本期Nature发表的一篇论文可能会重新激发人们对DCC（在结肠直肠癌中被删除的基因）作为一个肿瘤抑制因子的兴趣。DCC是上个世纪90年代初根据其在18q位点上的位置而作为一个肿瘤抑制基因提出的，在70%以上的结肠直肠癌中被删除。它的一些性能似乎没有典型性，人们的兴趣也转移到了它作为向药性分子netrin-1的轴向引导受体的作用上。现在，一个用在肠道中强制表达netrin-1、阻止由DCC诱导的细胞死亡的小鼠所做的实验，显示在抑制DCC或其相关受体与早期阶段结肠直肠癌增加之间存在一个联系。该研究工作还引入了条件性肿瘤抑制因子这样一个新概念。
Netrin-1 controls colorectal tumorigenesis by regulating apoptosis
The expression of the protein DCC (deleted in colorectal cancer) is lost or markedly reduced in numerous cancers and in the majority of colorectal cancers due to loss of heterozygosity in chromosome 18q, and has therefore been proposed to be a tumour suppressor1. However, the rarity of mutations found in DCC, the lack of cancer predisposition of DCC mutant mice, and the presence of other tumour suppressor genes in 18q have raised doubts about the function of DCC as a tumour suppressor2. Unlike classical tumour suppressors, DCC has been shown to induce apoptosis conditionally: by functioning as a dependence receptor, DCC induces apoptosis unless DCC is engaged by its ligand, netrin-1 (ref. 3). Here we show that inhibition of cell death by enforced expression of netrin-1 in mouse gastrointestinal tract leads to the spontaneous formation of hyperplastic and neoplastic lesions. Moreover, in the adenomatous polyposis coli mutant background associated with adenoma formation, enforced expression of netrin-1 engenders aggressive adenocarcinomatous malignancies. These data demonstrate that netrin-1 can promote intestinal tumour development, probably by regulating cell survival. Thus, a netrin-1 receptor or receptors function as conditional tumour suppressors.
Figure 1 Netrin-1 is expressed in the mouse gastrointestinal tract. a, RT–PCR using specific mouse netrin-1 primers was performed using total RNA extracted from various tissues as indicated. NC, negative control. b, c, In situ hybridization was performed on 6-month-old mouse colons as described in the Supplementary Methods section using either an anti-sense netrin-1 probe (b) or control sense netrin-1 probe (c). Note the specific staining at the base of the colonic crypts. d, e, Immunohistochemistry on 6-month-old mouse small intestine using anti-netrin-1 antibody (d) or no primary antibody (e). f, g, Immunohistochemistry on 6-month-old mouse small intestine using anti-DCC antibody without (f) or with prior incubation with a competitive peptide (g). Original magnifications: b, c, 120; d–g, 100.
Figure 2 Netrin-1 overexpression inhibits intestinal cell apoptosis. a, Tg-netrin-1 mouse tail DNA was analysed by Southern blot after restriction by EcoRI and XhoI using netrin-1 probe. A fragment of 2.3 kilobase pairs is expected if the Fabpl–netrin-1 construct is inserted. Tg-1 to 5, transgenic lines; ntg, non-transgenic line; wt, parental line. b, Total RNA from various tissues from either parental mice or Tg-netrin-1 mice were analysed by northern blot using a chick netrin-1 probe. c, Immunoblot performed after immunoprecipitation of c-Myc-tagged netrin-1 from dissociated villi and crypts of the small intestine from either non-transgenic mice or Tg-netrin-1 mice. d, e, Immunohistochemistry of tagged netrin-1 on colon section from Tg-netrin-1 mice (e) or control mice (d). f, g, h, Tg-netrin-1 mice showed a decrease in intestinal cell death. Representative microscopic images from hematoxylin-eosin-saffron staining of 20-month-old Tg-netrin-1 mouse (f) or wild-type mouse (g) colon section showing apoptotic cell death. Arrows indicate cells with apoptotic morphology. Quantitative analysis of apoptotic cells in either wild-type or Tg-netrin-1 mice performed from the observations of 14 animals from each type (h). Standard deviations are indicated (P < 0.0001). Original magnifications: d, 100; e, 120; f, g, 450.
Figure 3 Netrin-1 overexpression enhances the early stage of tumour development. a, Table showing the number of mice studied and the proportion of hyperplasia and adenoma in the small intestine and colon. 5 lines of Tg-netrin-1 mice were analysed. b, c, d, e, Hematoxylin-eosin-saffron staining of the small intestine and colon on Tg-netrin-1 and wild type animals. Compared with the normal colonic mucosa shown in b, in 43% Tg-netrin-1 animals, diffuse hyperplasia of the colonic mucosa (c) was present; it was characterized by an increase in the height of the mucosal layer and by the formation of pseudo-villous structures. m, mucosa; sm, submucosa; ms, muscularis. In some animals, low-grade adenomas were present, either in the small intestine (d, low-grade polypoid adenoma, arrows) or in the colon (e, low-grade sessile adenoma, arrows). f–i, -catenin immunohistolocalization on normal colonic mucosa (f), Tg-netrin-1 colonic mucosa (g), hyperplasia of Tg-netrin-1 (h) or adenoma of Tg-netrin-1 (i). Original magnifications: b–e, 60; f–i, 350.
Figure 4 Netrin-1 overexpression enhances the adenoma to adenoma-carcinoma transition. a, Table showing the number of APC+/1638N or APC+/1638N/Tg-netrin-1 mice studied and the proportion of low-grade adenoma and high-grade adenoma. Two independent lines of Tg-netrin-1 mice were backcrossed into the APC+/1638N background and analysed for tumour development. b, c, d, Hematoxylin-eosin-saffron staining of: polypoid high-grade adenoma (b, arrows); a high power view of a low-grade adenoma (c); and of focal invasion of the muscularis mucosae (mm) by small carcinomatous glands in the deep part of a mucosal adenocarcinomatous lesion (d). Original magnifications: b, 100; c, 280; d, 300.
