RHOA and PRKCZ control different aspects of cell motility in pancreatic cancer metastatic clones

Abstract

Background: Our understanding of the mechanism regulating pancreatic cancer metastatic phenotype is limited. We analyzed the role of RHOA and PRKCZ in the motility attitude of two subclones of the pancreatic adenocarcinoma cell line SUIT-2 (S2), with different in vivo metastatic potential in nude mice: S2-m with a low metastatic potential and highly metastatic S2-CP9 using RHOA and PRKCZ cell-permeable inhibitory peptides.

Methods: Adhesion assays, cell permeable peptides, RHOA activity assay, western blotting

Results: When used in combination cell-permeable inhibitory peptides partially inhibited cell adhesion by about 50% in clone S2-CP9. In clone S2-m, the effect was limited to 15% inhibition. In a wound healing assay, S2-CP9 was sensitive only to treatment with the combination of both RHOA and PRKCZ inhibitory peptides. Conversely, S2-m was unable to migrate toward both ends of the wound in basal conditions. Migration of cells through a membrane with 8 mum pores was completely abolished in both clones by individual treatment with RHOA and PRKCZ inhibitory peptides.

Conclusion: Herein, we demonstrate a critical role for RHOA and PRKCZ in the regulation of different aspects of cell motility of pancreatic adenocarcinoma and demonstrate the need to inhibit both pathways to obtain a functionally relevant effect in most assays. These results indicate that RHOA and PRKCZ, and their downstream effectors, can represent important pharmacological targets that could potentially control the highly metastatic attitude of PDAC.

Figure 1

Expression of RHOA and PRKCZ. Panel A: The level of expression of RHOA and PRKCZ in S2-m and S2-CP9 subclones of the SUIT-2 pancreas cancer cell line is overlapping. Actin (ACTB) served for normalization. Panel B: RHOA activity assay in S2-m and S2-CP9 clones show that not only expression but also the activity of RHOA is overlapping in both clones (n = 2).

Figure 2

RHOA and PRKCZ cell permeable inhibitory peptides. A: Site organization of RHOA and PRKCZ showing the effector regions of RHOA (aa 23-40, 75-92 and 92-119), and the inhibitory pseudosubstrate region of PRKCZ (aa 113-129). B: Representation of the the plasma membrane translocating peptides. The 23-40 RHOA effector region was fused to Penetratin-1 (left). A myristic acid was added at the N-terminal of the pseudosubstrate region of PRKCZ (right).

Figure 3

Role of RHOA and PRKCZ on cell adhesion. Percentage of the variation of the signal intensity of adherent S2-m and S2-CP9 clones between control and inhibitory peptides-treated cells, with 100% the absorbance found after Crystal Violet staining in the cell lines without treatment. S2-CP9 appear to respond to the individual inhibitory peptides and to their combination while S2-m clone adhesion is unaffected by all the treatments.

Figure 4

In vitro wound-healing assay uncover a selective role for both RHOA and PRKCZ in S2-CP9. Only the combined effect of 50 μM RHOA and 50 μM PRKCZ peptides (as indicated on the left) are effective in inhibiting wound healing selectively in S2-CP9 clone. S2m appear incapable to cover the wound, demonstrating a defect in random movement, therefore the effect of the inhibition of the indicated pathways can not be evaluated in S2m. Photomicrographs at 10× magnification were taken at the beginning and after 6 hours from the start of treatment to assess cell migration and were mounted together following appropriate reference marks. One representative of 3 individual experiments.

Figure 5

In vitro migration assays reveal a role for both RHOA and PKRCZ. Individual and combined inhibition of PKRCZ and RHOA pathways strongly inhibits the capability of all the clones to migrate through 8 μm pores. OD values determined by Crystal Violet staining of the subclones migrated through the membrane of the Transwell Assay, without and in the presence of the inhibitory peptides indicated. The values of OD are proportional to the number of cells present on the lower surface of the transwell (average values from three individual experiments).