Peptide-guided targeting of GPR55 for anti-cancer therapy

Purpose: Expression of the lysophosphatidylinositol receptor GPR55 correlates with invasive potential of metastatic cells and bone metastasis formation of different types of tumors. These findings suggest a role for GPR55 signaling in cancer progression. Methods: Here, we screened an M13-phage-displayed random library using the bait of HEK293 cells that heterologously expressed full-length HA-GPR55. We selected a set of phagotopes that carried 7-mer insert peptides flanked by a pair of cysteine residues, which resulted in cyclized peptides. Sequencing of selected phagotopes dictated the primary structure for the synthetic FITC-labeled peptide P1, which was analyzed for binding specificity to immunoprecipitated HA-GPR55, and to endogenously expressed GPR55 using cells interfered or not for the receptor. Results and Discussion: Synthetic peptide P1 showed binding specificity towards immunoprecipitated GPR55, with a Kd of 15 ?M. A similar binding affinity was extrapolated in binding assays with intact HeLa cells expressing the endogenous receptor. Loss of peptide-P1 binding was observed upon transient or stable GPR55 RNA interference in HeLa cells, with a recovery of peptide binding upon GPR55 re-expression, indicating that peptide-P1 binding to these cells depends on the GPR55 expression levels. Moreover, peptide P1 binding to GPR55 increased the rate of receptor internalization under steady-state as well as upon agonist stimulation. This led us to speculate that peptide P1 binding to GPR55 can inhibit receptor signaling by decreasing GPR55 content at the plasma membrane or by allosteric modulation of GPR55. Indeed, peptide P1 inhibited the proliferation of two lymphoblastoid cell lines. Conclusions: Our data support the potential therapeutic application of peptide ligands of GPR55 for targeting and inhibiting the growth of neoplastic cells, which overexpress GPR55 and are dependent on GPR55 signaling for their proliferation.