V L Martins1, M P Caley1, K Moore1, Z Szentpetery1, S T Marsh1, D F Murrell1, M H Kim1, M Avari1, J A McGrath1, R Cerio1, A Kivisaari1, V M Kähäri1, K Hodivala-Dilke1, C H Brennan1, M Chen1, J F Marshall1, E A O'Toole2. 1. Centre for Cell Biology and Cutaneous Research, Blizard Institute (VLM, MPC, ZS, STM, MA, RC, EOT), Barts Cancer Institute (KM, KHD, JFM), Barts and the London School of Medicine and Dentistry and School of Biological and Chemical Sciences (CHB), Queen Mary University of London, London, UK; Department of Dermatology, St George Hospital, University of NSW, Sydney, NSW, Australia (DFM, MHK); St John's Institute of Dermatology, Kings College London (Guys Campus), London, UK (JAM); Department of Dermatology and MediCity Research Laboratory, University of Turku, and Turku University Hospital, Turku, Finland (AK, VMK); Department of Dermatology, University of Southern California, Los Angeles, CA (MC). 2. Centre for Cell Biology and Cutaneous Research, Blizard Institute (VLM, MPC, ZS, STM, MA, RC, EOT), Barts Cancer Institute (KM, KHD, JFM), Barts and the London School of Medicine and Dentistry and School of Biological and Chemical Sciences (CHB), Queen Mary University of London, London, UK; Department of Dermatology, St George Hospital, University of NSW, Sydney, NSW, Australia (DFM, MHK); St John's Institute of Dermatology, Kings College London (Guys Campus), London, UK (JAM); Department of Dermatology and MediCity Research Laboratory, University of Turku, and Turku University Hospital, Turku, Finland (AK, VMK); Department of Dermatology, University of Southern California, Los Angeles, CA (MC). e.a.otoole@qmul.ac.uk.
Abstract
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 TGFβ 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 TGFβ 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 α2β1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFβ signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFβ markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFβ markers, n = 6; VEGF, n = 21). Inhibition of TGFβ 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 siTβRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFβ signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.
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 TGFβ 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 TGFβ 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 α2β1 integrin and Col7 increased TGFB1 mRNA expression 1.8-fold and p-Smad2 levels two-fold. Increased TGFβ signaling and VEGF expression were observed in Col7 KD xenografts (n = 4) compared with control (n = 4) and RDEB tumors (TGFβ markers, n = 6; VEGF, n = 17) compared with sporadic SCC (TGFβ markers, n = 6; VEGF, n = 21). Inhibition of TGFβ 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 siTβRII = 29.7, SD = 6.1, P = .02). CONCLUSIONS: Type VII collagen suppresses TGFβ signaling and angiogenesis in cutaneous SCC. Patients with RDEB SCC may benefit from anti-angiogenic therapy.
Authors: Qianghua Hu; Takeshi Hisamatsu; Monika Haemmerle; Min Soon Cho; Sunila Pradeep; Rajesha Rupaimoole; Cristian Rodriguez-Aguayo; Gabriel Lopez-Berestein; Stephen T C Wong; Anil K Sood; Vahid Afshar-Kharghan Journal: Clin Cancer Res Date: 2017-06-13 Impact factor: 12.531