Current evidence suggests that Rac activity is crucial for migration and invasion of cancer cells, and aberrant Rac signaling is often seen in cancer metastasis (Sun et al. 2006
). Since Rac mutations are rarely detected in cancer, research is focused on the factors that govern Rac activation. RTKs and GPCRs are among the plasma membrane proteins capable of initiating responses that lead to Rac activation through RacGEFs. Here, we report that P-Rex1, a RacGEF that functions downstream of both GPCR and RTK signaling, is markedly upregulated in metastatic prostate cancer and may function as one of the important molecules in metastatic signaling pathways in prostate cancer cells.
P-Rex1 is a relatively abundant protein normally expressed in cells of a hematopoietic or neuronal lineage (Welch et al. 2002
). We found that endogenous P-Rex1 expression levels are very low in normal prostate epithelial cells and nonmetastatic prostate cancer cells, but remarkably elevated in the metastatic prostate cancer cells. In addition, our studies using human prostate cancer specimens showed a progressive increase of P-Rex1 protein in cells from non-cancerous prostate tissues, localized prostate carcinoma and prostate cancer that has metastasized to a lymph node. Using a mouse xenograft model, we demonstrated that P-Rex1 expression induced lymph node metastasis of nonmetastatic prostate cancer CWR22Rv1 cells without an effect on primary tumor development and growth. These results suggest that upregulated P-Rex1 may contribute to prostate cancer metastasis.
P-Rex1 is characterized by a DH domain possessing its catalytic activity in tandem with a pleckstrin-homology (PH) domain, two adjacent DEP (disheveled, EGL-10 and pleckstrin homology) domains, two adjacent PDZ (postsynaptic density disc-large zo-1) domains and a tail with polyphosphate 4-phosphatase homology (Welch et al. 2002
). The PH domain of RacGEFs binds to PIP3 in the cell membrane, which can either directly activate the RacGEF or anchor the molecule to facilitate its activation by other proteins. P-Rex1 is among the few RacGEFs also known to be selectively activated by Gβγ subunits (Welch et al. 2002
; Rosenfeldt et al. 2004
; Hill et al. 2005
). Moreover, the binding of cytosolic P-Rex1 to both Gβγ subunits and PIP3 leads to a synergistic activation of this RacGEF (Welch et al. 2002
; Barber et al. 2007
). Interestingly, P-Rex1 has recently been shown to interact with the cellular nutrient sensor mTOR via a DEP domain and functions as an effector mediating Rac activation through the rapamycin-insensitive mTORC2 pathway (Hernandez-Negrete et al. 2007
). Thus, P-Rex1 appears to be positioned as a coincidence detector, something that could be crucial for a cancer cell navigating through a range of chemotactic signal gradients reaching it from different directions.
Indeed, our data indicate endogenous P-Rex1 can regulate metastatic prostate cancer cell migration and invasion. For instance, silencing endogenous P-Rex1 in metastatic PC-3 cells reduced their migration and invasion abilities toward chemoattractants in NIH3T3 CM by approximately 50%. As expected, silencing endogenous P-Rex1 also largely decreased Rac1 activity without a significant effect on RhoA activity. The loss of Rac1 activity is important for the reduction of the PC-3 cell migration since NSC, which blocks the activation of Rac1 by P-Rex1, also significantly reduced PC-3 cell migration. Those results indicate a significant contribution of endogenous P-Rex1 to the maintenance of Rac activity that is important for PC-3 cells to migrate in response to chemoattractants in NIH3T3 CM. Furthermore, silencing P-Rex1 attenuated the cell migratory response to CXCL12, an agonist of G-protein coupled CXCR4, by over 80% while RTK agonist EGF-stimulated cell migration was only reduced about 40%, suggesting that P-Rex1 is especially important for chemotactic factors that stimulate GPCRs. The relative insensitivity of EGF-stimulated responses to P-Rex1 silencing may be due to activation of a greater range of RacGEFs following stimulation of RTK pathways.
A wide range of evidence has appeared over the past several years supporting a role for activation of G-proteins by GPCRs during cancer growth and metastasis (Daaka 2004
; Dorsam and Gutkind 2007
). The Gα subunit of the G12 subfamily of G-proteins is directly linked to intracellular pathways that promote prostate cancer invasion (Kelly et al. 2006
). Pertussis toxin (PTx), which selectively inactivates Gi
-proteins, also suppresses prostate cancer metastasis in a mouse model (Bex et al. 1999
), demonstrating the importance of Gi
-proteins. Several prostate cancer metastasis-associated GPCRs including CXCR4 (Arya et al. 2004
) and CC chemokine receptor 2 (Lu et al. 2007
) are coupled to Gi
-proteins (Moepps et al. 1997
; Jimenez-Sainz et al. 2003
is not required for neutrophil chemotaxis mediated by Gi
-coupled receptors (Neptune et al. 1999
). Instead, disruption of Gβγ signaling inhibits this neutrophil chemotaxis (Lehmann et al. 2008
). Inhibition of Gβγ-mediated signaling has been shown to inhibit prostate cancer cell growth in vitro
and prostate tumor formation in vivo
(Bookout et al. 2003
). In addition, we found (Qin, unpublished observations) that the migration and invasion of P-Rex1-expressing metastatic prostate cancer cells are significantly attenuated by PTx and M119, a Gβγ inhibitor (Bonacci et al. 2006
). It therefore appears that chemotactic factors that stimulate GPCRs promote prostate cancer metastasis via a Gβγ-dependent P-Rex1/Rac activation pathway. Further investigation is in progress.
Characteristically, the DH domain accounts for the GEF activity leading to Rac activation (Hoffman and Cerione 2002
). We found that transient expression of recombinant P-Rex1, but not a DH-deleted P-Rex1 mutant, increases cell migration in both CWR22Rv1 cells and PC3-LN4 cells normally having undetectable and mid-range levels of endogenous P-Rex1, respectively. These results prompted us to establish stable CWR22Rv1 cells expressing P-Rex1 or its “GEF-dead” mutant. Compared to control cells, cells stably expressing P-Rex1 have 3-fold higher migration and invasion abilities that could be largely abolished by NSC, the inhibitor of Rac activation. The failure of the P-Rex1 “GEF-dead” mutant to activate Rac caused a corresponding loss of its ability to induce lamellipodia formation in prostate cancer cells. Consequently, the P-Rex1 “GEF-dead” mutant failed to stimulate prostate cancer cell migration and invasion in vitro
and spontaneous metastasis in an orthotopic xenograft model. Altogether, our studies demonstrated the importance of the classical action of P-Rex1 as a RacGEF in prostate cancer metastasis.
Other chemotactic factors acting through RTKs can increase PIP3 production through the PI3K signaling pathway (Cantley 2002
) and contribute to prostate cancer metastasis (Mimeault and Batra 2006
). In fact, the silencing of P-Rex1 reduced EGF stimulated migration by 40%, suggesting the importance of PIP3-dependent P-Rex1 activation in RTK-promoted prostate cancer metastasis. Furthermore, mTOR, the nutrient sensor in cells (Fingar and Blenis 2004
), also uses P-Rex1 to stimulate cell migration (Hernandez-Negrete et al. 2007
). Thus, by functioning as a molecule that integrates the simultaneous but separate inputs from GPCRs, RTKs, and mTOR nutrient sensing in its local microenvironment, the synergistic activation of P-Rex1 could function as a coincidence detector that helps regulate the direction of prostate cancer cell movement. Unlike Rac1, P-Rex1 has limited tissue expression and its knockout is not embryonically lethal (Welch et al. 2005
). P-Rex1 contributes to the regulation of neutrophil function, but is not essential for either chemotaxis or degranulation (Welch et al. 2005
). Thus, P-Rex1 could be an attractive drug target since blockade of its expression or actions may impair metastatic prostate cancer cell navigation with less destructive side effects than traditional chemotherapy, thereby making the cancer a better target for surgery, radiation and immunotherapy.