The communication between the tumor cells and the surrounding cells helps drive the process of tumor progression. Since the microenvironment of breast cancer includes CCL20 chemokine, the purpose of this study was to determine whether CCL20 modulates the physiology of healthy breast epithelial cells in areas adjacent to the tumor. Therefore, primary cultures of mammary cells taken from normal peritumoral areas were used. We assessed that breast cells expressed CCR6 CCL20 receptor. Using molecular (siRNA) and pharmacological (inhibitors) techniques, we found multiple signaling kinases to be activated by CCR6 and involved in CCL20-induced breast cell proliferation and migration. The binding of 10 ng/ml CCL20 to CCR6 induced cell migration whilst higher concentrations (from 15 to 25 ng/ml) led to cell proliferation. CCL20 controlled cell migration and MMP-9 expression by PKC-alpha that activated Src, which caused the activation of downstream Akt, JNK, and NF-kB pathways. Furthermore, higher CCL20 concentrations increased cycE and decreased p27Kip expression ending in enhanced cell proliferation. Cell proliferation occurred through PKC-epsilon activation that transactivated EGFR and ERK1/2/MAPK pathway. Although activated by different CCL20 concentrations, these pathways function in parallel and crosstalk to some extent, inasmuch as Akt activation was responsible for ERK1/2 nuclear translocation and enhanced the transcription of of c-fos and c-myc, involved in cell proliferation. In summary, tumor cells exchange signals with the surrounding healthy cells modifying the extracellular matrix through enzyme secretion; thus, CCL20 might be a factor involved in the ontogeny of breast carcinoma.

Following peripheral nerve injury, remnant Schwann cells adopt a migratory phenotype and remodel the extracellular matrix allowing axonal regrowth. Although much evidence has demonstrated that TGF-b1 promotes glioma cell motility and induces the expression of extracellular matrix proteins, the effects of TGF-b1 on Schwann cell migration has not yet been studied. We therefore investigated the cellular effects and the signal transduction pathways evoked by TGF-b1 in rattus norvegicus neuronal Schwann RSC96 cell. TGF-b1 significantly increased migration and invasion of Schwann cells assessed by the wound-healing assay and by cell invasion assay. TGF-b1-enhanced migration/invasion was blocked by inhibition of MMP-2 and MMP-9. Consistently, by real-time and western blot analyses, we demonstrated that TGF-b1 increased MMP-2 and MMP-9 mRNA and protein levels. TGF-b1 also increased MMPs activities in cell growth medium, as shown by gelatin zymography. The selective TGF-b Type I receptor inhibitor SB431542 completely abrogated any effects by TGF-b1. Indeed, TGF-b1 Type I receptor activation provoked the cytosol-to-nucleus translocation of SMAD2 and SMAD3. SMAD2 knockdown by siRNA blocked MMP-2 induction and cell migration/invasion due to TGF-b1. TGF-b1 also provoked phosphorylation of MAPKs extracellular regulated kinase 1/2 and JNK1/2. Both MAPKs were upstream to p65/NF-kB inasmuch as both MAPKs' inhibitors PD98059 and SP600125 or their down-regulation by siRNA significantly blocked the TGF-b1-induced nuclear translocation of p65/NF-kB. In addition, p65/NF-jB siRNA knockdown inhibited the effects of TGF-b1 on both MMP-9 and cell migration/invasion. We conclude that TGF-b1 controls RSC96 Schwann cell migration and invasion through MMP-2 and MMP-9 activities. MMP-2 is controlled by SMAD2 whilst MMP-9 is controlled via an ERK1/2-JNK1/2-NF-jB dependent pathway. Abbreviations used: ERK1/2, extracellular regulated kinase 1/2; FBS, fetal bovine serum; MEK, MAPK/ERK kinase; MMP (2-9), matrix metalloproteinases (2-9); MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide; NF-kB, nuclear factor-kappa B; siRNAs, small interfering RNAs; SMAD (2-3), small mother against decapentaplegic (2-3); TGF-b, transforming growth factor beta.

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Background and purpose:We showed previously that a new Pt(II) complex ([Pt(O,O′-acac)(g-acac)(DMS)]) exerted high and fast apoptotic processes in MCF-7 cells. The objective of this study was to investigate the hypothesis that [Pt(O,O′-acac)(gacac)(DMS)] is also able to exert anoikis and alter the migration ability of MCF-7 cells, and to show some of the signalling events leading to these alterations. Experimental approach: Cells were treated with sublethal doses of [Pt(O,O′-acac)(g-acac)(DMS)], and the efficiency of colony initiation and anchorage-independent growth was assayed; cell migration was examined by in vitro culture wounding assay. Gelatin zymography for MMP-2 and -9 activities, Western blottings of MMPs, MAPKs, Src, PKC-e and FAK, after [Pt(O,O′-acac)(g-acac)(DMS)] treatment, were also performed. Key results: Sub-cytotoxic drug concentrations decreased the: (i) anchorage-dependent and -independent growth; (ii) migration ability; and (iii) expression and activity of MMP-2 and MMP-9. [Pt(O,O′-acac)(g-acac)(DMS)] provoked the generation of reactive oxygen species (ROS), and the activation of p38MAPK, Src and PKC-e. p38MAPK phosphorylation, cell anoikis and migration due to [Pt(O,O′-acac)(g-acac)(DMS)] were blocked by PKC-e inhibition. Furthermore, Src inhibition blocked the [Pt(O,O′-acac)(g-acac)(DMS)]-provoked activation of PKC-e, while ROS generation blockage inhibited the activation of Src, and also the decrement of phosphorylated FAK observed in detached [Pt(O,O′-acac)(g-acac)(DMS)]-treated cells. Conclusions and implications:Sublethal concentrations of [Pt(O,O′-acac)(g-acac)(DMS)] induced anoikis and prevented events leading to metastasis via alterations in cell migration, anchorage independency, stromal interactions and MMP activity. Hence, [Pt(O,O′-acac)(g-acac)(DMS)] may be a promising therapeutic agent for preventing growth and metastasis of breast cancer.

We previously found that CCL20 induced primarily cultured healthy breast cell proliferation and migration. The objective of this study was to investigate the hypothesis that CCL20 modulated the epithelial-mesenchymal transition (EMT) of primarily cultured healthy breast epithelial cells and the angiogenesis in areas adjacent to the tumor. Key results showed that CCL20 (a) down-regulated E-cadherin and ZO-1; (b) up-regulated N-cadherin, vimentin, and Snail expressions; (c) increased mRNA and secretion of VEGF and (d) increased angiogenic micro vessel sprouting. Thus, the signal transduction pathways evoked by CCL20 were investigated. We showed that NF-kB p65 down-regulation (by small interfering RNA, siRNA) reversed CCL20-induced Snail and blocked the up-regulation of vimentin and N-cadherin mRNAs. Furthermore, PI3K/AKT inhibition (by LY294002) completely blocked CCL20-induced Snail and NF-kB activation. Inhibition of JNK1/2 (by SP60125) or PKC-α (by siRNA) or src (by PP1) blocked NF-kB activation and Snail expression suggesting that these kinases are all upstream of NF-kB/Snail. Inhibition of mTOR (by rapamycin) abolished the effects of CCL20 on N-cadherin and vimentin protein synthesis. Furthermore, siRNA of PKC-δ inhibited the phosphorylation of CCL20-induced mTOR and S6, increased vimentin and N-cadherin expressions and, finally, blocked the CCL20 induced-EMT. CCL20 increased mRNA and secretion of VEGF by healthy breast cells by using PKC-α, src, Akt, NF-kB, and Snail signalling. In summary, tumor cells signal to the surrounding healthy cells through CCL20 inducing the modulation of the expression of molecules involved in EMT and promoting angiogenesis directly and indirectly through the secretion of VEGF, a major contributor to angiogenesis. © 2015 Wiley Periodicals, Inc.