Children diagnosed with sonic hedgehog-activated MB (SHH MB) displaying desmoplastic histology have a good prognosis, while those with non-desmoplastic histology have higher rates of metastasis and an intermediate prognosis, indicating that additional factors to SHH activation account for the clinical dichotomy observed [2–4, 17]. Recent evidence shows that CXCR4 signaling, which is critical to the proliferation and migration of granule neuron precursors during development, is dependent on SHH for its activation in MB [13, 18, 30, 31]. This finding, coupled to CXCR4’s reported role in tumor progression [14, 16, 32, 33], point towards dysregulated CXCR4 signaling as a possible key determinant of SHH MB clinical behavior. We observe high co-expression levels of CXCR4 and CXCL12, as well as PDGFRA, distinctly associated with SHH MB. Herein, we demonstrate that GF receptor/PDGFR and Src, a major GF/PDGFR downstream effector, act to suppress the expression and stability of the G protein-coupled receptor kinase GRK6 in MB cells, which in turn functions to maintain CXCR4 signaling and promote cell migration, thereby identifying a new mechanism for the dysregulation of this signaling axis in MB. This finding has important implications for our understanding of SHH MB clinical behavior and potential translation to therapeutic targeting.
GPCRs are desensitized by agonist-induced GRK-mediated phosphorylation, whereby the receptors are uncoupled from heterotrimeric G-protein signaling. Suppression of GRKs, and loss of GRK-mediated desensitization, can result in the prolonged activation of GPCRs. The involvement of CXCR4 in cancer metastasis appears to be due to dysregulation of the receptor leading to enhanced CXCR4 signaling [14, 24]. In breast cancer cells, a similar functional relationship between the PDGFR pathway and CXCR4 has been reported, whereby overexpression of PDGF-D, which specifically binds to and activates PDGFRB, was shown to induce CXCR4 expression and promote lymph node metastasis . GRKs can also regulate EGFR and PDGFR activity, and in turn, GRKs may be regulated at the mRNA and protein level by altered oncogenic receptor signaling .
The description of GRK6 expression and its functional role in cancer is very limited, and until now, has not been reported in MB. In this study, we found that the percentage of GRK6 expression is lower in MB tumors with metastasis (22%), compared to those without metastasis (43%); however, these data revealed only a trend in MB, with the difference not being statistically significant due to the small sample size. GRK6 typically has a negative regulatory role in CXCR4 activation and CXCL12-induced cell migration [24, 26]. For example, GRK6 deficiency is associated with impaired desensitization and enhanced CXCR4-mediated neutrophil migration and has been implicated in the pro-inflammatory response seen in rheumatoid arthritis [26, 36]. However, in HeLa cells, siRNA-based functional screening identified GRK6 as a critical positive regulator of integrin-mediated cell adhesion and migration . Similarly, GRK6 silencing in myeloma cells induced a tumor suppressor effect by inhibiting STAT3 phosphorylation and decreasing tumor cell survival .
To date, we have little knowledge regarding the regulation of GRK6. Herein, we demonstrate that GF/PDGFR-Src activation results in decreased expression of GRK6 at the transcriptional and post-translational level to maintain optimal CXCR4 signaling. In fact, we found that Src, a key mediator of PDGFR signaling and other GF-induced pathways , can independently regulate GRK6 expression, indicating that Src could be a critical therapeutic target in MB, especially given its additional role as a central node in other pro-migratory and pro-survival signals. This therapeutic potential is further illustrated in our study, which shows that targeting CXCR4 with the inhibitor AMD3100, can be potentiated by the overexpression of GRK6. Since we show that Src suppresses GRK6, Src inhibitors could potentially be used to elevate levels of suppressive GRK6. Given that we have previously shown that PDGFR can regulate Rac1-Pak1 signaling important for cytoskeletal rearrangements required for MB cell migration , it remains to be seen whether the combined inhibition of PDGFR-Src-CXCR4 may act synergistically to suppress SHH MB growth and progression. In our study, the observation that specific alteration of GRK6 did not itself impact MB cell growth indicates that GRK6 is a critical mediator of GF receptor/PDGFR-Src oncogenic signaling for CXCR4-mediated migration, but is not essential for maintaining CXCR4-mediated growth. Rather, other mechanisms, and perhaps other GRKs may be necessary to regulate growth. Although we focused on GRK6 in this study because of its apparent dysregulated expression in metastatic MB, it is possible that other GRKs that we found expressed by MB (i.e. GRK4 and 5) may also play a role in MB growth and progression. For example, PDGFR/Src has been shown to regulate GRK2 activity in other cell types and the suppression of GRK3 appears necessary for maximal glioblastoma cell growth [35, 39]. Further studies will be necessary to investigate the potential functional role and regulation of GRK4 and GRK5 in MB as well as the effect of targeting GRKs and PDGFR-Src dysregulation of the CXCR4 signaling axis on MB progression in vivo.