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Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and caveolin-1.

Lin M, DiVito MM, Merajver SD, Boyanapalli M, van Golen KL - Mol. Cancer (2005)

Bottom Line: Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression.Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion.In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology-Oncology, Department of Internal Medicine, The University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109, USA. liming@yahoo.com

ABSTRACT

Background: In the current study we investigated the role of caveolin-1 (cav-1) in pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-1 is the major structural protein in caveolae; small Omega-shaped invaginations within the plasma membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding protein to organize multiple molecular complexes regulating a variety of cellular events. Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and metastasis; however, the molecular mechanisms have not been described. The small monomeric GTPases are among several molecules which associate with cav-1. Classically, the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion. RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif.

Results: Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines derived from primary tumors and low expression in those derived from metastases. Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation. Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression.

Conclusion: Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.

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Caveolin-1 expression in human pancreatic adenocarcinoma cell lines. Panel A is a comparison of caveolin-1 protein expression in human ductal pancreatic epithelial (HPDE) and 10 pancreatic cancer cell lines. Aliquots of 50 μg total protein were probed by immunoblotting with a monoclonal antibody specific for caveolin-1. Both the α and β isoforms of caveolin-1 were detected. Immunoblot analysis of β-actin served as a loading control. B. Comparison of BxPC-3 and HPAF-II migration in a colloidal gold random migration assay. PC cells were seeded onto coverslips coated with colloidal gold, stimulated by the addition of 10% FBS and allowed to migrate for 16 h at 37°C. Phagokinetic tracks were photographed and areas measured. Invasion was measured by a Matrigel invasion assay using 10% serum as a chemoattractant. C. HPAF-II cells were transiently transfected with GFP-cav-1 or GFP only and their migratory capabilities measured in a blue-fluorescent bead migration assay in a manner identical to what was described for the colloidal gold assay. A representative Western blot demonstrating that ectopic cav-1 protein levels in the HPAF-II cells were similar to the BxPC-3 cell line. Phagokinetic tracks from GFP-expressing cells were imaged and areas measured. GFP-cav-1 cells were significantly less migratory (*p = 0.0032) and less invasive (*p = 0.002) than the controls.
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Figure 1: Caveolin-1 expression in human pancreatic adenocarcinoma cell lines. Panel A is a comparison of caveolin-1 protein expression in human ductal pancreatic epithelial (HPDE) and 10 pancreatic cancer cell lines. Aliquots of 50 μg total protein were probed by immunoblotting with a monoclonal antibody specific for caveolin-1. Both the α and β isoforms of caveolin-1 were detected. Immunoblot analysis of β-actin served as a loading control. B. Comparison of BxPC-3 and HPAF-II migration in a colloidal gold random migration assay. PC cells were seeded onto coverslips coated with colloidal gold, stimulated by the addition of 10% FBS and allowed to migrate for 16 h at 37°C. Phagokinetic tracks were photographed and areas measured. Invasion was measured by a Matrigel invasion assay using 10% serum as a chemoattractant. C. HPAF-II cells were transiently transfected with GFP-cav-1 or GFP only and their migratory capabilities measured in a blue-fluorescent bead migration assay in a manner identical to what was described for the colloidal gold assay. A representative Western blot demonstrating that ectopic cav-1 protein levels in the HPAF-II cells were similar to the BxPC-3 cell line. Phagokinetic tracks from GFP-expressing cells were imaged and areas measured. GFP-cav-1 cells were significantly less migratory (*p = 0.0032) and less invasive (*p = 0.002) than the controls.

Mentions: Ten pancreatic adenocarcinoma (PC) cell lines were obtained from ATCC and compared with immortalized human pancreatic ductal epithelial (HPDE) cells for caveolin-1 (cav-1) expression. Figure 1A are the results of immunoblot analysis for total cellular cav-1 protein expression. Cav-1 expression was variable among the cell lines, with high cav-1 levels detected in the HPDE and PC cells derived from primary tumors (BxPC-3 and MiaPaCa-2). Comparatively, cav-1 levels in immortalized HPDE cells were lower than the BxPC-3 and MiaPaCa-2 cell lines. Expression was low or absent in cell lines derived from metastatic tumors or ascites fluid (HPAF-II, Capan-1, SW1990, SU86.86, Capan-2 and AsPc-1). The exception to this is the CFPAC-1 cell line, which was derived from a liver metastasis in a patient that had chronic cystic fibrosis. Interestingly, the Panc-1 cell line, which was derived from an invasive intraductal extension of a primary tumor, had an intermediate expression level. These results suggest that high cav-1 expression may be associated with primary tumors, while loss of cav-1 may be associated with metastases.


Regulation of pancreatic cancer cell migration and invasion by RhoC GTPase and caveolin-1.

Lin M, DiVito MM, Merajver SD, Boyanapalli M, van Golen KL - Mol. Cancer (2005)

Caveolin-1 expression in human pancreatic adenocarcinoma cell lines. Panel A is a comparison of caveolin-1 protein expression in human ductal pancreatic epithelial (HPDE) and 10 pancreatic cancer cell lines. Aliquots of 50 μg total protein were probed by immunoblotting with a monoclonal antibody specific for caveolin-1. Both the α and β isoforms of caveolin-1 were detected. Immunoblot analysis of β-actin served as a loading control. B. Comparison of BxPC-3 and HPAF-II migration in a colloidal gold random migration assay. PC cells were seeded onto coverslips coated with colloidal gold, stimulated by the addition of 10% FBS and allowed to migrate for 16 h at 37°C. Phagokinetic tracks were photographed and areas measured. Invasion was measured by a Matrigel invasion assay using 10% serum as a chemoattractant. C. HPAF-II cells were transiently transfected with GFP-cav-1 or GFP only and their migratory capabilities measured in a blue-fluorescent bead migration assay in a manner identical to what was described for the colloidal gold assay. A representative Western blot demonstrating that ectopic cav-1 protein levels in the HPAF-II cells were similar to the BxPC-3 cell line. Phagokinetic tracks from GFP-expressing cells were imaged and areas measured. GFP-cav-1 cells were significantly less migratory (*p = 0.0032) and less invasive (*p = 0.002) than the controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Caveolin-1 expression in human pancreatic adenocarcinoma cell lines. Panel A is a comparison of caveolin-1 protein expression in human ductal pancreatic epithelial (HPDE) and 10 pancreatic cancer cell lines. Aliquots of 50 μg total protein were probed by immunoblotting with a monoclonal antibody specific for caveolin-1. Both the α and β isoforms of caveolin-1 were detected. Immunoblot analysis of β-actin served as a loading control. B. Comparison of BxPC-3 and HPAF-II migration in a colloidal gold random migration assay. PC cells were seeded onto coverslips coated with colloidal gold, stimulated by the addition of 10% FBS and allowed to migrate for 16 h at 37°C. Phagokinetic tracks were photographed and areas measured. Invasion was measured by a Matrigel invasion assay using 10% serum as a chemoattractant. C. HPAF-II cells were transiently transfected with GFP-cav-1 or GFP only and their migratory capabilities measured in a blue-fluorescent bead migration assay in a manner identical to what was described for the colloidal gold assay. A representative Western blot demonstrating that ectopic cav-1 protein levels in the HPAF-II cells were similar to the BxPC-3 cell line. Phagokinetic tracks from GFP-expressing cells were imaged and areas measured. GFP-cav-1 cells were significantly less migratory (*p = 0.0032) and less invasive (*p = 0.002) than the controls.
Mentions: Ten pancreatic adenocarcinoma (PC) cell lines were obtained from ATCC and compared with immortalized human pancreatic ductal epithelial (HPDE) cells for caveolin-1 (cav-1) expression. Figure 1A are the results of immunoblot analysis for total cellular cav-1 protein expression. Cav-1 expression was variable among the cell lines, with high cav-1 levels detected in the HPDE and PC cells derived from primary tumors (BxPC-3 and MiaPaCa-2). Comparatively, cav-1 levels in immortalized HPDE cells were lower than the BxPC-3 and MiaPaCa-2 cell lines. Expression was low or absent in cell lines derived from metastatic tumors or ascites fluid (HPAF-II, Capan-1, SW1990, SU86.86, Capan-2 and AsPc-1). The exception to this is the CFPAC-1 cell line, which was derived from a liver metastasis in a patient that had chronic cystic fibrosis. Interestingly, the Panc-1 cell line, which was derived from an invasive intraductal extension of a primary tumor, had an intermediate expression level. These results suggest that high cav-1 expression may be associated with primary tumors, while loss of cav-1 may be associated with metastases.

Bottom Line: Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression.Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion.In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Hematology-Oncology, Department of Internal Medicine, The University of Michigan Comprehensive Cancer Center, Ann Arbor, Michigan 48109, USA. liming@yahoo.com

ABSTRACT

Background: In the current study we investigated the role of caveolin-1 (cav-1) in pancreatic adenocarcinoma (PC) cell migration and invasion; initial steps in metastasis. Cav-1 is the major structural protein in caveolae; small Omega-shaped invaginations within the plasma membrane. Caveolae are involved in signal transduction, wherein cav-1 acts as a scaffolding protein to organize multiple molecular complexes regulating a variety of cellular events. Recent evidence suggests a role for cav-1 in promoting cancer cell migration, invasion and metastasis; however, the molecular mechanisms have not been described. The small monomeric GTPases are among several molecules which associate with cav-1. Classically, the Rho GTPases control actin cytoskeletal reorganization during cell migration and invasion. RhoC GTPase is overexpressed in aggressive cancers that metastasize and is the predominant GTPase in PC. Like several GTPases, RhoC contains a putative cav-1 binding motif.

Results: Analysis of 10 PC cell lines revealed high levels of cav-1 expression in lines derived from primary tumors and low expression in those derived from metastases. Comparison of the BxPC-3 (derived from a primary tumor) and HPAF-II (derived from a metastasis) demonstrates a reciprocal relationship between cav-1 expression and p42/p44 Erk activation with PC cell migration, invasion, RhoC GTPase and p38 MAPK activation. Furthermore, inhibition of RhoC or p38 activity in HPAF-II cells leads to partial restoration of cav-1 expression.

Conclusion: Cav-1 expression inhibits RhoC GTPase activation and subsequent activation of the p38 MAPK pathway in primary PC cells thus restricting migration and invasion. In contrast, loss of cav-1 expression leads to RhoC-mediated migration and invasion in metastatic PC cells.

Show MeSH
Related in: MedlinePlus