A PDK1-dependent regulation of PLCγ1 activation is essential for cancer cell migration and invasion

Phospholipase Cγ1 (PLCγ1) is highly expressed in several tumours such as breast carcinomas and has been found overexpressed in metastases compared to primary tumour in breast cancer patients, indicating that PLCγ1 may be important in tumourigenesis and metastasis dissemination. PLCγ1 involvement in cancer cell motility and in cancer cell Matrigel invasion was investigated. Downregulation of PLCγ1 protein expression by siRNA or shRNA inhibited Matrigel cell invasion of highly invasive breast and melanoma cancer cell lines. Furthermore PLCγ1 protein downregulation inhibits intracellular calcium mobilisation upon EGF stimulation demonstrating the essential role of PLCγ1 in EGF-induced calcium release. In the effort to identify specific PLCγ1 inhibitors, Inositol (1,3,4,5,6) pentakisphosphate (InsP5) and 2-O-Bn-InsP5, a synthetic compound based on InsP5 structure, were tested on Matrigel cell invasion and PLCγ1 activity. I found that InsP5 and 2-O-Bn-InsP5 reduce cell migration and Matrigel invasion in breast and melanoma cancer cell lines, with a complete inhibition displayed by 2-O-Bn-InsP5 treated cells. Furthermore InsP5 and 2-O-Bn-InsP5 treatment reduces calcium release upon EGF stimulation, with a complete inhibition showed by 2-O-Bn-InsP5 treated cells, suggesting a potential inhibition on PLCγ1 activity. Analysis of PLCγ1 phosphorylation in tyrosine 783 residue revealed that 2-O-Bn-InsP5 inhibits EGF-induced PLCγ1 tyrosine phosphorylation. Kinase profile assay, performed in vitro to test the inhibitory effect of InsP5 and 2-O-Bn-InsP5 on different kinases, showed a specific inhibition by 2-O-Bn-InsP5 of the 3-phosphoinositide-dependent-protein kinase 1 (PDK1) with an IC50 of 26 nM. Knock down of PDK1 using siRNA and shRNA in MDA-MB-231 showed impairment in cell migration and Matrigel invasion and inhibition of EGF-induced calcium mobilisation. Co-immunoprecipitation and FRET analyses showed that PLCγ1 and PDK1 associate in a protein complex. My finding identified a novel pathway which involves PDK1 in PLCγ1 activation. Furthermore this work highlights PLCγ1 as a potential therapeutic target to prevent metastases spreading and identified 2-O-Bn- InsP5 as a leading compound for development of anti-metastatic drugs.EThOS - Electronic Theses Online ServiceGBUnited Kingdo