RESULTS Snail1 depletion ameliorates UUO-induced fibrosis Supplementary Fig. 1), impedes Snail1 expression in Cadherin-16 positive cells. Snail1 expression is silent in the adult kidney but it is reactivated after UUO in the cortex and in the medulla (Supplementary Fig. 1c). During renal development, Snail1 is a strong Cadh16 repressor 5 , and it is only when Snai1 is downregulated that renal cells express Cadherin-16 and epithelialization occurs. As Snail1 is silenced in the adult, preventing Snail1 activation in Cadherin-16 positive cells in the kidney does not have any impact in healthy mice. As expected, while Snail1 was highly reactivated in the kidney upon UUO, this was not the case in recombined renal epithelial cells in SFKC mice (Fig. 1a-c). We analyzed overall morphology, collagen deposition and expression of alpha smooth muscle actin ( -SMA) and vimentin 7 or 15 days after UUO and found that kidneys from SFKC mice were protected from the development of overt fibrosis, although signs of tubular distension resulting from the obstruction 5 were readily evident (Fig. 1d,e and Supplementary Fig. 2a,b). We confirmed the attenuation of fibrosis by analysis of the expression of epithelial and mesenchymal markers and this was particularly evident 2 weeks after UUO (Fig. 1f). Quantification of Sirius Red staining (Fig. 1g) showed a 35% reduction in fibrosis in the cortex of obstructed kidneys from SFKC mice when compared to obstructed kidneys from WT or control (Ksp1.3-Cre-only) mice ( Fig. 1g and Supplementary Fig. 3a,b). In our SFKC model, Snail1 reactivation was also prevented in the collecting ducts, and we observed a much better preserved morphology and lower collagen deposition in the medulla compared to WT animals ( Supplementary Fig. 3c) indicating that the overall protection from fibrotic degeneration is higher than that reflected by the quantification of SiriusRed in the cortex.We next assessed the contribution of epithelial cells to the interstitium after UOO in our system. We generated a mouse model harboring Cre-loxP mediated expression of the Tomato fluorescent protein driven by the Ksp 1.3 promoter (Ksp1.3-Cre; Rosa-LSL-TdTomato). These mice allow the visualization and fate mapping of renal epithelial cells. Notably, we could not detect tdTomato+ cells in the interstitium 7 days after obstruction ( Supplementary Fig. 4a) and found less than 1% after 15 days (Fig. 2a). One of those rare cells leaving the tubule is shown in Supplementary Fig. 4b. Thus, renal epithelial cells do not delaminate from the tubules to contribute to myofibroblasts or other interstitial cells, also in keeping with the observation that the total number of tubular cross sections did not differ between kidneys from WT or SFKC mice 15 days after UUO (Fig. 2b), indicative of tubular integrity. 6Snail1 expression was reactivated after UUO in over 80% of tubular cells and also in activated interstitial cells as reported in cancer models 16 (Fig. 2c).Although tubular cells did not significantly contribute to myofibroblasts, UUO pr...

SummaryThe melanogenic actions of the melanocortins are mediated by the melanocortin-1 receptor (MC1R). MC1R is a member of the G-protein-coupled receptors (GPCR) superfamily expressed in cutaneous and hair follicle melanocytes. Activation of MC1R by adrenocorticotrophin or a-melanocyte stimulating hormone is positively coupled to the cAMP signaling pathway and leads to a stimulation of melanogenesis and a switch from the synthesis of pheomelanins to the production of eumelanic pigments. The functional behavior of the MC1R agrees with emerging concepts in GPCR signaling including dimerization, coupling to more than one signaling pathway and a high agonist-independent constitutive activity accounting for inverse agonism phenomena. In addition, MC1R displays unique properties such as an unusually high number of natural variants often associated with clearly visible phenotypes and the occurrence of endogenous peptide antagonists. Therefore MC1R is an ideal model to study GPCR function. Here we review our current knowledge of MC1R structure and function, with emphasis on information gathered from the analysis of natural variants. We also discuss recent data on the regulation of MC1R function by paracrine and endocrine factors and by external stimuli such as ultraviolet light.

We generated cell lines resistant to BRAF inhibitors and show that the EGF receptor (EGFR)-SRC family kinase (SFK)-STAT3 signaling pathway was upregulated in these cells. In addition to driving proliferation of resistant cells, this pathway also stimulated invasion and metastasis. EGFR inhibitors cooperated with BRAF inhibitors to block the growth of the resistant cells in vitro and in vivo , and monotherapy with the broad specifi city tyrosine kinase inhibitor dasatinib blocked growth and metastasis in vivo . We analyzed tumors from patients with intrinsic or acquired resistance to vemurafenib and observed increased EGFR and SFK activity. Furthermore, dasatinib blocked the growth and metastasis of one of the resistant tumors in immunocompromised mice. Our data show that BRAF inhibitor-mediated activation of EGFR-SFK-STAT3 signaling can mediate resistance in patients with BRAF-mutant melanoma. We describe 2 treatments that seem to overcome this resistance and could deliver therapeutic effi cacy in patients with drug-resistant BRAF-mutant melanoma. SIGNIFICANCE:Therapies that target the driver oncogenes in cancer can achieve remarkable responses if patients are stratifi ed for treatment. However, as with conventional therapies, patients often develop acquired resistance to targeted therapies, and a proportion of patients are intrinsically resistant and fail to respond despite the presence of an appropriate driver oncogene mutation. We found that the EGFR/SFK pathway mediated resistance to vemurafenib in BRAF -mutant melanoma and that BRAF and EGFR or SFK inhibition blocked proliferation and invasion of these resistant tumors, providing potentially effective therapeutic options for these patients. Cancer Discov; 3(2);

Rounded-amoeboid cancer cells use actomyosin contractility driven by Rho-ROCK and JAK-STAT3 to migrate efficiently. It has been suggested that rounded-amoeboid cancer cells do not require matrix metalloproteinases (MMPs) to invade. Here we compare MMP levels in rounded-amoeboid and elongated-mesenchymal melanoma cells. Surprisingly, we find that rounded-amoeboid melanoma cells secrete higher levels of several MMPs, including collagenase MMP-13 and gelatinase MMP-9. As a result, rounded-amoeboid melanoma cells degrade collagen I more efficiently than elongated-mesenchymal cells. Furthermore, using a non-catalytic mechanism, MMP-9 promotes rounded-amoeboid 3D migration through regulation of actomyosin contractility via CD44 receptor. MMP-9 is upregulated in a panel of rounded-amoeboid compared with elongated-mesenchymal melanoma cell lines and its levels are controlled by ROCK-JAK-STAT3 signalling. MMP-9 expression increases during melanoma progression and it is particularly prominent in the invasive fronts of lesions, correlating with cell roundness. Therefore, rounded-amoeboid cells use both catalytic and non-catalytic activities of MMPs for invasion.