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Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Known mutations account for only a subset of MB cases. We hypothesized that CpG island methylation-mediated tumour suppressor gene (TSG) silencing contributes to MB pathogenesis, either alone, or in combination with genetic events such as loss of heterozygosity (LOH). We performed a microarray-based genome-wide screen of MB cell lines treated with 5-aza-2’deoxycytidine, identifying genes exhibiting increased expression following treatment. Using this strategy, we identified inhibitors of WNT signalling (SFRP1, SFRP2, and SFRP3) and an inhibitor of the HGF/MET signalling pathway (SPINT2) as putative TSGs silenced by promoter region methylation in MB. Methylation of the WNT signalling inhibitors SFRP1, SFRP2, and SFRP3 was identified using bisulfite sequencing and methylation-specific PCR (MSP). Stable re-expression of SFRP1, SFRP2, and SFRP3 reduced proliferation, impaired anchorage-independent growth, and limited WNT signalling activity. SFRP1 re-expression reduced tumour growth in vivo in xenograft models. Aberrant WNT signalling plays a role in the pathogenesis of a subset of sporadic human MB, as well as MB in cases of Turcot syndrome with germline mutations of APC. Activating mutations of β-catenin are also implicated in a subset of MB. We have identified for the first time an additional mechanism – loss of normal pathway inhibition by SFRP gene silencing – that contributes to MB pathogenesis. SPINT2 methylation was confirmed with bisulfite sequencing and MSP. Stable re-expression of SPINT2 reduced proliferation, impaired cell migratory ability, and decreased the capacity for anchorage-independent growth. In vivo, re-expression of SPINT2 reduced tumour formation in xenograft models. This study identified for the first time SPINT2 as a putative TSG in human MB, and further implicated aberrant HGF/MET oncogenic signalling in the pathogenesis of this disease. The efficacy of targeting the HGF/MET pathway as a novel therapeutic strategy was tested in vitro using the small molecule MET kinase inhibitor PHA665752. Treatment of MB cell lines with PHA665752 reduced cell proliferation, anchorage-independent growth, migration, and limited downstream signalling via the MAPK and PI3K/AKT pathways.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. Aberrant pathway activity can result in various developmental disorders and cancers. The Hh receptor Patched1 (Ptc1) is a negative regulator of the pathway and acts as a tumor suppressor. Our lab and others have shown that Suppressor of fused (Su(fu)) and Kinesin family member 7 (Kif7) are major negative regulators of the pathway that function downstream of Ptc1. Medulloblastoma (MB) is the most common malignant pediatric brain tumor originating from the cerebellum. Several forms of MB have been identified, with abnormal activation of the Hh pathway associated with one major subtype. These tumors commonly show inactivating mutations in PTCH1, whereas mutations in SU(FU) are more rare. Mouse models with deletion of Ptc1 or expression of a constitutively active form of Smoothened (Smo) exhibit elevated Hh pathway activity, leading to MB formation. In this study, I examined the complex roles of Su(fu) in the formation of MB alongside the inactivation of Ptc1, Kif7 and p53 in the cerebellum. Unlike Ptc1+/- mice, Su(fu)+/- mice do not develop MB, even upon exposure to DNA damaging agents (X-ray irradiation or chemical carcinogenesis), which dramatically increases MB incidence in Ptc1+/- mutants. However, Su(fu)+/-;p53-/- mice develop MB and these MB tumor cells exhibit loss of heterozygosity of Su(fu), suggesting a protective role of p53 in tumor suppression in Su(fu) deficiency. Kif7+/- mice are not prone to tumorigenesis, even with deletion of p53. To bypass the embryonic lethality of Su(fu)- and Kif7-null mice, I generated neural stem cell-specific knockout mice, GFAP-Cre;Su(fu)f/f and GFAP-Cre;Kif7f/f. GFAP-Cre;Su(fu)f/f mice exhibit a severely disorganized cerebellum, with drastic up-regulation of p53 expression, and they survive past 1 year of age and do not develop MB. In contrast, GFAP-Cre;Kif7f/f mice appear grossly normal with only subtle cerebellar defects. These observations indicate that neither Su(fu) nor Kif7 inactivation is sufficient to drive tumorigenesis in the cerebellum. To investigate whether Su(fu) and Kif7 possess any overlapping tumor suppressor functions, I generated GFAP-Cre;Kif7f/f;Su(fu)f/f mice. Indeed, simultaneous loss of these negative regulators resulted in MB formation, which is correlated with increased Hh pathway activity as well as a lower level of p53 expression. Furthermore, I discovered a novel positive role for Su(fu) in MB development, as Su(fu) activity is required for robust formation of MB in Ptc1-deficient mice. Together, these data illustrate that Su(fu) plays a dual role in the genesis of MB, and suggest that the rare human MBs with SU(FU) mutations could be caused by simultaneous deletion of TP53 or KIF7.

Medulloblastoma is the most common pediatric brain tumour, yet many of the underlying genetic and epigenetic factors have yet to be discovered. After a genome wide screen of a large cohort of primary medulloblastomas, we discovered that many of the genes within the cell adhesion family are affected by either copy number loss and/or decreased expression unexplained by copy number change. This led us to believe that both genetic and epigenetic factors were affecting this gene family. Through methylation-specific PCR, RT-PCR and high-throughput methylation status analysis, we have concluded that promoter CpG methylation plays a role in the expression of the PCDH10 protein in both medulloblastoma cell lines and primary tumours. Through functional validation with a stable cell line re-expressing PCDH10, I show that cell cycle and proliferation remain unchanged but migration is decreased in cells with PCDH10 re-expression. This suggests that PCDH10 has characteristics of a tumour suppressor in medulloblastoma.

Medulloblastoma is the most common pediatric brain tumour, yet many of the underlying genetic and epigenetic factors have yet to be discovered. After a genome wide screen of a large cohort of primary medulloblastomas, we discovered that many of the genes within the cell adhesion family are affected by either copy number loss and/or decreased expression unexplained by copy number change. This led us to believe that both genetic and epigenetic factors were affecting this gene family. Through methylation-specific PCR, RT-PCR and high-throughput methylation status analysis, we have concluded that promoter CpG methylation plays a role in the expression of the PCDH10 protein in both medulloblastoma cell lines and primary tumours. Through functional validation with a stable cell line re-expressing PCDH10, I show that cell cycle and proliferation remain unchanged but migration is decreased in cells with PCDH10 re-expression. This suggests that PCDH10 has characteristics of a tumour suppressor in medulloblastoma.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. Aberrant pathway activity can result in various developmental disorders and cancers. The Hh receptor Patched1 (Ptc1) is a negative regulator of the pathway and acts as a tumor suppressor. Our lab and others have shown that Suppressor of fused (Su(fu)) and Kinesin family member 7 (Kif7) are major negative regulators of the pathway that function downstream of Ptc1. Medulloblastoma (MB) is the most common malignant pediatric brain tumor originating from the cerebellum. Several forms of MB have been identified, with abnormal activation of the Hh pathway associated with one major subtype. These tumors commonly show inactivating mutations in PTCH1, whereas mutations in SU(FU) are more rare. Mouse models with deletion of Ptc1 or expression of a constitutively active form of Smoothened (Smo) exhibit elevated Hh pathway activity, leading to MB formation. In this study, I examined the complex roles of Su(fu) in the formation of MB alongside the inactivation of Ptc1, Kif7 and p53 in the cerebellum. Unlike Ptc1+/- mice, Su(fu)+/- mice do not develop MB, even upon exposure to DNA damaging agents (X-ray irradiation or chemical carcinogenesis), which dramatically increases MB incidence in Ptc1+/- mutants. However, Su(fu)+/-;p53-/- mice develop MB and these MB tumor cells exhibit loss of heterozygosity of Su(fu), suggesting a protective role of p53 in tumor suppression in Su(fu) deficiency. Kif7+/- mice are not prone to tumorigenesis, even with deletion of p53. To bypass the embryonic lethality of Su(fu)- and Kif7-null mice, I generated neural stem cell-specific knockout mice, GFAP-Cre;Su(fu)f/f and GFAP-Cre;Kif7f/f. GFAP-Cre;Su(fu)f/f mice exhibit a severely disorganized cerebellum, with drastic up-regulation of p53 expression, and they survive past 1 year of age and do not develop MB. In contrast, GFAP-Cre;Kif7f/f mice appear grossly normal with only subtle cerebellar defects. These observations indicate that neither Su(fu) nor Kif7 inactivation is sufficient to drive tumorigenesis in the cerebellum. To investigate whether Su(fu) and Kif7 possess any overlapping tumor suppressor functions, I generated GFAP-Cre;Kif7f/f;Su(fu)f/f mice. Indeed, simultaneous loss of these negative regulators resulted in MB formation, which is correlated with increased Hh pathway activity as well as a lower level of p53 expression. Furthermore, I discovered a novel positive role for Su(fu) in MB development, as Su(fu) activity is required for robust formation of MB in Ptc1-deficient mice. Together, these data illustrate that Su(fu) plays a dual role in the genesis of MB, and suggest that the rare human MBs with SU(FU) mutations could be caused by simultaneous deletion of TP53 or KIF7.

Medulloblastomas (MB) are the most common pediatric nervous system malignancy. Known mutations account for only a subset of MB cases. We hypothesized that CpG island methylation-mediated tumour suppressor gene (TSG) silencing contributes to MB pathogenesis, either alone, or in combination with genetic events such as loss of heterozygosity (LOH). We performed a microarray-based genome-wide screen of MB cell lines treated with 5-aza-2’deoxycytidine, identifying genes exhibiting increased expression following treatment. Using this strategy, we identified inhibitors of WNT signalling (SFRP1, SFRP2, and SFRP3) and an inhibitor of the HGF/MET signalling pathway (SPINT2) as putative TSGs silenced by promoter region methylation in MB. Methylation of the WNT signalling inhibitors SFRP1, SFRP2, and SFRP3 was identified using bisulfite sequencing and methylation-specific PCR (MSP). Stable re-expression of SFRP1, SFRP2, and SFRP3 reduced proliferation, impaired anchorage-independent growth, and limited WNT signalling activity. SFRP1 re-expression reduced tumour growth in vivo in xenograft models. Aberrant WNT signalling plays a role in the pathogenesis of a subset of sporadic human MB, as well as MB in cases of Turcot syndrome with germline mutations of APC. Activating mutations of β-catenin are also implicated in a subset of MB. We have identified for the first time an additional mechanism – loss of normal pathway inhibition by SFRP gene silencing – that contributes to MB pathogenesis. SPINT2 methylation was confirmed with bisulfite sequencing and MSP. Stable re-expression of SPINT2 reduced proliferation, impaired cell migratory ability, and decreased the capacity for anchorage-independent growth. In vivo, re-expression of SPINT2 reduced tumour formation in xenograft models. This study identified for the first time SPINT2 as a putative TSG in human MB, and further implicated aberrant HGF/MET oncogenic signalling in the pathogenesis of this disease. The efficacy of targeting the HGF/MET pathway as a novel therapeutic strategy was tested in vitro using the small molecule MET kinase inhibitor PHA665752. Treatment of MB cell lines with PHA665752 reduced cell proliferation, anchorage-independent growth, migration, and limited downstream signalling via the MAPK and PI3K/AKT pathways.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Medulloblastoma is the most common brain tumor in children and one third of the patients remain incurable. Tumor metastasis is one of the primary reasons for its high mortality rate. Despite evidence of overexpression of PDGFRα and PDGFRβ in metastatic medulloblastoma, their individual roles remain controversial and equivocal. Analysis of their specific signaling pathway in medulloblastoma cells revealed that PDGFRα and PDGFRβ signaling events lead to distinct cellular functions: while PDGFRβ stimulated cell proliferation and invasion, the expression of CD44 to regulate progression via c-Myc and inhibited cell death, PDGFRα displayed the opposite effects. Studies also revealed that c-Myc plays an intermediary role by regulating the downstream molecules in PDGFRβ signal pathway such as CD44 and NFB. NFB activity was found to be down- regulated in the absence of PDGFRβ pathway, with its activity restored by the overexpression of c-Myc. Analysis of medulloblastoma patient tissues without a prior knowledge of their metastatic nature further confirmed that PDGFRβ-CD44 axis regulate medulloblastoma metastasis. Co-inhibition studies performed by simultaneous inhibition of both PDGFRβ and c-Myc either by using siRNAs or by using pharmacological inhibitors demonstrated an enhanced inhibitory effect on medulloblastoma cell proliferation and migration. Using miRNA profiling of Daoy cells lacking either PDGFRβ or c-Myc alone or both, a set of miRNAs regulated by both PDGFRβ and c-Myc in common were identified. Integrative analysis of these miRNAs and their targets revealed that activation of PDGFRβ signaling and overexpression of c-Myc may enhance medulloblastoma progression via modulating the expression of several miRNAs such as miR-1280, -1260 and consequently regulating the expression of oncogenic molecules, such as Jagged 2 and CDC25A, respectively. Specific inhibition of miRNAs, miR-1280 and -1260, and JAG2 demonstrated their vital roles in medulloblastoma cell proliferation and migration. These findings suggest that the PDGFRβ-CD44 is a regulatory axis modulating medulloblastoma progression via c-Myc and targeting PDGFRβ/c-Myc/CD44 may provide a novel therapeutic strategy for the treatment of metastatic medulloblastoma.