PKCf Induced by HGF Increases CXCR4 Expression Figure 6. HGF enhances CXCR4 expression via PKCf and promotes the invasion and metastasis of breast cancers in BALB/c-nu mice. (A) Micrographs of H&E-stained xenografts demonstrating the presence or absence of margin invasion by the MDA-MB-436 tumors untransduced (Un) or transduced with GFP-shRNA or PKCf-shRNA (original magnification, 2006). (B) Representative micrographs of lung (upper) or liver (lower) tissue sections with H&E staining and PCNA immunohistochemical staining (original magnification, 2006). Each group from two independent experiments. (C) Mean 6 SD wet lung weight in tumor-bearing mice. The number of mice in each group is indicated. * P,0.05 as compared to PBS- treated mice. (D) Expression of human HPRT mRNA relative to mouse 18S rRNA as determined by qRT-PCR in the lungs and livers of tumor-bearing mice. Data were normalized to PBS-treated mice. # P,0.01 as compared to PBS-treated mice. (E–F) Immunoblot of total PKCf and p-PKCf or total CXCR4 and p-CXCR4 protein, respectively, in breast tumor xenografts in female nude mice that were inoculated in the mammary fat pads with MDA- MB-436 cells treated as indicated. The experiment was repeated twice with similar results. A representative study is shown. (G) Representative microphotos of immunohistochemical results for p-PKCf (middle) or p-CXCR4 (lower) expression in MDA-MB-436 tumors (original magnification, 4006). doi:10.1371/journal.pone.0029124.g006 then to PKCf. These were further supported by the fact that multiple chemical inhibitors and PSf can block signal transmis- sion. MT1-MMP, also known as MMP-14, has a single transmem- brane domain and is an integral membrane protein with an extracellular catalytic domain suggested to be a key enzyme in tumor metastasis and angiogenesis. Numerous studies implied that MT1-MMP in breast tumors are correlated with the outcome of patients with breast cancer [39]. Reduction of MT1-MMP from the breast cancer cells resulted in significant reduction of in vitro invasiveness and loss of response to an invasion stimulus. An association between MT1-MMP and the PKCf activation has been reported. PKCf may be responsible for the increased binding of Sp1 to MT1-MMP promoter site in shear-stressed endothelial cell (EC) and thus the induction of MT1-MMP protein [40]. MT1-MMP in coordination with CXCR4 promotes invasion and dissemination of the tumor [41]. HGF has also been shown to actively promote production of MT1-MMP in MDA-MB 231 cells, which further activated MMP2 and enhanced the invasive- ness of breast cancer cells [39]. Thus, we hypothesized that PKCf may be involved in the signaling pathway that mediates MT1- MMP expression in cells exposed to HGF. Our data were consistent with the requirement of PKCf phosphorylation and activation for Rac1 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. Therefore, the proper function of MT1-MMP may depend on both cellular expression and specific membrane localization. This study demonstrated that PKCf is critical to regulation of MT1-MMP expression. Activation of PKCf is also required in both EGFR- and chemokine receptor-mediated chemotaxis, suggesting the use of PKCf as a promising target for more potent anti-metastasis therapeutic strategies. Because PKCf-deficient mice are grossly normal, the pharmacologic inhibition of this enzyme should not be lethal to the hosts [57]. The identification of PKCf as a convergence point of EGFR- and chemokine receptor- mediated chemotaxis provided us with a potential novel target for anticancer drugs. We postulated that blocking PKCf may completely impair the chemotactic activities of some cancer cells and result in reduced in tumor invasion and spreading. Taken together, the results of our study provided new insights to molecular regulation of CXCR4 and to certain distinct regulatory molecules that can be targeted to modulate the CXCR4 signaling in breast cancer. Materials and Methods Patients and tissue samples Samples of primary ductal carcinoma of the breast were obtained from 197 female patients (median age 43.7 years, range 23–75) at the Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, from January 2002 to October 2007. Both the pathological diagnosis and estrogen receptor (ER)/Her2 status were verified independently by two pathologists. Patients with invasive carcinomas other than ductal carcinomas in-situ (DCIS) underwent 6 cycles of postoperative adjuvant chemotherapy with the FAC regimen (500 mg/m2 5-fluorouracil, 50 mg/m2 doxoru- bicin and 500 mg/m2 cyclophosphamide). Patients with ER+ tumors subsequently underwent endocrine therapy according to the NCCN guidelines. Distal metastasis was not found in these patients upon diagnosis but was identified in 45 cases during the postoperative follow-up. Additionally, benign breast tissue samples were collected from 20 cases of benign diseases of the breast with or without atypical epithelial hyperplasia. All samples were collected with patient informed consent according to the Internal Review and Ethics Boards of the Sun Yat-Sen Memorial Hospital of Sun Yat-Sen University. Cell cultures The human breast cancer cell lines MDA-MB-436 and MCF-7 were obtained from the American Type Culture Collection. They were cultured in L-15 or RPMI 1640 (Gibco, Birmingham, USA) with 10% fetal bovine serum (FBS) (Gibco, Birmingham, USA). Chemotaxis chambers and membranes were purchased from Costar. Wound healing assay The wound healing assay was mainly performed as described previously [53] with a slight modification. MDA-MB-436 breast cancer cells were grown were grown to confluence in 6-well tissue culture plates. The cells were pretreated with mitomycin (10 mg/ ml) for 2 h to block proliferation and then cell-free area introduced by scratching with a pipette tip. Cancer cells were allowed for a 24-hour recovery period to close the wound and were photo- graphed using phase-contrast microscopy. Chemotaxis assays The chemotaxis assays were performed as described by the manufacturer (Costar, Corning, NY, USA). Briefly, MDA-MB- 436 cells (56105 cells/ml) were pretreated with mitomycin (10 mg/ml) for 2 h to block proliferation and then suspended in medium (L-15, 0.1% bovine serum albumin, and 25 mM HEPES) were added to the upper chamber. The two chambers were separated by a 10-mm filter pretreated with 0.001% fibronectin in L-15 at 4uC overnight. The chambers were incubated in 5% CO2 at 37uC for 12 hours. The filter was then washed, fixed, and stained. The number of migrating cells was counted in three high- power fields (2006). In the inhibitor assay, MDA-MB-436 cells were pretreated with inhibitors at the indicated concentration for 60 minutes before HGF (Sigma, St. Louis, USA) were added at 37uC and then loaded into the upper chamber. PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e29124