HGF-Induced PKCf Activation Increases Functional CXCR4 Expression in Human Breast Cancer Cells Songyin Huang1., Nengyong Ouyang2., Ling Lin3,4., Lili Chen 3 , Wei Wu3, Fengxi Su 3 , Yandan Yao3*, Herui Yao3,5* 1 Department of Laboratory, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China, 2 Department of Gynaecology and Obstetrics, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China, 3 Breast Tumor Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China, 4 Department of Rheumatology, The First Affiliated Hospital, Shantou University Medical College, Shantou City, Guangdong, China, 5 Department of Oncology, Sun Yat- Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, China Abstract The chemokine receptor CXCR4 and its ligand CXCL12 have been shown to mediate the metastasis of many malignant tumors including breast carcinoma. Interaction between hepatocyte growth factor (HGF) and the Met receptor tyrosine kinase mediates development and progression of cancers. HGF is able to induce CXCR4 expression and contributes to tumor cell invasiveness in breast carcinoma. However, the mechanism of the CXCR4 expression modulated by c-Met-HGF axis to enhance the metastatic behavior of breast cancer cells is still unclear. In this study, we found that HGF induced functional CXCR4 receptor expression in breast cancer cells. The effect of HGF was specifically mediated by PKCf activity. After transfection with PKCf-siRNA, the phosphorylation of PKCf and CXCR4 was abrogated in breast cancer cells. Interference with the activation of Rac1, a downstream target of HGF, prevented the HGF-induced increase in PKCf activity and CXCR4 levels. The HGF-induced, LY294002-sensitive translocation of PKCf from cytosol to plasma membrane indicated that HGF was capable of activating PKCf, probably via phosphoinositide (PI) 3-kinases. HGF treatment also increased MT1- MMP secretion. Inhibition of PKCf, Rac-1 and phosphatidylinositol 3-kinase may attenuate MT1-MMP expression in cells exposed to HGF. Functional manifestation of the effects of HGF revealed an increased ability for migration, chemotaxis and metastasis in MDA-MB-436 cells in vitro and in vivo. Our findings thus provided evidence that the process of HGF-induced functional CXCR4 expression may involve PI 3-kinase and atypical PKCf. Moreover, HGF may promote the invasiveness and metastasis of breast tumor xenografts in BALB/c-nu mice via the PKCf-mediated pathway, while suppression of PKCf by RNA interference may abrogate cancer cell spreading. Citation: Huang S, Ouyang N, Lin L, Chen L, Wu W, et al. (2012) HGF-Induced PKCf Activation Increases Functional CXCR4 Expression in Human Breast Cancer Cells. PLoS ONE 7(1): e29124. doi:10.1371/journal.pone.0029124 Editor: Wael El-Rifai, Vanderbilt University Medical Center, United States of America Received February 13, 2011; Accepted November 21, 2011; Published January 5, 2012 Copyright: 2012 Huang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the National Natural Science Foundation of China (81102020, 30973505, 81172526, 81072177), Guangdong Natural Science Foundation (S2011040000385), the Foundation of the Health Department of the Guangdong Province (A2009182, B2009067), the Science and Technology Foundation of the Guangdong Province (2009B030801005, 2009B030801080, 2008B030301092, 2008B030301238), the special financial grant from the China Postdoctoral Science Foundation (201104379), and the Foundation of Guangzhou Science and Technology Bureau (2009Y-C011-1). This work was also supported by the Key Laboratory of malignant tumor gene regulation and target therapy of Guangdong Higher Education Institutes, Sun Yat-sen University (KLB09001). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yaoherui@163.com (HY); yyandan@126.com (YY) . These authors contributed equally to this work. Introduction Identification of novel therapeutic targets is critical for the treatment of malignant tumors, breast cancers in particular [1,2]. A quite exciting one of such targets is the chemokine (C-X-C motif) receptor 4 (CXCR4) expressed in various types of tumors [3]. In breast cancer cells, CXCR4 is a major chemokine receptor with possible but undefined roles in metastatic cell diffusion and homing to secondary sites [4]. Most models of CXCR4 functions in tumor focus on its potential role as a mediator of motility, invasiveness and metastatic behaviors [5,6]. Blocking CXCR4 may attenuate breast cancer metastasis to regional lymph nodes and the lungs [7], and activation of CXCR4 is believed to be primarily ligand-dependent, as is supported by the antitumor efficacy of AMD 3100, an antagonist of chemokine (C-X-C motif) ligand 12 (CXCL12) binding [8]. CXCR4 is phosphorylated in response to ligand binding in a G protein-coupled receptor kinase 2-dependent fashion [9]. Phosphorylation of CXCR4 can also occur in response to the activation of other receptors and can involve additional kinases, such as protein kinase C f (PKCf) [10,11] or tyrosine kinases [12]. Regulation of phosphorylation and internalization has a significant effect on CXCR4-mediated cell responses [10]. Thus, understanding the mechanisms and molecular pathways that affect CXCR4 expression and cellular signaling might have important implications for breast cancer cell metastasis. Hepatocyte growth factor/scatter factor (HGF/SF) is an important fibroblast-secreted protein that mediates development and progression of cancers [13]. HGF has been demonstrated to transduce its biological activities through the Met receptor tyrosine kinase by activating a number of intracellular pathways which in turn transmit the HGF signal to cytosol and to nucleus [14,15,16]. PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29124