The HGF/Met couple controls the expression of a panel of genes that are important for these processes, including HIF-1a (the inducible subunit of HIF-1 transcription factor), members of the plasminogen activation system and the C-X-C motif receptor 4 (CXCR4) [17,18]. The receptor tyrosine kinases Met and CXCR4 are associated with the morphogenesis and functional differenti- ation of normal mammary gland epithelium and play an important role in malignant transformation [19]. Previously, both CXCR4 and c-Met were shown to be upregulated by hypoxia in glioma cells and breast cancer cells [17,18,19,20]. HGF up- regulates CXCR4 expression via NF-kappaB and contributes to cell invasion. Knock-down of NF-kappaB expression inhibited the induction of CXCR4 expression in response to HGF [21,22]. In the cases of liver injury, HGF can promote the expression of CXCR4 in human CD34 stem cells, and subsequently, CD34 stem cells migrate to the injury site under the directional guidance of chemokine stromal cell-derived factor-1 (SDF-1, also named CXCL12). The protein kinase C family (PKC) has been shown to be involved in the expression and function of CXCR4, and is also linked to HGF-induced intracellular signal transduction in tumor cells. It is therefore vitally important to understand whether PKC pathway is associated with the expression and function of CXCR4 induced by HGF [13,23]. A specific isoform of the PKC family, the atypical PKCf, is an important class of secondary messengers that mediates a number of cellular responses to exogenous stimuli and stress agents. Unlike conventional (a,b,c) and novel (d, e, g, h) PKC isoenzymes, atypical PKCs(aPKCs) include PKCf and PKCl/i which are not activated by calcium and diacylglycerol [24]. The atypical PKCf is a key regulator of CXCL12/CXCR4-activated signaling in human hematopoietic progenitors. Petit I et al have demonstrated that ectopic PKCf expression increases SDF-1 induced motility, whereas inhibition of PKCf activity impairs survival, proliferation, adhesion and engraftment of immature CD34+ progenitors [25]. Findings of other studies [26,27] have positioned PKCf in the center of chemoattractant and immunoregulator-induced respons- es. Activation/phosphorylation of PKCf by HGF is an essential intracellular signaling requirement for growth factor-inducedb-cell proliferation [28]. Recent reviews also described the involvement of atypical PKCf/i in HGF-induced Ras-related C3 botulinum toxin substrate (Rac) activation and membrane ruffling of colony growing epithelial cells [23]. Therefore, we speculated that PKCf may be involved in HGF-induced CXCR4 expression in breast cancer, and may affect the behaviors of migration and invasion in breast cancer cells. In the present study, we demonstrated that HGF-induced activation of PKCf increases CXCR4 expression and the migratory capacity of MDA-MB-436 breast cancer cells. A molecular analysis of these events indicated that augmented CXCR4 expression was regulated by PKCf activity. The phosphatidylinositol 3 (PI 3)-kinase and protein kinase B (PKB/ AKT) pathways were involved in CXCR4 expression and the HGF-induced activation of PKCf. The functional importance of HGF-induced PKCf activation in breast cancer metastasis was further demonstrated in a xenograft experiment in which the suppression of PKCf abrogates HGF-induced metastasis of breast cancer to the lung and liver. Results HGF/c-Met or CXCR4 expression in breast carcinomas correlated with tumor invasiveness High level of HGF/c-Met expression is considered as a possible indicator of metastasis, earlier recurrence and shortened survival PKCf Induced by HGF Increases CXCR4 Expression in breast cancer patients [29]. In the tumor microenvironment, HGF regulates the expression of c-Met and CXCR4 through autocrine or paracrine actions [13,17]. Moreover, the chemokine receptor CXCR4 has been shown to be one of the vital factors for metastasis in breast cancer patients [7]. Initially, we intended to establish whether c-Met and CXCR4 are also expressed on breast cancer cells in clinically resected tissues. To do this, we determined the expression of HGF, c-Met and CXCR4 in breast cancer tissues by immunohistochemistry using corresponding antibodies, which revealed positive immunostaining of HGF, c-Met or CXCR4 in 197 cases of invasive breast carcinoma. In contrast, c-Met+ and CXCR4+ cells were not found to be present in any of the benign breast tissues with or without atypical epithelial hyperplasia (Figure 1A). When the c-Met+ and CXCR4+ cell counts in breast cancer were calculated according to the clinicopathology of these patients (Table S1), the numbers of HGF + , c-Met+ or CXCR4+ cells increased along with the histopathological grading of the tumor (P,0.001) (Figure 1B, Table S1). In addition, c-Met+ or CXCR4+ cell infiltration appeared to be more intense in those with axillary lymph node (P,0.001) or distal metastasis (P,0.001) (Table S1). These findings linked the enhanced expression of c-Met and CXCR4 to invasiveness and metastasis of breast cancers. HGF upregulated CXCR4 protein expression and membrane presentation in human breast cancer cells Tumor development involves an intricate set of molecular events driven by different environmental stimuli. Changes in stromal cell-released cytokines (proinflammatory cytokines, che- mokines and growth factors) seem to influence the gene expression profile in malignancy and to determine the cell fate [30]. To further study the effect of HGF on CXCR4 expression, MDA- MB-436 and MCF-7 breast cancer cell lines with different grades of malignancy were cultured in the presence or absence of HGF or SDF-1 for 24 hours. Treatment with HGF was found to result in a 2- to 7-fold increase in CXCR4 mRNA and protein expression in the MDA-MB-436 and MCF-7 cells (Figures 2A–2B, Figure S1A). To study the level of Met expression and the effect of HGF on Met expression, MDA-MB-436 and MCF-7 breast cancer cell lines were cultured in the presence or absence of HGF or SDF-1 for 24 hours. Treatment with HGF was found to result in increased Met phosphorylation but not Met expression in the MDA-MB-436 and MCF-7 cells (Figure 2B, Figure S1B). To study the possible correlation between the patterns of CXCR4 mRNA and protein expression, we evaluated mRNA or protein expression of CXCR4 after HGF treatment by quantita- tive RT-PCR (qRT-PCR) or immunoblotting (Figures 2C–2D) and flow cytometry (Figure 2E). The level of CXCR4 mRNA increased by approximately 2 to 9 folds between 4 and 12 hours after HGF treatment, and then declined gradually (Figure 2C). The level of CXCR4 protein began to increase by 4 hours, doubled between 8 and 16 hours, and was approximately 2- to 6- fold higher than in the starved cells at 24 hours after HGF treatment (Figures 2D–2E, Figure S1B). The difference in the time course was probably consistent with the time required for protein synthesis, although we cannot exclude the possible involvement of post-transcriptional mechanisms therein. Our study also found that HGF treatment promoted the expression of CXCR4 and led to CXCR4 phosphorylation. In contrast, SDF-1, the ligand of CXCR4, another important cytokine functioning in a different way, was also found to cause CXCR4 phosphorylation but did not increase expression of CXCR4 mRNA and protein in MDA-MB- 436 and MCF-7 cells (Figures 2A–2B, Figure S1A). Changes in the membrane and intracellular CXCR4 levels were then compara- PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29124