Gonadotropin-releasing hormone receptor activates GTPase RhoA and inhibits cell invasion in the breast cancer cell line MDA-MB-231

Abstract

BackgroundGonadotropin-releasing hormone (GnRH) and its receptor (GnRHR) are both expressed by a number of malignant tumors, including those of the breast. In the latter, both behave as potent inhibitors of invasion. Nevertheless, the signaling pathways whereby the activated GnRH/GnRHR system exerts this effect have not been clearly established. In this study, we provide experimental evidence that describes components of the mechanism(s) whereby GnRH inhibits breast cancer cell invasion.MethodsActin polymerization and substrate adhesion was measured in the highly invasive cell line, MDA-MB-231 transiently expressing the wild-type or mutant DesK191 GnRHR by fluorometry, flow cytometric analysis, and confocal microscopy, in the absence or presence of GnRH agonist. The effect of RhoA-GTP on stress fiber formation and focal adhesion assembly was measured in MDA-MB-231 cells co-expressing the GnRHRs and the GAP domain of human p190Rho GAP-A or the dominant negative mutant GAP-Y1284D. Cell invasion was determined by the transwell migration assay.ResultsAgonist-stimulated activation of the wild-type GnRHR and the highly plasma membrane expressed mutant GnRHR-DesK191 transiently transfected to MDA-MB-231 cells, favored F-actin polymerization and substrate adhesion. Confocal imaging allowed detection of an association between F-actin levels and the increase in stress fibers promoted by exposure to GnRH. Pull-down assays showed that the effects observed on actin cytoskeleton resulted from GnRH-stimulated activation of RhoA GTPase. Activation of this small G protein favored the marked increase in both cell adhesion to Collagen-I and number of focal adhesion complexes leading to inhibition of the invasion capacity of MDA-MB-231 cells as disclosed by assays in Transwell Chambers.ConclusionsWe here show that GnRH inhibits invasion of highly invasive breast cancer-derived MDA-MB-231 cells. This effect is mediated through an increase in substrate adhesion promoted by activation of RhoA GTPase and formation of stress fibers and focal adhesions. These observations offer new insights into the molecular mechanisms whereby activation of overexpressed GnRHRs affects cell invasion potential of this malignant cell line, and provide opportunities for designing mechanism-based adjuvant therapies for breast cancer.

SND1 Acts Downstream of TGFβ1 and Upstream of Smurf1 to Promote Breast Cancer Metastasis

“…Correlated with the expression of SND1 and Smurf1 (Fig. 6E) RhoA plays essential roles in maintaining the assembly of focal adhesions and contractile actin-myosin stress fibers (26). The lack of RhoA can lead to F-actin cytoskeleton disorganization.…”

Roles of Ras Homolog A in Invasive Ductal Breast Carcinoma

“…Rho GTPases regulate cytoskeleton dynamics, cell migration, malignant transformation, cell polarity, invasion, and metastasis [17, 28], and the overexpression of these proteins has been observed in various human neoplasms, suggesting aberrant regulatory mechanisms [17]. Poor prognosis in breast cancer is attributed to invasion and metastases, particularly distant metastases [2, 7, 34]. EMT is one theory explaining tumor invasion and metastasis [35].…”

“…Although RhoA activation downregulates E-cadherin expression and induces EMT in metastatic cancer cells [33], the correlation between RhoA expression and EMT, which is suggested by the negative E-cadherin expression and the positive vimentin expression, has not been found. The metastatic process comprises an ordered series of events in which the acquisition of a motile and invasive phenotype to penetrate the ECM is an important determinant of the invasive potential of tumor cells [2, 4]. Actin polymerization (F-actin), which is induced by RhoA, a small GTPase, is involved in cell migration in a breast cancer cell line [2].…”

“…The metastatic process comprises an ordered series of events in which the acquisition of a motile and invasive phenotype to penetrate the ECM is an important determinant of the invasive potential of tumor cells [2, 4]. Actin polymerization (F-actin), which is induced by RhoA, a small GTPase, is involved in cell migration in a breast cancer cell line [2]. The mechanisms of Rho regulation, including a kinase cascade triggered by growth factor stimuli and integrin-ECM interaction-mediated focal adhesion and stress fiber formation, have been described [5].…”

Characterization of 12 GnRH peptide agonists – a kinetic perspective

“…This decapeptide binds to a class A GPCR, namely, the GnRH receptor located mainly on pituitary gonadotrophs. Along with the pituitary, GnRH receptors are expressed in reproductive tissues, both normal and malignant, such as those of the prostate and mammary gland (Kakar et al ., ; von Alten et al ., ; Angelucci et al ., ; Aguilar‐Rojas et al ., ). Upon receptor activation, the gonadotropins luteinizing hormone (LH) and follicle‐stimulating hormone (FSH) are synthesized and secreted from gonadotrophic cells.…”