The development of novel imidazopyridines, which includes both tetrahydroimidazo[1,5-a]pyridine (rIMP) and imidazo[1,5-a]pyridine (IMP) was investigated using conventional and microwave induced procedures that afforded compounds at high yield of 88-96%. rIMP was synthesized using a two-step procedure that involved the microwave synthesis of IMP, then the reduction of the pyridine moiety of the fused imidazopyridine rings using 10% Pd/C and hydrazine monohydrate. The microwave synthesis of imidazopyridines involved the one pot reaction of 2-benzoylpyridine, substituted benzaldehyde and ammonium formate in acetic acid under open vessel microwave conditions, which resulted in products within 40 minutes. Novel PEG-IMP development, involved the synthesis of ethylene glycol tethered benzaldehydes and IMPs using traditional Williamson etherification synthesis, which afforded products at a high yield of 92-95%. We have then shown IMP and rIMP roles in its antiproliferative property towards PCa cells, specificity in inhibiting PI3K isoforms, and structural motif’s interaction with different residues in the kinase binding domain of the class I PI3K isoforms. The antiproliferative property towards PC3 cells shows increased activity with compounds containing pyridyl group on carbon 3 of the imidazo[1,5-a]pyridine parent moiety with signs of toxicity to PC3 within 24 hours of incubation and at 1 μM of the parent compound. Furthermore, the IMPs were tested against five prostate cellular lines: PC3, RWPE1, D145, LNCaP and LNCaP C81. IMPs showed little activity towards RWPE1 and increased activity towards PC3 cells. We determined that functionalizing the phenyl group at position 1 increased the efficacy of rIMP compared to the IMP. After showing increased toxicity to PC3 cells, it was important to investigate the mechanism in which IMP pose toxicity towards PC3 cells. The biochemical assay showed that rIMP was more effective in inhibiting PI3Kα isoform compared to both pan inhibitor wortmannin and IMP. Both IMP and rIMP inhibited more than 60% of PI3Kγ isoform activity at nanomolar concentrations. After showing IMPs affinity to PI3K isoforms, we investigated the binding interactions rIMP and IMP towards the PI3K isoforms using MOE molecular modeling software.

A common phenotype observed in most cancers is chromosomal instability. This includes both structural and numerical chromosomal aberrations, which can promote carcinogenesis. The fusion gene CBFB/MYH11 is created by the structural chromosomal inversion(16)(p13.1q22), resulting in the fusion protein CBFβ-SMMHC, which blocks differentiation in hematopoietic progenitor cells. This mutation alone, however, is not sufficient for transformation, and at least one additional cooperating mutation is necessary. The role of wildtype Cbfb in modulating the oncogenic function of the fusion protein Cbfβ-SMMHC in mice was examined. Transgenic mice expressing the fusion protein, but lacking a wild-type copy of Cbfb, were created to model the effects of these combined mutations. It was found that wild-type Cbfb is necessary for maintaining normal hematopoietic differentiation. Consequently, complete loss of wild-type Cbfb accelerates leukemogenesis in Cbfb/MYH11 mice compared to mice expressing both the fusion and wild-type proteins. While there is no evidence in human patient samples that loss of wild-type Cbfb expression cooperates with the fusion protein to cause transformation, it is apparent from these experiments that wild-type Cbfβ does play a role in maintaining genomic integrity in the presence of Cbfβ-SMMHC. Experiments have also shown that loss of Cbfb leads to accumulation of hematopoietic progenitor cells, which may acquire additional cooperating mutations. Not unlike CBFB/MYH11, the human papillomavirus (HPV) E6 and E7 proteins are not sufficient for cellular transformation. Instead, high risk HPV E7 causes numerical chromosomal aberrations, which can lead to accumulation of additional cooperating mutations. Expression of HPV-16 E7 and subsequent downregulation of the retinoblastoma protein (Rb) has been shown to induce polyploidy in human keratinocytes. Polyploidy predisposes cells to aneuploidy and can eventually lead to transformation in HPV positive cells. There are several possible mechanisms through which E7 may lead to polyploidization, including abrogation of the spindle assembly checkpoint, cleavage failure, abrogation of the postmitotic checkpoint, and re-replication. Rb-defective mouse and human cells were found to undergo normal mitosis and complete cytokinesis. Furthermore, DNA re-replication was not found to be a major mechanism to polyploidization in HPV-E7 cells upon microtubule disruption. Interestingly, upon prolonged mitotic arrest, cells were found to adapt to the spindle assembly checkpoint and halt in a G1-like state with 4C DNA content. This post-mitotic checkpoint is abrogated by E7-induced Rb-downregulation leading to S-phase induction and polyploidy. This dissertation explores two examples of the multi-step pathway in human cancers. While certain genes or genetic mutations are often characteristic of specific cancers, those mutations are often not sufficient for transformation. The genetic or chromosomal abnormalities that they produce often stimulate the additional mutations necessary for oncogenesis. The studies with Cbfb/MYH11 and HPV E7 further exemplify the significance of numerical and structural chromosomal aberrations in multi-step carcinogenesis.

Cancer is the second leading cause of mortality in the United States and the World, therefore, early effective prevention, diagnosis, and therapy is needed. Estrogens play a major role in the initiation and progression of breast cancer. Elevated lifetime exposure to estrogens is associated with an increased risk of developing breast cancer. Estrogens through influencing mitochondria contribute to estrogen induced breast carcinogenesis; however, the exact mitochondrial mechanisms underlying the estrogen carcinogenic effect in breast tissue are not clearly understood. For this dissertation, the mitotoxic and cytotoxic effects of triphenylphosphonium cation (TPP) and Origanum majorana organic extract (OME) as well as PEGylated bioconjugate of OME with TPP (P-OME-TPP) against human breast epithelial and cancer cell lines was investigated. Initially, TPP, a lipophilic cation, was used to check whether an imbalance in mitochondrial bioenergetics, in part, may be responsible for estrogen induced growth of breast cancer. The results showed that exposure of estrogen-dependent MCF-7 cells to 17 β-estradiol (E2) induced the metabolic activity, proliferation, mitochondrial bioenergetics, DNA damage, and formation of cellular and mitochondrial reactive oxidant species (ROS). These E2-induced endpoints were inhibited by co-treatment with TPP, indicating mitochondrial mechanisms, in part, may contribute to the development of breast cancer. Furthermore, O. majorana, widely used in the Middle East as a culinary aromatic medicinal herb, has been shown to possess an extensive range of biological activity including antioxidant, anti-inflammatory, and anti-tumor growth effects. Interestingly, the anticancer potential of O. majorana against breast cancer remains largely unexplored; therefore, the anticancer effect of O. majorana on breast cell lines was investigated. The results showed that E2-induced metabolic activity and growth were inhibited by OME in MCF-7 cells. The results also demonstrated that synthesized P-OME-TPP conjugate, compared to OME, was far more effective in exerting its cytotoxic effect through the inhibition of growth and mitochondrial metabolic activity in both highly metastatic, triple negative MDA-MB-231 and estrogen-dependent MCF-7 breast cancer cells. Altogether, these findings offer a new perspective on the utility of mitochondria-targeted lipophilic TPP cation and the potential of O. majorana extract to be developed as a new therapy against breast tumors.

A major challenge of cancer treatment is successful discrimination of cancer cells from healthy cells. Nanotechnology offers multiple venues for efficient cancer targeting. Magnetoelectric nanoparticles (MENs) are a novel, multifaceted, physics-based cancer treatment platform that enables high specificity cancer targeting and externally controlled loaded drug release. The unique magnetoelectric coupling of MENs allows them to convert externally applied magnetic fields into intrinsic electric signals, which allows MENs to both be drawn magnetically towards the cancer site and to electrically interface with cancer cells. Once internalized, the MEN payload release can be externally triggered with a magnetic field. MENs uniquely allow for discrete manipulation of the drug delivery and drug release mechanisms to allow an unprecedented level of control in cancer targeting. In this study, we demonstrate the physics behind the MEN drug delivery platform, test the MEN drug delivery platform for the first time in a humanized mouse model of cancer, and characterize the biodistribution and clearance of MENs. We found that MENs were able to fully cure the model cancer, which in this case was human ovarian carcinoma treated with paclitaxel. When compared to conventional magnetic nanoparticles and FDA approved organic PLGA nanoparticles, MENs are the highest performing treatment, even in the absence of peripheral active targeting molecules. We also mapped the movement through peripheral organs and established clearance trends of the MENs. The MENs cancer treatment platform has immense potential for future medicine, as it is generalizable, personalizable, and readily traceable in the context of treating essentially any type of cancer.

I proposed the study of two distinct aspects of Ten-Eleven Translocation 2 (TET2) protein for understanding specific functions in different body systems. In Part I, I characterized the molecular mechanisms of Tet2 in the hematological system. As the second member of Ten-Eleven Translocation protein family, TET2 is frequently mutated in leukemic patients. Previous studies have shown that the TET2 mutations frequently occur in 20% myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN), 10% T-cell lymphoma leukemia and 2% B-cell lymphoma leukemia. Genetic mouse models also display distinct phenotypes of various types of hematological malignancies. I performed 5-hydroxymethylcytosine (5hmC) chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq) of hematopoietic stem/progenitor cells to determine whether the deletion of Tet2 can affect the abundance of 5hmC at myeloid, T-cell and B-cell specific gene transcription start sites, which ultimately result in various hematological malignancies. Subsequent Exome sequencing (Exome-Seq) showed that disease-specific genes are mutated in different types of tumors, which suggests that TET2 may protect the genome from being mutated. The direct interaction between TET2 and Mutator S Homolog 6 (MSH6) protein suggests TET2 is involved in DNA mismatch repair. Finally, in vivo mismatch repair studies show that the loss of Tet2 causes a mutator phenotype. Taken together, my data indicate that TET2 binds to MSH6 to protect genome integrity. In Part II, I intended to better understand the role of Tet2 in the nervous system. 5-hydroxymethylcytosine regulates epigenetic modification during neurodevelopment and aging. Thus, Tet2 may play a critical role in regulating adult neurogenesis. To examine the physiological significance of Tet2 in the nervous system, I first showed that the deletion of Tet2 reduces the 5hmC levels in neural stem cells. Mice lacking Tet2 show abnormal hippocampal neurogenesis along with 5hmC alternations at different gene promoters and corresponding gene expression downregulation. Through the luciferase reporter assay, two neural factors Neurogenic differentiation 1 (NeuroD1) and Glial fibrillary acidic protein (Gfap) were down-regulated in Tet2 knockout cells. My results suggest that Tet2 regulates neural stem/progenitor cell proliferation and differentiation in adult brain.

Chronic myeloid leukemia (CML) is a disease characterized by the expansion of granulocytic cells. The BCR-ABL tyrosine kinase inhibitor imatinib, the frontline treatment for Ph+ leukemias, can induce complete hematologic and cytogenetic response in most chronic phase CML patients. Despite the remarkable initial clinic effects, it is now recognized that imatinib will unlikely cure patients because a small cell population containing leukemic stem cells (LSCs) with self-renewal capacity is insensitive to tyrosine kinase inhibitors. In Chapter I, I briefly review the BCR-ABL kinase and its related signaling pathways. BCR-ABL kinase activates several signaling pathways including MAPK, STAT, and JNK/SAPK. BCR-ABL also mediates kinase-independent pathways through SRC family kinases. I will also discuss pathways involving β-catenin, hedgehog, FoxO and Alox5 are critical to the regulation of self-renewal and differentiation in LSC of CML. As detailed in Chapter II, I describe our work evaluating the effects of omacetaxine, a novel CML drug inducing cell apoptosis by inhibition of protein synthesis, on self-renewal and differentiation of LSCs and BCR-ABL-induced CML and acute lymphoblastic leukemia (B-ALL) in mice. We found that treatment with omacetaxine decreased the number of LSCs and prolonged the survival of mice with CML or B-ALL. In chapter III, I describe that Alox5 is an essential gene in the function of LSCs and CML development. We show evidence that Alox5 affects differentiation, cell division, and survival of long-term LSCs. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly and prolonged survival. In chapter IV, I present evidence of our work showing a further dissection the Alox5 pathway by comparing the gene expression profiles of wild type and Alox5-/- LSCs. We show that Msr1 deletion causes acceleration of CML development. We also show that Msr1 affects CML development by regulating the PI3K-AKT pathway and β-catenin. Taken together, these results demonstrate that some pathways including Alox5 and Msr1 play an important role in regulating the self-renewal and differentiation of LSC. More efforts should be put into developing the novel strategies that may effectively target LSCs and thus cure CML.

Pancreatic ductal adenocarcinoma (PDAC), one of the most aggressive human malignancies, is thought to be initiated by KRAS activation. Here, we find that transcriptional activation mediated by the GLI family of transcription factors, although dispensable for pancreatic development, is required for KRAS induced pancreatic transformation. Inhibition of GLI using a dominant-negative repressor (Gli3T) inhibits formation of precursor Pancreatic Intraepithelial Neoplasia (PanIN) lesions in mice, and significantly extends survival in a mouse model of PDAC. Further, ectopic activation of the GLI1/2 transcription factors in mouse pancreas accelerates KRAS driven tumor formation and reduces survival, underscoring the importance of GLI transcription factors in pancreatic tumorigenesis. Interestingly, we find that although canonical GLI activity is regulated by the Hedgehog ligands, in the context of PDAC, GLI transcription factors initiate a unique ligand-independent transcriptional program downstream of KRAS, that involves regulation of the RAS, PI3K/AKT, and NF-кB pathways. We identify I-kappa-B kinase epsilon (IKBKE) as a PDAC specific target of GLI, that can also regulate GLI transcriptional activity via positive feedback mechanism involving regulation of GLI subcellular localization. Using human PDAC cells, and an in vivo model of pancreatic neoplasia, we establish IKBKE as a novel regulator pf pancreatic tumorigenesis that acts as an effector of KRAS/GLI, and mediates pancreatic transformation. We show that genetic knockout of Ikbke leads to a dramatic inhibition of initiation and progression of pancreatic intraepithelial viii neoplasia (PanIN) lesions in mice carrying pancreas specific activation of oncogenic Kras. Furthermore, we find that although IKBKE is a known NF-кB activator, it only modestly regulates NF-кB activity in PDAC. Instead, we find that IKBKE strongly promotes AKT phosphorylation in PDAC in vitro and in vivo, and that IKBKE mediates reactivation of AKT post-inhibition of mTOR. We also show that while mTOR inhibition alone does not significantly affect pancreatic tumorigenesis, combined inhibition of IKBKE and mTOR has a synergistic effect leading to significant decrease tumorigenicity of PDAC cells. Together, our findings identify GLI/IKBKE signaling as an important oncogenic effector pathway of KRAS in PDAC that regulates tumorigenicity, cell proliferation, and apoptosis via regulation of AKT and NF-кB signaling. We provide proof of concept for therapeutic targeting of GLI/IKBKE in PDAC, and support the evaluation of IKBKE as a therapeutic target in treatment of pancreatic cancer, and IKBKE inhibition as a strategy to improve efficacy of mTOR inhibitors in the clinic.

Integrins have the ability to impact major aspects of epithelial biology including adhesion, migration, invasion, signaling and differentiation, as well as the formation and progression of cancer (Hynes 2002; Srichai and Zent 2010; Anderson et al. 2014). This thesis focuses on how integrins are regulated and function in the context of mammary epithelial biology and breast cancer with a specific focus on the α6 integrin heterodimers (α6β1 and α6β4). These integrins function primarily as receptors for the laminin family of extracellular matrix (ECM) proteins and they have been implicated in mammary gland biology and breast cancer (Friedrichs et al. 1995; Wewer et al. 1997; Mercurio et al. 2001; Margadant and Sonnenberg 2010; Muschler and Streuli 2010; Nistico et al. 2014). The first project investigates how alternative splicing of the α6 subunit impacts the genesis and function of breast cancer stem cells (CSCs). This work revealed that the α6Bβ1 splice variant, but not α6Aβ1, is necessary for the function of breast CSCs because it activates the Hippo transducer TAZ (Zhao et al. 2008a), which is known to be essential for breast CSCs (Cordenonsi et al. 2011). My work also led to the discovery that laminin (LM) 511 is the specific ligand for α6Bβ1 and that autocrine LM511, which is mediated by TAZ, is needed to sustain breast CSCs by functioning as a ‘ECM niche’. An important aspect of this study is the finding that surface-bound LM511 characterizes a small population of cells in human breast tumors with CSC properties. The second project of my thesis concentrated on identifying transcription factors that regulate expression of the β4 subunit. The expression of the α6β4 integrin is repressed during the epithelial-mesenchymal transition (EMT) (Yang et al. 2009) but the contribution of specific transcription factors to this repression is poorly understood. This study revealed that Snai1 is a transcriptional repressor of β4, which is responsible for establishing the PRC2 (Polycomb complex 2)- associated repressive histone mark H3K27Me3. However, I also found that the ability of Snai1 to repress transcription is abrogated by its interaction with Id2. Specifically, I identified the biochemical mechanism for how Id2 regulates Snai1. Id2 binds the SNAG domain of Snai1 that is the docking site for several corepressors (Peinado et al. 2004; Lin et al. 2010b; Dong et al. 2012a). One important consequence of Id2 interacting with Snai1 on the β4 promoter is that it prevents repressive epigenetic modifications. This finding may explain why some epithelial cells express Snai1 and β4 because they also express Id2 (Vincent et al. 2009; Bastea et al. 2012). The repression of the α6β4 integrin during the EMT is consistent with data indicating that this integrin is not expressed in CSCs (Mani et al. 2008; Goel et al. 2012; Goel et al. 2013; Goel et al. 2014). An important question going forward is to understand how the α6β4 integrin contributes to tumor formation. In summary, my thesis provides novel insights into the biology of the α6 integrins that has important implications for the function of these integrins in mammary gland biology and breast cancer, especially our understanding of breast CSCs.

Werner syndrome (WS) is an autosomal recessive disorder associated with premature aging and cancer predisposition. WS cells show increased genomic instability and are hypersensitive to DNA-damaging agents. WS is caused by mutations of the WRN gene. WRN protein is a member of RecQ DNA helicase family. In addition to a conserved 3’–5’ helicase activity, the WRN protein contains unique 3’–5’ exonuclease activity. WRN recognizes specific DNA structures as substrates that are intermediates of DNA metabolism. WRN physically and functionally interacts with many other proteins that function in telomere maintenance, DNA replication, and DNA repair. The function of WRN is regulated by post–translational modifications that include phosphorylation, acetylation, and sumoylation. SIRT1 is a NAD-dependent histone deacetylase (HDAC) that deacetylates histones and a numbers of cellular proteins. SIRT1 regulates the functions of many proteins, which are important for apoptosis, cell proliferation, cellular metabolism, and DNA repair. SIRT1 is also regulated by other proteins or molecules from different levels to activate or inhibit its deacetylase activity. In this study, we found that SIRT1 interacts with and deacetylates WRN. We further identified the major acetylation sites at six lysine residues of the WRN protein and made a WRN acetylation mutant for functional analysis. We found that WRN acetylation increases its protein stability. Deacetylation of WRN by SIRT1 reverses this effect. CREB-binding protein (CBP) dramatically increased the half-life of wild-type WRN, while this increase was abrogated with the WRN acetylation mutant. We further found that WRN stability is regulated by the ubiquitination pathway, and that WRN acetylation by CBP dramatically reduces its ubiquitination level. We also found that acetylation of WRN decreases its helicase and exonuclease activities, and that SIRT1 reverses this effect. Acetylation of WRN alters its nuclear distribution. Down-regulation of SIRT1 increases WRN acetylation level and prevents WRN protein translocating back to nucleolus after DNA damage. Importantly, we found that WRN protein is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells that were stably expressing WRN acetylation mutant display significantly higher sensitivity to MMC than the cells expressing wild-type WRN. Taken together, these data demonstrated that acetylation pathway plays an important role in regulating WRN function in response to DNA damage. A model has been proposed based on our discoveries.

Higher-order genome organization is important for the regulation of gene expression by bringing different cis-regulatory elements and promoters in proximity. The establishment and maintenance of long-range chromatin interactions occur in response to cellular and environmental cues with the binding of transcription factors and chromatin modifiers. Understanding the organization of the nucleus in differentiation and cancer has been a long standing challenge and is still not well-understood. In this thesis, I explore the dynamic changes in the higher-order chromatin structure in bone differentiation and breast cancer. First, we show dynamic chromatin contact between a distal regulatory element and the promoter of Runx2 gene, which encodes the Runtrelated transcription factor 2 (RUNX2) that is essential for bone development. Next, via using a genome-wide approach, we show that breast cancer cells have altered long-range chromatin contacts among small, gene-rich chromosomes and at telomeres when compared with mammary epithelial cells. Furthermore, we assess the changes in nuclear structure and gene expression of breast cancer cells following Runt-related transcription factor 1 (RUNX1) deficiency, an event frequently observed in breast cancer. Finally, I present the role of the central ATPase subunit of the SWI/SNF complex, SMARCA4 (BRG1), in mediating nuclear structure and gene expression. Taken together, the research presented in this thesis reveals novel insight and paradigm for the dynamic changes in disease and differentiation, as well as uncovers previously unidentified roles for two chromatin regulatory proteins, RUNX1 and SMARCA4.