A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of G␣ and G␤␥ subunits. Here we evaluated the role of G␤␥ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking G␤␥ signaling with G␣ t or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of G␣ t in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking G␤␥ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that G␤␥ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of G␤␥ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of G␤␥ signaling in promoting breast tumor growth and metastasis and suggest that targeting G␤␥ may represent a novel therapeutic approach for breast cancer.Breast cancer is the most frequently diagnosed cancer in women (1). Despite recent advances in its diagnosis and treatment with adjuvant targeted therapies, breast cancer remains the second most common cause of cancer death among women in the United States (2). As many as 90% of cancer deaths are caused by metastatic spread from primary tumors because the majority of currently marketed anticancer drugs have little effect on tumors at this stage. In light of this situation, efforts to better understand the mechanisms underlying tumor metastasis and to identify novel treatment strategies are warranted.

PLEKHG2 is a Dbl family Rho guanine nucleotide exchange factor (RhoGEF) whose gene was originally identified as being upregulated in a leukemia mouse model and was later shown to be activated by heterotrimeric G protein ␤␥ (G␤␥) subunits. However, its function and activation mechanisms remain elusive. Here we show that, compared to its expression in primary human T cells, its expression is upregulated in several leukemia cell lines, including Jurkat T cells. Downregulation of PLEKHG2 in Jurkat T cells by small interfering RNAs (siRNAs) specifically inhibited G␤␥-stimulated Rac and Cdc42, but not RhoA, activation. Consequently, suppressing PLEKHG2 expression blocked actin polymerization and SDF1␣-stimulated lymphocyte migration. Additional studies indicate that G␤␥ likely activates PLEKHG2, in part by binding the N terminus of PLEKHG2 to release an autoinhibition imposed by its C terminus, which interacts with a region encompassing the catalytic Dbl homology (DH) domain. As a result, overexpressing either the N terminus or the C terminus of PLEKHG2 blocked G␤␥-stimulated Rac and Cdc42 activation and prevented Jurkat T cells from forming membrane protrusions and migrating. Together, our studies have provided the first evidence for the endogenous function of PLEKHG2, which may serve as a key G␤␥-stimulated RhoGEF that regulates lymphocyte chemotaxis via Rac and Cdc42 activation and actin polymerization.

A spice variant of Gβ3, termed Gβ3s, has been associated with the C825T polymorphism in the Gβ3 gene and linked with many human disorders. However, the biochemical properties and functionality of Gβ3s remain controversial. Here, using multidisciplinary approaches including co-immunoprecipitation analysis and bioluminescence resonance energy transfer (BRET) measurements, we showed that unlike Gβ3, Gβ3s failed to form complexes with either Gγ or Gα subunits. Moreover, using a mutant Gγ2 deficient in lipid modification to purify Gβ3s from Sf9 cells without the use of detergents, we further showed that the failure of Gβ3s to form dimers with Gγ was not due to the instability of the dimers in detergents, but rather, reflected the intrinsic properties of Gβ3s. Additional studies indicated that Gβ3s is unstable, and unable to localize properly to the plasma membrane and to activate diverse Gβγ effectors including PLCβ2/3, PI3Kγ, ERKs and the Rho guanine exchange factor (RhoGEF) PLEKHG2. Thus, these data suggest that the pathological effects of Gβ3 C825T polymorphism may result from the downregulation of Gβ3 function. However, we found that the chemokine SDF1α transmits signals primarily through Gβ1 and Gβ2, but not Gβ3, to regulate chemotaxis of several human lymphocytic cell lines, indicating the effects of Gβ3 C825T polymorphism are likely to be tissue and/or stimuli specific and its association with various disorders in different tissues should be interpreted with great caution.

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Chen. You have gone above and beyond the call of duty in helping me achieve my goals. Your time, patience, and effort in helping me develop the skills I will need to pursue my career, both on and off the bench, are greatly appreciated. I must also thank my thesis