The SmgGDS Splice Variant SmgGDS-558 Is a Key Promoter of Tumor Growth and RhoA Signaling in Breast Cancer

Abstract

Breast cancer malignancy is promoted by the small GTPases RhoA and RhoC. SmgGDS is a guanine nucleotide exchange factor that activates RhoA and RhoC in vitro. We previously reported that two splice variants of SmgGDS, SmgGDS-607 and SmgGDS-558, have different characteristics in binding and transport of small GTPases. To define the role of SmgGDS in breast cancer, we tested the expression of SmgGDS in breast tumors, and the role of each splice variant in proliferation, tumor growth, Rho activation, and NF-κB transcriptional activity in breast cancer cells. We show upregulated SmgGDS protein expression in breast cancer samples compared to normal breast tissue. Additionally, Kaplan-Meier survival curves indicated that patients with high SmgGDS expression in their tumors had worse clinical outcomes. Knockdown of SmgGDS-558, but not SmgGDS-607, in breast cancer cells decreased proliferation, in vivo tumor growth, and RhoA activity. Futhermore, we found that SmgGDS promoted a Rho-dependent activation of the transcription factor NF-κB, which provides a potential mechanism to define how SmgGDS-mediated activation of RhoA promotes breast cancer. This study demonstrates that elevated SmgGDS expression in breast tumors correlates with poor survival, and that SmgGDS-558 plays a functional role in breast cancer malignancy. Taken together, these findings define SmgGDS-558 as a unique promoter of RhoA and NF-κB activity and a novel therapeutic target in breast cancer.

The Tumor-suppressive Small GTPase DiRas1 Binds the Noncanonical Guanine Nucleotide Exchange Factor SmgGDS and Antagonizes SmgGDS Interactions with Oncogenic Small GTPases

“…The proteins were transferred to PVDF and immunoblotted using antibodies against SmgGDS (BD Transduction Laboratories; 612511), GAPDH (Santa Cruz Biotechnology; sc-32233), Myc (Covance; PRB-150P), HA (Covance; MMS-101P), RhoA (Cytoskeleton; ARH03-A), and DiRas1 (Proteintech; 12634-AP). Bound antibodies were visualized using HRPlinked secondary antibodies (GE Healthcare), as previously described (6).…”

“…NF-B Transcriptional Activation Assays-U87, U251, T47D, and MCF7 cells were transfected with the indicated cDNAs, as well as both the pNifty-Luciferase NF-B luciferase reporter (Invivogen) and ␤-gal reporter plasmids in 48-well plates using Lipofectamine 2000 (Life Technologies), as previously described (6). HEK293T cells were transfected with the indicated cDNAs, as well as the pNifty-Luc NF-B luciferase reporter plasmid in 6-well plates.…”

“…We sought to determine whether SmgGDS acts as a GEF for DiRas1, because there are no known GEFs for DiRas1. The GEF activity of SmgGDS for RhoA is not isoform-dependent, and SmgGDS-558 appears to promote malignant phenotypes more potently than the full-length SmgGDS protein (6). Therefore, we utilized SmgGDS-558 for the remainder of our studies.…”

“…Recent studies have demonstrated that SmgGDS-558 plays a more significant role than SmgGDS-607 in activating RhoA in breast cancer cells, despite lower levels of endogenous SmgGDS-558 protein (6,7). In addition, SmgGDS promotes RhoA-mediated NF-B transcriptional activity (6,8,9), which is critical to cancer cell growth and proliferation (10).…”

SmgGDS as a Crucial Mediator of the Inhibitory Effects of Statins on Cardiac Hypertrophy and Fibrosis

“…20,[39][40][41] In this study, we found parallel increase in Rho-kinase activity and Rac1 in SmgGDS +/-CFs compared with WT CFs. Previous studies have demonstrated that SmgGDS activates RhoA, 42,43 which is the upstream activator of Rho kinase. 44 However, in this study, Rho-kinase activity was significantly increased in SmgGDS +/-CFs.…”