 1.6.4 Regulation of ErbB2 signaling by PTPs…………………….…30 1.7 Loss of E-cadherin and cancers……………………………………..…31 1.7.1 Functions of E-cadherin…………………………………………31 1.7.2 Loss of E-cadherin and cancers………………………………..32 1.7.3 Internalization of E-cadherin disrupts adherens junctions…...33 1.7.4 Regulation of E-cadherin internalization……………………….34 1.7.5 Regulation of E-cadherin recycling………………………….…35 1.8 Implications of this dissertation to understanding the function of the PTP family…………………………………………………………..……35 Chapter 2: Construction of the PTP shRNA library…………………………...……45 2.1 RNA Interference…………………………………………………………45 2.1.1 microRNAs……………………………………………………..…45 2.1.2 Biogenesis of miRNA………………………………………….…46 2.2 Application of RNAi in loss-of-function studies……………………..…47 2.3 Short hairpin RNA expression constructs………………………...……49 2.4 Results and Discussions……………………………………………...…50 vi 2.4.1 The design of the PTP shRNA library……………………….…50 2.4.2 The construction of the PTP shRNA library………………...…51 Chapter 3: Functional studies of PTPRO in MCF10A three-dimensional culture system……………………………………………………………………..59 3.1 Materials and methods……………………………………………..……61 3.2 Results……………………………………………………………………65 3.2.1 The expression of PTPRO is up-regulated in matured MCF10A acini………………………………………………………………65 3.2.2 Suppression of PTPRO increases the size of MCF10A acini ……………………………………………………………………………65 3.2.3 Suppression of PTPRO delays cell proliferation arrest, but does not affect cell apoptosis……………………………..……66 3.2.4 Suppression of PTPRO augments the effect of ErbB2 activation……………………………………………………….…67 3.2.5 Suppression PTPRO enhances ErbB2-induced cell proliferation………………………………………………………68 3.2.6 Suppression PTPRO enhances ErbB2 phosphorylation…….68 3.2.7 ErbB2 is a direct substrate of PTPRO…………………………69 3.2.8 Suppression of DEP1 induces the formation of multi-acinar structures independent of ErbB2 activation………………..…70 3.3 Discussion…………………………………………………………………71 Chapter 4: Identification of PTPs involved in MCF10A/ErbB2 cells motility and invasiveness………………………………………………………………83 4.1 Materials and methods………………………………………………..…85 4.2 Results………………………………………………………………….…92 4.2.1 Loss-of-function screening of the PTPome to identify regulators of mammary epithelial cell motility……………...………………………………………92 vii 4.2.2 Suppression of PTPRG, PTPN23 and PTPRR induce distinct stimulatory effects on mammary epithelial cell motility………93 4.2.3 Suppression of PTPN23, but not PTPRG or PTPRR, promotes mammary epithelial cell invasion………………………………94 4.2.4 Suppression of PTPN23 induces mammary epithelial cell scattering and impairs cell-cell adhesion………………...……95 4.2.5 Suppression of PTPN23 induces caveolin-1 mediated endocytosis of E-cadherin and causes accumulation of E- cadherin in early endosomes……………………………...……97 4.2.6 Suppression of PTPN23 induces expression of the mesenchymal proteins……………………………………..……98 4.2.7 Suppression of PTPN23 enhances downstream signaling through activation of Src Family Kinases……………………100 4.2.8 Src is a direct substrate of PTPN23………………………..…101 4.2.9 Inhibition of SFKs activity impairs shN23-induced phenotypes ……………………………………………………………………102 4.2.10 Characterization of PTP1B as a negative regulator of mammary epithelial cell motility………………………………103 4.3 Discussion……………………………………………………………….105 Chapter 5: Conclusion and future directions………………………………………134 5.1 Construction of the PTP shRNA library and the design of RNAi- mediated loss-of-function screen…………………………………..…135 5.2 Validation of the identified PTPs………………………………………137 5.3 Characterization of the Identified PTPs individually…………...……138 5.4 Future directions………………………………………………….….…139 5.4.1 PTPRO……………………………………………………..……140 5.4.2 PTPN23………………………………………………….………142 5.4.3 DEP1……………………………………………………….….…144 viii 5.4.4 PTPRG and PTPRR……………………………………………145 5.4.5 Systematic characterization of downstream signaling of PTPs by quantitative phosphoproteomics-SILAC………………….145 5.4.6 Study the function of PTPs in vivo by using mosaic mouse
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