Blockade of Gq Coupled P2Y6 Receptor Inhibited Myeloma Cells Proliferation and Survival Via Suppression of Camkiiγ-NF-Κb Pathway

Background:

The purinergic receptor P2Y6 receptor (P2Y6R) is a member of the G protein-coupled receptors family, which is widely expressed in various cell types and plays a critical role in physiological and pathological processes, where it is activated by extracellular uridine diphosphate (UDP) and mobilizes Ca²⁺ via the Gαq/11 protein pathway. Growing lines of evidence indicate that an aberrant P2Y6 receptor-mediated Ca²⁺ signaling may be involved in numerous oncogenic processes, including cancer tumorigenesis, metastasis and chemotherapeutic drug resistance. Unfortunately, the roles of P2Y6R and corresponding Ca²⁺ signaling in multiple myeloma (MM) have not been explored so far. Our previous studies have demonstrated that P2Y6R is abnormally highly expressed in MM and promotes the proliferation of myeloma cells. However, the effects of P2Y6R on the pathogenesis of MM and its possible mechanisms remain unclear.

Aims:

We aimed to assess for the first time the effects of P2Y6R on the proliferation and survival of MM cells and elucidate the molecular mechanism by which P2Y6R/Ca²⁺ regulates the growth of MM cells.

Methods:

We examined the expression levels of P2Y6R on MM cells using our patient cohort and myeloma cell lines by quantitative real-time PCR, western blotting, and immunofluorescence. P2Y6shRNA, specific antagonist MRS2578 and P2Y6R overexpressed MM cell lines were used to explore the functional roles of P2Y6R in MM pathogenesis. In order to clarify the underlying mechanism, human UDP ELISA kit was used to detect the concentration of UDP in bone marrow plasma of MM patients and healthy donor and the intracellular calcium signals activated by UDP were detected by Ca²⁺ indicator Calbryte™ 520. RNA sequencing was performed to investigate the downstream mechanisms and in vivo, the mouse xenograft model was used to determine the role of P2Y6R in the proliferation of MM cells.

Results:

Our findings demonstrated that P2Y6R was highly expressed in MM and inhibition of P2Y6R with antagonist MRS2578 or shRNA inhibited the proliferation of MM cells in vitro and in vivo. Moreover, overexpression of P2Y6 receptor promoted the growth of MM cells in vitro and in vivo. RNA seq results performed on P2Y6R knockdown MM cells showed that calcium signaling pathway and NF-κB signaling pathway were significantly inhibited. Furthermore, the inhibition of NF-κB signaling pathway related proteins by P2Y6shRNA and MRS2578 were determined by western blotting. In comparison with healthy donor, the concentration of UDP was significantly higher in bone marrow plasm of MM patients. UDP activated the P2Y6R inducing elevation of intracellular Ca²⁺ levels in MM cells, which can be reversed by P2Y6 shRNA. In addition, UDP treatment or overexpression of P2Y6R induced the phosphorylation of NF-κB as well as Ca²⁺/calmodulin-dependent protein kinase II γ (CaMKIIγ) and proliferation in MM cells. CaMKIIγ inhibition decreased the phosphorylation of NF-κB and MM cells growth.

Conclusion:

In conclusion, our study uncovers the possible roles of P2Y6R prevented tumor apoptosis via inducing intracellular Ca²⁺ elevation, enhancing the phosphorylation of CaMKIIγ and regulating the activity of NF-κB. Thus, P2Y6R might be a potential target against multiple myeloma.