Medulloblastoma, the most common malignant pediatric brain tumor, arises from multiple cerebellar cell lineages and can be driven by several genetic abnormalities. Recent gene expression profiling and immunohistochemical studies have elucidated 4 distinct molecular subgroups with unique expression profiles (3
). Three subgroups are characterized by abnormal activation of the Wnt, SHH, or Myc pathways. The fourth subgroup includes tumors whose molecular foundations are not yet specified.
Clinically, Wnt tumors carry the best prognosis and those medulloblastoma with MYC alterations, the worst (34
). SHH tumors exhibit an intermediate outcome (1
). Identifying the pathways altered in each of these tumor subgroups is necessary for the development of novel and molecularly targeted therapies and in addition, promises to improve outcome through molecular stratification of patients for clinical trials. In this regard, identification of a medulloblastoma as belonging to the SHH subgroup may not provide complete characterization. SHH pathway activation alone does not determine medulloblastoma biology. SHH medulloblastoma can include low-risk desmoplastic tumors or more aggressive anaplastic disease. Hence, secondary pathways, acting in concert with SHH must also determine biology within the SHH subtype. In the current study, we show that the level of CXCR4 expression further divides the SHH group of medulloblastoma into relevant subsets. We identified 2 medulloblastoma populations with either high or low CXCR4 expression. These 2 subsets of SHH-driven medulloblastoma possess distinct molecular, epidemiologic, and histologic profiles. CXCR4-high tumors are predominantly either desmoplastic or classic histology and occur most frequently in the youngest patients. In contrast, CXCR4-low tumors more commonly occur in older patients and exhibit classic or large cell anaplastic histology. Thus, CXCR4 expression may distinguish populations of SHH medulloblastoma with different prognoses.
The impact of CXCR4 in the SHH subgroup of medulloblastomas appears to be a consequence of unique molecular interactions that occur between these pathways. Our data suggest that SHH regulates CXCR4 activity at one of the earliest stages of its signaling. While SHH did not affect overall CXCR4 levels, prolonged inhibition of the SHH pathway reduced the cell surface fraction of CXCR4 and abrogated cellular responses to CXCL12. Cell surface localization of CXCR4, like other GPCRs, is regulated through desensitization, involving receptor phosphorylation and arrestin-mediated internalization, followed by either degradation or recycling to the cell surface (35
). SHH could potentially affect any of these processes. Recent studies have highlighted the complexity of such heterologous regulation of GPCR internalization/ recycling. Yu and colleagues showed that ligand-induced activation of Neurokinin 1 receptors (NK1R) produced a nonreciprocal inhibition of ligand-induced mu-opioid receptor endocytosis as a consequence of NK1R-dependent sequestration of arrestins on endosome membranes (38
). In addition, nerve growth factor (NGF) has been shown to modulate the recycling of delta-opioid receptors to the surface membrane of central synaptic terminals (39
We previously described alterations in CXCR4 signaling as a consequence of reduced desensitization in astrocytes null for the tumor suppressor neurofibromin
; ref. 22
). Loss of Nf1
resulted in hyperactivation of the RAS-MAP kinase pathway and resultant ERK-dependent inhibition of GRK2, a kinase involved in CXCR4 desensitization. GRK2 inhibition was correlated with reduced phosphorylation of CXCR4 and sustained CXCL12-induced suppression of cAMP. Interestingly, GRK2 and β-arrestin 2 also directly interact with Smo and regulate SHH-signaling (40
). Thus, activation of the SHH pathway might modulate CXCR4 signaling through regulation of the desensitization machinery, including GRK2 and β-arrestin 2.
The biologic significance of interactions between the SHH and CXCR4 pathways may lie in the unique transcriptional profile of the CXCR4-high tumors. PPP2R2C is the regulatory subunit of the phosphatase PP2A, which enhances SHH-driven proliferation by stabilizing N-myc (42
). In addition, PP2A blocks differentiation within the granule lineage by inactivating S6 kinase (33
). SHH directly induces expression of the catalytic subunit of PP2A and here we show that CXCR4 suppresses expression of the regulatory subunit of PP2A. Both effects could increase PP2A activity and promote proliferation.
Cyclin D1 is a regulator of cyclin-dependent kinases such as CDK4/CDK6, which are critical for G1 to S transition and cell-cycle progression. Induction of cyclin D1 expression is essential for SHH-induced proliferation (43
). CXCR4 enhanced cyclin D1 expression in the SHH-medulloblastomas and this is likely to be a critical component of CXCR4's positive effects on tumor growth. Furthermore, CXCR4's effect on cyclin D1 expression suggests that high CXCR4 expression and/or activation may feed back and augment SHH signaling.
Significantly, CXCR4 activation appeared to be necessary for maximal proliferation of SHH-driven tumors. In SmoA1
tumor models, the CXCR4 antagonist AMD3100 significantly inhibited tumor growth. These data indicate that dual inhibition of SHH and the CXCR4 pathways may provide additional benefit in treating those SHH subtype medulloblastomas that also express CXCR4. A recent clinical report described a single patient with medulloblastoma whose tumor was initially sensitive to SHH antagonist therapy, but who subsequently experienced an aggressive recurrence because of mutation in Smo
). The ability of AMD3100 to inhibit the growth of SmoA1
-driven tumors suggests that perhaps CXCR4 antagonism might address this mechanism of resistance.
The interplay between the SHH and CXCR4 pathways could function in normal development and cancer biology in any circumstances where the elements of both pathways are coexpressed. In addition to medulloblastoma, components of the CXCR4 and SHH pathways are expressed in primary specimens of breast, pancreatic, and prostate cancers as well as in gliomas and rhabdomyosarcomas (24
). It will be important to determine whether coexpression of these pathways provides additional prognostic value and whether combined antagonism of these pathways may have added benefit in the treatment of these cancers. Defining how SHH and CXCR4 modulate each other's signaling is likely to open up new avenues of inquiry in each of these fields and new approaches to treating any cancer in which both pathways are operative.