β-Adrenergic receptors suppress Rap1B prenylation and promote the metastatic phenotype in breast cancer cells

Abstract

Keywords: adenosine receptor, breast cancer, cholera toxin, G protein-coupled receptor, protein isoprenylation, protein kinase A (PKA), Ras-related protein 1 (Rap1), b-adrenergic receptor Abbreviations: Adenosine 2B Receptor (A 2 BR), b-adrenergic receptor (bAR), Bay 60-6583 (Bay), Cholera toxin (Ctx), half time (T 1/2 ), Isoproterenol (Iso), G protein-coupled receptors (GPCR), Phosphate buffered saline (PBS), Protein kinase A (PKA), Propranolol (Prop).A greater understanding of the molecular basis of breast cancer metastasis will lead to identification of novel therapeutic targets and better treatments. Rap1B is a small GTPase that suppresses the metastasis of breast cancer cells by increasing cell-cell adhesion. In breast cancer, a decrease in Rap1B prenylation and subsequent loss of Rap1B at the plasma membrane decreases cell-cell adhesion and increases cell scattering, which promotes the metastatic phenotype. Protein kinase A (PKA) was recently found to phosphorylate Rap1B and inhibit its prenylation. PKA is activated by G protein-coupled receptors (GPCR) that stimulate Ga s . In this study, we investigated whether the general Ga s activator, cholera toxin, and agonists of the b-adrenergic receptor (bAR), which is a Ga s -coupled GPCR, promote Rap1B phosphorylation and inhibit its prenylation. We show here that cholera toxin and bAR activation phosphorylate Rap1B and inhibit its prenylation and membrane localization, reducing cell-cell adhesion and promoting cell scattering. Furthermore, we report that breast cancer cell migration is decreased by the FDA-approved b-blocker, propranolol. Pharmacological targeting of GPCRs, especially those such as the bAR that are regulated by FDA-approved drugs, to increase cell adhesion and decrease cell scattering could provide a promising therapeutic approach to reduce breast cancer metastasis.

Differences in the Phosphorylation-Dependent Regulation of Prenylation of Rap1A and Rap1B

“…Since prenylated Rap1 migrates faster than non-prenylated Rap1 in SDS-PAGE, the prenylated and non-prenylated forms of Rap1 can be detected by a migration shift in immunoblots 7, 16 . In agreement with our previous results 7, 8 , myc-Rap1B is approximately 75% prenylated (Figure 7A, lane 5), while phosphodeficient myc-Rap1B-AA is almost fully prenylated (Figure 7A, lane 6). Phosphomimetic myc-Rap1B-EE stays in a non-prenylated form (Figure 7A, lane 7) that migrates with the prenylation deficient Rap1B-SAAX mutant (Figure 7A, lane 8).…”

“…As expected, the mutation of S179 (Rap1B-ES) to the phosphomimetic form inhibits the prenylation of Rap1B, as it runs with the higher non-prenylated band of myc-Rap1B-WT and the single non-prenylated band for myc-Rap1B-EE, which is always non-prenylated. Intriguingly, the mutation of S180 (Rap1B-SE) to the phosphomimetic form also inhibits prenylation of Rap1B, supporting our previous studies showing the double mutation Rap1B-EE inhibits prenylation of Rap1B 7, 8 but conflicting with our model in Figure 5B showing that only S-179, and not S-180, is involved in binding to SmgGDS-607. As further support that phosphorylation of both amino acids are important for binding to SmgGDS-607, only the double mutant Rap1B-EE trends towards a significant decrease in binding to SmgGDS-607, while neither individual phosphomimetic mutant has any significant change in binding to SmgGDS-607 (Figure 7C).…”

“…We reported that S-179 and S-180 in Rap1B are phosphorylated by treating cells with the general Gα s activator Cholera toxin (Ctx), or by activating Gα s protein-coupled receptors (GPCRs) using the adenosine receptor agonist 5′- N -Ethylcarboxamidoadenosine (NECA), or the β-adrenergic receptor agonist Isoproterenol (Iso). This phosphorylation inhibits binding of newly synthesized Rap1B to SmgGDS-607, prevents Rap1B prenylation, and diminishes the membrane localization of Rap1B 7, 8 . In contrast to Rap1B, which has two serines that can be phosphorylated in its PBR, Rap1A contains only one serine in its PBR, S-180, which is also phosphorylated by PKA 9, 10 .…”

Repurposing Drugs in Oncology (ReDO)—Propranolol as an anti-cancer agent

“…There is evidence to suggest that loss of Rap1B at the plasma membrane decreases cell-cell adhesion and may promote a metastatic phenotype [117–118]. MDA-MB-231 breast cancer cells showed reduced cell-cell adhesion in response to isoproterenol and an increase in cell migration, whereas treatment with PRO reduced the level of migration [119]. …”

Propranolol and survival from breast cancer: a pooled analysis of European breast cancer cohorts

“…Numerous in-vitro studies have demonstrated that beta-blockers can disrupt migratory activity and inhibit angiogenesis of cancer cells [4, 5]. In particular, propranolol appears to have potent anti-migratory and anti-angiogenic properties as demonstrated in cancer cell lines and animal models [4–10]. This preclinical evidence has led to calls for randomised controlled trials of propranolol as adjuvant therapy in breast cancer patients [11]; however, although early phase trials are underway [12, 13], phase 3 trials have not been conducted to date.…”