The Rho-family of small GTPase specific guanine nucleotide exchange factor, GEFT, is expressed at high levels in adult human excitable tissues including the brain, heart, and skeletal muscle. Previously, we demonstrated that GEFT is specifically expressed in the adult mouse hippocampus and cerebellum, and that overexpression of this protein can result in neurite and dendrite remodeling. This finding prompted us to explore the expression of GEFT in other tissues, which share common developmental ancestry to the nervous system, specifically the ocular system. Using immunohistochemical analysis specific for GEFT protein expression, we observed the highest ocular expression of GEFT occurring in the neuroblastic layer and differentiating lens fibers of the late-stage mouse embryo, and in the postnatal corneal epithelium, lens epithelium, and throughout the retina. Exogenous expression of GEFT in N/N1003A rabbit lens epithelial cells induced lens fiber differentiation as reflected by cell elongation and lentoid formation, as well as a strong increase in β-crystallin and filensin expression. Moreover, transfection of lens epithelial cells with GEFT resulted in a Rac-1 mediated up-regulation of αA-, αB-, βB-, γC-, or γF-crystallin promoter activities that is in part dependent on the nuclear localization of Rac1. Furthermore, pharmacological inhibition of Rac1 blocked GEFT-induced N/N1003A lens fiber differentiation and βB-crystallin expression in ex vivo mouse lens explants. These results demonstrate for the first time a role for GEFT in lens cell differentiation and mouse eye development. Moreover, GEFT regulation of lens differentiation and eye development occurs through a Rac1-dependent mechanism.
Abstract Development of cutting edge molecular techniques greatly enhanced the knowledge of the biology of the prostate cancer (CaP). These results helped us for early detection of the disease and also developing targeted therapies of CaP. It is a fact that other than Androgen Receptor (AR), there is no specific gene candidate that could be either linked for initiation, progression or treatment of CaP. Emerging evidences suggest that a small set of microRNAs (miRNA) linked to pathogenesis of CaP especially leading to castration resistant prostate cancer (CRPC). The current conventional therapies have not proven to be successful for CRPC that compelled us to identify new targets or potent small molecules to efficiently suppress the growth of advance stages of CaP. In genome wide miRNA profiling, we have found that hsa-miR-301a-3p is expressed at stage specific manner in prostate tumor as compared to controls. These results encouraged us to dissect the role of miRNA-301a in preclinical models of CaP. miR-301-3P was highly expressed (10-14 folds) in CaP cell lines (LNCaP, C4-2B, DU-145 and PC-3) when compared to normal prostate epithelial cell lines (PZHPV-7 and PrEC). Silencing specifically miR-301a-3p resulted in inhibition of proliferation, colony forming abilities, invasion, migration and cell adhesion of CaP cells. At molecular levels we found, inhibition of miR-301a-3p upregulated its direct target of tumor suppressor TXNIP/VDUP1 expression. In addition, silencing miR-301a-3p negatively activated Rho GTPases family proteins; specifically Rho1 and Rac-1 ubiquitous signaling, resulted in transcriptional down-regulation of Epithelial-Mesenchymal-Transition (EMT) markers like slug and β-catenin. Our immunofluorescence studies suggest E-cadherin is highly expressed in the membranes and at the cellular junctions when miR-301a-3p were inhibited in CRPC cells. On the contrary, over expression of miR-301-3P, increased proliferation of CaP cells and activated Rho GTPase signaling (Rho A/B/C, Rac1 and CDC42) and mesenchymal markers like slug, b-catenin resulted in invasion and migration of CaP cells. Currently, we are interrupting Rho signaling pathways, especially upstream events of RhoA effectors Rho-associated kinases (ROCK) in miR301 over expressed CaP cells and dissecting the downstream events and correlate with phenotypic changes of CaP cells. Our in vivo assays may reveal whether silencing miR-301-3p could be a potential target for the treatment of CRPC cells. Citation Format: Vittal Kurisetty, Trinath P. Das, Rama S. Reddy, Jessica Stiles, Brad Bryan, Chendil Damodaran. The role of miR-301-3P in the regulation of Rho GTPases mediated EMT signaling in castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5329. doi:10.1158/1538-7445.AM2013-5329 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.
If you have been following current news in the pharmaceutical industry, you have likely heard about over 3500 product liability lawsuits that were filed against Merck in the past several years asserting claims arising from the use of the NuvaRing, an intravaginal contraceptive ring that releases ethinyl estradiol and etonogestrel. These claims alleged that the manufacturers failed to design and produce the NuvaRing with adequate testing and study, that use of the NuvaRing leads to injuries including blood clots, cardiovascular dysfunction, and death, and that the drug companies failed to adequately warn doctors and patients of the increased risk of blood clots posed by use of the NuvaRing. As a result of these allegations, Merck has agreed to settle with the claimants for $100 million. But is the NuvaRing any more dangerous than other hormonal birth control options?
The non-selective beta-blocker propranolol is a leading candidate for repurposing as a novel anti-cancer agent.Emerging evidence, including human data, suggests that there are multiple mechanisms of action particularly relevant to breast cancer.This editorial reviews a number of recent studies that show it has anti-metastatic activity that warrants clinical investigation, including investigation as a potential perioperative therapy in breast cancer.
Abstract The Rho-associated coiled-coil protein kinases (ROCK 1 & 2) are serine/threonine kinases serving as key modulators of processes involving cytoskeletal rearrangement, focal adhesion formation, cell motility, and tumor cell invasion. Deregulation of ROCK signaling has been reported across diverse tumors types and overexpression of ROCK proteins in metastatic tumors directly contributes to their aggressive behavior through stimulating cell migration and epithelial-to-mesenchymal transition. Indeed, a number of published reports from our lab and others indicate that inhibiting ROCK signaling effectively disrupts solid tumor formation, progression, and metastasis. Interestingly, over the past decade more than 20 publications from different groups have reported diverse and often conflicting roles for the ROCK proteins in cell survival in differing tumor types, bringing into question what the overall response would be of a cancer patient who is taking standard chemotherapy combined with ROCK inhibitors. Despite the widespread interest in targeting ROCK signaling for cancer therapy, the molecular mechanisms explaining these conflicting pleiotropic roles for ROCK signaling in cell fate decisions is almost exclusively unknown. In this study, we examined the effects of ROCK inhibition combined with standard chemotherapy regimens on angiosarcoma tumor progression. Pharmacological inhibition of ROCK's kinase activity or shRNA knockdown of ROCK1 or 2 gene expression resulted in decreased angiosarcoma tumor formation and invasion, however led to significantly increased tumor cell survival following cisplatin or busulfan treatment. To determine the molecular mechanisms controlling the pro-survival response of inhibiting ROCK signaling following cytotoxic injury, we performed whole genome microarray analysis revealing that pharmacological inhibition or gene expression knockdown of ROCK proteins led to a statistically significant ablation in the expression of a number of cisplatin and busulfan responsive genes known to be downstream targets of p53 induction, DNA damage response and repair, and cell cycle control. This study provides one of the most detailed explanations at the molecular level as to how ROCK signaling modulates cell survival by regulating gene expression at a global level—a paradox that must be solved before inhibition of ROCK activity can be clinically used as an effective anticancer therapeutic, lest we develop an excellent antimetastatic drug that unfortunately (for some tumor types) enhances tumor resistance to chemotherapies.
Rho family guanine nucleotide exchange factors (GEFs) regulate diverse cellular processes including cytoskeletal reorganization, cell adhesion, and differentiation via activation of the Rho GTPases.However, no studies have yet implicated Rho-GEFs as molecular regulators of the mesenchymal cell fate decisions which occur during development and repair of tissue damage.In this study, we demonstrate that the steady-state protein level of the Rho-specific GEF GEFT is modulated during skeletal muscle regeneration and that gene transfer of GEFT into cardiotoxin-injured mouse tibialis anterior muscle exerts a powerful promotion of skeletal muscle regeneration in vivo.In order to molecularly characterize this regenerative effect, we extrapolate the mechanism of action by examining the consequence of GEFT expression in multipotent cell lines capable of differentiating into a number of cell types, including muscle and adipocyte lineages.Our data demonstrate that endogenous GEFT is transcriptionally upregulated during myogenic differentiation and downregulated during adipogenic differentiation.Exogenous expression of GEFT promotes myogenesis of C2C12 cells via activation of RhoA, Rac1, and Cdc42 and their downstream effector proteins, while a dominantnegative mutant of GEFT inhibits this process.Moreover, we show that GEFT inhibits insulin-induced adipogenesis in 3T3L1 preadipocytes.In summary, we provide the first evidence that the Rho family signaling pathways act as potential regulators of skeletal muscle regeneration and provide the first reported molecular mechanism illustrating how a mammalian Rho family GEF controls this process by modulating mesenchymal cell fate decisions.
