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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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RhoC GTPase expression in BxPC-3 and HPAF-II cell lines. A. Total GDP/GTP-bound RhoC was measured in a panel of 10 PC cell lines. Protein (30 μg) was harvested from actively growing PC cell lines. B. Expression of RhoC in BXPC-3 and HPAF-II cells was measured by RT-PCR and immunoblot analysis using RhoC-specific primers and antibody, respectively. To determine the amount of active, GTP-bound RhoC a Rho activation assay was performed. GST-rhotekin is used to pull down RhoC-GTP from total cell lysates followed by immunoblotting with a RhoC-specific antibody. C. The RhoC activation assay was performed on the GFP-cav-1 HPAF-II transfectants 24 h after transfection. Differences in active and total RhoC levels were measured by densitometry and are represented.
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Figure 2: RhoC GTPase expression in BxPC-3 and HPAF-II cell lines. A. Total GDP/GTP-bound RhoC was measured in a panel of 10 PC cell lines. Protein (30 μg) was harvested from actively growing PC cell lines. B. Expression of RhoC in BXPC-3 and HPAF-II cells was measured by RT-PCR and immunoblot analysis using RhoC-specific primers and antibody, respectively. To determine the amount of active, GTP-bound RhoC a Rho activation assay was performed. GST-rhotekin is used to pull down RhoC-GTP from total cell lysates followed by immunoblotting with a RhoC-specific antibody. C. The RhoC activation assay was performed on the GFP-cav-1 HPAF-II transfectants 24 h after transfection. Differences in active and total RhoC levels were measured by densitometry and are represented.

Mentions: Due to their role in cellular migration, invasion and metastasis the Rho GTPases were logical molecular candidates to interact with cav-1 to mediate cell migration and invasion. RhoC GTPase is prevalent in metastatic tumors, particularly in PC [20-28]. Hence, we chose to study RhoC GTPase in relationship to cav-1. As shown in Figure 2A RhoC GTPase is expressed on the protein level to varying degrees in the panel of 10 pancreatic cancer cell lines that were analyzed for cav-1 expression. However, more important than expression is the activation state of the GTPase. Figure 2B is a comparison of RhoC expression and activation in the BxPC-3 and HPAF-II cell lines. RT-PCR and immunoblot analysis confirm that RhoC is highly expressed on the mRNA and protein levels both in the BxPC-3 and HPAF-II cells. To determine the relative levels of active RhoC in these cell lines, a GST fusion protein of the Rho-binding domain of the downstream Rho effector protein, rhotekin, was used to selectively pull out GTP-bound RhoC. Although levels of total GDP/GTP-bound RhoC were similar for both cell lines, levels of active RhoC was considerably higher in the HPAF-II cell line.


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)

RhoC GTPase expression in BxPC-3 and HPAF-II cell lines. A. Total GDP/GTP-bound RhoC was measured in a panel of 10 PC cell lines. Protein (30 μg) was harvested from actively growing PC cell lines. B. Expression of RhoC in BXPC-3 and HPAF-II cells was measured by RT-PCR and immunoblot analysis using RhoC-specific primers and antibody, respectively. To determine the amount of active, GTP-bound RhoC a Rho activation assay was performed. GST-rhotekin is used to pull down RhoC-GTP from total cell lysates followed by immunoblotting with a RhoC-specific antibody. C. The RhoC activation assay was performed on the GFP-cav-1 HPAF-II transfectants 24 h after transfection. Differences in active and total RhoC levels were measured by densitometry and are represented.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: RhoC GTPase expression in BxPC-3 and HPAF-II cell lines. A. Total GDP/GTP-bound RhoC was measured in a panel of 10 PC cell lines. Protein (30 μg) was harvested from actively growing PC cell lines. B. Expression of RhoC in BXPC-3 and HPAF-II cells was measured by RT-PCR and immunoblot analysis using RhoC-specific primers and antibody, respectively. To determine the amount of active, GTP-bound RhoC a Rho activation assay was performed. GST-rhotekin is used to pull down RhoC-GTP from total cell lysates followed by immunoblotting with a RhoC-specific antibody. C. The RhoC activation assay was performed on the GFP-cav-1 HPAF-II transfectants 24 h after transfection. Differences in active and total RhoC levels were measured by densitometry and are represented.
Mentions: Due to their role in cellular migration, invasion and metastasis the Rho GTPases were logical molecular candidates to interact with cav-1 to mediate cell migration and invasion. RhoC GTPase is prevalent in metastatic tumors, particularly in PC [20-28]. Hence, we chose to study RhoC GTPase in relationship to cav-1. As shown in Figure 2A RhoC GTPase is expressed on the protein level to varying degrees in the panel of 10 pancreatic cancer cell lines that were analyzed for cav-1 expression. However, more important than expression is the activation state of the GTPase. Figure 2B is a comparison of RhoC expression and activation in the BxPC-3 and HPAF-II cell lines. RT-PCR and immunoblot analysis confirm that RhoC is highly expressed on the mRNA and protein levels both in the BxPC-3 and HPAF-II cells. To determine the relative levels of active RhoC in these cell lines, a GST fusion protein of the Rho-binding domain of the downstream Rho effector protein, rhotekin, was used to selectively pull out GTP-bound RhoC. Although levels of total GDP/GTP-bound RhoC were similar for both cell lines, levels of active RhoC was considerably higher in the HPAF-II cell line.

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