The Role of Ect2 Nuclear RhoGEF Activity in Ovarian Cancer Cell Transformation

Abstract

Ect2, a Rho guanine nucleotide exchange factor (RhoGEF), is atypical among RhoGEFs in its predominantly nuclear localization in interphase cells. One current model suggests that Ect2 mislocalization drives cellular transformation by promoting aberrant activation of cytoplasmic Rho family GTPase substrates. However, in ovarian cancers, where Ect2 is both amplified and overexpressed at the mRNA level, we observed that the protein is highly expressed and predominantly nuclear and that nuclear but not cytoplasmic Ect2 increases with advanced disease. Knockdown of Ect2 in ovarian cancer cell lines impaired their anchorage-independent growth without affecting their growth on plastic. Restoration of Ect2 expression rescued the anchorage-independent growth defect, but not if either the DH catalytic domain or the nuclear localization sequences of Ect2 were mutated. These results suggested a novel mechanism whereby Ect2 could drive transformation in ovarian cancer cells by acting as a RhoGEF specifically within the nucleus. Interestingly, Ect2 had an intrinsically distinct GTPase specificity profile in the nucleus versus the cytoplasm. Nuclear Ect2 bound preferentially to Rac1, while cytoplasmic Ect2 bound to RhoA but not Rac. Consistent with nuclear activation of endogenous Rac, Ect2 overexpression was sufficient to recruit Rac effectors to the nucleus, a process that required a functional Ect2 catalytic domain. Furthermore, expression of active nuclearly targeted Rac1 rescued the defect in transformed growth caused by Ect2 knockdown. Our work suggests a novel mechanism of Ect2-driven transformation, identifies subcellular localization as a regulator of GEF specificity, and implicates activation of nuclear Rac1 in cellular transformation.

Ect2-Dependent rRNA Synthesis Is Required for KRAS-TRP53 -Driven Lung Adenocarcinoma

“…Cellular fractionation revealed that RhoA and Rac1 localized to both the cytoplasm and nucleus of A549 cells, whereas Cdc42 was exclusively cytoplasmic (Figure 6A), consistent with published results (Huff et al, 2013; Lanning et al, 2003). Similar results were observed in H358 and H23 cells (Figure S5A).…”

“…However, we recently showed that nuclear Ect2 GEF activity is necessary for transformed growth of ovarian cancer cells (Huff et al, 2013). We therefore generated a GEF-deficient Ect2 mutant (DH mut Ect2) (E428A and N608A within the Dbl homology (DH) domain); and NLS mut Ect2 (five key arginine residues in the Ect2 NLS changed to alanines; R348,349,350,370,372A) (Rossman et al, 2005) (Figure 3A).…”

“…Early dogma was that nuclear Ect2 was auto-inhibited and physically sequestered from predominantly cytoplasmic GTPases (Hara et al, 2006). However, we recently reported that nuclear Ect2 GEF activity is required for transformed growth of ovarian cancer cells (Huff et al, 2013). Here, we validate and extend these observations, and establish that nuclear Ect2-mediated rDNA transcription is required for LADC transformation.…”

“…In lung cancer cells, a pool of Ect2 becomes mislocalized to the cytoplasm where it associates with the PKCι-Par6 complex and participates in transformed growth by activating Rac1, a process distinct from its role in cytokinesis (Justilien and Fields, 2009). More recently we demonstrated that nuclear Ect2 participates in transformed growth of ovarian cancer cells (Huff et al, 2013). However, neither the molecular mechanism(s) by which nuclear Ect2 participates in oncogenesis, nor a role for Ect2 in tumorigenesis in vivo has been elucidated.…”

ERK/MAPK Signaling Drives Overexpression of the Rac-GEF, PREX1, in BRAF- and NRAS-Mutant Melanoma

“…Increased expression and activation of these RhoGEFs result in enhanced activity of their Rho family small GTPase substrates in a context-dependent manner. For example, we recently identified overexpression of ECT2 protein in ovarian cancer, mediated by gene amplification, that resulted in activation of RHOA in the cytosol and RAC1 in the nucleus (3). The critical importance of RAC1 in cancer cell migration and invasion (4) has further focused attention on the mechanisms regulating activators of RAC1, such as the Dbl family of RhoGEFs.…”