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P120 Catenin Is a Key Effector of a Ras-PKC&Epsiv

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P120 Catenin Is a Key Effector of a Ras-PKC&Epsiv Oncogene (2014) 33, 1385–1394 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE p120 catenin is a key effector of a Ras-PKCe oncogenic signaling axis SG Dann1, J Golas1, M Miranda1, C Shi1,JWu2, G Jin1, E Rosfjord1, E Upeslacis1 and A Klippel1 Within the family of protein kinase C (PKC) molecules, the novel isoform PRKCE (PKCe) acts as a bona fide oncogene in in vitro and in vivo models of tumorigenesis. Previous studies have reported expression of PKCe in breast, prostate and lung tumors above that of normal adjacent tissue. Data from the cancer genome atlas suggest increased copy number of PRKCE in triple negative breast cancer (TNBC). We find that overexpression of PKCe in a non-tumorigenic breast epithelial cell line is sufficient to overcome contact inhibition and results in the formation of cellular foci. Correspondingly, inhibition of PKCe in a TNBC cell model results in growth defects in two-dimensional (2D) and three-dimensional (3D) culture conditions and orthotopic xenografts. Using stable isotope labeling of amino acids in a cell culture phosphoproteomic approach, we find that CTNND1/p120ctn phosphorylation at serine 268 (P-S268) occurs in a strictly PKCe-dependent manner, and that loss of PKCe signaling in TNBC cells leads to reversal of mesenchymal morphology and signaling. In a model of Ras activation, inhibition of PKCe is sufficient to block mesenchymal cell morphology. Finally, treatment with a PKCe ATP mimetic inhibitor, PF-5263555, recapitulates genetic loss of function experiments impairing p120ctn phosphorylation as well as compromising TNBC cell growth in vitro and in vivo. We demonstrate PKCe as a tractable therapeutic target for TNBC, where p120ctn phosphorylation may serve as a readout for monitoring patient response. Oncogene (2014) 33, 1385–1394; doi:10.1038/onc.2013.91; published online 1 April 2013 Keywords: protein kinase C; epithelial to mesenchymal transition; triple negative breast cancer; p120 catenin; drug discovery INTRODUCTION Here, we report that PKCe expression in normal mammary ducts Protein kinase C (PKC) molecules belong to the AGC kinase is confined to myoepithelial cells, and this compartmentalization superfamily and are divided into one of three classes (conven- is lost in breast cancer tissue. Exogenous expression of PKCe is tional, novel or atypical) determined by dependence on the sufficient to overcome contact inhibition in the non-tumorigenic second messengers Ca2 þ and diacylgycerol for activation.1 breast epithelial cell line MCF10A. We also show that PKCe activity Although several PKC isoforms have been implicated in cancer, in triple negative breast cancer (TNBC) cells is tightly correlated only the novel isoform e is causative for tumor formation in in vitro with phosphorylation of p120ctn on S268. Furthermore, activation and in vivo cancer models.2 Expression of the novel isoform PRKCE of oncogenic Ras induces a cell morphology and signaling (PKCe) is elevated in breast, prostate and lung tumors compared response that is dependent on PKCe activity. We present data with normal adjacent tissue.3 Pioneering studies in immortalized validating PKCe as a small molecule inhibitor target for oncology, murine cells showed that overexpression of PKCe is sufficient to and propose that high p120ctn P-S268 may reflect PKCe activity in promote growth in soft agar and tumor formation in nude mouse cancer cells. xenografts.4,5 Further, transgenic overexpression of PKCe in the mouse prostate leads to invasive prostatic intraepithelial neo- plasia, while knockout of even a single prkce allele is sufficient RESULTS to impair tumorigenesis in a genetically engineered mouse model PKCe expression drives focal growth in non-tumorigenic breast of prostate cancer.6,7 epithelial cells Recent data suggest that PKCe is indispensable for wound In order to evaluate PKCe expression by immunohistochemistry closure, cytokinesis and migration; all of which point toward (IHC), we qualified a rabbit monoclonal antibody against normal involvement in epithelial to mesenchymal transition (EMT).8,9 PKCe tissues from prkce À / À and À / þ mice as well as TNBC cells has also been implicated in several steps required for the EMT harboring a doxycycline (Dox)-inducible short hairpin RNA (shRNA) migratory phenotype.10,11 EMT of epithelial cancers is linked to a targeting PKCe (Supplementary Figure 1a and b). Satisfied with the broad transcriptional network induced by a set of embryonic fact that immunoreactivity was dependent on PKCe expression, transcription factors.12 The activity of this transcriptional network we surveyed normal and malignant mammary tissue samples by is generally scored by loss of epithelial markers, such as IHC. Normal breast tissue stains positive for PKCe in the E-cadherin, and gain of mesenchymal-specific markers, such as vasculature and myoepithelial cell layer surrounding epithelial N-cadherin and vimentin.13 Although the transcriptional ducts (Figure 1a and Supplementary Figure 1c). By comparison, network of EMT has been extensively studied, post-translational breast tumor tissue samples present with intense stromal staining mechanisms that mediate the effects on cell morphology are not for PKCe and with tumor epithelia-specific PKCe expression in a well understood.14 subset of cases (Figure 1a and Supplementary Figure 1c). 1Pfizer Oncology Research Unit, Pearl River, NY, USA and 2Pfizer Structural Biology and Biophysics, Groton, CT, USA. Correspondence: Dr SG Dann, Pfizer Oncology Research Unit, 401 N Middletown road, Pearl River, NY 10965, USA. E-mail: stephen.dann@pfizer.com Received 16 August 2012; revised 24 January 2013; accepted 4 February 2013; published online 1 April 2013 Oncogenic Ras-PKCe signaling requires p120ctn SG Dann et al 1386 Figure 1. PKCe overexpression is associated with altered morphology of breast epithelia and disruption of tissue organization. (a) Representative images taken at  20 magnification of normal breast (upper panels) and cancerous breast (lower panels) matched tissue stained for PKCe and p120ctn expression. (b) Analysis of the cancer genome atlas data for invasive breast cancer comparing PRKCE copy number in TNBC versus all other biomarker status. Oncomine (Compendia Bioscience, Ann Arbor, MI, USA) was used for analysis and visualization. (c) Relative PKCe expression levels in a panel of breast cancer cell lines. Protein extracts of comparable cell numbers from the indicated tumor lines were analyzed for PKCe expression and activation state by immunoblotting. (d) Ten days Dox-inducible expression of PKCe in MCF10A cells results in foci formation photographed at  10 magnification. Western blots showing relative PKCe expression are shown next to each panel. Quantitation was performed by bright-field phase contrast microscopy as described in the Materials and methods section. Consequently, analysis of the TNBC subgroup within the invasive (Figure 1d and Supplementary Figure 2). Focal growth was breast cancer genome atlas finds PRKCE in the top 2% for all copy quantified using phase contrast bright-field microscopy as per the number gains (Figure 1b). In agreement with this data set, we find Materials and methods (Supplementary Figure 2 and Figure 1d). that immortalized but non-tumorigenic MCF10A breast epithelial cells express low levels of PKCe compared with a panel of cell lines derived from breast malignancies. Moreover, the TNBC cell lines PKCe is required for the growth of triple negative MDA-MB-231 MDA-MB-231 and BT549 express constitutively phosphorylated, or breast cancer cells in vitro and in vivo primed, PKCe, indicating hyperactivation of the kinase in this To further explore the role of PKCe in a disease relevant model, we context (Figure 1c). engineered MDA-MB-231 cells to stably express Dox-inducible To test the hypothesis that PKCe may drive tumorigenesis in shRNAs targeting PKCe. MDA-MB-231 cells are derived from a breast epithelial cells, we engineered MCF10A cells to express TNBC with activating mutations in KRAS and BRAF. Induction of wild-type PKCe (WTe), the kinase-defective mutant (K437R) or an PKCe knockdown for an extended period (10 days) resulted in empty vector control upon exposure to Dox. Dox-mediated reduced cell density over time, while the uninduced cells and induction of WTe and K437R mutant expression in each stable those harboring the non-targeting (NT)-shRNA displayed no line is shown in Figure 1d. Plated in the absence of Dox, each obvious growth or survival defect (Supplementary Figure 3). The stable MCF10A variant reached confluency and became contact PKCe loss of function (LOF) phenotype in two-dimensional (2D) inhibited (Figure 1d) as did the empty vector control and K437R in growth conditions prompted us to explore more stringent three- the presence of Dox. Upon induction of WTe, however, cells dimensional (3D) culture conditions by growing the cells overcame contact inhibition and cellular foci developed through- embedded in an extracellular matrix (ECM). In this environment out the plating surface, a hallmark of transformation in culture MDA-MB-231 cells form invasive structures,15 and expression of Oncogene (2014) 1385 – 1394 & 2014 Macmillan Publishers Limited Oncogenic Ras-PKCe signaling requires p120ctn SG Dann et al 1387 Figure 2. PKCe activity can be monitored through p120ctn S268 phosphorylation and is required for MDA-MB-231 malignant cell growth in vitro and in vivo.(a) Stably transduced MDA-MB-231 cells with Dox-inducible expression of two independent PKCe or
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