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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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Establishment of stable, dominant negative RhoC HPAF-II transfectants. A. Results of a RhoC activation assay and total RhoC Western blot comparing wildtype, C3 exotransferase treated, vector control and dominant negative RhoC (dnRhoC) transfected HPAF-II cells. B. Comparison of total (dark grey) and active (light grey) RhoA levels utilizing a RhoA-specific antibody. C. Results of the effect of dnRhoC transfection and C3 treatment on HPAF-II cells in a colloidal gold migration assay. Inhibition of RhoC lead to a significant decrease in migration (*p = 0.001) and invasion (*p = 0.023).
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Figure 3: Establishment of stable, dominant negative RhoC HPAF-II transfectants. A. Results of a RhoC activation assay and total RhoC Western blot comparing wildtype, C3 exotransferase treated, vector control and dominant negative RhoC (dnRhoC) transfected HPAF-II cells. B. Comparison of total (dark grey) and active (light grey) RhoA levels utilizing a RhoA-specific antibody. C. Results of the effect of dnRhoC transfection and C3 treatment on HPAF-II cells in a colloidal gold migration assay. Inhibition of RhoC lead to a significant decrease in migration (*p = 0.001) and invasion (*p = 0.023).

Mentions: Next, to directly implicate RhoC in PC cell migration and invasion, we generated stable HPAF-II dominant negative RhoC (dnRhoC) transfectants. For clarity and simplicity the results shown are from a polyclonal population which is representative of three individual clones that were tested. Figure 3A shows a 57% decrease of active RhoC in the HPAF-II/dnRhoC transfectants compared with the untransfected and vector transfected controls. As a positive control the HPAF-II cells were treated with C3 exotransferase. C3 exotransferase is a toxin derived from Clostridium botulinum and is effective at inhibiting RhoA, -B and -C activity with virtually no effect on Rac1 or Cdc42 [34,35]. C3 exotransferase treatment reduced active RhoC levels by 85%. As shown, both C3 exotransferase treatment and expression of dnRhoC did not significantly alter total levels of RhoC protein.


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)

Establishment of stable, dominant negative RhoC HPAF-II transfectants. A. Results of a RhoC activation assay and total RhoC Western blot comparing wildtype, C3 exotransferase treated, vector control and dominant negative RhoC (dnRhoC) transfected HPAF-II cells. B. Comparison of total (dark grey) and active (light grey) RhoA levels utilizing a RhoA-specific antibody. C. Results of the effect of dnRhoC transfection and C3 treatment on HPAF-II cells in a colloidal gold migration assay. Inhibition of RhoC lead to a significant decrease in migration (*p = 0.001) and invasion (*p = 0.023).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1173138&req=5

Figure 3: Establishment of stable, dominant negative RhoC HPAF-II transfectants. A. Results of a RhoC activation assay and total RhoC Western blot comparing wildtype, C3 exotransferase treated, vector control and dominant negative RhoC (dnRhoC) transfected HPAF-II cells. B. Comparison of total (dark grey) and active (light grey) RhoA levels utilizing a RhoA-specific antibody. C. Results of the effect of dnRhoC transfection and C3 treatment on HPAF-II cells in a colloidal gold migration assay. Inhibition of RhoC lead to a significant decrease in migration (*p = 0.001) and invasion (*p = 0.023).
Mentions: Next, to directly implicate RhoC in PC cell migration and invasion, we generated stable HPAF-II dominant negative RhoC (dnRhoC) transfectants. For clarity and simplicity the results shown are from a polyclonal population which is representative of three individual clones that were tested. Figure 3A shows a 57% decrease of active RhoC in the HPAF-II/dnRhoC transfectants compared with the untransfected and vector transfected controls. As a positive control the HPAF-II cells were treated with C3 exotransferase. C3 exotransferase is a toxin derived from Clostridium botulinum and is effective at inhibiting RhoA, -B and -C activity with virtually no effect on Rac1 or Cdc42 [34,35]. C3 exotransferase treatment reduced active RhoC levels by 85%. As shown, both C3 exotransferase treatment and expression of dnRhoC did not significantly alter total levels of RhoC protein.

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