- Email: [email protected]
Possible role of cell adhesion molecules in tumour growth and metastasis
April 1995
Gastrointestinal Oncology
A443
Gastrointestinal Oncology
• EXPRESSION OF CADHERINS (E & P), CATENINS ((x & 13) AND DESMOSOME IN NORMAL HUMAN COLORECTAL MUCOSA AND IN COLORECTAL TUMOURS. AM Abbasi. R Day, IC Talbot, EM McKeegan*, A Forbes. St. Mark's Hospital, London, ECl, UK, and *ICRF Unit, St Thomas' Hospital, London, SE1, UK. Epithelial cell adhesion is principally regulated by expression of junctional proteins. Their loss or decrease correlates with impaired intercellular adhesion, and may be important in tumor development. We have investigated the relative expression of adherens junction molecules (E- & P-cadherins), desmosomal junction molecule (DG1), and the catenins (c~ & 13), which bind to and regulate the function of cadherins. Normal colonic mucosa was compared to adenomas, and to adenocarcinomas, using immunohistochemistry. E-cadherin, o~- and J3-catenin, and DG1 were strongly expressed at the lateral borders of normal epithelial cells. P-cadherin, in contrast, was either not detected or only weakly expressed in the lower part of normal crypts. E-cadherin and o~-catenin were detected in 9/9 adenomas, !(3catenin in 8•9, DG1 in 7/9, and P,cadherin in 5/9. In each case the staining pattern was similar to that in normal cells, but cytoplasmic staining occurred also in more dysplastic sections. E-cadherin, or- and 13-catenin, and DG1 were expressed in 2 well-differentiated aden0carcinomas, in 1 of which P-cadherin staining was weakly and heterogeneously positive. In 17 moderately differentiated carcinomas E-cadherin, (~-catenin, 13-catenin, DG1, and P-cadherin, were expressed in 15, 12, 11, 8 and 9 cases respectively. The pattern of staining changed from lateral to predominantly cytoplasmic with worsening degrees of differentiation. In 6 poorlydifferentiated carcinomas E-cadherin, c~-catenin, ~-catenin, DG1, and P-cadherin, were heterogeneously expressed in 2, 1, 1, 0, and 1 cases respectively. The expression of junctional molecules and catenins altered both in cellular distribution and intensity with the progression from normal to undifferentiated tumor, which suggests that changes in the pattern of expression may prove critical in tumor development.
• POSSIBLE ROLE OF CELL ADHESION MOLECULES IN TUMOUR GROWTH AND METASTASIS. Atta M AbbasL lan C Talbot*, Alastair Forbes. St Mark's Hospital, and *ICRF Colorectal Cancer Unit, City Road, London, ECIV 2PS, UK. Abnormal cell adhesion has been proposed as a critical event in neoplasia, but most investigation has been of experimental systems. Cell adhesion molecules (CAMs) representative of the 3 principal surfaces of the normal colonocyte have therefore been studied in human colorectal carcinoma. CEA is normally exPressed at the apical (luminal) surface, E-cadherin at the lateral surface (adherens junction), and CD44 at the cell base. Immunofluorescence and western blotting were performed on frozen sections from 3 polyps, 29 carcinomas (25 well to moderately-, 4 poorly-differentiated), biopsies from normal colon, and on 2 colonic cell lines: LIM 1863 (which forms glandular like structures in vitro) and HT 29 (which grows as a monolayer). There was apical staining for CEA in normal colon, in the polyps, in 20 of the differentiated carcinomas, and in LIM 1863 cells. Basal staining was observed in the other 5 differentiated tumors. Uniform cytoplasmic staining was observed in the poorly differentiated tumors, and in HT29 cells. E-cadherin was expressed at lateral cell borders in normal colon, polyps, differentiated tumors and LIM 1863 cells, and was not detected in poorly differentiated tumors or HT29 cells. There was strong cytoplasmic staining for CD44 in normal colon, polyps, tumors, and cell lines, corresponding to areas of cell proliferation as supported by staining for Ki-67 (a well recognised marker for proliferation) in adjacent sections. The present results, combined with prior evidence of the importance of down regulation of E-cadherin in emigration of cells from tumors, of CD44 and CEA in protection of cells in circulation, and of CEA in implantation, promote a hypothesis for colorectal metastasis in which the 3 CAMs have a crucial role.
OEXPRESSlON OF CD44v6 AND 17-1A IN DEVELOPMENT AND PROGNOSIS OF COLORECTAL CANCER. Atta M Abbasi, Richard Day, lan C Talbot*, Alastair Forbes. St Mark's Hospital, and *ICRF Colorectal Cancer Unit, City Road, London, ECl, UK. The v6 splice variant of the cell adhesion molecule CD44 confers metastatic potential in rodent tumors. Homologous sequences exist in human carcinoma cell lines, and expression of CD44v6 has been thought restricted to metastatic tumors. Immunotherapy with antibody to 17-1A (a ubiquitous human epithelial cell adhesion molecule) extends life and prolongs remission in patients with Dukes' C colorectal cancer. Study of the adenoma-carcinoma sequence has been lacking in both contexts. We have examined CD44v6 and 17-1A immunohistochemicatly in normal colon, adenomas, and colorectal Carcinomas, comparing expression to differentiation, Dukes' staging and survival in the latter. There was uniform expression of CD44v6 in the lower two thirds of normal colonic crypts, and of 17-1A at luminal and lateral borders of normal epithe{ial cells. CD44v6 and 17-1A were similarly located in 7/9 and 9/9 adenomas respectively. 17-1A was detected in 18/19 primary colorectal carcinomas. The intensity a n d distribution of staining was unrelated to differentiation, Dukes' staging, survival, and CD44v6 expression. CD44v6 was expressed in 16/19 carcinomas. Arbitrary division to 4 groups on the basis of the percentage of cancer cells expressing CD44v6, placed 3 patients in group I (>60% cells +re), 7 in group I] (30-60%), 6 in group ;[[~[ (<30%), and 3 in group IV (-re). Survival at 5 years was 0/3, 4/7, 5/6, and 3/3 in groups [, H, IH and IV respectively, and appeared independent of differentiation and Dukes' staging. Expression of CD44v6 is not therefore restricted to metastatic tumors but, like 17-1A, is also present in normal colon, adenomas and primary adenocarcinomas. There was no relationship of CD44v6 or 17-1A to Dukes' staging or differentiation, but the persistence of the CD44v6 variant may be an independent adverse prognostic marker once malignancy has occurred.
O SULINDAC SULFIDE (SS) AND SULFONE(FGN-1) BOTH INHIBITTHE GROWTHOF COLONCANCERCELLUNESBY INDUCINGAPOPTOSIS. D. Ahnen. G. Piazza, D. Alberts, N. Paranka, R. Burr, R. Pamukcu. Dept. of Medicine, Univs. of CO, AZ, and UT and the VA MedicalCenter, DenverCO. Nonsteroidal anti-inflammatory drugs (NSAIDs) are potentially useful for chemoprevention of colon cancer. Several NSAIDs are known to inhibit the growth of colon cancer cell lines but the mechanism of the growth inhibition is not known. Sulindac is a prodrug that is converted in-vivo to sulfide (SS) and sulfone (FGN-1) metabolites. SS is an active NSAID and inhibits prostaglandin synthesis but FGN-1 does not. Goal: To determine if SS or FGN-1 inhibit colon cancer cell growth by either inhibition of proliferation or induction of necrosis or apopto'~is. Methods: Subconfluent or confluent cultures of HT-29 cells were grown for 1-7 days (d) in the presence or absence of either SS or FGN-1 (2-480 p.M). Cell growth was measured by the sulforhodamine B (SRB) protein binding assay; viable celt number was measured with the neutral red assay; cell proliferation was measured by bromodeoxyuddine incorporation and by flow cytometric analysis of DNA content (% of cells in S phase); apoptosis was measured by DNA fragmentation, by the acridine orange/ethidium bromide (AO/EB) fluorescence assay and by chromatin condensation in Wright stained preparations; necrosis was measured by the AO/EB assay. Results: In sub-confluent cells both drugs inhibited cell growth in a time dependant (effect by 4 d, maximal at 7 d) and dose dependant (50% Inhibitory Concentration [IC-50]=90 tJ.M for SS and 300 pM for FGN-1) manner. Neither of the drugs inhibited proliferation or induced necrosis but both drugs significantly (p<0,001) induced apoptosis in a dose dependant manner with maximal (3-7 fold) induction for SS at 120 pM and for FGN1 at 480 ~M. In confluent cells both drugs decreased cell number in a dose dependant manner (IC-50 for neutral red assay 45 p.M for SS and 180 ~LM for FGN-1; IC-50 for SRB assay 30 ~M for SS and 100 u.M for FGN1). Neither drug inhibited cell proliferation or induced necrosis but both drugs induced apoptosis in a dose (maximal for SS at 240 ,uM and for FGN1 at 480 p.M) and time (maximal at 4 days for SS and at 7 days for FGN-1) dependant manner. All 3 assays detecteda comparableincrease in SS and FGN-1 induced apoptosis (10 fold for SS and 2-3 fold for FGN-1). Drug DNA Frag. AO/EB (%+SEM) Wright's(%+SEM) None 0.02 9.7+1.9 11.6-+1.3 120p.M SS 0.22* 83.3+5.7* 91.7_+3.5* 2401zM FGN-1 0.06* 27.7+4.4* 21.1-+4.4" (*p<0.05 vs control) Conclusions: SS and FGN-1 inhibit colon cancer cell growth by inducing apoptosis. This may be the mechanism of the in-vivo chemopreventive activity of these drugs. Supported in part by Cell Pathways Inc., Denver CO.
Gastrointestinal Oncology
A443
Gastrointestinal Oncology
• EXPRESSION OF CADHERINS (E & P), CATENINS ((x & 13) AND DESMOSOME IN NORMAL HUMAN COLORECTAL MUCOSA AND IN COLORECTAL TUMOURS. AM Abbasi. R Day, IC Talbot, EM McKeegan*, A Forbes. St. Mark's Hospital, London, ECl, UK, and *ICRF Unit, St Thomas' Hospital, London, SE1, UK. Epithelial cell adhesion is principally regulated by expression of junctional proteins. Their loss or decrease correlates with impaired intercellular adhesion, and may be important in tumor development. We have investigated the relative expression of adherens junction molecules (E- & P-cadherins), desmosomal junction molecule (DG1), and the catenins (c~ & 13), which bind to and regulate the function of cadherins. Normal colonic mucosa was compared to adenomas, and to adenocarcinomas, using immunohistochemistry. E-cadherin, o~- and J3-catenin, and DG1 were strongly expressed at the lateral borders of normal epithelial cells. P-cadherin, in contrast, was either not detected or only weakly expressed in the lower part of normal crypts. E-cadherin and o~-catenin were detected in 9/9 adenomas, !(3catenin in 8•9, DG1 in 7/9, and P,cadherin in 5/9. In each case the staining pattern was similar to that in normal cells, but cytoplasmic staining occurred also in more dysplastic sections. E-cadherin, or- and 13-catenin, and DG1 were expressed in 2 well-differentiated aden0carcinomas, in 1 of which P-cadherin staining was weakly and heterogeneously positive. In 17 moderately differentiated carcinomas E-cadherin, (~-catenin, 13-catenin, DG1, and P-cadherin, were expressed in 15, 12, 11, 8 and 9 cases respectively. The pattern of staining changed from lateral to predominantly cytoplasmic with worsening degrees of differentiation. In 6 poorlydifferentiated carcinomas E-cadherin, c~-catenin, ~-catenin, DG1, and P-cadherin, were heterogeneously expressed in 2, 1, 1, 0, and 1 cases respectively. The expression of junctional molecules and catenins altered both in cellular distribution and intensity with the progression from normal to undifferentiated tumor, which suggests that changes in the pattern of expression may prove critical in tumor development.
• POSSIBLE ROLE OF CELL ADHESION MOLECULES IN TUMOUR GROWTH AND METASTASIS. Atta M AbbasL lan C Talbot*, Alastair Forbes. St Mark's Hospital, and *ICRF Colorectal Cancer Unit, City Road, London, ECIV 2PS, UK. Abnormal cell adhesion has been proposed as a critical event in neoplasia, but most investigation has been of experimental systems. Cell adhesion molecules (CAMs) representative of the 3 principal surfaces of the normal colonocyte have therefore been studied in human colorectal carcinoma. CEA is normally exPressed at the apical (luminal) surface, E-cadherin at the lateral surface (adherens junction), and CD44 at the cell base. Immunofluorescence and western blotting were performed on frozen sections from 3 polyps, 29 carcinomas (25 well to moderately-, 4 poorly-differentiated), biopsies from normal colon, and on 2 colonic cell lines: LIM 1863 (which forms glandular like structures in vitro) and HT 29 (which grows as a monolayer). There was apical staining for CEA in normal colon, in the polyps, in 20 of the differentiated carcinomas, and in LIM 1863 cells. Basal staining was observed in the other 5 differentiated tumors. Uniform cytoplasmic staining was observed in the poorly differentiated tumors, and in HT29 cells. E-cadherin was expressed at lateral cell borders in normal colon, polyps, differentiated tumors and LIM 1863 cells, and was not detected in poorly differentiated tumors or HT29 cells. There was strong cytoplasmic staining for CD44 in normal colon, polyps, tumors, and cell lines, corresponding to areas of cell proliferation as supported by staining for Ki-67 (a well recognised marker for proliferation) in adjacent sections. The present results, combined with prior evidence of the importance of down regulation of E-cadherin in emigration of cells from tumors, of CD44 and CEA in protection of cells in circulation, and of CEA in implantation, promote a hypothesis for colorectal metastasis in which the 3 CAMs have a crucial role.
OEXPRESSlON OF CD44v6 AND 17-1A IN DEVELOPMENT AND PROGNOSIS OF COLORECTAL CANCER. Atta M Abbasi, Richard Day, lan C Talbot*, Alastair Forbes. St Mark's Hospital, and *ICRF Colorectal Cancer Unit, City Road, London, ECl, UK. The v6 splice variant of the cell adhesion molecule CD44 confers metastatic potential in rodent tumors. Homologous sequences exist in human carcinoma cell lines, and expression of CD44v6 has been thought restricted to metastatic tumors. Immunotherapy with antibody to 17-1A (a ubiquitous human epithelial cell adhesion molecule) extends life and prolongs remission in patients with Dukes' C colorectal cancer. Study of the adenoma-carcinoma sequence has been lacking in both contexts. We have examined CD44v6 and 17-1A immunohistochemicatly in normal colon, adenomas, and colorectal Carcinomas, comparing expression to differentiation, Dukes' staging and survival in the latter. There was uniform expression of CD44v6 in the lower two thirds of normal colonic crypts, and of 17-1A at luminal and lateral borders of normal epithe{ial cells. CD44v6 and 17-1A were similarly located in 7/9 and 9/9 adenomas respectively. 17-1A was detected in 18/19 primary colorectal carcinomas. The intensity a n d distribution of staining was unrelated to differentiation, Dukes' staging, survival, and CD44v6 expression. CD44v6 was expressed in 16/19 carcinomas. Arbitrary division to 4 groups on the basis of the percentage of cancer cells expressing CD44v6, placed 3 patients in group I (>60% cells +re), 7 in group I] (30-60%), 6 in group ;[[~[ (<30%), and 3 in group IV (-re). Survival at 5 years was 0/3, 4/7, 5/6, and 3/3 in groups [, H, IH and IV respectively, and appeared independent of differentiation and Dukes' staging. Expression of CD44v6 is not therefore restricted to metastatic tumors but, like 17-1A, is also present in normal colon, adenomas and primary adenocarcinomas. There was no relationship of CD44v6 or 17-1A to Dukes' staging or differentiation, but the persistence of the CD44v6 variant may be an independent adverse prognostic marker once malignancy has occurred.
O SULINDAC SULFIDE (SS) AND SULFONE(FGN-1) BOTH INHIBITTHE GROWTHOF COLONCANCERCELLUNESBY INDUCINGAPOPTOSIS. D. Ahnen. G. Piazza, D. Alberts, N. Paranka, R. Burr, R. Pamukcu. Dept. of Medicine, Univs. of CO, AZ, and UT and the VA MedicalCenter, DenverCO. Nonsteroidal anti-inflammatory drugs (NSAIDs) are potentially useful for chemoprevention of colon cancer. Several NSAIDs are known to inhibit the growth of colon cancer cell lines but the mechanism of the growth inhibition is not known. Sulindac is a prodrug that is converted in-vivo to sulfide (SS) and sulfone (FGN-1) metabolites. SS is an active NSAID and inhibits prostaglandin synthesis but FGN-1 does not. Goal: To determine if SS or FGN-1 inhibit colon cancer cell growth by either inhibition of proliferation or induction of necrosis or apopto'~is. Methods: Subconfluent or confluent cultures of HT-29 cells were grown for 1-7 days (d) in the presence or absence of either SS or FGN-1 (2-480 p.M). Cell growth was measured by the sulforhodamine B (SRB) protein binding assay; viable celt number was measured with the neutral red assay; cell proliferation was measured by bromodeoxyuddine incorporation and by flow cytometric analysis of DNA content (% of cells in S phase); apoptosis was measured by DNA fragmentation, by the acridine orange/ethidium bromide (AO/EB) fluorescence assay and by chromatin condensation in Wright stained preparations; necrosis was measured by the AO/EB assay. Results: In sub-confluent cells both drugs inhibited cell growth in a time dependant (effect by 4 d, maximal at 7 d) and dose dependant (50% Inhibitory Concentration [IC-50]=90 tJ.M for SS and 300 pM for FGN-1) manner. Neither of the drugs inhibited proliferation or induced necrosis but both drugs significantly (p<0,001) induced apoptosis in a dose dependant manner with maximal (3-7 fold) induction for SS at 120 pM and for FGN1 at 480 ~M. In confluent cells both drugs decreased cell number in a dose dependant manner (IC-50 for neutral red assay 45 p.M for SS and 180 ~LM for FGN-1; IC-50 for SRB assay 30 ~M for SS and 100 u.M for FGN1). Neither drug inhibited cell proliferation or induced necrosis but both drugs induced apoptosis in a dose (maximal for SS at 240 ,uM and for FGN1 at 480 p.M) and time (maximal at 4 days for SS and at 7 days for FGN-1) dependant manner. All 3 assays detecteda comparableincrease in SS and FGN-1 induced apoptosis (10 fold for SS and 2-3 fold for FGN-1). Drug DNA Frag. AO/EB (%+SEM) Wright's(%+SEM) None 0.02 9.7+1.9 11.6-+1.3 120p.M SS 0.22* 83.3+5.7* 91.7_+3.5* 2401zM FGN-1 0.06* 27.7+4.4* 21.1-+4.4" (*p<0.05 vs control) Conclusions: SS and FGN-1 inhibit colon cancer cell growth by inducing apoptosis. This may be the mechanism of the in-vivo chemopreventive activity of these drugs. Supported in part by Cell Pathways Inc., Denver CO.