GRK2 promotes growth of medulloblastoma cells and protects them from chemotherapy-induced apoptosis

Anup S Pathania,Xiuhai Ren,Gregory M Shackleford,Anat Erdreich-Epstein,Min Y Mahdi

doi:10.1038/s41598-019-50157-5

Anup S Pathania, Xiuhai Ren + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-50157-5

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Abstract

G-protein coupled receptor kinase 2 (GRK2; ADRBK1, BARK1) is most known as a regulator of G-protein coupled receptors. However, GRK2 also has other functions. Medulloblastomas are the most common malignant brain cancers in children. GRK2 has not been implicated in medulloblastoma biology. Here we report that GRK2 knockdown slowed cell growth, diminished proliferation, and enhanced cisplatin- and etoposide-induced apoptosis in medulloblastoma cell lines UW228-2 and Daoy. Reciprocally, GRK2 overexpression attenuated apoptosis induced by these chemotherapy drugs. Cisplatin and etoposide increased phosphorylation of AKT (S473) and GRK2 knockdown mitigated this increase. Cisplatin and etoposide attenuated ERK phosphorylation, but GRK2 knockdown did not alter this effect. Wildtype GRK2 reversed the increase in cisplatin- and etoposide-induced apoptosis caused by GRK2 knockdown. GRK2-K220R (kinase dead) and GRK2-S670A (unphosphorylated, constitutively active) conferred protection from cisplatin that was similar to wildtype GRK2, suggesting that this protection may be mediated though a kinase-independent activity of GRK2. These data demonstrate that GRK2 contributes to proliferation and survival of these medulloblastoma cell lines and to their protection from cisplatin- and etoposide-induced apoptosis.

Highlights

G-protein coupled receptor kinase 2 (GRK2) belongs to the GRK family of serine/threonine kinases and is largely known as a regulator of G-protein coupled receptor (GPCR) signaling, including the β2 adrenergic receptor[1,2,3,4,5]
The decreased S-phase following GRK2 knockdown in both cell lines indicates decrease in proliferation compared to controls (Fig. 1D,E). siGRK2 knockdown did not affect protein level of its closest family member, GRK3 (Suppl Fig. S2), strengthening our conclusion that GRK2 contributes to growth and proliferation of medulloblastoma cell lines UW228 and Daoy
We examined the reciprocal scenario of GRK2 knockdown followed by treatment with cisplatin using cells infected with RDAV virus carrying miRE.GRK2 knockdown constructs compared to miRE.Renilla controls: cisplatin induced apoptosis (AnnexinV/7AAD) of control UW228 and Daoy cells, and this apoptosis was augmented in cells with GRK2 knockdown (Fig. 3)

Summary

Introduction

G-protein coupled receptor kinase 2 (GRK2) belongs to the GRK family of serine/threonine kinases and is largely known as a regulator of G-protein coupled receptor (GPCR) signaling, including the β2 adrenergic receptor[1,2,3,4,5]. Group 4 medulloblastomas lack a defining single somatic gene mutation, and like Group 3, are associated with a variety of molecular alterations and signaling pathways, some of which overlap with Group 3 tumors[37,41,43,46,47]. In the present study we demonstrate for the first time a pro-survival role for GRK2 in two SHH subgroup medulloblastoma cell lines and GRK2-mediated mitigation of their cisplatin- and etoposide-induced apoptosis

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