Caveolin-1 Expression in Pancreatic Cancer Cell Lines
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 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.
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 (more ...)
To determine whether cav-1 expression plays a central role in cell migration and invasion we chose the BxPC-3 and HPAF-II cell lines to represent tumor cells derived from primary tumors and metastases, respectively. Morphologically, both of the cell lines are similar and have a well-differentiated, epithelial appearance. We first tested the cells in a colloidal gold random motility assay to assess basal, non-ECM mediated migratory capabilities of cells. Figure shows that after 16 h the BxPC-3 cell line was essentially non-migratory with an average phagokinetic track area of 357 ± 107 square pixels in contrast to the HPAF-II cell line which was highly migratory with an average track area of 1567 ± 227 square pixels. Similarly, when tested for their ability to invade through a Matrigel coated filter in response to a serum chemoattractant, the HPAF-II cells were 10-fold more invasive than the BxPC-3 cells.
Next we sought to establish whether loss of cav-1 was responsible for the increased migratory and invasive capabilities of the HPAF-II cells. To accomplish this we transiently transfected the HPAF-II cells with a GFP-tagged cav-1 expression vector. Using a variation of the colloidal gold assay, we compared untransfected, GFP-vector control and GFP-cav-1 transfected HPAF-II cells in a blue fluorescent bead random migration assay. This assay is performed the same as the colloidal gold assay, but allows us to fluorescently identify GFP-transfected cells. Figure demonstrates that ectopic re-expression of cav-1 to levels similar to that of the BxPC-3 cells significantly decreases HPAF-II migration (890 ± 67 square pixels; p = 0.0032) compared with the untransfected or GFP-vector control HPAF-II cells (1630 ± 183 and 1435 ± 235 square pixels, respectively).
Similarly in a Matrigel invasion assay, re-expression of cav-1 in the HPAF-II cells decreased the invasiveness of the cells nearly 2-fold compared with the controls. The results from the motility and invasion experiments suggest that HPAF-II migration and invasion is mediated by cav-1.
RhoC GTPase Induces PC Cell Migration
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
]. Hence, we chose to study RhoC GTPase in relationship to cav-1. As shown in Figure 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 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.
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 (more ...)
To determine if cav-1 expression affected RhoC activation, levels of GTP-bound RhoC was measured in the GFP-cav-1 and control HPAF-II cells (Figure ). Compared with the controls, transient ectopic re-expression of cav-1 decreased levels of active RhoC by 6-fold without effecting total RhoC protein levels, suggesting regulation of RhoC activation by cav-1.
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 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
]. 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.
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 (more ...)
Dominant negative Rho GTPases work by entering into a non-productive interaction with Rho Guanine Exchange Factors (RhoGEFs), the proteins which catalyze the exchange of GDP for GTP. Due to the close homology between RhoC and RhoA (91% on the protein level), the possibility exists that both GTPases can be activated by the same RhoGEFs in vivo. As shown in Figure , RhoA activity was not significantly altered by expression of dnRhoC. Again, as a positive control HPAF-II cells were treated with C3 exotransferase; this reduced RhoA activity an average of 63%. Therefore, RhoC activity was specifically decreased in the HPAF-II cells expressing dominant negative RhoC.
As shown in Figure , inhibition of RhoC activity, either by dnRhoC or by treatment with C3 exotransferase, significantly reduced HPAF-II cell migration by nearly 4-fold (p = 0.001) and invasion by 3-fold (p = 0.023), suggesting a key role for RhoC GTPase mediating HPAF-II cell migration and invasion.
Association of Cav-1 and RhoC GTPase in PC cells
Next, we considered the possibility that RhoC activity and PC migration and invasion are regulated by cav-1 through physical interaction of the GTPase with the scaffolding protein. Proteins that associate with cav-1 contain the canonical cav-1 binding domain, ΦXΦXXXXΦ or ΦXXXXΦXXΦ (where Φ= Trp, Phe or Tyr) [36
]. A review of the RhoC protein sequence revealed a putative cav-1 binding sequence at amino acid residues 35–43 (Y
Figure are the results of immunoprecipitation assays using either a polyclonal or monoclonal antibody specific for cav-1 followed by immunoblotting for RhoC. Cav-1 and RhoC proteins co-immunoprecipitated in the BxPC-3 cells but not in the HPAF-II cell line. Unexpectedly, the association between cav-1 and RhoC was restored in the HPAF-II/dnRhoC cell line suggesting that inhibition of RhoC activity leads to re-expression of cav-1 protein.
Figure 4 Interaction of cav-1 and RhoC in BxPC-3 and HPAF-II PC cell lines. A. Aliquots of 500 μg total protein from BxPC-3, HPAF-II and HPAF-II/dnRhoC transfectants were immunoprecipitated with either a monoclonal or polyclonal antibody to cav-1. Proteins (more ...)
The results of a cav-1 immunoblot for total cellular protein are shown in Figure . Expression of cav-1 was increased 3.1-fold in the HPAF-II/dnRhoC cells compared with the parental HPAF-II and vector control cells. Actin was used as a loading control. Together with the immunoprecipitation data, these data suggest that cav-1 expression is in a reciprocal relationship with RhoC activation. High cav-1 protein expression leads to inhibition of RhoC activation while inhibition of RhoC activity leads to partial re-expression of cav-1. Furthermore, interaction of cav-1 and RhoC may result in decreased RhoC activation, limiting cell migration and invasion.
PC Cell Migration Involves the Mitogen Activated Protein Kinase (MAPK) Pathway
Previous studies demonstrated that activation of p42/p44 extracellular regulated kinase (Erk) decreased cav-1 protein levels in constitutively-active Ras transformed NIH3T3 cells [38
]. In the same set of studies it was shown that inhibition of oncogenic Ras-induced Erk activation with PD98059 increased cav-1 expression 5-fold [38
]. That same group also demonstrated that ectopic re-expression of cav-1 decreased Erk activation in Ras-transformed CHO cells [39
]. Our laboratory has previously demonstrated that RhoC can mediate inflammatory breast cancer cell migration and invasion through co-activation of the p42/p44 Erk and p38 arms of the MAPK pathway [40
]. With these studies in mind we next examined whether RhoC can activate p42/p44 Erk in PC, subsequently decreasing cav-1 expression and leading to increased cellular migration. Also, we examined the potential involvement of p38 MAPK in this process.
Figure is a comparison of active (phospho-) and total levels of p42/p44 Erk and p38 MAPK in BxPC-3, HPAF-II and HPAF-II/dnRhoC cells that were serum starved for 16 h and stimulated with 10% serum alone or after pre-treatment with C3 exotransferase. Although active phospho-Erk levels were stimulated in both cell lines, active p42/p44 Erk was considerably higher in the BxPC-3 cell line compared with the HPAF-II cell line. Pretreatment with C3 exotransferase slightly decreased active Erk in serum stimulated cell lines. Similar results were previously demonstrated and are unexpected since the HPAF-II cell line harbors an activating G12D K-Ras mutation, while the BxPC-3 cell line has a wildtype K-Ras [41
]. In the HPAF-II/dnRhoC cells, the overall phospho-p42/p44 levels were higher compared with the parental HPAF-II cell line suggesting that inhibition of RhoC leads to increased Erk activation.
Figure 5 Analysis of the mitogen activated protein kinase pathway (MAPK) in the BxPC-3 and HPAF-II cells. A. Actively growing PC cells were serum starved for 24 h and left untreated or pretreated for 1 h with 5 μg C3 exotransferase, stimulated by the addition (more ...)
Converse to what was observed for p42/p44 Erk the levels of phospho-p38 MAPK were low in the BxPC-3 and HPAF-II/dnRhoC cells and higher in the HPAF-II cells. C3 treatment decreased p38 activity in the BxPC-3 and HPAF-II cell lines. The levels of active p38 in the HPAF-II/dnRhoC and C3 treated HPAF-II cells were comparable; demonstrating an approximate 52% decrease in activity compared with the serum stimulated HPAF-II cells. Taken together, an inverse relationship between p42/p44 Erk and p38 MAPK signaling is suggested in the PC cells.
Levels of active Erk and p38 MAPK were assessed in the HPAF-II/GFP-cav-1 transfectants. As shown in Figure , levels of active phospho-p42/p44 Erk increased in the GFP-cav-1 transfectants compared with the untransfected and control GFP-vector transfectants. Conversely, levels of phosphorylated p38 decreased in the cav-1 transfectants, mirroring what is observed in the HPAF-II/dnRhoC cells.
To tease out the individual roles of the p42/p44 Erk and p38 MAPK pathways in PC cellular migration, the BxPC-3 and HPAF-II cells were treated with the pharmacologic inhibitors PD98059 (to inhibit MEK1 and subsequently Erk) or SB220025 (to inhibit p38) and tested in migration and invasion assays (Figure ). SB220025 treatment significantly reduced HPAF-II migration and invasion (p = 0.001). PD98059 treatment had no effect on the cells ability to move in either assay suggesting that migration and invasion occurs through signaling of the p38 MAPK pathway.
Changes in cav-1 expression due to inhibitor treatment are also shown in Figure . Cav-1 expression was slightly less after PD98059 treatment in both PC cell lines. Treatment with SB220025 increased cav-1 expression dramatically in the HPAF-II and slightly in the BxPC-3 cells, implying a reciprocal relationship between cav-1 expression and p38 activation. Furthermore, RhoC activity was appreciably reduced in the SB220025 treated HPAF-II cells strengthening the notion that cav-1 is in a reciprocal relationship with RhoC and p38 activity.
Methyl-β-cyclodextrin increases PC cell motility
Lastly, we treated the BxPC-3 cell line with methyl-β-cyclodextrin (MβCD), which sequesters cholesterol, disrupts caveolae and mis-localizes cav-1 in the cell [43
]. As shown in Figure , MβCD treatment significantly increased the area of BxPC-3 migration (p = 0.0001). Increased cellular migration was accompanied by a significant increase in active GTP-bound RhoC (Figure ; p = 0.0001). Consistent with previous observations, Figure demonstrates that levels of phospho-p42/p44 Erk in the MβCD treated cells decreased while levels of phospho-p38 MAPK increased. The changes in MAPK proteins approached but did not achieve statistical significance. These data further suggest that activation of RhoC GTPase is regulated by cav-1 and that RhoC signals through the p38 arm of the MAPK pathway to induce cellular migration.
Figure 6 Effect of disruption of caveolae and mis-localization of caveolin-1 after treatment of BxPC-3 cells with MβCD. A. Results of a colloidal gold random motility assay performed after a 1 h pre-treatment of BxPC-3 cells with 5 mM MβCD in growth (more ...)