Medulloblastoma/Primitive Neuroectodermal Tumors in the Brains of Children

Rapid adoption of technologies for the investigation of the molecular make-up of tumors such as array comparative genomic hybridization as well as mRNA and miRNA expression profiling has occurred in the study of embryonal tumors including medulloblastoma, CNS PNET, and atypical teratoid/rhabdoid tumor. These tools have been used to demonstrate unique genetic signatures not only between histologically distinct tumors, but also in subgroups established by unbiased grouping of expressed genes or chromosomal gains and losses when comparison is made between samples of a specific tumor type derived from different patients.

Currently, medulloblastoma is classified according to histological features into four different types: classic, desmoplastic/nodular, extensive nodularity, and large cell/anaplastic (35). Specific and critical cellular signaling pathways have been linked to distinct histopathological subgroups, giving insight into both tumor pathogenesis and potential new therapies.

 

 

Classical medulloblastoma has been associated with expression of Wnt family genes, nuclear translocation of beta-catenin (36), and loss of chromosome 6 (37).

Desmoplastic/nodular types are found to harbor aberrant expression of hedgehog pathway genes. Large cell/anaplastic medulloblastoma has been uniquely linked to enhanced MYC expression and a photoreceptor type of gene expression profile. Heterogeneity in gene expression within individual histological subtypes is present, as expression of neuronal differentiation genes can be observed in some tumors within classic, desmoplastic/nodular, or large cell/anaplastic histological subtypes.

The finding of mutations in members of the Wnt signaling pathway in sporadic medulloblastoma (38) has led to further characterization of this pathway in medulloblastoma. As noted above, a specific subgroup of patients with medulloblastoma harbor tumors that have changes in Wnt family gene expression, specifically involving a combination of increased expression of inhibitors of Wnt signaling (e.g., WIF1, DKK1, DKK2, AXIN2) and potential promoters of Wnt signaling (e.g., WNT16, LEF1) (41). Activation of Wnt signaling, which occurs in up to 25% of medulloblastomas, has been correlated with good outcome (42); however, this does not mean that altered Wnt pathway signaling in medulloblastoma is impairing tumor aggressiveness. The role of concurrent chromosome 6 loss, which is intimately related to activated Wnt signaling in this subgroup of good-prognosis patients, remains undefined. Activated Wnt pathway signaling is believed to play a role in metastatic potential in prostate cancer and is associated with tumor progression in non-small cell lung cancer (45). Furthermore, Wnt pathway activation can result in downstream expression of VEGF, which plays an important role in tumor angiogenesis.

Wnt signaling can occur through two distinct pathways; one that is dependent on beta-catenin (canonical) and the other that is beta-catenin independent (non-canonical). The Wnt family of proteins is a group of secreted glycoproteins (19 human genes) that bind a heterodimeric cell surface receptor formed by the Fizzled protein and the low-density lipoprotein receptor-related proteins 5/6 (LRP5/6). Binding of the ligands results in recruitment of the protein disheveled (Dvi) to the membrane, where it is activated by phosphorylation. Activation of Dvi causes the dissociation of glycogen synthase kinase 3β (GSK-3β) from Axin. Axin is a scaffolding protein that anchors GSK-3B to adenomatous polyposis coli (APC) and casein kinase 1α (CK1α) in a complex that phosphorylates beta-catenin, marking it for recognition by B-transducin-repeat-containing protein (β-TrCP) and destruction by the ubiquitin-proteosome pathway. Lack of formation of the beta-catenin phosphorylation complex therefore results in reduced beta-catenin destruction. Beta-catenin is able to translocate into the nucleus where it regulates the activity of the Tcf/Lef (T cell factor and lymphoid-enhancing factor) transcription factors. In patients with familial adenomatous polyposis, inactivating mutations in the APC gene predispose not only to colorectal cancer, but also to CNS tumors such as medulloblastoma (48). APC, through its inactivating role in Wnt signaling, is thought to be critical for regulation of apoptosis (49). In the non-canonical pathway ligand binding to the Frizzled –LPR5/6 receptor complex results in heterotrimeric G protein-mediated activation of phospholipase C, which cleaves inositol triphosphate into diacylglycerol and phosphatidylinositol diphosphate and results in increased intracellular calcium. The link between non-canonical Wnt signaling and regulation of intracellular calcium is important as calcium is known to participate in multiple cellular processes including proliferation, differentiation, and apoptosis. Multiple tumor-promoting pathways can be triggered by rising intracellular calcium, and there is growing interest in calcium modulation as a potential anti-cancer strategy (36). A Wnt-mediated increase in intracellular calcium has been shown to signal nuclear factor of activated T-cells (NF-AT) transcriptional regulation in a calcium-camodulin kinase II/calcineurin-dependent manner as well as activate TAK1-NLK kinases. The heterotrimeric G protein can also stimulate p38 kinase and activates phosphodiesterase 6, which hydrolyzes cyclic guanosine monophosphate (cGMP), resulting in inactivation of protein kinase G. Cross-talk exists between the canonical and non-canonical pathways such that Wnt5a activation of the non-canonical pathway can trigger the inhibition of canonical pathway signaling (52). Inhibition of Wnt signaling by secreted Frizzled-related proteins has been demonstrated to suppress medulloblastoma growth in vitro and in flank and intracranial tumor xenograft models (53). The genes encoding these proteins are also found to be epigenetically silenced by promoter methylation in medulloblastoma. Wnt signaling in medulloblastoma can also be antagonized by the secreted protein Dikkopf1 (Dkk1), which is also under epigenetic regulation (54).

Targeting the canonical pathway has been proposed as a viable anti-tumor strategy. Potential therapeutics that have demonstrated inhibitory activity in this pathway include nonsteroidal anti-inflammatory drugs, quercetin (57), monoclonal anti-Wnt1 antibody (58), soluble Wnt receptor (59), and small molecule and peptide inhibitors of the Fz-Dvl interaction. Co-expression of lactate dehydrogenase B and beta-catenin identifies medulloblastoma patients with poor outcome (62). Furthermore, lactate dehydrogenase B expression has been shown to be a downstream target of mTOR signaling in human cancer (63). These findings highlight the potentially complex interaction between multiple signaling pathways driving medulloblastoma aggressiveness and also point to the potential role for targeting multiple pathways such as the RTK/PI3K/AKT/mTOR concurrently with Wnt signaling.

Genetic analysis of patients with Gorlin’s syndrome who have increased risk of medulloblastoma has revealed germline mutations in a gene known to be important for cell fate, patterning, and growth regulation in Drosophila called the human homologue (ptch-1) of Drosophila patched. This gene is located on chromosome 9q22.3 and is expressed in mammalian embryonic development in the ventral neural tube, somites, and limb buds (66). The gene product (PTCH) is a transmembrane protein whose loss of function is associated with the spectrum of anomalies in Gorlin’s syndrome including congenital defects (bifid ribs, ectopic calcification, spina bifida occulta), basal cell carcinoma, rhabdomyosarcoma, and medulloblastoma. Ptch-1 was the first tumor suppressor gene identified in human tumors, and its gene product is involved in a complex signaling pathway known as the hedgehog pathway. Three extracellular lipid modified ligands for PTCH exist – sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh) – and these factors play a key role in embryonic development (67). Upon binding to PTCH these ligands relieve the inhibition of PTCH on a seven transmembrane domain protein called Smoothened (SMO). The disinhibited SMO protein is thought to migrate into primary cilia, undergoes a conformational change, and signals the activation of Gli transcription factors (68).

 

 

Sonic hedgehog pathway

Gli activation can be regulated by multiple kinases (69) with inactivating mutation identified in one of these regulators called suppressor of fused (SUFU) in medulloblastoma (70). As zinc-finger DNA binding proteins, Gli transcription factors recognize the 5’-GACCACCCA -3’ consensus sequence in the promoter region of target genes (71). There are three mammalian Gli transcription factors, with Gli1 and Gli2 having gene-activating function while Gli 3 has a mainly gene-repressing function (69). Hedgehog pathway gene regulation positively regulates proliferation by activation of cyclins and cyclin-dependent kinases (CDK), which are molecules that drive the cell cycle (72). B-cell lymphoma 2 protein, which signals inhibition of apoptosis is also upregulated (73). Activation of hedgehog pathway by loss of function mutations in Ptch-1 or SUFU is found in desmoplasmic and large cell/anaplastic medulloblastoma. Tumors harboring these mutations are found in young patients and are exclusive from activating beta-catenin mutations (37). Downstream effects of Sonic hedgehog pathway activation may involve transforming growth factor beta activation as observed in gastric cancer (74) and induction of Wnt family gene (75).

Because of the multiple downstream targets of the activated hedgehog pathway and their potential for driving important tumor characteristics such as growth, invasiveness, and metastasis, antagonists of the pathway have been proposed in the treatment of medulloblastoma. Preclinical studies on the plant-derived alkyloid, cyclopamine, which is capable of blocking activation of hedgehog signaling and abnormal cell growth in the presence of patch-1 or smo mutations by binding directly to SMO have demonstrated its growth inhibiting effect in cultured medulloblastoma cells, mouse-derived medulloblastoma, and human medulloblastoma xenografts. Other agents that block hedgehog signaling and have been shown to have antitumor effects in medulloblastoma in preclinical studies include the cyclopamine analogue IPI-926 (80), itraconazole (81), and curcumin (52). While prolonged hedgehog pathway inhibition with GDC-0449 has been reported to be tolerated in adults with basal cell carcinoma (84), there are concerns over the use of these inhibitors in children in which disruption of developmental pathways may result in unexpected adverse effects. The CNS availability of hedgehog pathway inhibitors remains poorly defined. Additionally, treatment of medulloblastoma with the currently available hedgehog pathway inhibitors will be challenging, as only a fraction of tumors harboring hedgehog pathway mutations at or above the level of SMO will be responsive to small molecule inhibitors that act as SMO antagonists (69).

The key convergence points in Sonic hedgehog, Wnt, and PI3K/Akt signal transduction are MYC and NMYC. These proteins act as transcriptional regulators modulating chromatin modifications and RNA polymerase III pause release (85) and have been implicated in a number of cellular functions including cell cycle control, apoptosis, differentiation, metabolism, and angiogenesis (86). MYC protein levels are controlled by GSKβ-3-mediated, phosphorylation-dependent, protein degradation (87). Increased expression of MYC is found in the Wnt and group C molecular subclasses in the genetic analysis conducted by Northcott et al. (41). Furthermore, robust MYCN expression is observed in the Shh group and are required for Sonic hedgehog-driven medulloblastoma tumorigenesis (88). Amplification of the MYC and MYCN loci has been correlated with poor survival in medulloblastoma (89). MYC has also been demonstrated to be important in the growth, cell adhesion, and migration of CNS-PNET (90), and low levels of MYC mRNA predict prolonged progression-free survival in patients with cerebellar PNET/medulloblastoma (70, 91). During embryonic development MYC levels are elevated, and increased MYC function is required for embryonic stem cell self-renewal and pleuripotency (92). In adult tissues MYC expression is low or undetectable, a finding that suggests that medulloblastomas recapitulate an embryonic gene expression profile, thereby potentiating dysregulated proliferation.  Several signal transduction pathways have been implicated in transcriptional activation of MYC including canonical Wnt, PI3K/Akt, Sonic hedgehog, Notch, and transforming growth factor β signaling. Therefore, inhibition of these specific activating pathways as well as direct inhibition of MYC have been proposed as viable therapeutic strategies for treatment of medulloblastoma.

Identification of the cell of origin in medulloblastoma remains elusive, but the observation of neuronal progenitor gene patterns and the overexpression of Notch 2 in a subset of medulloblastomas points to the potential role of transformed proliferating cerebellar granule cell precursors. Notch signal transduction results in the positive regulation of the transcription of HES1 (93), a transcription factor that complexes with FOXG1 (94), blocking differentiation of neural progenitor cells. HES1 expression is associated with reduced survival in medulloblastoma (95); therefore, targeted disruption of Notch signaling could be an important target for treatment of tumors with Notch 2 overexpression. Treatment of medulloblastoma cell lines with the Notch pathway inhibitor gamma secretase has resulted in depletion of a stem cell-like population characterized by CD133 surface marker, resulting in impaired growth of the tumor cells when implanted into animals (96). One possible explanation for the negative effects of Notch signaling on patient outcome in medulloblastoma may be enhanced tumor stem cell resistance to radiation, as it has been reported that inhibition of Notch signaling sensitizes glioma stem cells to radiation (97).