CCN Proteins in Metastatic Melanoma

Melanoma cells recruit host tissue to become a part of the activated tumour stroma. This stromal microenvironment is similar to that seen in fibrotic tissue. CCN1 and CCN2 are tightly spatiotemporally regulated matricellular proteins involved in development and wound healing, and are abberantly expressed in fibrosis. Additionally they have been seen to be abnormally highly expressed in several cancers, including melanoma. Recent evidence has shown that deletion of CCN2 in the fibroblasts renders mice resistant to several models of fibrosis. Given this, I tested the hypothesis that deletion of CCN1 and CCN2 from fibroblasts could similarly impede the formation of the activated stromal microenvironment in melanoma. I used B16F(10) murine melanoma cells and syngeneic C57 BL6 mice with a tamoxifen-dependent conditional deletion of CCN1 or CCN2 in their fibroblasts. First I determined that loss of CCN2 in the fibroblasts prevents the metastasis of melanoma to the lungs of the mice, while loss of CCN2 in the tumour alone does not. Second I determined that loss of CCN2 from the fibroblasts prevented the expression of myofibroblast marker α SMA and reduced the expression of pluripotency marker SOX2. This loss of CCN2 was accompanied by a reduced tumour vascularisation, and a reduction in tumour cell vasculogenic mimicry. Finally I determined that loss of CCN1 in the fibroblasts results in highly disorganized collagen in the skin, which results in reduced metastasis of the melanoma cells. These observations were supported by in vitro experiments showing that deletion of CCN1 or CCN2 from melanoma cells reduce their ability to invade through a collagen basement membrane, and that deletion of CCN2 impedes the ability of melanoma cells to form tubule networks in nutrient-deficient environments. The results presented here suggest that CCN1 and CCN2 in the stromal microenvironment mediate the metastasis of melanoma through different mechanisms, with CCN2 being required for the activated stromal microenvironment and tumour vascularisation, and CCN1 being required for formation of a stiff and organized collagen network that facilitates tumour cell invasion, and thus they might both present novel targets for therapies to improve patient outcome

TAK1 Is Required for Dermal Wound Healing and Homeostasis

Dermal connective tissue is a supportive structure required for skin’s barrier function; dysregulated dermal homeostasis results in chronic wounds and fibrotic diseases. The multifunctional cytokine transforming growth factor (TGF) β promotes connective tissue deposition, repair, and fibrosis. TGF-β acts through well-defined canonical pathways; however, the non-canonical pathways through which TGF-β selectively promotes connective tissue deposition are unclear. In dermal fibroblasts, we show that inhibition of the non-canonical TGF-β-activated kinase 1 (TAK1) selectively reduced the ability of TGF-β to induce expression of a cohort of wound healing genes, such as collagens, CCN2, TGF-β1, and IL-6. Fibroblast-specific TAK1-knockout mice showed impaired cutaneous tissue repair and decreased collagen deposition, α-smooth muscle actin and CCN2 expression, proliferating cell nuclear antigen staining, and c-Jun N-terminal kinase and p38, but not Smad3, phosphorylation. TAK1-deficient fibroblasts showed reduced cell proliferation, migration, cell attachment/spreading, and contraction of a floating collagen gel matrix. TAK1-deficient mice also showed progressively reduced skin thickness and collagen deposition. Thus, TAK1 is essential for connective tissue deposition in the dermis

ACTIVATION OF LATENT TGFβ BY ΑVΒ1 INTEGRIN: OF POTENTIAL IMPORTANCE IN MYOFIBROBLAST ACTIVATION IN FIBROSIS

ABSTRACT Cell--mediated activation of latent TGF--β1 is intimately involved with tissue repair and fibrosis in all organs. Previously, it was shown that the integrin β1 subunit was requred for activation of latent TGF--β1 and skin fibrosis. A recent study by Henderson and colleagues (Nature Medicine 19,1617-1624, 2013) used three different in vivo models of fibrosis to show that integrin αv subunit was required for fibrogenesis. Through a process of elimination, the authors conclude that in vivo, the little--studied αvβ1 could be the major integrin responsible for TGF--β activation by myofibroblasts. Thus targeting this integrin might be a useful therapy for fibrosis

Cancer-associated Fibroblast-specific Expression of the Matricellular Protein CCN1 Coordinates Neovascularization and Stroma Deposition in Melanoma Metastasis.

Melanoma is the leading cause of skin cancer-related death. As prognosis of patients with melanoma remains problematic, identification of new therapeutic targets remains essential. Matricellular proteins are nonstructural extracellular matrix proteins. They are secreted into the tumor microenvironment to coordinate behavior among different cell types, yet their contribution to melanoma is underinvestigated. Examples of matricellular proteins include those comprising the CCN family. The CCN family member, CCN1, is highly proangiogenic. Herein, we show that, in human patients with melanoma, although found in several tumor cell types, CCN1 is highly expressed by a subset of cancer-associated fibroblasts (CAF) in patients with melanoma and this expression correlates positively with expression of proangiogenic genes and progressive disease/resistance to anti-PD1 checkpoint inhibitors. Consistent with these observations, in a syngeneic C57BL6 mouse model of melanoma, loss of CCN1 expression from Col1A2-Cre-, herein identified as "universal," fibroblasts, impaired metastasis of subcutaneously injected B16F10 tumor cells to lung, concomitant with disrupted neovascularization and collagen organization. Disruption of the extracellular matrix in the loss of CCN1 was validated using a novel artificial intelligence-based image analysis platform that revealed significantly decreased phenotypic fibrosis and composite morphometric collagen scores. As drug resistance is linked to matrix deposition and neoangiogenesis, these data suggest that CCN1, due to its multifaceted role, may represent a novel therapeutic target for drug-resistant melanoma. Our data further emphasize the essential role that cancer-associated, (universal) Col1A2-Cre-fibroblasts and extracellular matrix remodeling play in coordinating behavior among different cell types within the tumor microenvironment.SignificanceIn human patients, the expression of proangiogenic matricellular protein CCN1 in CAFs correlates positively with expression of stroma and angiogenic markers and progressive disease/resistance to checkpoint inhibitor therapy. In an animal model, loss of CCN1 from CAFs impaired metastasis of melanoma cells, neovascularization, and collagen deposition, emphasizing that CAFs coordinate cellular behavior in a tumor microenvironment and that CCN1 may be a novel target

(5Z)-7-oxozeaenol reduces TGFβ1-induced collagen expression.

<p>A) Western Blot Analysis. Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment for 24 hours with or without TGFβ1 (4ng/ml). As described in methods, proteins were harvested and subjected to Western blot analysis with anti-collagen type I and anti-β-actin antibodies, as indicated. A representative blot is shown. Experiments were performed on 3 separate occasions. (N = 4, averages+/-SEM are shown; * = p<0.05, Student’s t-test. CCN2 expression in response to TGFβ was taken to represent 1). (B) mRNA analysis Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1 (90 pM)). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown; * = p<0.05, One-Way ANOVA).</p

(5Z)-7-oxozeaenol reduces TGFβ1 induced gingival fibroblast proliferation.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol ((5Z)-7-oxo; 400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml-1 (90 pM)) ligand or left untreated. Cultures were grown in the presence of BrdU for up to 72 hours as described in methods. One of three representative experiments is shown; (N = 4; averages+/-SEM are shown * p<0.05 for: DMSO vs TGFβ1, (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1; ** p<0.05 for: DMSO vs TGFβ1; (5Z)-7-oxo vs TGFβ1, TGFβ1 vs (5Z)-7-oxo+TGFβ1. Two-Way ANOVA followed by Tukey's Post Hoc analysis).</p

Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.

<p>Genes involved with a hyperproliferative response are shown.</p><p>Cluster analysis of TAK1 depended mRNAs with over 1.7 fold induction (average of two arrays) in response to TGFβ-1 treatment.</p

(5Z)-7-oxozeaenol reduces TGFβ1-induced CCN2 mRNA expression in human gingival fibroblasts.

<p>Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with or without TGFβ1 (4ngml^-1). Total RNA was harvested six hours later and subjected to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. Values are expressed relative to untreated control. (N = 3; averages+/-SEM are shown; **** = p<0.0001, * = p<0.05 One-Way ANOVA).</p

(5Z)-7-oxozeaenol inhibits TGFβ1-induced mRNA expression in human gingival fibroblasts.

<p>(A) Human gingival fibroblasts were serum starved overnight and pre-treated with (5Z)-7-oxozeaenol (400 nM) or DMSO for 45 min followed by treatment with TGFβ1 (4ngml^-1 (90 pM)) ligand or left untreated. Total RNA was harvested six hours later and subjected to gene expression profiling using GeneChip Human Gene 1.0 ST arrays (N = 2) as described in Methods. 147 genes were up-regulated in response to TGFβ1 (1.7 fold induction compared to DMSO control group) and 139 genes of the latter group were found to be (5<i>Z</i>)-7-Oxozeaenol sensitive. (B) Human gingival fibroblasts were treated as in (A) and subject to TaqMan RT-qPCR analysis using the indicated probe/primer set. 18S RNA was used as the internal control. (N = 3; averages+/-SEM are shown. * = p<0.05; ** = p<0.01; *** = p<0.001, One-Way ANOVA).</p