Molecular Genetics And Targeted Therapeutics

The molecular biological basis of medulloblastoma continues to be elucidated [82-85]. Current molecular genetic data indicates that the pattern of oncogene expression and tumor suppressor gene dysfunction in medulloblastoma differs from that in the more common gliomas of adulthood. In adult high-grade gliomas, there is a predilection for amplification of platelet derived growth factor receptors (PDGFR) and epidermal growth factor receptors (EGFR), excessive or constitutive activity of internal signal transduction systems (e.g., ras), and loss or reduced expression of tumor suppressor genes (e.g., p53, Rb, PTEN) [82,86]. In medulloblastoma, different molecular abnormalities are associated with cellular transformation. For example, although there is an elevated frequency of isochromosome 17q and loss of alleles on chromosome 17p, mutations and deletions of the p53 gene are infrequent [3,82,87]. It is theorized that a separate tumor suppressor gene must exist on 17p, distal to the site of p53 [88]. It has also been shown that amplification and over expression of EGF, PDGF, and their associated receptors occurs infrequently in medulloblastoma cell lines and tumor resection specimens. In contrast, amplification and over expression has been demonstrated for members of the myc family of oncogenes [89-94]. Amplification of C-myc has been reported by several groups and ranges from 5 to 20 per cent of tested specimens. Abnormalities of C-myc expression are more frequent and range from 42 to 90 per cent of biopsy specimens and cell lines. In addition, recent laboratory experiments have demonstrated that overexpression of C-myc into medullo-blastoma cell lines resulted in a more aggressive phenotype [95]. When these C-myc cell lines were grown as xenografts in SCID mice, they grew 75 per cent larger over 8 weeks than non-transfected cell lines and histologically resembled large cell/ anaplastic medulloblastoma. Although amplification and over expression of N-myc have been described, they are much less common.

A recent analysis of the insulin-like growth factor I receptor (IGF-IR) has determined that a constitutively active form of the receptor is frequently present in surgical specimens and cell lines [96]. In addition, when compared to normal cerebellum, higher concentrations of the major substrate for the receptor (IRS-1) were also noted in the majority of cells, suggesting the possibility of an autocrine and paracrine stimulatory mechanism. Several reports by Gilbertson and co-workers have described the expression patterns of HER (ErbB) receptors in medulloblastoma cell lines, tumor specimens, and normal cerebellum [85,97,98]. The HER2 receptor is undetectable in the developing and mature cerebellum. However, in a series of 70 medulloblastoma patients, HER2 was present in 86 per cent of the tumors and was co-expressed with HER4 in 54 per cent of tumors. The co-expression of HER2 and HER4 imparted a poor prognosis for survival (p = 0.006), independent of age and tumor stage. Expression of the NRG1-^^ligand (binding strongly to the HER2/HER4 heterodimer) was also noted in 87.5 per cent of tumor samples, supporting the presence of an autocrine loop. Co-expression of HER2, HER4, and NRG1-^Vwas significantly correlated (p < 0.05) with the presence of central nervous system metastases at diagnosis.

Another important pathway involves the ^-catenin gene product, which interacts with other proteins (i.e., glycogen synthase kinase 3, APC) to influence the rate of cell proliferation (see Fig. 29.1) [85,99,100]. In normal cells, ^-catenin levels are kept low through constitutive interaction with several multiprotein complexes. The most well-characterized complex contains axin, casein kinase 1a, APC, and glycogen synthase kinase 3. Casein kinase 1a^phosphorylates ^-catenin on serine 45 (S45), which primes ^-catenin for further phosphorylation by glycogen synthase kinase 3 on S41, S37, and S33. These residues provide binding sites for the ^-transducin repeat-containing protein, which promotes polyubiquitination of ^-catenin and proteasome degradation. The other multiprotein complex contains protein kinase A, presenilin, and glycogen synthase kinase 3 and functions in a similar manner. Oncogenic mutations of ^-catenin eliminate serine phosphorylation sites (i.e., S33, S37) for glycogen synthase kinase 3^. Hypophosphorylation of ^-catenin reduces its ubiquitin-mediated degradation, leading to higher cytoplasmic concentrations and more frequent binding to T-cell factor (Tcf-1). The complex of ^-catenin and Tcf-1 functions as a transcriptional transactivator and allows constitutive expression of genes that promote cell proliferation (e.g., cyclin D1, C-myc). Several groups have noted oncogenic mutations in the ^-catenin pathway in approximately 15 per cent of sporadic medulloblastomas [101,102]. These mutations lead to increased transcription of several genes, including cyclin D1, C-myc, and Tcf-1.

Neuropeptides and associated receptors have also been implicated in the cellular transformation of medulloblastoma. Somatostatin (SS-14) and vaso-active intestinal polypeptide (VIP) are both neuromo-dulators and growth regulators of the developing nervous system [103,104]. Fruhwald and colleagues found that somatostatin and the sst2 receptor subtype were highly expressed in medulloblastoma tumor specimens and cell lines. They postulated that somatostatin was involved in the proliferation and differentiation of these tumors. The same group has reported similar results for VIP and its receptors (VIPR1 and VIPR2). There was high expression of VIP, VIPR1, and VIPR2 in resected specimens, while in cell lines, only the receptors were highly expressed. Application of VIP to the cell lines resulted in significant growth inhibition. The neurotropins are another important class of trophic factors that influence the nervous system. Grotzer and co-workers analyzed the expression of TrkC, the specific receptor for Neurotropin-3, in a series of 81 patients with medulloblastoma [105]. TrkC expression was common in their cohort, and the level of expression was highly prognostic (p < 0.00005) for extended survival in univariate and multivariate analyses.

An exciting new area of research involves the PTCH gene and the Sonic Hedgehog (SHH) signaling pathway. This pathway was originally described in Drosophila and was discovered during a screen for genes involved in embryonic patterning defects

Wnt Pathway Trcp

FIGURE 29.1 The Wnt/S-catenin signaling pathway. In normal cells, there is limited binding of WNT to the Frizzled receptor. Without stimulation by WNT, Frizzled does not activate Disheveled (DSH), thereby allowing the multiprotein complex of axin, casein kinase 1ai/(CK1), adenomatous polyposis coli (APC), and glycogen synthase kinase 3 (GSK3) to phosphorylate S-catenin, which provides binding sites for S-transducin repeat-containing protein ( S TRCP). A similar phosphorylation and ubiquitination pathway is mediated by protein kinase A (PKA), presenilin (PRSN), and GSK3). S TRCP mediates poly-ubiquitination of S-catenin, with subsequent proteasome-dependent degradation. With WNT binding, Frizzled phosphorylates and activates DSH, which inhibits the multiprotein complex, leading to hypophosphorylation of S-catenin and an increase in its cytosolic concentrations. S-catenin then shuttles to the nucleus and interacts with T-cell factor 1 (Tcf-1), turning on target genes such as cyclin D1 and C-myc. See Plate 29.1 in Color Plate Section.

FIGURE 29.1 The Wnt/S-catenin signaling pathway. In normal cells, there is limited binding of WNT to the Frizzled receptor. Without stimulation by WNT, Frizzled does not activate Disheveled (DSH), thereby allowing the multiprotein complex of axin, casein kinase 1ai/(CK1), adenomatous polyposis coli (APC), and glycogen synthase kinase 3 (GSK3) to phosphorylate S-catenin, which provides binding sites for S-transducin repeat-containing protein ( S TRCP). A similar phosphorylation and ubiquitination pathway is mediated by protein kinase A (PKA), presenilin (PRSN), and GSK3). S TRCP mediates poly-ubiquitination of S-catenin, with subsequent proteasome-dependent degradation. With WNT binding, Frizzled phosphorylates and activates DSH, which inhibits the multiprotein complex, leading to hypophosphorylation of S-catenin and an increase in its cytosolic concentrations. S-catenin then shuttles to the nucleus and interacts with T-cell factor 1 (Tcf-1), turning on target genes such as cyclin D1 and C-myc. See Plate 29.1 in Color Plate Section.

[85,106-108]. These genes have been found to be highly conserved and are also important for normal development and morphogenesis in vertebrates. SHH is a secreted protein that undergoes auto-catalytic cleavage into a 19 kDa N-terminal active portion (SHH-N). PTCH, located on chromosome 9q22.3, codes for a transmembrane protein that acts as the receptor for SHH-N (see Fig. 29.2). It now appears that PTCH functions as a tumor suppressor gene through its interactions with another transmembrane protein, smoothened (Smo), which transduces the SHH-N signal within the cell. When PTCH is not bound by SHH-N, it inhibits signal transduction by Smo, thereby down-regulating transcriptional activity. Once SHH-N binds to PTCH, the activity of Smo is no longer inhibited and the signal is transduced through activation of the Gli genes (Gli1, Gli2, Gli3). Modulators of Gli activation include fused, which has a positive influence, and protein kinase A, suppressor of fused, and costal-2, which are all inhibitory. The Gli genes act synergistically as transcription factors to induce downstream expression of several genes, including PTCH, WNT-1, and members of the bone morphogenetic proteins (BMPs).

It is now clear that dysfunction of the PTCH gene and the SHH pathway can cause abnormalities of development and induce a predisposition to several different forms of cancer [85,106-109]. Patients with familial and sporadic Gorlin's syndrome (i.e., nevoid basal cell carcinoma syndrome) have germline mutations of PTCH. Affected patients display multiple basal cell carcinomas, jaw cysts, dyskeratotic palmar and plantar pits, skeletal abnormalities, and various other tumors. Approximately 3 per cent of patients with Gorlin's syndrome develop medullo-blastoma, often of the desmoplastic variant [109].

Genetic analysis of these tumors reveals frequent mutations or loss of heterozygosity of the PTCH gene. In transgenic mouse models, the homozygous animals (PTCH —/—) die early in utero, while hemizygous animals (PTCH +/—) survive, with 14-19 per cent developing medulloblastoma [110-114]. Analysis of the remaining PTCH allele showed that mutations were rare and that expression of PTCH was not deficient. Therefore, in this model, PTCH was not behaving as a classical tumor suppressor gene, which requires the inactivation of both alleles before transformation occurs. However, levels of Gli1 mRNA and protein were increased in the tumors, suggesting that the SHH pathway could be activated despite the persistence of PTCH expression. If PTCH (+/—) mice were crossed with mice deficient for p53 (—/—), the tumor rate increased to 95 per cent and all mice died before 12 weeks, implying that loss of p53 may enhance genomic instability and the rate of secondary mutations [115]. Microarray analysis of tumor cells from PTCH (+/—) mice suggests that SHH activation promotes overexpression of cyclin D1 and N-myc, and that these factors are important mediators of SHH-induced proliferation and tumorigenesis [116]. A more recent transgenic mouse model activated the SHH pathway by expressing a constitutively active form of Smo in cerebellar granule neuron precursors (i.e., ND2:SmoA1 mice), resulting in tumors in 48 per cent of high-expressing mice by a median 6 months of age [117]. Gene expression analysis of tumors demonstrated an increase in Gli1 and N-myc, as well as Notch2 and the Notch target gene HESS. The ND2:SmoA1 model suggests that aberrant Notch signaling contributes to medulloblastoma cell proliferation and survival. Pharmacological inhibition of both the SHH and Notch signaling pathways was shown to induce widespread apoptosis in tumor cells and was more effective than inhibition of either pathway alone.

Mutations of PTCH have also been described in 10-15 per cent of sporadic human medulloblastomas [85,118-122]. Although the mutations seem to be more frequent in the desmoplastic variant, they can occur in the classic type as well. Missense, nonsense, and frameshift deletions/insertions have all been described throughout the PTCH gene; no hot spots have been identified. The most common mutations predict the generation of a truncated protein. PTCH2 may also play a role in the transformation of some medulloblastomas, since a truncating mutation was recently described in a sporadic tumor [123]. Onco-genic mutations of other genes involved in the SHH signaling pathway, such as SHH, Smo, GLI, and PKA have also been studied, but appear to be uncommon events [85,121,124,125]. Mutations in SuFu, which has been mapped to chromosome 10q24.3, appear to be more common and were identified in approximately 9 per cent of tumor samples in a series of 46 medulloblastoma patients [126]. In most cases, truncating mutations were noted, along with a few of the missense variety. All of the truncating SuFu mutations were present in tumors of the desmoplastic subtype. Although mutations of Smo are relatively uncommon, many tumors may demonstrate increased expression of Smo mRNA when compared to normal skin or brain tissue [127]. In a similar expression analysis of PTCH and Smo, levels of mRNA were found to have an inverse correlation with grade of malignancy in astrocytic tumors [128]. These results implicated the expression of PTCH and Smo in the transformation process of astrocytic tumors. Expression of the Gli genes did not correlate with astrocytic malignancy.

The SHH/PTCH signaling pathway is frequently abnormal and overactive in Gorlin's syndrome-associated and sporadic medulloblastoma and, in addition, may be involved in the transformation of a subset of astrocytic tumors. Due to the relative simplicity of the pathway, it is now under evaluation for "targeted" therapy [85,129]. Small molecule approaches to "targeted" therapy of medulloblastoma are under investigation [85,130-134]. Cyclopamine is a teratogenic steroidal alkaloid derived from the Veratrum californicum lily, that can inhibit SHH/ PTCH signaling through a mechanism that involves binding to Smo and an alteration of its conformation (see Fig. 29.2). The conformational change is induced when cyclopamine binds to the heptahelical domain of Smo, thereby reducing its internal signal transduc-tion capacity. This alteration of conformation and function is similar to the inhibitory action of PTCH. In vitro, cyclopamine can inhibit proliferation and induce apoptosis of murine and human medulloblas-toma cells. Cyclopamine can reduce the growth of tumor allografts in nude mouse models and is also able to inhibit growth and reduce cell viability of freshly cultured human medulloblastoma cells [133]. Because cyclopamine has a relatively low affinity for binding to Smo, second generation inhibitors are under investigation. High-throughput cell-based screening assays for inhibitors of the pathway have identified Hh-Antag691, a benzimidazole derivative with a greater than 10-fold higher binding affinity for Smo than cyclopamine [135]. Hh-Antag691 is able to penetrate the blood-brain barrier after oral delivery, making it an excellent candidate for brain tumor treatment. Using a medulloblastoma mouse model (PTCH+/— <p53—/—), Romer and colleagues demonstrated that Hh-Antag691 was able to

Ptch Expression

FIGURE 29.2 The SHH/PTCH signaling pathway. In the resting state, without binding of SHH, PTCH inhibits Smo and does not allow internal signaling. Once SHH binds to PTCH, repression of Smo activity is released and the intracellular cascade proceeds, culminating in the expression of the Gli genes. Smo expression of Gli is facilitated by fused (fu) and inhibited by protein kinase A (PKA), costal-2, and suppressor of fused (sup-fu). The Gli genes induce transcription of several target genes, including PTCH, WNT-1, and BMP's. New molecular therapeutics such as cyclopamine and Hh-Antag can specifically inhibit the activity of Smo. Adapted from reference [151]. Used with permission from Ashley Publications. See Plate 29.2 in Colour Plate Section.

FIGURE 29.2 The SHH/PTCH signaling pathway. In the resting state, without binding of SHH, PTCH inhibits Smo and does not allow internal signaling. Once SHH binds to PTCH, repression of Smo activity is released and the intracellular cascade proceeds, culminating in the expression of the Gli genes. Smo expression of Gli is facilitated by fused (fu) and inhibited by protein kinase A (PKA), costal-2, and suppressor of fused (sup-fu). The Gli genes induce transcription of several target genes, including PTCH, WNT-1, and BMP's. New molecular therapeutics such as cyclopamine and Hh-Antag can specifically inhibit the activity of Smo. Adapted from reference [151]. Used with permission from Ashley Publications. See Plate 29.2 in Colour Plate Section.

reduce Gli1 expression and inhibit tumor growth in a dose-dependent manner. Treatment of tumor-bearing mice (20 or 100 mg/kg, twice daily for four days, by oral gavage) resulted in inhibition of cellular proliferation and increased apoptosis. If treatment was extended over two weeks, the 20 mg/kg dose resulted in reduction of tumor volume, while the 100 mg/kg dose induced complete or near-complete eradication of tumors (p = 0.0159). In addition, long-term exposure of tumor-bearing mice to 100 mg/kg per day resulted in a significant extension in tumor-free survival (Log-rank test = 0.0001). Further pre-clinical testing of Hh-Antag691 is ongoing and phase I clinical trials are under development.

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