PKCf Induced by HGF Increases CXCR4 Expression Figure 3. HGF-induced increase in CXCR4 expression depends on PKCf activity. (A). Time course of relative p-PKCf levels as determined by immunoblot in MDA-MB-436 and MCF-7 cells following stimulation with 50 ng/ml HGF. The experiment was repeated three times with similar results. A representative study is shown. (B) and (C). 100 mM PKCf-siRNA1 (si1) or PKCf-siRNA2 (si2) transient transfections and siRNA-mediated PKCf protein silencing in MDA-MB-436 cells. p-CXCR4 and p-PKCf expression levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF or/and 100 mM PKCf-siRNA1 (si1) or PKCf-siRNA2 (si2). The experiment was repeated three times with similar results. A representative study is shown. (D). Western blot analysis of total CXCR4 expression and p-CXCR4 levels in MDA-MB-436 cells cultured for 24 hours with 50 ng/ml HGF, 2 mM cholesterol sulfate (Choles sul), or PBS. As indicated, 10 mM PS of PKCf (PSf), PKCe (PSe), or PKCa/b (PSa/b) or 25 mM NSC23766 (Rac1 inhibitor, Rac1 inh) was used. The experiment was repeated three times with similar results. A representative study is shown. (E). Membrane CXCR4 expression in MDA-MB-436 cells incubated for 24 hours with 50 ng/ml HGF, 2 mM Choles Sulfate, or PBS. Where indicated, 10 mM PS of PSf, PSe, or PSa/b was used. The flow cytometry analysis data are shown in arbitrary units (AU) normalized to PBS as the mean 6 SD of three independent experiments. # P,0.01 as compared to PBS. (F). Western blot analysis of Rac1-GTP and total-Rac1 in MDA-MB-436 and MCF-7 cells treated with 50 ng/ml HGF with or without 25 mM NSC23766. The experiment was repeated three times with similar results. A representative study is shown. (G). Dose-dependent inhibition of HGF-induced p-PKCf was achieved using NSC23766 in HGF-treated MDA-MB-436 cells; the cells were assayed by western blot. The experiment was repeated three times with similar results. A representative study is shown. (H). Western blot analysis of Rac1, PKCf and CXCR4 expression in MDA-MB- 436 cells cultured for 48 hours with 100 mM Rac1-siRNA. The experiment was repeated three times with similar results. A representative study is shown. (I). Cellular distribution of PKCf (left panel) and CXCR4 (right panel) before and after HGF (50 ng/ml) or NSC23766 (50 mM) stimulation for 10 minutes. Representative of two independent experiments was shown. doi:10.1371/journal.pone.0029124.g003 [38]. Moreover, the HGF-induced invasion was inhibited by PKCf-siRNAs, as well as the effect of PSf. Conversely, PSe and PSa/b had no effect on HGF-induced invasion (Figure 4F, Figure S3A–3B). In agreement with our findings implicating Rac1 activity in HGF-induced PKCf activation (Figures 3H–3I), we found that Rac1 inhibited the effect of HGF-induced invasion of MDA-MB 436 cells. To further define the role of PKCf in HGF-stimulated processes, we looked at other possible targets of PKCf that could stimulate metastasis of these cells. The experiments were based on the reports that HGF is a potent activator of membrane type-1 matrix metalloproteinase (MT1-MMP) in breast cancer cells [39], in a PKCf-dependent manner [40], is a proteolytic enzyme known to be involved in degrading extracellular matrix and assist in cancer invasion and progression. MT1-MMP in coordination with CXCR4 promotes invasion and dissemination of the tumor [41]. Surprisingly, we found that HGF treatment also increased MT1- MMP expression by MDA-MB 436 cells. Inhibition of PKCf, Rac-1 and phosphatidylinositol 3-kinase by their respective inhibitors PSf, NSC23766 and LY290042 attenuated MT1- MMP expression in MDA-MB 436 cells (Figure 4G–4H, Figure S3E). These data were consistent with previous reports [42,43]. Therefore, the proper function of MT1-MMP may depend on both cellular expression and specific membrane localization. Rac1 may be involved in both processes. These findings implied that up-regulation of CXCR4 by HGF was essential for in vitro migration and invasion via the CXCR4/ PKCf-mediated pathway. HGF induction of Rac1 activation and MT1-MMP production implied that cell migration and proteolysis are two essential processes during tumor invasion and metastasis. Our data suggested that PKCf stimulation and CXCR4 expression increased the in-vitro migration and invasion of HGF-treated MDA-MB-436 cells. HGF-induced CXCR4 expression was mediated via activation of the PI 3-kinase and AKT signaling modules In an attempt to identify the pathway leading to PKCf activation, we investigated the role of phosphatidylinositol triphosphate-kinase (PI 3K) which has been proposed to activate PKCf [44]. Akt is a pivotal effector immediately downstream of the PI 3K and has been implicated in G protein-coupled receptor- mediated chemotaxis [45]. The HGF-induced Akt activation is reflected by an increase in phosphorylated Akt [46]. To verify whether HGF activates Akt phosphorylation in MDA-MB-436 cells, we performed western blot analyses of extracts from treated and unstimulated cells, and found that HGF had a major effect on Akt activity. Adding PI 3K inhibitors LY294002 and wortmannin was shown to partially prevent the phosphorylation of AKT in MDA-MB-436 cells (Figure 5A, Figure S4A). Furthermore, LY294002 and AKT inhibitor III inhibited PKCf phosphoryla- tion and membrane CXCR4 expression, which suggested that PKCf exerted its function downstream of the PI 3-kinases signaling pathway (Figures 5B–5C, Figure S4B). Pretreatment with LY294002 was also found to prevent translocation of PKCf and CXCR4 to the plasma membrane (Figure 5D), demonstrating that PKCf activation depended on PI3K activity. Rac1 reportedly acts as a downstream effector of PI3-kinase in several growth factor-stimulated pathways [47]. Rac1 activation is often dependent on PI3-kinase activity, and inhibitors of PI3- kinase block Rac1 activation. To verify whether HGF activates Rac1 activities in MDA-MB-436 cells and the relationship of Rac1 and PI3K pathway, we performed Rac1 activity assay analyses of extracts from treated and unstimulated cells, and found that HGF can increase Rac1 activities. Adding PI 3K inhibitors LY294002 and wortmannin was shown to partially decrease the activity of Rac1 in MDA-MB-436 cells (Figure 5A, Figure S4A). Our studies conformed that PI3-kinase inhibitors can suppress Rac1 activities, which further suggested that the PI3-kinase regulate the activity of small GTPases Rac1 [48]. These findings implied that functional CXCR4 expression induced by HGF might involve a pathway of activation of the PI 3K/Akt and PKCf, and that signals stimulated by HGF passed from PI 3K/Akt to Rac1 and then to PKCf. HGF enhanced CXCR4 expression via PKCf and promoted the invasion and metastasis of breast cancers in vivo To further examine the effects of HGF-induced CXCR4 expression via PKCf on breast cancer invasion and metastasis in vivo, we inoculated the mammary fat pads of athymic nude mice with MDA-MB-436 cells. Cancer metastasis to the lung and liver of the mice was evaluated when the xenografts reached 1.5 cm in diameter. Biweekly intratumoral injection with HGF at a dosage of 30 mg/kg for 4 consecutive weeks enhanced the penetration of MDA-MB-436 cancer cells into the adjacent normal tissues, which was not seen with PBS injection (Figure 6A). Moreover, intratumoral injection of HGF conspicuously increased the number of mice with lung and liver metastasis (Table S2) but not the size of breast cancer xenografts (Figure S5). As observed with hematoxylin and eosin (H&E) and PCNA staining, HGF injection led to more intense infiltration of cancer cells into the lung and liver of MDA-MB-436 xenograft-bearing mice than did the PBS injection (Figure 6B). The mean wet lung weight in HGF-injected tumor-bearing mice was greater as compared with those injected with PBS or PKCf-shRNA PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29124