The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer.

BACKGROUND: Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial. METHODS: Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells. RESULTS: Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo. CONCLUSIONS: This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs.

Analysis of the phospholipase C-δ1 pleckstrin homology domain using native polyacrylamide gel electrophoresis.

The phospholipase C (PLC)-δ1 pleckstrin homology (PH) domain has a characteristic short α-helix (α2) from residues 82 to 87. The contributions of the α2-helix toward the inositol 1,4,5-trisphosphate (IP(3)) binding activity and thermal stability of the PLC-δ1 PH domain were investigated using native polyacrylamide gel electrophoresis (PAGE). Native PAGE analyses of gel migration shift induced by IP(3) binding and of protein aggregation induced by heating indicated that disruption of the α-helical conformation by replacement of Lys86 with proline resulted in reduced affinity for IP(3) and in thermal destabilization of the IP(3)-binding state. Although the mutant protein with replacement of Lys86 with alanine showed a slight reduction in thermal stability, the IP(3)-binding affinity was similar to that of the wild-type protein. Replacement of Phe87 with alanine, but not with tyrosine, also resulted in reduced affinity for IP(3) and in thermal instability. These results indicated that the helical conformation of the α2-helix and the phenyl ring of Phe87 play important roles in the IP(3)-binding activity and thermal stability of the PLC-δ1 PH domain. Based on these results, the biological role of the α2-helix of the PLC-δ1 PH domain is discussed in terms of membrane binding.

Pleckstrin homology-phospholipase C-δ1 interaction with phosphatidylinositol 4,5-bisphosphate containing supported lipid bilayers monitored in situ with dual polarization interferometry.

We have determined the kinetics and affinity of binding of PH-PLCδ(1) to the PIP(2) headgroup lipids using an optical surface-sensitive technique in a time-resolved manner. The use of dual polarization interferometry to probe supported lipid bilayers (SLBs) of different compositions allowed determination of accurate affinity constants and a layer structure of the peptide binding to the model membrane platform. In addition, the platform enabled us to monitor the detailed adsorption kinetics characterized by a strong initial electrostatic attraction of the peptide to the SLB surface followed by rearrangement and loss of possibly clustered peptides upon specific binding to the phosphoinositide headgroup. These kinetics differed substantially from adsorption kinetics for nonspecific binding to similarly charged control SLBs.

The small GTPase Ral couples the angiotensin II type 1 receptor to the activation of phospholipase C-delta 1.

The angiotensin II type 1 receptor (AT(1)R) plays an important role in cardiovascular function and as such represents a primary target for therapeutic intervention. The AT(1)R has traditionally been considered to be coupled to the activation of phospholipase C (PLC) beta via its association with G alpha(q/11), leading to increases in intracellular inositol phosphate (IP) and release of calcium from intracellular stores. In the present study, we investigated whether the small GTPase RalA contributed to the regulation of AT(1)R endocytosis and signaling. We find that neither RalA nor RalB is required for the endocytosis of the AT(1)R, but that RalA expression is required for AT(1)R-stimulated IP formation but not 5-HT(2A) receptor-mediated IP formation. AT(1)R-activated IP formation is lost in the absence of Ral guanine nucleotide dissociation stimulator (RalGDS), and requires the beta-arrestin-dependent plasma membrane translocation of RalGDS. G alpha(q/11) small interfering RNA (siRNA) treatment also significantly attenuates both AT(1)R- and 5-HT(2A) receptor-stimulated IP formation after 30 min of agonist stimulation. PLC-delta1 has been reported to be activated by RalA, and we show that AT(1)R-stimulated IP formation is attenuated after PLC-delta 1 siRNA treatment. Taken together, our results provide evidence for a G protein-coupled recepto-activated and RalGDS/Ral-mediated mechanism for PLC-delta 1 stimulation.