Water soluble platinum complexes are a recent area of emphasis of cisplatin chemistry. The water soluble complexes could have a reduced toxicity compared with cisplatin. Oxaliplatin, which has an oxalate leaving group, has previously been shown to have less nephro-toxicity and higher water solubility than cisplatin. [Pt(en)(oxalate)] (en = ethylenediamine) has been prepared from Pt(en)Cl2 and silver oxalate. This complex has been reacted with methionine and N-acetylmethionine at different molar ratios. At high Pt: methionine ratios, chelates with the sulfur and nitrogen atoms of the methionine are dominant; at lower Pt: methionine ratios, a bis-methionine product is formed. The en ligand is displaced by methionine but not N-acetylmethionine.

Bladder cancer has been associated with chronic arsenic exposure. Monomethylarsonous acid [MMA(III)] is a metabolite of inorganic arsenic biotransformation and has been shown to transform an immortalized urothelial cell line (UROtsa) at a concentration 20-fold less than arsenite. MMA(III) was used as a model arsenical to examine the mechanisms of arsenical-induced transformation of the urothelium. A microarray analysis was performed to assess the transcriptional changes in UROtsa during the critical window of chronic MMA(III) exposure that leads to transformation at three months time. The analysis revealed only minor changes in gene expression at one and two months of exposure, contrasting with substantial changes observed at three months of exposure. The gene expression changes at three months were analyzed showing distinct alterations in biological processes and pathways such as a response to oxidative stress, enhanced cell proliferation, anti-apoptosis, MAPK signaling, as well as inflammation. To address the lack of information between two and three months of exposure -- the critical period of transformation -- the expression of selected pathway marker genes were measured by PCR array analysis on a weekly and monthly basis. A very similar pattern of altered expression of these genes was observed when compared to microarray results, and suggested early perturbations in cell signaling cascades, immunological pathways, cytokine expression, and MAPK pathway, are particularly important in driving malignant transformation. These results showed a strong association between the acquired phenotypic changes that occurred as early as one to two months of chronic MMA(III) exposure, and gene expression patterns that are indicative of the earliest stages in carcinogenesis. Additionally, studies on the effects of withdrawal of arsenical were also conducted and showed that phenotypic changes persisted even in the absence of arsenical; that gene expression patterns of pathway marker genes, those that showed significant alterations between 3 and 6 months of exposure, appeared to normalize after withdrawal of the arsenical.

Inflammation and aberrant microRNAs expressions promote colon cancer growth and progression. However, the molecular mechanisms that link these pathways remain to be determined. In this dissertation, the causal relationship between inflammation and aberrant microRNAs expressions were explored. Elevated expression of prostaglandin E₂ (PGE₂) receptor EP4 has been seen in human colon cancer. However, the mechanism by which EP4 receptor protein is deregulated is not known. Experiments in this dissertation demonstrate, for the first time, that the EP4 receptor is negatively regulated by miR-101.In previous work, we show that S100P is induced by stimulation of the PGE₂/EP4 receptor signaling pathway. S100P is a ligand for Receptor for Advance Glycation End-products (RAGE). However, little is known about the downstream targets of S100P/RAGE signaling. Here, we demonstrated that S100P/RAGE receptor signaling induces expression of miR-155 via the transcription factor AP-1. In addition, we investigated the genes that are downstream of S100P/RAGE/miR-155 pathway. Our microarrays and bioinformatics analyses identified two novel miR-155 targets, WNK1 and ZNF493 that are down-regulated upon activation of the S100P/RAGE/miR-155 pathway. Lastly, we investigated whether inhibition of S100P/RAGE signaling pathway would be beneficial as a cancer therapy using methyl-2-acetamidoacrylate (M2AA). M2AA treatments decreased colon cancer cells viability and also suppressed colon tumor growth and metastasis in vitro and also in the CAM assay in vivo. Taken together, our results suggest that modulation of S100P/RAGE signaling by M2AA offers therapeutic potential as anti-metastatic agents. In summary, this dissertation provides new insights on the molecular events that link inflammation pathways and microRNAs to colon cancer as well as show that therapeutic strategies targeting these pathways could be effective in treatment of neoplasia.

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.

Inhibitor of DNA binding/differentiation protein 4 (ID4) is a dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. ID4 shares the homology of HLH domain with other ID proteins (ID1, ID2, and ID3) and lack the basic DNA binding region. Evidence suggested that unlike ID1, ID2 and ID3, ID4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promoting apoptosis. Consistent with these observations ID4 is epigenetically silenced in DU145 prostate cancer cell line. In this study we investigated whether ID4 is also epigenetically silenced in prostate cancer. We also examined association between ID4 promoter hyper-methylation and its expression in prostate cancer cell lines. ID4 protein expression was analyzed in human prostate adenocarcinoma samples by Immunohistochemistry (IHC). ID4 promoter methylation pattern on prostate cancer cell lines was examined by methylation specific PCR. In addition, we performed methylation specific PCR on the human prostate tissues and genomic DNA to correlate cell line studies with clinical studies. IHC demonstrated decreased ID4 protein expression in human prostate tissue samples, whereas higher nuclear ID4 expression was found in normal prostate tissues. ID4 methylation specific PCR (MSP) on prostate cancer cell lines, showed ID4 methylation in DU145, but not in LNCaP and C33 cells. C81 and PC3 cells showed partial methylation. Increased ID4 methylation in C81 as compared to LNCaP suggests its epigenetic silencing as cells acquire androgen independence. Tumors with ID4 promoter hyper-methylation showed distinct loss of ID4 expression. However, the underlying mechanism involved in epigenetic silencing of ID4 is currently unknown. We hypothesized that ID4 promoter methylation is initiated by an EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27Me3). ID4 expressing (LNCaP) and non-expressing (DU145 and C81) prostate cancer cell lines were used to investigate EZH2, H3K27Me3 and DNMT1 enrichment on ID4 promoter by Chromatin immuno-precipitation (ChIP). Increased enrichment of EZH2, H3K27Me3 and DNMT1 in DU145 and C81 cell lines was compared to ID4 expressing LNCaP cell line. Knockdown of EZH2 in DU145 cell line led to re-expression of ID4 and decrease in enrichment of EZH2, H3K27Me3 and DNMT1 demonstrating that ID4 is regulated in an EZH2 dependent manner. ChIP on prostate cancer tissue specimens and cell lines suggested EZH2 occupancy and H3K27Me3 marks on the ID4 promoter. Collectively, our data indicate a PRC2 dependent mechanism in ID4 promoter silencing in prostate cancer through recruitment of EZH2 and a corresponding increase in H3K27Me3. Increased EZH2, but decreased ID4 expression in prostate cancer strongly supports this model.

MYC is overexpressed in most types of tumors, but a means to selectively decrease its expression is yet to be found. Our recent findings on modulation of BCL2 gene expression through protein interactions with the BCL2 i-motif have provided a basis for further investigation of MYC gene control. It is proposed that the MYC i-motif could function by a similar molecular switch mechanism as in BCL2.Binding sites for heterogeneous nuclear ribonucleoprotein K (hnRNP K) within the MYC promoter also exist in the i-motif-forming sequence. Circular dichroism and bromine footprinting confirmed that this DNA sequence is able to form an i-motif, and systematic mutation of the cytosine residues in this sequence has revealed a 5:5:5 loop configuration. Indeed, all loops of the i-motif, when folded into a 5:5:5 loop configuration, contain the hnRNP K consensus sequence (CCCT). Previous studies show that hnRNP K binds to this i-motif-forming sequence, but it was assumed to be single-stranded. Binding studies revealed that hnRNP K has more binding affinity to its consensus sequence in the i-motif compared to a mutant sequence where the i-motif cannot form. Further investigation of the MYC promoter revealed an additional two runs of cytosine seven bases downstream of the MYC i-motif. Biophysical studies showed that the additional two runs were not involved in i-motif formation, however recent studies describe their importance for transcriptional activation. We found that hnRNP K preferred the longer 5CT sequence compared to the i-motif forming 4CT sequence when using a competitive binding assay. Utilizing luciferase reporters containing either the 4CT or 5CT sequence validated that hnRNP K required both the i-motif and 5th CT element for maximum transcriptional activation. Competition binding studies and bromine footprinting showed that hnRNP K bound to the downstream 5th CT element and the central and lateral loops of the i-motif.Additionally, we found that co-overexpression of Sp1 and hnRNP K induced a 10-fold increase in luciferase activity in the 5CT reporter only. We hypothesize that Sp1 continuously primes the promoter to initiate transcription inducing more negative superhelicity and increasing the melting of duplex DNA. This increased melting grants hnRNP K’s three KH domains access to the i-motif loops and the 5Th CT element. Confirmation by ChIP analysis validated that Sp1 overexpression causes an increase in hnRNP K occupancy at the MYC promoter. These findings provide new insight into the mechanisms of MYC transcriptional control by the i-motif and G-quadruplex.Recently, our group has demonstrated that two small molecules IMC-48 and IMC-76 can interact with the i-motif and can be an effective means to modulate BCL2 expression. Based on these results with the BCL2 i-motif, we employed a similar strategy and screened and identified small drug-like molecules that interact with MYC i-motif, using a FRET high-throughput assay. We then further validated that IMC-16 stabilizes the MYC i-motif through the interactions with the loops of the i-motif. No stabilization by IMC-16 treatment was observed with the MYC G-quadruplex and the BCL2 and PDGFRβi-motifs demonstrating selectivity for the MYC i-motif.Finally, we investigated the effects of IMC-16 on MYC expression in three lymphoma cell lines all expressing different levels of MYC. In the case of both Daudi and RAJI Burkitt’s lymphoma cell lines we demonstrated that selectively stabilizing the i-motif by IMC-16 could increase MYC expression. Furthermore, we demonstrated that the MYC G-quadruplex stabilizing compound GQC-05 and IMC-16, which stabilizes the MYC i-motif, have antagonistic effects on MYC expression, providing further evidence of a molecular switch mechanism in the NHEIII1. Directly targeting MYC expression through the i-motif offers advantages over targeting the G-quadruplex, because of the reduced stability and dynamic nature of the i-motif, additionally the i-motif is only found in DNA. The use of such i-motif interactive compounds is the first step into the development of new innovative approaches to treat cancers.

The epidermal growth factor receptor (EGFR) belongs to the erbB family of receptor tyrosine kinases which consists of four members (EGFR, ErbB2, ErbB3 and ErbB4). Upon ligand binding, the EGFR is capable of dimerization with other erbB receptors and propagates signals regulating a diverse array of cellular physiologies, including cell growth, migration and survival. Dysregulation of the EGFR is important for development and progression of different types of cancers, including breast cancer. Breast cancer is the second leading cause of cancer death in women. EGFR overexpression has been observed in about 15% of all breast cancers. Moreover, in triple negative breast cancer (TNBC), which is a more aggressive type of breast cancer and lacks effective therapies, up to 50% of tumors are found to overexpress EGFR. Targeted therapy against EGFR has been used in TNBC. However, limited efficacy has been observed in TNBC due to intrinsic and acquired resistant mechanisms. In order to overcome this issue, we have developed two novel therapeutic peptides derived from the nuclear localization signal (NLS) sequence and juxtamembrane domain of EGFR and investigated their efficacy in regard to inhibiting EGFR translocation and activation in TNBC. EGFR has been found to translocate into the nucleus and nuclear EGFR can affect gene transcription, cell proliferation, stress response and DNA repair through interacting with different components in the nucleus. Importantly, these functions of nuclear EGFR correlate with cancer prognosis and therapeutic resistance. We found that an EGFR NLS-derived peptide (ENLS peptide) could inhibit activated EGFR (pY845) undergoing nuclear translocation. We also showed that this ENLS peptide sensitized breast cancer cells to AG1478 (EGFR tyrosine kinase inhibitor) treatment. The juxtamembrane domain of EGFR regulates its trafficking to the nucleus and mitochondria, interaction with calmodulin and calcium signaling, and participates in dimerization and activation of EGFR. These non-traditional kinase related functions of EGFR represent a novel target for EGFR therapy. We found that a mimetic peptide of the juxtamembrane domain of EGFR (EJ1 peptide) could effectively inhibit EGFR activation through promoting inactive dimer formation. It could also effectively kill cancer cells through processes of apoptosis and necrosis. Mechanistically, this EJ1 peptide affects membrane integrity thereby leading to calcium influx, disruption of mitochondrial membrane potential and reactive oxygen species (ROS) accumulation. Importantly, EJ1 peptide appeared to be effective in inhibition of tumor growth and metastasis in a transgenic mouse model of breast cancer and showed no observable toxicity. ErbB3, another member of the erbB family, represents an important driver of the parallel signaling pathway to EGFR as well as a key regulator of PI3K/AKT activity which is important for therapeutic resistance. ErbB3 has been shown to interact with MUC1. The interaction between MUC1 and EGFR promotes EGFR stability through recycling of receptors. We found that MUC1 expression also affected ErbB3 activity and stability through ErbB3/EGFR/MUC1 complex formation. In conclusion, we demonstrated that two EGFR-derived peptides, working through novel strategies, represent a new foundation of effective therapeutic agents to breast cancer. ErbB3/EGFR/MUC1 complex formation under MUC1 expression also represents a druggable target for ErbB3 activity and stability.

Prostate and breast cancer are among the most commonly diagnosed cancers and leading causes of cancer-related deaths in men and women worldwide. It is therefore evident that enhanced understanding of tumorigenesis is required to improve diagnostic tools, improve prognostics and identify novel therapeutic targets. The goal of this dissertation was to elucidate the role of primary cilia in prostate and breast cancer. Little is known about the role primary cilia may play in these cancers. Primary cilia are microtubule-based organelles which aid in sensing the extracellular environment and participate in signal transduction. Important developmental signaling pathways, such as Hedgehog (Hh) and Wnt signaling pathways, involve cilia. These pathways have also been implicated in prostate and breast cancer. In this work, we demonstrate that cilia are lost through prostate cancer progression. The few remaining cilia on prostate cancers appeared to be dysfunctional, as assessed by quantifying cilia lengths, an indirect measure of functionality. We also investigated a link between the observed cilia loss and canonical Wnt signaling in prostate cancers. Primary cilia have been determined to have a suppressive role in Wnt signaling, therefore we predicted loss of cilia to correlate with increased Wnt signaling. A link between cilia loss or shortened cilia and activated Wnt signaling was suggested in a subset of prostate cancers. Our lab has established that cilia are similarly lost in breast cancer. These data suggested the hypothesis that cilia may act as tumor suppressor organelles in the prostate and breast. To test this hypothesis, we knocked down cilia in an oncogenic mammary mouse model and assessed changes in tumor growth and characteristics. We observed enhanced tumor growth with cilia loss. The data supports the hypothesis that primary cilia may be playing a tumor suppressor role in the prostate and breast, and provides promising avenues for identifying novel therapeutic approaches for cancer patients.

A major limitation in developing a successful cancer treatment is the need for a distinction between normal and cancerous tissue. For solid tumors, this distinction can be made on a spatial basis, and successful treatments have been developed accordingly; however, many of these treatments cause pathologies in healthy tissue, much to the detriment of patient health. To address this issue for solid tumours, a conceptual approach would be to administer the chemotherapeutic drug locally, such that the intra-tumour concentration was high, while the systemic exposure to the drug remained low, thus, minimizing side effects. The present research focuses on providing proof-of-concept for the electrochemical generation of a toxicant from a prodrug, and subsequent use to elicit cytotoxicity in cancer cells, in attempts to electrochemically mimic monooxygenase-catalyzed bioactivation. Electro-oxidation of acetaminophen and cyclophosphamide substrates at graphite and Ti/RuO2 anodes was successful in generating their respective toxicants; however, the graphite anode was superior with respect to current efficiency and toxicant yield. Electrolyses conducted in batch and flow reactors produced effluents which reduced EMT-6 cell viability to the IC99 level. This thesis provides proof-of-concept for electrochemical prodrug activation as a viable area for further cancer research.

Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.