Regulator of G Protein Signaling 6 (RGS6) Induces Apoptosis via a Mitochondrial-dependent Pathway Not Involving Its GTPase-activating Protein Activity

Abstract

Regulator of G protein signaling 6 (RGS6) is a member of a family of proteins called RGS proteins, which function as GTPase-activating proteins (GAPs) for G␣ subunits. Given the role of RGS6 as a G protein GAP, the link between G protein activation and cancer, and a reduction of cancer risk in humans expressing a RGS6 SNP leading to its increased translation, we hypothesized that RGS6 might function to inhibit growth of cancer cells. Here, we show a marked down-regulation of RGS6 in human mammary ductal epithelial cells that correlates with the progression of their transformation. RGS6 exhibited impressive antiproliferative actions in breast cancer cells, including inhibition of cell growth and colony formation and induction of cell cycle arrest and apoptosis by mechanisms independent of p53. RGS6 activated the intrinsic pathway of apoptosis involving regulation of Bax/Bcl-2, mitochondrial outer membrane permeabilization (MOMP), cytochrome c release, activation of caspases-3 and -9, and poly(ADP-ribose) polymerase cleavage. RGS6 promoted loss of mitochondrial membrane potential (⌬⌿ m ) and increases in reactive oxygen species (ROS). RGS6-induced caspase activation and loss of ⌬⌿ m was mediated by ROS, suggesting an amplification loop in which ROS provided a feed forward signal to induce MOMP, caspase activation, and cell death. Loss of RGS6 in mouse embryonic fibroblasts dramatically impaired doxorubicin-induced growth suppression and apoptosis. Surprisingly, RGS6-induced apoptosis in both breast cancer cells and mouse embryonic fibroblasts does not require its GAP activity toward G proteins. This work demonstrates a novel signaling action of RGS6 in cell death pathways and identifies it as a possible therapeutic target for treatment of breast cancer. Regulator of G protein signaling (RGS)2 proteins comprise a family of proteins, defined by the presence of a semiconserved region called the RGS domain, which negatively regulate heterotrimeric G protein signaling (1-3). These proteins were discovered as essential negative regulators of heterotrimeric G protein signaling by genetic studies in yeast followed by similar studies in Caenorhabditis elegans (4 -6). RGS proteins function as GTPase-activating proteins (GAPs) for heterotrimeric G protein subunits (7,8), an activity bestowed by their RGS domain (9) thereby enhancing the shut-off mechanism for G protein signaling. Thirty mammalian genes encode proteins with the hallmark RGS domain or less closely related versions of this domain, and RGS proteins have been classified into subfamilies based upon sequence similarities within the RGS domain that extend to sequences outside of this domain (2, 8). RGS6 is a member of the R7 subfamily of proteins that possess, in addition to their RGS domain, a GGL (G ␥ subunit-like) domain that allows for association with G␤ 5 and stabilization of RGS6 and a Drosophila/EGL10/Pleckstrin homology/DEP helical extension (DEP/DHEX) domain that allows for association with one of two additional proteins, R7BP or R9AP, and is thought to pr...

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

“…An RGS6 enhancer could also be beneficial as a novel cancer therapeutic. RGS6 has been proposed as a tumour suppressor in several types of cancer, including bladder, lung and breast cancer (Berman et al, 2004;Gu et al, 2006;Maity et al, 2011;Maity et al, 2013). While the effects of RGS6 in the CNS seem to mainly be mediated through is canonical GAP activity, its action as a tumour suppressor is mediated through non-G protein mechanisms (Maity et al, 2011).…”

“…RGS6 has been proposed as a tumour suppressor in several types of cancer, including bladder, lung and breast cancer (Berman et al, 2004;Gu et al, 2006;Maity et al, 2011;Maity et al, 2013). While the effects of RGS6 in the CNS seem to mainly be mediated through is canonical GAP activity, its action as a tumour suppressor is mediated through non-G protein mechanisms (Maity et al, 2011). These and other studies not only further demonstrate the potential for RGS proteins as potential targets in drug development for a wide range of therapeutic indications but also highlights the complexity and challenges facing investigators that wish to pursue this avenue.…”

RGS6 as a Novel Therapeutic Target in CNS Diseases and Cancer

“…First, the same RGS6 SNP, identified in the previous bladder cancer study, was similarly linked to a reduced risk of lung cancer (99,100). Second, and likely the best evidence for the role of RGS6 as a tumor suppressor, RGS6 expression was found to be negatively correlated with breast cancer progression in humans (28,29). Furthermore, mice lacking RGS6 displayed both accelerated carcinogenesis in response to carcinogen exposure and developed spontaneous mammary tumors at an increased rate (further described below) (28).…”

“…In particular, RGS6−/− mice chronically exposed to alcohol lacked alcohol-induced cardiac hypertrophy and fibrosis, hepatic steatosis, and gastrointestinal barrier dysfunction and endotoxemia. This reduction in alcohol-induced peripheral tissue damage is believed to involve RGS6's direct or indirect regulation of reactive oxygen species (ROS) production and the apoptotic cascade (21) similar to its functions in cancer suppression (27)(28)(29)(30).…”

“…RGS6 specific modulation of Gα i/o protein activity has been implicated in the regulation of several disease states, particularly in the central nervous system (CNS), including the following: alcoholism (21), anxiety/depression (22), Parkinson's disease (23), and potentially Alzheimer's disease (24), schizophrenia (25), and vision (26). However, RGS6 is also unique in that it remains the only member of the R7 protein family that has been demonstrated to regulate G protein-independent pathways, as evidenced by its compelling pro-apoptotic and tumor suppressor actions in cancer (27)(28)(29)(30).…”