TGFbeta induces fibrogenic responses in fibroblasts. Reactive oxygen species (ROS)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) may contribute to fibrogenic responses. Here, we examine if the antioxidant N-acetylcysteine (NAC), the NOX inhibitor diphenyleneiodonium (DPI) and the selective NOX1/NOX4 inhibitor GKT-137831 impairs the ability of TGFbeta to induce profibrotic gene expression in human gingival (HGF) and dermal (HDF) fibroblasts. We also assess if GKT-137831 can block the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts. We use real-time polymerase chain reaction and Western blot analysis to evaluate whether NAC and DPI impair the ability of TGFbeta1 to induce expression of fibrogenic genes in fibroblasts. The effects of GKT-137831 on TGFbeta-induced protein expression and the persistent fibrotic phenotype of lesional scleroderma (SSc) fibroblasts were tested using Western blot and collagen gel contraction analyses. In HDF and HGF, TGFbeta1 induces CCN2, CCN1, endothelin-1 and alpha-smooth muscle actin (SMA) in a fashion sensitive to NAC. Induction of COL1A1 mRNA was unaffected. Similar results were seen with DPI. NAC and DPI impaired the ability of TGFbeta1 to induce protein expression of CCN2 and alpha-SMA in HDF and HGF. GKT-137831 impaired TGFbeta-induced CCN2 and alpha-SMA protein expression in HGF and HDF. In lesional SSc dermal fibroblasts, GKT-137831 reduced alpha-SMA and CCN2 protein overexpression and collagen gel contraction. These results are consistent with the hypothesis that antioxidants or NOX1/4 inhibition may be useful in blocking profibrotic effects of TGFbeta on dermal and gingival fibroblasts and warrant consideration for further development as potential antifibrotic agents.

Metastatic melanoma is highly fatal. Within the tumor microenvironment, the role of cancer-associated fibroblasts (CAFs) in melanoma metastasis and progression is relatively understudied. Matricellular proteins of the CCN family are emerging targets for cancer therapy. Anti-CCN2 (CTGF) strategies are under clinical development; however, the role of CCN1 (cyr61) in cancers is unclear. Herein, we use bioinformatic analyses to report that the CCN1 is overexpressed, in a fashion independent of BRAF mutational status, in melanoma. In human melanoma patients, CCN1 expression negatively correlates with overall survival, disease-free, and metastasis-free survival (p<0.03). CCN1 expression positively correlates with expression of stroma and neovascularization markers. To assess the role of CAFs in melanoma progression, we used C57BL/6 mice expressing a tamoxifen-dependent cre recombinase expressed under the control of a fibroblast-specific promoter/enhancer (COL1A2) to delete CCN1 postnatally in fibroblasts. Mice deleted or not for CCN1 in fibroblasts were injected subcutaneously with B16-F10 melanoma cells. Loss of CCN1 in CAFs did not result in reduced CAF activation, as detected by staining with anti-α-smooth muscle actin antibodies, but resulted in reduced extracellular matrix surrounding tumors, as detected by trichrome stain, and reduced tumor-induced neovascularization, as detected by staining with anti-CD31 antibodies. CCN1-deficient skin showed reduced expression of mRNAs encoding collagen crosslinking enzymes (PLOD2 and LOX) and impaired formation of collagen fibers, as determined by electron microscopy. Tumor growth was not affected by loss of CCN1 from tumor stroma; however, micrometastasis to the lung was severely impaired (p<0.001). Our results provide new insights into the crosstalk among different cell types in the tumor microenvironment and confirm the essential role of CAFs in metastasis and tumor neovascularization. Our data are consistent with the hypothesis that CAFs are essential for melanoma metastasis and that CCN1 is a therapeutic target for melanoma. Citation Format: James Hutchenreuther, Katherine Quensel, Krista Vincent, Lynne-Marie Posrovit, Andrew Leask. Ccn1 expression by fibroblasts is required for melanoma metastasis [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A18.

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