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Mislocalization of the Cell Polarity Protein Scribble Promotes Mammary Tumorigenesis and Is Associated with Basal Breast Cancer

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Mislocalization of the Cell Polarity Protein Scribble Promotes Mammary Tumorigenesis and Is Associated with Basal Breast Cancer Published OnlineFirst March 24, 2014; DOI: 10.1158/0008-5472.CAN-13-3415 Cancer Tumor and Stem Cell Biology Research Mislocalization of the Cell Polarity Protein Scribble Promotes Mammary Tumorigenesis and Is Associated with Basal Breast Cancer Michael E. Feigin1, S. Dipikaa Akshinthala1, Kiyomi Araki2, Avi Z. Rosenberg1, Lakshmi B. Muthuswamy3, Bernard Martin2, Brian D. Lehmann5, Hal K. Berman2, Jennifer A. Pietenpol5, Robert D. Cardiff4, and Senthil K. Muthuswamy1,2 Abstract Scribble (SCRIB) localizes to cell–cell junctions and regulates establishment of epithelial cell polarity. Loss of expression of SCRIB functions as a tumor suppressor in Drosophila and mammals; conversely, over- expression of SCRIB promotes epithelial differentiation in mammals. Here, we report that SCRIB is frequently amplified, mRNA overexpressed, and protein is mislocalized from cell–cell junctions in human breast cancers. High levels of SCRIB mRNA are associated with poor clinical prognosis, identifying an unexpected role for SCRIB in breast cancer. We find that transgenic mice expressing a SCRIB mutant [Pro 305 to Leu (P305L)] that fails to localize to cell–cell junctions, under the control of the mouse mammary tumor virus long terminal repeat promoter, develop multifocal hyperplasia that progresses to highly pleomorphic and poorly differentiated tumors with basal characteristics. SCRIB interacts with phosphatase and tensin homolog (PTEN) and the expression of P305L, but not wild-type SCRIB, promotes an increase in PTEN levels in the cytosol. Overexpression of P305L, but not wild-type SCRIB, activates the Akt/mTOR/S6K signaling pathway. Human breast tumors overexpressing SCRIB have high levels of S6K but do not harbor mutations in PTEN or PIK3CA, identifying SCRIB amplification as a mechanism of activating PI3K signaling in tumors without mutations in PIK3CA or PTEN. Thus, we demonstrate that high levels of mislocalized SCRIB functions as a neomorph to promote mammary tumorigenesis by affecting subcellular localization of PTEN and activating an Akt/mTOR/S6kinase signaling pathway. Cancer Res; 74(11); 1–15. Ó2014 AACR. Introduction suppressive mechanism. Although the precise mechanism of Scribble (SCRIB) was identified in a Drosophila screen for tumor suppression is unclear, SCRIB is known to inhibit b maternal effects mutants that displayed defects in epithelial apoptosis in a -PIX (PAK-interacting exchange factor b polarization and morphogenesis. Loss of SCRIB results in )/Rac/JNK pathway-dependent manner, and promote prolif- uncontrolled proliferation and tissue growth, identifying it as eration in a Ras/MAPK-dependent manner in mammary and tumor suppressor (1). SCRIB is a scaffold protein containing 16 prostate epithelia, respectively (2, 3). Conversely, overexpres- N-terminal leucine rich repeat (LRR) domains and four PDZ sion of SCRIB in mammary epithelial cells promotes epithelial (PSD-95/Discs-large/ZO-1) domains, conserved from Drosoph- differentiation by suppressing expression of epithelial mesen- ila to humans (1). In mammals, loss of SCRIB induces dysplastic chymal transition regulators in a Ras/MAPK-dependent man- growth in vivo (2, 3), identifying loss of expression as a tumor ner (4). Conditional deletion of SCRIB in the corneal epithelium decreases E-cadherin expression and promotes mesenchymal transition, suggesting that SCRIB is required for maintaining epithelial cell identity (5). In addition, SCRIB also regulates the fi 1 Authors' Af liations: Cold Spring Harbor Laboratory, Cold Spring Har- Hippo signaling pathway (6), and signal transducer and acti- bor, New York; 2Princess Margaret Cancer Center, Campbell Family Institute for Breast Cancer Research, Department of Medical Biophysics, vator of transcription (STAT; ref. 7). SCRIB interacts with the University of Toronto; 3Ontario Institute for Cancer Research, Ontario, Akt phosphatase PHLPP1 (8), the planar cell polarity protein Canada; 4Center for Comparative Medicine, University of California, Davis, Davis, California; and 5Department of Biochemistry, Vanderbilt University, VANGL1 (9), and the neuronal nitric oxide synthase adaptor Nashville, Tennessee protein NOS1AP (10) to regulate cancer cell migration and Note: Supplementary data for this article are available at Cancer Research axon morphogenesis. Online (http://cancerres.aacrjournals.org/). In epithelial cells, SCRIB localizes to cell–cell junctions and Corresponding Author: Senthil K. Muthuswamy, Princess Margaret Can- mislocalization of SCRIB phenocopies a complete null pheno- cer Center, 610 University Avenue, Toronto, ON M5G 2M9, Canada. Phone: type in Drosophila (11), demonstrating that subcellular local- 416-581-8569; Fax: 416-946-2984; E-mail: [email protected] ization is critical for SCRIB function. Flies carrying the scrib 1 doi: 10.1158/0008-5472.CAN-13-3415 allele, in which a conserved leucine within the tenth LRR Ó2014 American Association for Cancer Research. domain is mutated to glutamine (L223Q), display epithelial www.aacrjournals.org OF1 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst March 24, 2014; DOI: 10.1158/0008-5472.CAN-13-3415 Feigin et al. defects, including disrupted cell shape and multilayered orga- tories), pTSC2 (T1462) (Cell Signaling), TSC2 (Cell Signaling), nization. Interestingly, overexpression of SCRIBL223Q fails to pPRAS40 (T246) (Cell Signaling), PRAS40 (Cell Signaling), rescue the scrib null phenotype, demonstrating the importance pp70S6K (T389) (Cell Signaling), p70S6K (Cell Signaling), pS6 of basolateral localization for normal function. In mammals, a (S235/236) (Cell Signaling), S6 (Cell Signaling), Ki67 (Invitro- genetic screen for cortical defects in mice uncovered a novel gen), cleaved caspase 3 (Cell Signaling), vimentin (Abcam), SCRIB allele, encoding an isoleucine to lysine mutation within CD49f-PE (eBioscience), CD61-647 (BioLegend), CD31-Biotin an LRR domain, displaying an open neural tube and disorga- (BD Biosciences), CD140a-Biotin (eBioscience), FITC-Strepta- nized and hyperplastic neuroepithelium (12). A specific point vidin (AnaSpec), Biotin mouse lineage panel (BD Biosciences) mutation within LRR13 (Prol 305 to Leu, P305L) of the human and p-(S/T)-AKT Substrate (Cell Signaling). The T7 antibody SCRIB protein (hSCRIB) has also been shown to abrogate was a kind gift from Dr. A. Krainer (Cold Spring Harbor membrane localization (13), disrupting recruitment of bPIX, Laboratory) and the caspase-3 antibody was from Dr. Y. and exocytosis. Consistently, overexpression of wild-type Lazebnik (Cold Spring Harbor Laboratory). hSCRIB in MCF-10A mammary epithelial cells grown in 3- dimension (3D) had no impact on morphogenesis, whereas Transgenic mouse generation expression of hSCRIBP305L displayed defective morphogene- Full length SCRIB cDNA (from pBluescript SCRIBibble sis (2). These observations demonstrate that basolateral mem- P305L) containing a P305L mutation (2) was subcloned into brane localization is required for normal SCRIB function in the MMTV p206 vector. Mice were generated by pronuclear both Drosophila and mammals. injection. Screening of founder mice for insertion was con- In addition to changes in SCRIB gene expression, hSCRIB firmed by PCR from genomic DNA using the MMTV forward – 0 0 protein is mislocalized from cell cell junctions in multiple primer (5 -GGCCCCGGCCCCAAGCTT-3 ) and SCRIB reverse human cancers, including breast, prostate, and colon (2, 3). In primers (50-GCAGAACTTGATGCTCTC-30) and (50-ATCATCC- prostate cancer, hSCRIB mislocalization is correlated with TCCGTCTGGAAC-30) with predicted 300 and 1,200 bp PCR poor patient survival (3). Mislocalization of other polarity products. proteins, including LGL and DLG, has also been associated with cancer progression (14), suggesting that mislocalization Cell culture of polarity proteins is likely to have important implications for MCF-10A cells were grown as previously described (2). cancer in addition to changes in gene expression levels. How- Stable cell lines were generated by retroviral infection as ever, it is not known if mislocalization of SCRIB is a conse- previously described (15). HEK293 cells were cultured in quence or if mislocalized SCRIB has a causal role in the cancer Dulbecco's Modified Eagle Medium (DMEM) supplemented process. with 10% FBS. HEK293 cells were transfected using Lipofecta- To investigate the effect of expressing Scribble that does not mine 2000 (Invitrogen) according to the manufacturer's sug- localize to cell–cell junctions, we generated a transgenic mouse gested protocol. All cell lines were obtained from the American expressing hSCRIBP305L within the mammary epithelium Type Culture Collection. under the control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. We show that expres- sion of hSCRIBP305L induces mammary tumors, providing Immunoprecipitation and fractionation direct evidence that SCRIB mislocalization can have a causal All immunoprecipitations were carried out on cells lysed role during tumorigenesis. with 0.7% CHAPS buffer. One milligram of protein was incubated with 1 mgantibodyand10mL Protein G sepharose Materials and Methods beads (GE Healthcare) overnight at 4C. The beads were Plasmids and reagents washed 3 times in lysis buffer and the bound proteins were The hSCRIBP305L vector was previously described (2). The eluted with sample buffer. The samples were subjected to T7-SCRIB construct was generated by subcloning the SCRIB SDS-PAGE on 8% acrylamide
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