Targeted therapy of cancer Targeted therapy of cancer relies on the identification of new molecular targets for anti-cancer drugs. This study is aimed at verifying whether the G16 signalling protein is involved in cancerogenesis and hence it can represent a potential target for cancer therapeutics. Human G16 (and murine orthologue G15) belongs to the superfamily of heterotrimeric G proteins. It couples seven transmembrane receptors (GPCRs) to the stimulation of phospholipase Cβ (PLCβ) activity, thereby leading to the intracellular accumulation of inositol tris-phosphate (IP3), Ca2+, and diacylglycerol. Compared to other G proteins G16/15 displays unique features. Firstly, it shows an expression pattern restricted only to hematopoietic cell precursors. Secondly, it is capable of indiscriminate coupling to most GPCRs. Thus, even GPCRs that do not normally signal through PLCβ do so when co-expressed in transfected COS-7 cells with the alpha-subunit of G15 (Galpha15). More intriguingly, we have recently found that GPCRs co-transfected with Galpha15 /16 become refractory to desensitization induced by agonist exposure or arrestin over-expression. In addition, we have detected the expression of Galpha16 mRNA and protein, using RT-PCR and western blotting, in human cancer cell lines, such as pancreatic and mammary carcinomas, melanoma, colon-rectal cancer, and higher levels of Galpha16 mRNA in 7 out of 8 pancreatic tumor biopsies than adjacent healthy tissues.
Based on these findings the objectives of the project are:
(1) to verify whether the signalling of GPCRs via G16 in cancer cells is resistant to agonist- or arrestin-induced desensitization, thus generating a sustained activation of PLC-gamma and downstream effectors instead of a ‘normal’ transient signalling;
(2) to study the biological consequences of G16 signalling, by comparing proliferation rate, degrees of neoplastic transformation and invasiveness, between G16-positive and knocked down cancer cells and between G16-negative and -transfected cells;
(3) to clarify the molecular basis underlying G15/16-induced resistance to desensitization and to identify the downstream signalling pathway(s) activated by G16 in cancer cells (e.g. MAPK, NFkB, etc.), through comparisons of G16-negative and -positive cells.
Our study combines a variety of experimental approaches, ranging from siRNA technology to knock down G16 gene expression, to fluorescence-resonance energy transfer (FRET) technique coupled to microscopy, to visualize protein-protein interactions in living cells, to assays of second messenger accumulation (cAMP, IPs and Ca2+) and protein phosphorylation (using phospho-specific antibodies).
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