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A loss of function mutation in WISP3 derived from microsatellite unstable gastric carcinoma
November 2003
CORRESPONDENCE
1563
citis could be correlated with any particular genetic modification involved for patients with Crohn’s disease. GIAN LUIGI ADANI UMBERTO BACCARANI ANDREA RISALITI ANNIBALE DONINI University Hospital Udine Department of Surgery and Transplantation Udine, Italy TAKESHI AOKI ITZHAK AVITAL Cedars-Sinai Medical Center Department of Surgery UCLA School of Medicine Los Angeles, California 1. Andersson RE, Olaison G, Tysk C, Ekbom A. Appendectomy is followed by increased risk of Crohn’s disease. Gastroenterology 2003;124:40 – 46. 2. Su C, Lichtenstein GR. Recent development in inflammatory bowel disease. Med Clin North Am 2002;86:1497–1523. 3. Wyatt J, Vogelsang H, Hubl W, Waldhoer T, Lochs H. Intestinal permeability and the prediction of relapse in Crohn’s disease. Lancet 1993;341:1437–1439. 4. Yacyshyn BR, Meddings JB. CD45RO expression on circulating CD 19⫹ B cells in Crohn’s disease correlates with intestinal permeability. Gastroenterology 1996;110:132–137. 5. Toyoda H, Wang SJ, Yang HJ, Landers C, Tyan D, McElree CK, Pressman SR, Shanahan F, Targan SR, Rotter JI. Distinct associations of HLA class II genes with inflammatory bowel disease. Gastroenterology 1993;104:741–748. 6. Danze PM, Colombel JF, Jacquot S, Loste MN, Heresbach D, Ategbo S, Khamassi S, Perichon B, Semana G, Charron D, Cezard JP. Association of HLA Class II genes with susceptibility to Crohn’s disease. Gut 1996;39:62– 69. 7. Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998;115:182–205. doi:10.1053/S0016-5085(03)01466-5
A Loss of Function Mutation in WISP3 Derived From Microsatellite Unstable Gastric Carcinoma Dear Sir: Gastric carcinoma is the second most common malignancy worldwide, and poorly differentiated adenocarcinoma of the stomach with a scirrhous phenotype has the poorest prognosis as invasion is rapid. While the molecular mechanisms of such aggressiveness have not been clarified, microsatellite instability (MSI) is known to closely correlate to the poor differentiation of gastric carcinoma.1 Thorstensen et al. and we reported in GASTROENTEROLOGY,2,3 that WISP3 gene was mutated in 11%–20% of microsatellite unstable gastric carcinomas. TheWISP3 gene encodes 4 distinct domains (Figure 1A), and contains an (A)9 repeat within the coding region between the second and the third domain. One frameshift mutation (A)8 in this repeat leads to a premature stop codon at 69-bp downstream, resulting in a predicted truncation of both the third and the fourth domain. To evaluate the effects of the MSI mutation in WISP3, stable transfection of the cDNA was performed on human gastric carcinoma cell line Kato-III that expresses no WISP3 mRNA. Western blot analysis of cell lysates of the transfectants with WISP3(A)9 cDNA
Figure 1. (A) Schematic structures of WISP3(A)9 and WISP3(A)8. WISP3 protein is composed of the signal sequences (SS) (a black box), domain I homologue to IGF binding proteins (IGF-BP), domain II homologue to Von Willebrand factor type C repeats (VWC), domain III homologue to the thrombospondin type I domain (THBS), and domain IV of the cysteine knot domain (CK) (large boxes). Conserved cysteine residues are indicated by thin bars. The WISP3(A)8, derived from microsatellite unstable gastric carcinomas, lacks both the THBS and CK domain. The WISP3(A)8 sequences were deposited with GenBank, accession number AB075040. (B) Ectopic expression of WISP3 in Kato-III human gastric carcinoma cells. Stable transfectants of WISP3(A)9 cDNA or WISP3(A)8 cDNA were prepared with a bicistonic expression vector pIRESneo (Clontech, Palo Alto, CA), that contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus. The expression cassette contains a single promoter, which, in combination with the IRES, permits the translation of both the gene of interest and the neomycin-resistant gene from the same mRNA. Specific expression of WISP3 was detected in cell lysates of the transfectants using a polyclonal antibody against the WISP3 IGF-BP domain (sc-8871; Santa Cruz Biochemistry, Santa Cruz, CA). The mock-vector transfectants as a negative control. The molecular weights shown in the left lane. (C) Matrigel invasion assays on the transfectants or the parental gastric carcinoma Kato-III cells. The number of cells invading into the Matrigel matrix were determined for each clone, following 24-hour stimulation of chemotaxis by serum. To avoid clonal deviation, 3 independent clones of WISP3(A)9 or WISP3(A)8 transfectants were used, and the results were derived from 3 individual experiments. Error bars indicate the standard deviation from the mean.
and WISP3(A)8 cDNA identified specific proteins of approximately 40 kDa and 25 kDa, respectively (Figure 1B), using anti-N-terminus WISP3 antibody. Since WISP family proteins have been reported to regulate cellular invasiveness by us and others,4 – 6 invasion analysis of the transfected gastric carcinoma cells was assessed using Boyden Chamber methods. Compared with the parental or mock-transfected Kato-III cells, invasion potentials of WISP3(A)9-transfectants were remarkably reduced, as shown in Figure 1C. On the other hand, no inhibitory effect was not recognized in the transfectants with WISP3(A)8 missing both the third and fourth domain. The third domain is analogous to a thrombospondin type I (THBS) that is involved in the binding to sulfated glycosaminoglycans on the cell surface, and the fourth domain of a cysteine knot region (CK) may participate in its dimerization and the binding to the cell surface
1564
CORRESPONDENCE
receptor and/or heparan sulfate. The truncation of both the THBS and CK domain in the WISP3 protein cause a loss of function for transmitted signals in the microsatellite unstable gastric carcinomas. In addition, frequent mutations in the WISP3 gene were reported to be associated with autosomal recessive skeletal disorder progressive pseudorheumatoid dysplasia, characterized by unrestrained invasive phenotype of synovial fibroblasts into cartilage. A better understanding of the WISP3 function will likely increase awareness of its importance in the pathological cell invasion. Identification of such MSI targets, as well as the functional alteration should be warranted. SHINJI TANAKA KEISHI SUGIMACHI SHIN-ICHIRO MAEHARA MITSUO SHIMADA YOSHIHIKO MAEHARA Department of Surgery and Science Graduate School of Medical Sciences Kyushu University Fukuoka, Japan 1. Han HJ, Yanagisawa A, Kato Y, Park JG, Nakamura Y. Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer. Cancer Res 1993;53:5087–5089. 2. Thorstensen L, Holm R, Lothe RA, Trope C, Carvalho B, Sobrinho-
GASTROENTEROLOGY Vol. 125, No. 5
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Simoes M, Seruca R. WNT-inducible signaling pathway protein 3, WISP-3, is mutated in microsatellite unstable gastrointestinal carcinomas but not in endometrial carcinomas. Gastroenterology 2003;124:270 –271. Tanaka S, Sugimachi K, Shimada M, Maehara Y, Sugimachi K. Variant WISPs as targets for gastrointestinal carcinomas. Gastroenterology 2002;123:392–393. Tanaka S, Sugimachi K, Saeki H, Kinoshita J, Ohga T, Shimada M, Maehara Y, Sugimachi K. A novel variant of WISP1 lacking a Von Willebrand type C module overexpressed in scirrhous gastric carcinoma. Oncogene 2001;20:5525–5532. Kleer CG, Zhang Y, Pan Q, van Golen KL, Wu ZF, Livant D, Merajver SD. WISP3 is a novel tumor suppressor gene of inflammatory breast cancer. Oncogene 2002;21:3172–3180. Soon LL, Yie TA, Shvarts A, Levine AJ, Su F, Tchou-Wong KM. Overexpression of WISP-1 downregulated motility and invasion of lung cancer cells through inhibition of Rac activation. J Biol Chem 2003 (in press). Hurvitz JR, Suwairi WM, Van Hul W, El-Shanti H, Superti-Furga A, Roudier J, Holderbaum D, Pauli RM, Herd JK, Van Hul EV, RezaiDelui H, Legius E, Le Merrer M, Al-Alami J, Bahabri SA, Warman ML. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nat Genet 1999; 23:94 –98. doi:10.1053/S0016-5085(03)01467-7
CORRESPONDENCE
1563
citis could be correlated with any particular genetic modification involved for patients with Crohn’s disease. GIAN LUIGI ADANI UMBERTO BACCARANI ANDREA RISALITI ANNIBALE DONINI University Hospital Udine Department of Surgery and Transplantation Udine, Italy TAKESHI AOKI ITZHAK AVITAL Cedars-Sinai Medical Center Department of Surgery UCLA School of Medicine Los Angeles, California 1. Andersson RE, Olaison G, Tysk C, Ekbom A. Appendectomy is followed by increased risk of Crohn’s disease. Gastroenterology 2003;124:40 – 46. 2. Su C, Lichtenstein GR. Recent development in inflammatory bowel disease. Med Clin North Am 2002;86:1497–1523. 3. Wyatt J, Vogelsang H, Hubl W, Waldhoer T, Lochs H. Intestinal permeability and the prediction of relapse in Crohn’s disease. Lancet 1993;341:1437–1439. 4. Yacyshyn BR, Meddings JB. CD45RO expression on circulating CD 19⫹ B cells in Crohn’s disease correlates with intestinal permeability. Gastroenterology 1996;110:132–137. 5. Toyoda H, Wang SJ, Yang HJ, Landers C, Tyan D, McElree CK, Pressman SR, Shanahan F, Targan SR, Rotter JI. Distinct associations of HLA class II genes with inflammatory bowel disease. Gastroenterology 1993;104:741–748. 6. Danze PM, Colombel JF, Jacquot S, Loste MN, Heresbach D, Ategbo S, Khamassi S, Perichon B, Semana G, Charron D, Cezard JP. Association of HLA Class II genes with susceptibility to Crohn’s disease. Gut 1996;39:62– 69. 7. Fiocchi C. Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 1998;115:182–205. doi:10.1053/S0016-5085(03)01466-5
A Loss of Function Mutation in WISP3 Derived From Microsatellite Unstable Gastric Carcinoma Dear Sir: Gastric carcinoma is the second most common malignancy worldwide, and poorly differentiated adenocarcinoma of the stomach with a scirrhous phenotype has the poorest prognosis as invasion is rapid. While the molecular mechanisms of such aggressiveness have not been clarified, microsatellite instability (MSI) is known to closely correlate to the poor differentiation of gastric carcinoma.1 Thorstensen et al. and we reported in GASTROENTEROLOGY,2,3 that WISP3 gene was mutated in 11%–20% of microsatellite unstable gastric carcinomas. TheWISP3 gene encodes 4 distinct domains (Figure 1A), and contains an (A)9 repeat within the coding region between the second and the third domain. One frameshift mutation (A)8 in this repeat leads to a premature stop codon at 69-bp downstream, resulting in a predicted truncation of both the third and the fourth domain. To evaluate the effects of the MSI mutation in WISP3, stable transfection of the cDNA was performed on human gastric carcinoma cell line Kato-III that expresses no WISP3 mRNA. Western blot analysis of cell lysates of the transfectants with WISP3(A)9 cDNA
Figure 1. (A) Schematic structures of WISP3(A)9 and WISP3(A)8. WISP3 protein is composed of the signal sequences (SS) (a black box), domain I homologue to IGF binding proteins (IGF-BP), domain II homologue to Von Willebrand factor type C repeats (VWC), domain III homologue to the thrombospondin type I domain (THBS), and domain IV of the cysteine knot domain (CK) (large boxes). Conserved cysteine residues are indicated by thin bars. The WISP3(A)8, derived from microsatellite unstable gastric carcinomas, lacks both the THBS and CK domain. The WISP3(A)8 sequences were deposited with GenBank, accession number AB075040. (B) Ectopic expression of WISP3 in Kato-III human gastric carcinoma cells. Stable transfectants of WISP3(A)9 cDNA or WISP3(A)8 cDNA were prepared with a bicistonic expression vector pIRESneo (Clontech, Palo Alto, CA), that contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus. The expression cassette contains a single promoter, which, in combination with the IRES, permits the translation of both the gene of interest and the neomycin-resistant gene from the same mRNA. Specific expression of WISP3 was detected in cell lysates of the transfectants using a polyclonal antibody against the WISP3 IGF-BP domain (sc-8871; Santa Cruz Biochemistry, Santa Cruz, CA). The mock-vector transfectants as a negative control. The molecular weights shown in the left lane. (C) Matrigel invasion assays on the transfectants or the parental gastric carcinoma Kato-III cells. The number of cells invading into the Matrigel matrix were determined for each clone, following 24-hour stimulation of chemotaxis by serum. To avoid clonal deviation, 3 independent clones of WISP3(A)9 or WISP3(A)8 transfectants were used, and the results were derived from 3 individual experiments. Error bars indicate the standard deviation from the mean.
and WISP3(A)8 cDNA identified specific proteins of approximately 40 kDa and 25 kDa, respectively (Figure 1B), using anti-N-terminus WISP3 antibody. Since WISP family proteins have been reported to regulate cellular invasiveness by us and others,4 – 6 invasion analysis of the transfected gastric carcinoma cells was assessed using Boyden Chamber methods. Compared with the parental or mock-transfected Kato-III cells, invasion potentials of WISP3(A)9-transfectants were remarkably reduced, as shown in Figure 1C. On the other hand, no inhibitory effect was not recognized in the transfectants with WISP3(A)8 missing both the third and fourth domain. The third domain is analogous to a thrombospondin type I (THBS) that is involved in the binding to sulfated glycosaminoglycans on the cell surface, and the fourth domain of a cysteine knot region (CK) may participate in its dimerization and the binding to the cell surface
1564
CORRESPONDENCE
receptor and/or heparan sulfate. The truncation of both the THBS and CK domain in the WISP3 protein cause a loss of function for transmitted signals in the microsatellite unstable gastric carcinomas. In addition, frequent mutations in the WISP3 gene were reported to be associated with autosomal recessive skeletal disorder progressive pseudorheumatoid dysplasia, characterized by unrestrained invasive phenotype of synovial fibroblasts into cartilage. A better understanding of the WISP3 function will likely increase awareness of its importance in the pathological cell invasion. Identification of such MSI targets, as well as the functional alteration should be warranted. SHINJI TANAKA KEISHI SUGIMACHI SHIN-ICHIRO MAEHARA MITSUO SHIMADA YOSHIHIKO MAEHARA Department of Surgery and Science Graduate School of Medical Sciences Kyushu University Fukuoka, Japan 1. Han HJ, Yanagisawa A, Kato Y, Park JG, Nakamura Y. Genetic instability in pancreatic cancer and poorly differentiated type of gastric cancer. Cancer Res 1993;53:5087–5089. 2. Thorstensen L, Holm R, Lothe RA, Trope C, Carvalho B, Sobrinho-
GASTROENTEROLOGY Vol. 125, No. 5
3.
4.
5.
6.
7.
Simoes M, Seruca R. WNT-inducible signaling pathway protein 3, WISP-3, is mutated in microsatellite unstable gastrointestinal carcinomas but not in endometrial carcinomas. Gastroenterology 2003;124:270 –271. Tanaka S, Sugimachi K, Shimada M, Maehara Y, Sugimachi K. Variant WISPs as targets for gastrointestinal carcinomas. Gastroenterology 2002;123:392–393. Tanaka S, Sugimachi K, Saeki H, Kinoshita J, Ohga T, Shimada M, Maehara Y, Sugimachi K. A novel variant of WISP1 lacking a Von Willebrand type C module overexpressed in scirrhous gastric carcinoma. Oncogene 2001;20:5525–5532. Kleer CG, Zhang Y, Pan Q, van Golen KL, Wu ZF, Livant D, Merajver SD. WISP3 is a novel tumor suppressor gene of inflammatory breast cancer. Oncogene 2002;21:3172–3180. Soon LL, Yie TA, Shvarts A, Levine AJ, Su F, Tchou-Wong KM. Overexpression of WISP-1 downregulated motility and invasion of lung cancer cells through inhibition of Rac activation. J Biol Chem 2003 (in press). Hurvitz JR, Suwairi WM, Van Hul W, El-Shanti H, Superti-Furga A, Roudier J, Holderbaum D, Pauli RM, Herd JK, Van Hul EV, RezaiDelui H, Legius E, Le Merrer M, Al-Alami J, Bahabri SA, Warman ML. Mutations in the CCN gene family member WISP3 cause progressive pseudorheumatoid dysplasia. Nat Genet 1999; 23:94 –98. doi:10.1053/S0016-5085(03)01467-7