Colony-stimulating factor 1 receptor (CSF1R, Fms) is an integral transmembrane glycoprotein with tyrosine specific protein kinase activity that it is found on the mononuclear phagocytes to promote their survival, proliferation and differentiation. Colony-stimulating factor 1 (CSF-1), also known as M-CSF, is a protein ligand that acts on the CSF1R. There is a variable association of Fms with the stem cell marker CD34 on acute myeloid leukemia (AML) cells and this suggests different structures of the AML hierarchy in different patients. Mouse stromal cells (MS-5) were transduced with a plasmid containing human CSF-1 because mouse CSF-1 is inactive on human CSF1R. Results show that AML cells cultured with CSF-1-expressing stroma had a much better growth and survival than the control stroma, suggesting that CSF-1 might be a stimulating factor for the growth of leukemic stem cells.

In an attempt to identify agents that specifically target neuroblastoma (NB) tumour-initiating cells (TIC) we performed drug screens using libraries of bioactive compounds. Cardiac glycosides (CGs) were the largest class of drugs identified with antitumour activity. At high CG doses inhibitory effects on the Na+/K+-ATPase induce cardiotoxicity; therefore, CG analogues were designed in an attempt to separate the effects on NB cells from cardiotoxicity. We identified RIDK34 as our lead compound from a structure-activity-relationship analysis (IC50 8 nM). RIDK34 contains a unique oxime group and shows increasing potency against NB TICs. The Na+/K+-ATPase is a target for the apoptotic activity of digoxin and RIDK34, whereby a signaling cascade involving Src and ERK may induce apoptosis. Furthermore, we predict that signaling activation does not require inactivation of the Na+/K+-ATPase and subsequent deregulation of [Na+]i and [K+]I gradients. Thus CGs and particularly RIDK34 may be expected to display diminished cardiotoxicity and greater therapeutic potential.

Neuropilin (Nrp) overexpression is correlated with increased invasion and metastasis in many epithelial carcinomas including breast cancer. The exact molecular mechanism of how Nrp promotes cancer cell tumourigenicity is unknown. Nrp is a coreceptor for VEGF, hepatocyte growth factor (HGF), and also shown to activate TGF-beta on tumour cells. We hypothesize that binding of Nrp potentiates growth factor (GF) signalling and results in GF-dependent aggressive phenotype in breast cancer. In the current study, Nrp was shown to potentiate HGF signalling in vitro in MCF-7 cells by increasing phosphorylation of the MET receptor. However MDA-MB-231 cell line failed to show any differences after Nrp knockdown, due to constitutively activated MET. Nrp is also shown to increase the number and size of cancer stem cell (CSC) enriched mammospheres through NF-kB pathway activation. These results suggest a novel function of Nrp in CSCs and identify it as a potential target for effective cancer therapy.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

In an attempt to identify agents that specifically target neuroblastoma (NB) tumour-initiating cells (TIC) we performed drug screens using libraries of bioactive compounds. Cardiac glycosides (CGs) were the largest class of drugs identified with antitumour activity. At high CG doses inhibitory effects on the Na+/K+-ATPase induce cardiotoxicity; therefore, CG analogues were designed in an attempt to separate the effects on NB cells from cardiotoxicity. We identified RIDK34 as our lead compound from a structure-activity-relationship analysis (IC50 8 nM). RIDK34 contains a unique oxime group and shows increasing potency against NB TICs. The Na+/K+-ATPase is a target for the apoptotic activity of digoxin and RIDK34, whereby a signaling cascade involving Src and ERK may induce apoptosis. Furthermore, we predict that signaling activation does not require inactivation of the Na+/K+-ATPase and subsequent deregulation of [Na+]i and [K+]I gradients. Thus CGs and particularly RIDK34 may be expected to display diminished cardiotoxicity and greater therapeutic potential.

Neuropilin (Nrp) overexpression is correlated with increased invasion and metastasis in many epithelial carcinomas including breast cancer. The exact molecular mechanism of how Nrp promotes cancer cell tumourigenicity is unknown. Nrp is a coreceptor for VEGF, hepatocyte growth factor (HGF), and also shown to activate TGF-beta on tumour cells. We hypothesize that binding of Nrp potentiates growth factor (GF) signalling and results in GF-dependent aggressive phenotype in breast cancer. In the current study, Nrp was shown to potentiate HGF signalling in vitro in MCF-7 cells by increasing phosphorylation of the MET receptor. However MDA-MB-231 cell line failed to show any differences after Nrp knockdown, due to constitutively activated MET. Nrp is also shown to increase the number and size of cancer stem cell (CSC) enriched mammospheres through NF-kB pathway activation. These results suggest a novel function of Nrp in CSCs and identify it as a potential target for effective cancer therapy.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

Glioblastoma multiforme (GBM) is one of the most common and aggressive primary brain tumours, with a median patient survival time of 6-12 months in adults. It has been recently suggested that a typically small sub-population of brain tumour cells, in possession of certain defining properties of stem cells, is responsible for initiating and maintaining the tumour. More recent experiments have studied the interactions between this subpopulation of brain cancer cells and tumour microenvironmental factors such as hypoxia and high acidity. In this thesis a computational approach (based on Gillespie’s algorithm and cellular automata) is proposed to investigate the tumour heterogeneities that develop when exposed to various microenvironmental conditions of the cancerous tissue. The results suggest that microenvironmental conditions highly affect the characterization of cancer cells, including the self-renewal, differentiation and dedifferentiation properties of cancer cells.

Most of tumor consists of a heterogeneous population of tumor cells among a tumor initiating and chemo or radiation resistant subpopulation, called cancer stem cells (CSCs), which have become increasingly important new anticancer targets. CD133 has been recently identified as a prominent marker for CSCs in pancreatic and other tumors; however, the signaling cascade of this cancer stem cell marker has not been fully explored. This study shows increased cell proliferation, colony formation, adhesion, and migration following CD133 overexpression in pancreatic ductal adenocarcinoma (PDAC) cells. Signaling studies have indicated that CD133 overexpression increases the epidermal growth factor receptor (EGFR) activation and phosphorylation of PI3K/Akt and MAPK/ ERK pathways. An in vivo xenograft study confirmed that overexpression of CD133 has higher tumorgentic ability than control mice. Molecular studies have found that CD133 physically associates with EGFR and promotes EGFR protein level and its phosphorlyation, which might be critical for PDAC tumor progression and chemoresistance. The data also showed that CD133 overexpression suppresses the EGF mRNA expression, which may imply that CD133 induces EGFR activation through an EGF ligand-independent process. The findings here point to an important mechanism of action for CD133 in PDAC. The EGFR inhibitor has potent anti-CD133 activity, and the current results have important implications for developing targeting CD133 activity as a novel cancer therapy strategy and the inhibitor approach presented here identifies the inhibition of CD133 activity by the EGFR inhibitor and sheds light on developing a new cancer therapeutic that functions by targeting CD133 activity in human cancer.

Medulloblastomas represent a heterogeneous group of cerebellar tumors that constitute the most frequent primary pediatric solid malignancy. Molecular characterization of these tumors have led to the understanding that distinctsubtypes possess characteristic properties such as gene expression profile, histological classification, and degree of dissemination that are predictive of disease progression and prognosis. Fractionation of primary medulloblastomas has led to the appreciation of brain tumor stem cells (BTSC) that may be driving the more aggressive and malignant disease. However, the developmental origins of these cells as well as the influences of early mutations in tumor suppressors on development and tumorigenesisremain unclear. My work is geared towards understanding the impact of mutations in the key tumor suppressor genes Ptc1 and p53 on medulloblastoma formation. I first identified key differences in neural stem cell marker expression that distinguish between Ptc1 and Ptc1;p53 medulloblastomas, demonstrating that the Ptc1;p53 genotype may pre-dispose to a more malignant, stem-like tumor. Through the use of a somatic mosaic model, we describe a synergistic interaction between Ptc1 haploinsufficiency and p53 deficiency leading to developmental seeding of the cerebellar field by pre-malignant cells and term this phenomenon “developmental field cancerization.” Interestingly, we observed this premalignant colonization in the cerebellarstem cell compartment as well, resulting in an aberrant population of self-renewing cells. Upon loss-of-heterozygosity at the Ptc1 locus, the Ptc;p53 animals alone develop robust cerebellar tumorsthat possess a definable stem-like population of cells that can re-initiate metastatic secondary tumors. These findings demonstrate how early mutationsin the tumor suppressor genes, such as Ptc1 and p53, may lead to stem cell field cancerization and play an important role in determining future tumor character and prognosis.