Abstract
BackgroundProstate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13. However, the differential expression of G protein α, β, and γ subunits by PCa cell lines and the precise combination of these proteins with CXCR5 has not been elucidated.MethodsWe examined differences in G protein expression of normal prostate (RWPE-1) and PCa cell lines (LNCaP, C4-2B, and PC3) by western blot analysis. Further, we immunoprecipitated CXCR5 with different G protein subunits, and CXCR4, following CXCL13 stimulation. To investigate constitutive coupling of CXCR5 with CXCR4 and PAR-1 we performed invasion assay in PCa cells transfected with Gαq/i2 or Gα13 siRNA, following CXCL13 treatment. We also investigated Rac and RhoA activity by G-LISA activation assay in PCa cells following CXCL13/thrombin stimulation.ResultOf the 22 G proteins studied, Gαi1-3, Gβ1-4, Gγ5, Gγ7, and Gγ10 were expressed by both normal and PCa cell lines. Gαs was moderately expressed in C4-2B and PC3 cell lines, Gαq/11 was only present in RWPE-1 and LNCaP cell lines, while Gα12 and Gα13 were expressed in C4-2B and PC3 cell lines. Gγ9 was expressed only in PCa cell lines. Gα16, Gβ5, Gγ1-4, and Gγ13 were not detected in any of the cell lines studied. Surprisingly, CXCR4 co-immunoprecipitated with CXCR5 in PCa cell lines irrespective of CXCL13 treatment. We also identified specific G protein isoforms coupled to CXCR5 in its resting and active states. Gαq/11/Gβ3/Gγ9 in LNCaP and Gαi2/Gβ3/Gγ9 in C4-2B and PC3 cell lines, were coupled to CXCR5 and disassociated following CXCL13 stimulation. Interestingly, Gα13 co-immunoprecipitated with CXCR5 in CXCL13-treated, but not in untreated PCa cell lines. Inhibition of Gαq/i2 significantly decreased the ability of cells to invade, whereas silencing Gα13 did not affect CXCL13-dependent cell invasion. Finally, CXCL13 treatment significantly increased Rac activity in Gαq/i2 dependent manner, but not RhoA activity, in PCa cell lines.ConclusionsThese findings offer insight into molecular mechanisms of PCa progression and can help to design some therapeutic strategies involving CXCR5 and/or CXCL13 blockade and specific G protein inhibition to abrogate PCa metastasis.
Highlights
Prostate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13
We have shown that PCa cell lines differentially express CXCR5, and this expression positively correlates with the ability of cell lines to migrate and invade extracellular matrix components following interaction with CXCL13 [14,15]
Our results show that Gαi subunit are widely expressed by RWPE-1, LNCaP, C4-2B, and PC3 cell lines (Figure 1A)
Summary
Prostate cancer (PCa) cell lines and tissues differentially express CXCR5, which positively correlate with PCa progression, and mediate PCa cell migration and invasion following interaction with CXCL13. G protein-coupled receptors (GPCRs) are divided into three broad classes based on the similarity of the transmembrane sequences and the nature of their ligand [1]. The receptor elicits a conformational alteration resulting in the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) by the Gα subunit. This leads to heterotrimer dissociation and stimulation of downstream effector molecules to initiate intracellular signaling cascades [1,3,4]. Gα subunits are divided into four families Gαs, Gαi, Gαq/11, and Gα12/13 based on sequence homology and functional similarities. G protein β and γ subunits function as a tightly associated complex to modulate the activity of several effectors including AC, protein tyrosine kinases (e.g., Src family tyrosine kinases), phosphoinositide-3 kinase (PI3K) γ, GPCR kinases (GRKs), and Ca+2 as well as K+ ion channels [4,5]
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