CUB-domain-containing protein 1 regulates peritoneal dissemination of gastric scirrhous carcinoma - PubMed (original) (raw)

CUB-domain-containing protein 1 regulates peritoneal dissemination of gastric scirrhous carcinoma

Takamasa Uekita et al. Am J Pathol. 2008 Jun.

Abstract

CUB-domain-containing protein 1 (CDCP1) is a type-I transmembrane protein that is highly expressed in colon, breast, and lung cancers. We recently revealed that CDCP1 is associated with and phosphorylated by Src family kinases and is involved in the regulation of anchorage independence of certain lung cancer cell lines. In this study, we examined whether CDCP1 is involved in the regulation of tumor progression of scirrhous gastric cancer, which is a diffusely infiltrative carcinoma with high invasion potential. Expression and phosphorylation levels of CDCP1 correlated with the invasive potential of scirrhous gastric cancers. Reduction of CDCP1 expression by siRNA suppressed migration, invasion, and anchorage independence without affecting the proliferation of highly invasive scirrhous gastric cancer cells. However, CDCP1 overexpression promoted gastric cancer cell migration with low potential of invasion. Loss of CDCP1 suppressed invasion and dissemination of cancer cells that were orthotopically implanted in the gastric wall of nude mice. Expression and phosphorylation of CDCP1 were also detected in cancer cells of surgically resected tissues of human scirrhous gastric cancer by immunohistochemical analysis. Our results suggest that CDCP1 promotes invasion and peritoneal dissemination of cancer cells through the regulation of cell migration and anchorage independence. Therefore, it is both a potential prognostic and therapeutic target in certain types of gastrointestinal cancers, and suppression of its phosphorylation might be a useful strategy for modulating cancer metastasis.

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Figures

Figure 1

Figure 1

Expression and tyrosine phosphorylation of CDCP1 is higher in invasive gastric cancer cell lines. A: Lysates from cells as indicated were subjected to immunoblotting with anti-CDCP1 and anti-phospho-CDCP1 (Tyr734) in each cell lysate. HSC-59 and HSC-60 of noninvasive cell lines and HSC-44PE, 44As3, HSC-58, 58As9, and 58As1 of invasive cell lines were seeded onto each cell culture plate. B: Cellular levels of CDCP1 were analyzed 72 hours after treatment with siRNAs by Western blotting using α-tubulin as a loading control. Expression of CDCP1 was reduced in cells treated with CDCP1 siRNA (siRNA-1, -2) compared with siRNA control (siRNA-Ctr) and parental cell (parent). C: 44As3 and 58As9 cells treated with CDCP1 siRNA (siRNA-1, -2) or control siRNA (siRNA-Ctr) were detached by EDTA, and replated on the chamber slides coated with either collagen type I (100 μg/ml), fibronectin (50 μg/ml), or Matrigel (85 μg/ml). After incubation for 30 minutes, unattached cells were removed by washing the slides in PBS(−) several times, and the remaining cells were stained with Giemsa’s solution. The number of attached cells on each substrate was counted, and the results from three independent experiments, each in duplicate, are shown as the mean ± SD. The asterisks indicate differences from the control cells. *P < 0.01. D: Cell growth in 44As3 and 58As9 cells were shown by the number of cells. Approximately 1 × 104 cells were seeded onto cell culture plates with medium. The growth medium was changed every 2 days and cell numbers were counted by Coulter particle counter z1 (Beckman).

Figure 2

Figure 2

Tyrosine-phosphorylated CDCP1 regulates the invasiveness of gastric cancer cells via cell migration. A: 44As3 and 58As9 cells treated with either CDCP1 siRNA (siRNA-1, -2), control siRNA (siRNA-Ctr), or untreated parent cells (parent) were plated onto a Transwell membrane in serum-free medium. In the lower chamber, medium containing 10% FBS was added as a chemoattractant. After 14 hours of incubation for migration and 17 hours for invasion, the wells were harvested and cells that migrated to the lower surface of the membrane were counted. The results from three independent experiments, each in duplicate, are shown as the mean values ± SD. The asterisks indicate differences from the cells treated with control siRNA. *P < 0.01. B: CDCP1 or CDCP1 mutant (Y734F) tagged with FLAG were transiently transfected in HSC-59 and HSC-60 cells of noninvasive potential. After 24 hours, cells were treated EDTA and collected. The lysates were subjected to immunoblotting with the indicated antibody or immunoprecipitation of total CDCP1 and Y734F using FLAG M2 antibody and immunoblotting with anti-phosphotyrosine antibody (4G10). C: The transiently transfected cells as indicated were seeded onto a Transwell membrane in serum-free medium. In the lower chamber, medium containing 10% FBS was added. After 12 hours of incubation, the wells were harvested, and cells that migrated to the lower surface of the membrane were counted. The results from three independent experiments, each in duplicate, are shown as the mean values ± SD. The asterisks indicate differences from the cells transfected with mock. *P < 0.01.

Figure 3

Figure 3

CDCP1 confers anchorage independence in highly invasive gastric cancer cells. A: 44As3 and 58As9 cells of highly invasive potential cultured for 24 hours in both adhesion and suspension condition were collected and subjected to immunoblotting with anti-CDCP1, anti-phospho-CDCP1 (Tyr734), or anti-phosphotyrosine (4G10) antibody. A, adhesion; S, suspension. The black arrow indicates CDCP1. B: CDCP1-defective 44As3 clones (miCDCP-1 and miCDCP1-2) were generated by miR RNAi expression vector kit (Invitrogen). miLacZ-1 and miLacZ-2 were control clones. The expression of CDCP1 in each clone cultured for 24 hours in 2-methacryloyloxyethyl phosphorylcholine-coated plates was examined by Western blotting using CDCP1 antibody. The concentration of total protein in each clone was confirmed by the same membrane rehybridized with anti-α-tubulin antibody. C: Each CDCP1-defective clone and control clone was seeded onto soft agar plates (3 × 103 cells). Colonies equal to and larger than 0.5 mm in diameter were counted after 30 days. The results from three independent experiments, each in duplicate, are shown as the mean values ± SD. The asterisks indicate differences from the cells with control miLacZ-1. *P < 0.01.

Figure 4

Figure 4

Disruption of CDCP1 expression suppressed the peritoneal dissemination of 44As3 cells. Each CDCP1-defective clone (miCDCP1) and control clone (miLacZ) was injected intraperitoneally into nude mice (5 × 106 cells/mice). A: Representative appearance of peritoneal dissemination at 21 days after the injection of miLacZ-1 clone (a–c) or miCDCP1-1 clone (d–f) is shown. Carcinomatus peritonitis and abdominal distension because of bloody ascites was observed in miLacZ clones (a and b). c and f: The asterisk indicates dissemination of cancer nodules in the mesentery and arrows indicate the tumor mass, including omentum. Arrowheads indicate the tumor nodules of cancer cells disseminated around the rectouterine region. B: Representative dissected intestinal loops from two mice injected with miCDCP1-1 and miLacZ-1 clones, respectively. Top: Representative appearance of peritoneal dissemination 2 weeks after the injection of miLacZ-1 clone (a–c) or miCDCP1-1 clone (d–f) is shown. c and f show high magnification of the mesentery in the middle panels (yellow box). The red arrow indicates local invasion of tumor nodules in the mesentery. Bottom: Tumor nodules equal to and larger than 1.0 mm diameter were counted after 16 days (n = 6). Error bars indicate the SD. The asterisks indicate differences from the cells with control miLacZ-1. *P < 0.01. C: The effect of CDCP1 on the tumor growth in nude mice was determined as described under Materials and Methods. The data represent the weight (g) of tumors from miCDCP1 clones and miLacZ clones (n = 3). Error bars indicate the SD.

Figure 5

Figure 5

Disruption of CDCP1 suppressed the peritoneal dissemination of orthotopically implanted 44As3 cells. A: Each CDCP1-defective clone (miCDCP1) and control clone (miLacZ) was orthotopically implanted in submucosa of the gastric wall (n = 12) as described in the Materials and Methods. The representative macroscopic views of dissected organs 15 days (miLacZ) and 16 days (miCDCP1) after implantation including mesenterium (a–d) and liver (e–j) are shown. g and h show high magnification of the liver in the top panels (yellow box). Red arrow indicates tumor nodules. B: Stromal fibrosis of each miCDCP1 and miLacZ clone orthotopically implanted in mice submucosa was detected by azan staining. The region with stromal fibrosis was observed as blue stain.

Figure 6

Figure 6

Tyrosine phosphorylation of CDCP1 is up-regulated in the tumor nodules of peritoneal dissemination. A: Histology of the tumor nodules 14 days after peritoneal dissemination of 44As3 cells. Left: Representative macroscopic views of organs 14 days after implantation are shown (dissemination). Black arrowheads indicate the tumor of dissemination in nude mouse. The left panels show H&E staining. Middle: Staining of tumor nodules using anti-CDCP1 antibody and neutralized with CDCP1 peptide. Right: Staining of tumor nodules using anti-phospho-CDCP1 (pY734) antibody and neutralized with phospho-CDCP1 peptide. B: 44As3 cells were cultured onto cell culture plate (plate) or injected intraperitoneally into nude mice (tissue). Each lysate was collected and subjected to immunoblotting with anti-CDCP1, anti-phospho-CDCP1 (Tyr734), anti-pan-Src (Src2), anti-phospho-Src family (pY416), or anti-phosphotyrosine (4G10) antibody. α-Tubulin as a loading control. Black arrows indicate Src family kinases.

Figure 7

Figure 7

Expression and tyrosine phosphorylation of CDCP1 is detected in the invasive site of human scirrhous tissue. Human scirrhous tissue was stained with H&E (a) and either CDCP1 (c and d) or phospho-CDCP1 [p-CDCP1(pY734)] (e and f). Normal mucosa was stained with CDCP1 (b). Black arrowheads indicate the area of invasive tumor and white arrowheads indicate blood vessels (c and e). High magnification of the invasive site of the tumor is shown in d and f, respectively. Note that expression and phosphorylation of CDCP1 was detected in invasive sites of human scirrhous tissue but not detected in normal tissue including blood vessel (c–f). Original magnifications: ×200 (c, e); ×400 (d, f).

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