Immunohistochemical Analysis of Retinoblastoma and β-Catenin as an Assistant Tool in the Differential Diagnosis between Crohn's Disease and Ulcerative Colitis
In about 10–15% of patients with inflammatory bowel diseases (IBD) there is no clear definitive differential diagnosis between Crohn's disease (CD) and ulcerative colitis (UC) and the disease is classified as indeterminate colitis. Since pharmacological and surgical treatments differ in CD and UC, establishing a correct diagnosis is critical. The aim of this work was to access the expression profile of proteins involved in colonic inflammation and cancer in samples from CD and UC. For that, colon samples from 24 CD, 21 UC and 10 control patients were processed for immunohistochemistry using anti-phosphorylated RB at Ser807/811 and anti-β-catenin. Crypts were blinded, analyzed and counted for phosphorylated RB-positive (phospho-RB) cells or scored for positive β-catenin staining. Western blot was used for confirming immuhistochemical results: RB phosphorylation was significantly greater in colon samples from patients with CD compared with UC (p<0.005). In contrast, the expression of β-catenin was significantly increased in UC compared with CD (p<0.005) samples. Phospho-RB and β-catenin are negatively correlated (CC: ？0.573; p = 0.001). A positive phospho-RB test yielded high levels of sensitivity, specificity, negative and positive predictive values, and accuracy for the diagnosis of CD against UC. This work indicates that RB phosphorylation and β-catenin nuclear translocation are differently expressed in CD and UC, and provide novel insights into the pathogenic mechanisms of IBD. In particular, rates of phospho-RB-positive cells in mucosal samples emerge as a promising tool for the differential diagnosis of patients with IBD.
References
[1]
Kirsner JB (2006) The Historical Basis of the Idiopathic Inflammatory Bowel Diseases. Inflamm Bowel Dis 1: 2–26.
[2]
Lakatos PL (2006) Recent trends in the epidemiology of inflammatory bowel diseases: Up or down? World J Gastroenterol 2: 6102–6108.
[3]
van Assche G, Vermeire S, Rutgeerts P (2010) Mucosal healing and anti TNFs in IBD. Curr Drug Targets 11(2): 227–233.
[4]
Bouma G, Strober W (2003) The immunological and genetics basis of inflammatory bowel disease. Nature Rev 3: 521–533.
[5]
Cohen RD (2003) Inflammatory Bowel Diseases. Humana Press, Totowa.
Nikolaus S, Schreiber S (2007) Diagnostics of inflammatory bowel disease. Gastroenterology 133(5): 1670–1689.
[9]
Telakis E, Tsironi E (2008) Indeterminate colitis – definition, diagnosis, characteristics and management. Ann Gastroenterol 21(3): 173–179.
[10]
Iskandar HN, Ciorba MA (2012) Biomarkers in inflammatory bowel disease: current practices and recent advances. Transl Res 159(4): 313–325.
[11]
Van Assche G, Vermeire S, Rutgeerts P (2005) Medical treatment of inflammatory bowel diseases. Curr Opin Gastroenterol 21(4): 443–447.
[12]
Abraham C, Cho JH (2009) Inflammatory bowel disease. N Engl J Med 361(21): 2066–2078.
[13]
Lawrance IC, Fiocchi C, Chakravarti S (2001) Ulcerative colitis and Crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes. Hum Mol Genet 10(5): 445–456.
[14]
Christophi GP, Rong R, Holtzapple PG, Massa PT, Landas SK (2012) Immune markers and differential signaling networks in ulcerative colitis and Crohn's disease. Inflamm Bowel Dis 29.
[15]
Van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, et al. (2002) The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111: 241–250.
[16]
Van Dekken H, Wink JC, Vissers KJ, Franken PF, Ruud Schouten W, et al. (2007) Wnt pathway-related gene expression during malignant progression in ulcerative colitis. Acta Histochem 109(4): 266–272.
[17]
Sturm A, Leite AZ, Danese S, Krivacic KA, West GA, et al. (2004) Divergent cell cycle kinetics underlie the distinct functional capacity of mucosal T cells in Crohn's disease and ulcerative colitis. Gut 53(11): 1624–1631.
[18]
Chau BN, Borges HL, Chen TT, Chen TT, Masselli A, et al. (2002) Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb. Nat Cell Biol 4(10): 757–765.
[19]
Borges HL, Bird J, Wasson K, Cardiff RD, Varki N, et al. (2005) Tumor promotion by caspase-resistant retinoblastoma protein. Proc Natl Acad Sci U S A 102(43): 15587–15592.
[20]
Huang X, Masselli A, Frisch SM, Hunton IC, Jiang Y, et al. (2007) Blockade of tumor necrosis factor-induced Bid cleavage by caspase-resistant Rb. J Biol Chem 282(40): 29401–29413.
[21]
Pallone F, Blanco Gdel V, Vavassori P, Monteleone I, Fina D, et al. (2003) Genetic and pathogenetic insights into inflammatory bowel disease. Curr Gastroenterol Rep 5: 487–492.
[22]
Harvey RF, Bradshaw JM (1980) A simple index of Crohn's-disease activity. Lancet 1: 514.
[23]
Truelove SC, Witts LJ (1955) Cortisone in ulcerative colitis; final report on a therapeutic trial. Br Med J 4947: 1041–1048.
[24]
Nirmalan NJ, Harnden P, Selby PJ, Banks RE (2009) Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting. J Pathol 217: 497–506.
[25]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
[26]
Dieckgraefe BK, Stenson WF, Korzenik JR, Swanson PE, Harrington CA (2000) Analysis of mucosal gene expression in inflammatory bowel disease by parallel oligonucleotide arrays. Physiol Genomics 4: 1–11.
[27]
Uthoff SM, Eichenberger MR, Lewis RK, Fox MP, Hamilton CJ, et al. (2001) Identification of candidate genes in ulcerative colitis and Crohn's disease using cDNA array technology. Int J Oncol 19: 803–810.
[28]
Costello CM, Mah N, Hasler R, Rosenstiel P, Waetzig GH (2005) Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays identifies novel candidate disease genes. PLoS Med 2: e199.
[29]
Knudson AG Jr (1971) Mutation and cancer: a statistical study of retinoblastoma. Proc Natl Acad Sci U S A 68: 820–923.
[30]
Harbour JW, Dean DC (2000) The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev 14: 2393–2409.
[31]
Sherr CJ, McCormick F (2002) The RB and p53 pathways in cancer. Cancer Cell 2: 103–112.
[32]
Knudsen ES, Knudsen KE (2006) Retinoblastoma tumor suppressor: where cancer meets the cell cycle. Exp Biol Med (Maywood) 231: 1271–1281.
[33]
Gotoh Y, Noda T, Iwakiri R, Fujimoto K, Rhoads CA, et al. (2002) Lipid peroxide-induced redox imbalance differentially mediates CaCo-2 cell proliferation and growth arrest. Cell Prolif 35: 221–235.
[34]
Cicchillitti L, Fasanaro P, Biglioli P, Capogrosso MC, Martelli F (2003) Oxidative stress induces protein phosphatase 2A-dependent dephosphorylation of the pocket proteins pRb, p107, and p130. J Biol Chem 278: 19509–19517.
[35]
Ying L, Marino J, Hussain SP, Khan MA, You S, et al. (2005) Chronic inflammation promotes retinoblastoma protein hyperphosphorylation and E2F1 activation. Cancer Res 65(20): 9132–9136.
[36]
Ying L, Hofseth AB, Browning DD, Nagarkatti M, Nagarkatti PS, et al. (2007) Nitric oxide inactivates the retinoblastoma pathway in chronic inflammation. Cancer Res 67(19): 9286–9293.
[37]
Quinton JF, Sendid B, Reumaux D, Duthilleul P, Cortot A, et al. (1998) Anti-Saccharomyces cerevisiae mannan antibodies combined with antineutrophil cytoplasmic autoantibodies in inflammatory bowel disease: prevalence and diagnostic role. Gut 42: 788–791.
[38]
Joossens S, Reinisch W, Vermeire S, Sendid B, Poulain D, et al. (2002) The value of serologic markers in indeterminate colitis: a prospective follow-up study. Gastroenterology 122: 1242–1247.
[39]
Israeli E, Grotto I, Gilburd B, Balicer RD, Goldin E, et al. (2005) Anti-Saccharomyces cerevisiae and antineutrophil cytoplasmic antibodies as predictors of inflammatory bowel disease. Gut 54: 1232–1236.
[40]
Mokrowiecka A, Gasiorowska A, Malecka-Panas E (2007) pANCA and ASCA in the diagnosis of different subtypes of inflammatory bowel disease. Hepatogastroenterology 54(77): 1443–1448.
[41]
Desplat-Jégo S, Johanet C, Escande A, Goetz J, Fabien N, et al. (2007) Update on Anti-Saccharomyces cerevisiae antibodies, anti-nuclear associated anti-neutrophil antibodies and antibodies to exocrine pancreas detected by indirect immunofluorescence as biomarkers in chronic inflammatory bowel diseases: results of a multicenter study. World J Gastroenterol 13(16): 2312–8.
[42]
Torok HP, Glas J, Gruber R, Brumberger V, Strasser C, et al. (2004) Inflammatory bowel disease-specific autoantibodies in HLA-B27-associated spondyloarthropathies: increased prevalence of ASCA and pANCA. Digestion 70: 49–54.
[43]
Krause I, Monselise Y, Milo G, Weinberger A (2002) Anti-Saccharomyces cerevisiae antibodies–a novel serologic marker for Behcet's disease. Clin Exp Rheumatol 20: S21–S24.
[44]
Condino AA, Hoffenberg EJ, Accurso F, Penvari C, Anthony M, et al. (2005) Frequency of ASCA seropositivity in children with cystic fibrosis. J Pediatr Gastroenterol Nutr 41: 23–26.
[45]
Kinzler KW, Vogelstein B (2002) Colorectal tumors. In: Vogelstein B, Kinzler KW. The Genetic Basis of Human Cancer, ed. McGraw-Hill, New York 583–612.
[46]
Gavert N, Ben-Ze'ev A (2007) beta-Catenin signaling in biological control and cancer. J Cell Biochem 102(4): 820–828.
[47]
Morin PJ, Sparks AB, Korinek V, Barker N, Clevers H, et al. (1997) Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275: 1787–1790.
[48]
Iwamoto M, Ahnen DJ, Franklin WA, Maltzman TH (2000) Expression of β- catenin and full-length APC protein in normal and neoplastic colonic tissues. Carcinogenesis 21: 935–940.
[49]
Han J, Soletti RC, Sadarangani A, Sridevi P, Ramirez ME, et al. (2013) Nuclear expression of β-catenin promotes RB stability and resistance to TNF-induced apoptosis in colon cancer cells. Mol Cancer Res 11(3): 207–218.
[50]
Lee G, Goretsky T, Managlia E, Dirisina R, Singh AP, et al. (2010) Phosphoinositide 3-kinase signaling mediates beta-catenin activation in intestinal epithelial stem and progenitor cells in colitis. Gastroenterology 139(3): 869–881.
[51]
Brown JB, Lee G, Managlia E, Grimm GR, Dirisina R, et al. (2010) Mesalamine inhibits epithelial beta-catenin activation in chronic ulcerative colitis. Gastroenterology 138(2): 595–605.
