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.

Inhibition of mitochondrial translation as a therapeutic strategy for acute myeloid leukemia Marko Škrtić Doctor of Philosophy Institute of Medical Science University of Toronto 2012 Abstract Intro: Acute myeloid leukemia (AML) therapies have remained unchanged for 20 years, and thus new therapies are needed. Objective: To identify FDA-approved agents with anti-leukemia stem cell activity, we performed a screen and identified the antimicrobial tigecycline (TIG). Methods: Primary AML mononuclear cells were isolated by Ficoll centrifugation from peripheral blood. Flow cytometry dye; JC-1, Carboxy-H2DCFDA, Mitotracker GreenFM. Leukemia stem cell activity was assayed by human AML engraftment in NOD/SCID mice. Results: TIG induced cell death in primary AML patient samples (LD50, 3-6μM n=14), preferentially over normal hematopoietic cells. Likewise, in colony assays, TIG (5μM) reduced the clonogenic growth of AML samples (n=7) by 93%, demonstrating an effect on leukemia progenitor cells, but not normal hematopoietic cells (34% reduction, n=5). A yeast genome-wide screen identified mitochondrial translation inhibition as the mechanism of tigecycline-mediated cell death in eukaryotic cells. TIG decreased the expression of mitochondrial peptides, enzyme activity and membrane potential preferentially in AML cells over normal hematopoietic cells. ShRNA knockdown of TuFM mitochondrial translation factor in leukemia cells reproduced TIG anti-leukemia target effects previously described. We discovered that primary AML CD34+/CD38- stem cells have greater mitochondrial mass (3-fold, n=5) than normal CD34+ cells (n=4). Higher baseline mitochondrial mass in primary AML samples was predictive for tigecycline sensitivity in vitro (r=-0.71, p<0.05). We assessed the effect of TIG on primary AML stem cells defined by their ability to initiate leukemic engraftment in vivo. NOD/SCID mice treated with TIG had decreased human AML engraftment (n=3 AML patients) compared to control. Conclusions: We identified mitochondrial translation inhibition as a novel therapeutic strategy for AML. Currently, a Phase I clinical trial of tigecycline in hematological malignancies is underway.

Increased local tumor tissue expression of versican in breast cancer patients is predictive of relapse and has a negative impact on survival rates. It is recognized that bone is a common anatomic site of breast cancer metastasis. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. We exogenously expressed a G3 construct in mouse breast cancer cell line 66c14, and found that G3 expression enhanced breast cancer cell proliferation and migration, and spontaneous metastasis to bone in an orthotopic model by upregulating the EGFR-mediated signaling pathway. Possessing anti-apoptotic and drug resistant properties, overexpression of versican was accompanied by selective sensitization to several chemotherapeutic agents. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may, in part, explain breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. New knowledge gained by our experiments includes the understanding that GSK-3β (S9P) appears to function as a key check-point in this balance. In addition, versican G3 enhanced breast cancer cell self-renewal in vitro and in vivo. Versican was expressed at high levels in breast cancer mammosphere cells, which contained a high percentage of SP cells. Reduction of versican’s functionality through anti-versican shRNA or knocking out the EGF-like motifs using G3ΔEGF reduced the effect of versican on enhancing mammosphere and colony formation. Versican promoted breast cancer cell self-renew appears to play a role in enhanced chemotherapeutic drug resistance (including Docetaxel, Doxorubicin, and Epirubicin), which relates partly to its upregulated expression of EGFR signaling. Versican enhances breast cancer bone metastasis not only by enhancing tumor cell mobility, invasion, and survival in bone tissues, but also through mechanisms inhibiting osteoblast cell growth and differentiation, affording favorable microenvironments for tumor metastasis.

Glycogen Synthase Kinase-3 (GSK-3) is a highly conserved serine/threonine kinase comprised of two mammalian homologues, GSK-3α and β, encoded by independent genes. This thesis reports the characterization of GSK-3-null primary mouse embryonic fibroblasts (MEFs) generated by gene targeting to gain insight into the physiological functions of this protein kinase. Combined inactivation of both alleles of GSK-3α and GSK-β led to elevated sensitivity to TNFα-induced apoptosis, altered organization of focal adhesion complexes, defects in cell spreading on fibronectin, decreased cell growth associated with altered cell cycle progression through the G2/M phase and increased spontaneous apoptosis. Future work will focus on unraveling the molecular mechanisms responsible for these effects and identifying the common and distinct cellular roles for GSK-3α and β, and specific variants of these isoforms.

Transient Receptor Potential Melastatin 2 (TRPM2) is a widely expressed, non-selective cationic channel with implicated roles in cell death, chemokine production and oxidative stress. This study characterizes a novel interactor of TRPM2. Using fusion proteins comprised of the TRPM2 C-terminus we established that tubulin interacted directly with the predicted C-terminal coiled-coil domain of the channel. In vitro studies revealed increased interaction between tubulin and TRPM2 during LPS-induced macrophage activation and taxol-induced microtubule stabilization. We propose that the stabilization of microtubules in activated macrophages enhances the interaction of tubulin with TRPM2 resulting in the gating and/or localization of the channel resulting in a contribution to increased intracellular calcium and downstream production of chemokines.

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.

Protein phosphorylation is a central regulatory mechanism in countless cellular processes. Deletion of the PP1 serine/threonine phosphatase gene Ppp1cc in mice results in male infertility due to a severe impairment in spermatogenesis. This disruption in spermatogenesis is hypothesized to arise due to a deficiency of the testis specific Ppp1cc isoform PPP1CC2. To learn more about the function of PPP1CC2 in spermatogenesis, we have employed several proteomic approaches aimed at identifying both regulatory proteins and substrates that interact with PPP1CC2 in the testis. First, we created transgenic mouse embryonic stem cell lines expressing a tandem affinity tagged version of PPP1CC2. Tandem affinity purification using these cell lines identified a number of known PP1 interacting proteins, and one novel interactor DDOST (dolichyl-di-phosphooligosaccharide-protein glycotransferase) which we hypothesize to have a role in spermatogenesis. In a second approach, we conducted GST pull down assays from mouse testis lysates to identify PPP1CC2 interacting proteins. TSSK1 (testis-specific serine kinase 1) was identified as a novel PPP1CC2 interacting protein. We then demonstrated that TSSK1 interacts with PPP1CC2 in an indirect manner via a common interacting protein TSKS (testis-specific serine kinase substrate). Binding of TSKS to PPP1CC2 is regulated via phosphorylation of a PP1 docking motif on the TSKS surface, and localization of TSSK1 and TSKS in the testis is disrupted in Ppp1cc mutants. Finally, to identify candidate substrates of PPP1CC2 in the testis, we conducted a comparative phosphoproteomic analysis and identified 33 different peptides that were hyperphosphorylated in the testis of 3 week old Ppp1cc knockout mice. Amongst these candidate substrates are several proteins essential for mouse spermatogenesis—HMGA1 (high mobility group AT-hook 1), HSPA4 (heat shock protein 4), YBX2 (Y box protein 2) and SYCP2 (synaptonemal complex protein 2). Taken together, our results suggest that PPP1CC2 interacts with a number of different proteins in the testis, and is likely to play a role at several different stages of spermatogenesis, in both meiotic and post-meiotic spermatogenic cells.

TTumor hypoxia (poor oxygenation) adversely affects patient prognosis by promoting therapeutic resistance and an aggressive tumor phenotype. We aimed to understand how urokinase plasminogen activator receptor (uPAR), a cysteine-rich protein implicated in the malignant phenotype and poor patient prognosis, matures in hypoxia. We hypothesized that secretion of uPAR during hypoxia is conferred by a superior ability to form disulfide bonds without oxygen. A model and assay was established to monitor the oxygen-dependency of suPAR (a soluble secreted isoform of uPAR) folding and secretion. We found that suPAR maturation involves disulfide formation and N-linked glycosylation in normoxia. In anoxia, suPAR disulfide formation was impaired, but suPAR was nevertheless secreted. We propose that suPAR has low dependency on disulfide formation for efficient secretion in comparison to other disulfide-containing proteins. Mechanisms supporting protein expression during hypoxia may potentially be targeted to mitigate the adverse effects of tumor hypoxia and ultimately improve cancer therapy.

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.