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VII 型胶原对皮肤 SCC 中 TGFβ 和血管生成的抑制作用。

Suppression of TGFβ and Angiogenesis by Type VII Collagen in Cutaneous SCC.

机构信息

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).

出版信息

J Natl Cancer Inst. 2015 Oct 16;108(1). doi: 10.1093/jnci/djv293. Print 2016 Jan.

DOI:10.1093/jnci/djv293

PMID:26476432

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.

摘要

背景

严重的全身性隐性营养不良性大疱性表皮松解症（RDEB）是一种遗传性水疱性疾病，由 COL7A1 基因突变引起，患者会出现不明原因的侵袭性鳞状细胞癌（SCC）。本研究报道，SCC 中 VII 型胶原（Col7）的缺失会导致体外和体内 TGFβ 信号和血管生成增加。

方法

使用 shRNA 建立稳定的 Col7 敲低（KD），并将细胞用于小鼠异种移植模型。通过免疫组织化学、内皮管状形成测定和蛋白质组芯片评估血管生成。使用小鼠和斑马鱼模型来检查重组 Col7 对血管生成的影响。在匿名的、存档的人类组织中证实了发现：RDEB SCC 肿瘤、非 EB SCC 肿瘤、RDEB 皮肤、正常皮肤；以及两种人类 RDEB SCC 细胞系。使用免疫印迹、免疫组织化学、生化抑制和 siRNA 检查 TGFβ 途径。所有统计检验均为双侧。

结果

与对照组异种移植物（每组 4 至 7 个）相比，Col7 KD 中观察到更多的横切血管（n = 4 至 7 个），与散发 SCC（n = 24 个）相比，RDEB 肿瘤中观察到更多的横切血管（n = 21 个，P <.001）。体内 Col7 KD 异种移植物中重组人 Col7 逆转了增加的 SCC 血管生成（每组 n = 7 个，P =.04）。阻断 α2β1 整联蛋白与 Col7 的相互作用使 TGFB1 mRNA 表达增加 1.8 倍，p-Smad2 水平增加两倍。与对照组（n = 4 个）和 RDEB 肿瘤（TGFβ 标志物，n = 6 个；VEGF，n = 17 个）相比，Col7 KD 异种移植物中观察到 TGFβ 信号和 VEGF 表达增加（n = 4 个）与散发 SCC（TGFβ 标志物，n = 6 个；VEGF，n = 21 个）相比。使用 siRNA 抑制 TGFβ 受体信号会导致内皮细胞管形成减少（每组 n = 9 个，siC 组平均管数/孔为 63.6，SD = 17.1；siTβRII 组平均管数/孔为 29.7，SD = 6.1，P =.02）。