Suppression of TGF beta and Angiogenesis by Type VII Collagen in Cutaneous SCC

Background: Individuals with severe generalized recessive dystrophic epidermolysis bullosa (RDEB), an inherited blistering disorder caused by mutations in the COL7A1 gene, develop unexplained aggressive squamous cell carcinomas (SCC). Here we report that loss of type VII collagen (Col7) in SCC results in increased TGF beta signaling and angiogenesis in vitro and in vivo. Methods: Stable knockdown (KD) of Col7 was established using shRNA, and cells were used in a mouse xenograft model. Angiogenesis was assessed by immunohistochemistry, endothelial tube-forming assays, and proteome arrays. Mouse and zebrafish models were used to examine the effect of recombinant Col7 on angiogenesis. Findings were confirmed in anonymized, archival human tissue: RDEB SCC tumors, non-EB SCC tumors, RDEB skin, normal skin; and two human RDEB SCC cell lines. The TGF beta pathway was examined using immunoblotting, immunohistochemistry, biochemical inhibition, and siRNA. All statistical tests were two-sided. Results: Increased numbers of cross-cut blood vessels were observed in Col7 KD compared with control xenografts (n = 4 to 7 per group) and in RDEB tumors (n = 21) compared with sporadic SCC (n = 24, P < .001). Recombinant human Col7 reversed the increased SCC angiogenesis in Col7 KD xenografts in vivo (n = 7 per group, P = .04). Blocking the interaction between alpha 2 beta 1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGF beta signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGF beta markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGF beta markers, n = 6; VEGF, n = 21). Inhibition of TGF beta receptor signaling using siRNA resulted in decreased endothelial cell tube formation (n = 9 per group, mean tubes per well siC = 63.6, SD = 17.1; mean tubes per well siT beta RII = 29.7, SD = 6.1, P = .02). Conclusions: Type VII collagen suppresses TGF beta signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.