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. 2000 Nov;157(5):1563-73.
doi: 10.1016/S0002-9440(10)64793-1.

Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer

V J Wielenga  1 , R van der Voort, T E Taher, L Smit, E A Beuling, C van Krimpen, M Spaargaren, S T Pals
Affiliations

Expression of c-Met and heparan-sulfate proteoglycan forms of CD44 in colorectal cancer

V J Wielenga et al. Am J Pathol. 2000 Nov.

Abstract

In colorectal cancer patients, prognosis is not determined by the primary tumor but by the formation of distant metastases. Molecules that have been implicated in the metastatic process are the proto-oncogene product c-Met and CD44 glycoproteins. Recently, we obtained evidence for functional collaboration between these two molecules: CD44 isoforms decorated with heparan sulfate chains (CD44-HS) can bind the c-Met ligand, the growth and motility factor hepatocyte growth factor/scatter factor (HGF/SF). This interaction strongly promotes signaling through the receptor tyrosine kinase c-Met. In the present study, we explored the expression of CD44-HS, c-Met, and HGF/SF in the normal human colon mucosa, and in colorectal adenomas and carcinomas, as well as their interaction in colorectal cancer cell lines. Compared to the normal colon, CD44v3 isoforms, which contain a site for HS attachment, and c-Met, were both overexpressed on the neoplastic epithelium of colorectal adenomas and on most carcinomas. Likewise, HGF/SF was expressed at increased levels in tumor tissue. On all tested colorectal cancer cell lines CD44v3 and c-Met were co-expressed. As was shown by immunoprecipitation and Western blotting, CD44 on these cells lines was decorated with HS. Interaction with HS moieties on colorectal carcinoma (HT29) cells promoted HGF/SF-induced activation of c-Met and of the Ras-MAP kinase pathway. Interestingly, survival analysis showed that CD44-HS expression predicts unfavorable prognosis in patients with invasive colorectal carcinomas. Taken together, our findings indicate that CD44-HS, c-Met, and HGF/SF are simultaneously overexpressed in colorectal cancer and that HS moieties promote c-Met signaling in colon carcinoma cells. These observations suggest that collaboration between CD44-HS and the c-Met signaling pathway may play an important role in colorectal tumorigenesis.

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Figures

Figure 1.
Figure 1.
Expression of CD44v3 and c-Met by normal colon mucosa and colorectal carcinomas. Normal colon mucosa (a and c) and colorectal carcinoma (b and d) serial frozen tissue sections were stained for CD44v3 (a and b) or for c-Met (c and d) by immunohistochemistry. a: Normal colon mucosa showing weak focal expression of CD44v3 in the lower part of the crypts. b: Invasive colorectal carcinoma with strong CD44v3 expression. c: Normal colon mucosa showing weak expression of c-Met. d: Invasive colorectal carcinoma with strong c-Met expression. Tissues were counterstained with hematoxylin. Scale bars, 57 μm (a and c); 69 μm (b and d).
Figure 2.
Figure 2.
Expression of CD44v3 and c-Met by colon carcinoma cell lines. A: FACS analysis of the expression of CD44v3 on the colon carcinoma cell lines colo 320, HT29, SW480, SW620, colo 201, colo 205, and colo 320. Wild-type and CD44v3–10 transfected Namalwa cells are shown as negative or positive controls, respectively. Expression was analyzed with mouse anti-CD44v3 (filled histogram) or an isotype-matched control antibody (empty histogram), followed by RPE-conjugated goat anti-mouse. B: FACS analysis of the c-Met expression on the colon carcinoma cell lines shown in A. Wild-type or c-Met-transfected Namalwa cells are shown as negative and positive controls, respectively. Expression was analyzed with mouse anti-c-Met (filled histogram) or an isotype-matched control antibody (empty histogram), followed by RPE-conjugated goat anti-mouse.
Figure 3.
Figure 3.
CD44v3 isoforms on colon carcinoma cell lines are decorated with HS. CD44v3 was immunoprecipitated from the colon carcinoma cell lines SW480 and HT-29, and, as a positive control, from Namalwa cells transfected with CD44v3–10, with mouse anti-CD44v3. Before immunoprecipitation, the cells were treated with either PBS (−), 30 mU/ml heparitinase (HT), or 30 mU/ml chondroitinase ABC (CH) at 37°C for 3.5 hours. The Western blot of the precipitates was stained with the anti-pan CD44 mAb Hermes-3, stripped, and restained with the mAb 3G10 that detects ΔHS stubs after treatment of HS with heparitinase. CD44v3 isoforms decorated with HS are indicated with arrows.
Figure 4.
Figure 4.
HGF/SF mRNA and protein expression in normal colon mucosa and colorectal carcinomas. A: RT-PCR was performed on total RNA isolated from five pairs of normal colon (N) and primary colorectal carcinoma (T), on water (none), and on a plasmid containing full-length human HGF/SF cDNA (pHGF/SF). Primers used were HGF/SF-specific or, as a control, glyceraldehydephosphate dehydrogenase (GAPDH)-specific. The tumors analyzed were CD44- and Met-positive. B: HGF/SF protein expression in colorectal cancer was assessed by immunohistochemistry. a and b: Frozen sections from colorectal cancer tissue were stained with anti-human HGF/SF. This identified cells (arrows) in the tumor stroma as HGF/SF-producing cells.
Figure 5.
Figure 5.
The interaction of HGF with HS moieties of HS proteoglycans promotes Met signaling in HT29 cells. A: The effect of heparitinase treatment on HGF-induced Met signaling. HT29 cells were pretreated with 10 mU/ml heparitinase (HT) for 3.5 hours and subsequently stimulated with 100 ng/ml HGF for 10 minutes, as indicated. Met autophosphorylation was analyzed by immunoprecipitation (IP) of Met and immunoblotting (IB) with anti-phosphotyrosine (PY) antibody, and subsequent reprobing of the blot with anti-Met antibody (top). In addition, activation of the MAP kinases ERK1 (p44) and 2 (p42) was analyzed by immunoblotting total cell lysates with anti-phospho-ERK1/2 (P-ERK), and subsequent reprobing of the blot with anti-ERK antibody (bottom). B: Stimulation of Met autophosphorylation by wild-type HGF or a non-HS-binding HGF mutant. HT29 cells were stimulated for 10 minutes with either 100 ng/ml HGF or HP1, a non-HS-binding mutant form of HGF, as indicated, and Met autophosphorylation was analyzed by immunoprecipitation of Met and immunoblotting with anti-phosphotyrosine antibody.
Figure 6.
Figure 6.
Patient survival and expression of CD44v3. Kaplan-Meier curves showing the relation between the expression of CD44v3 on primary tumors and survival in patients with colorectal carcinoma. Dotted line, low expression of CD44v3; dashed line, intermediate expression; solid line, high expression; +, censored cases.
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