The relationship between MUC1 and EGFR has been characterized by our lab to be highly tumorigenic. A peptide therapeutic was developed in our lab to block the cytoplasmic interaction of MUC1 and EGFR by competing with the EGFR-binding domain of MUC1. The peptide, PMIP, reduced invasion and proliferation in vitro and reduced tumor growth and metastasis in vivo. These studies demonstrated the potency of MUC1/EGFR interactions in tumor progression, and we sought to explore this concept further. We wanted to clarify a mechanism by which MUC1 and EGFR together drive breast cancer metastasis, and we identified c-Met as a mediator of MUC1 and EGFR-driven cell motility. In two separate assays, we demonstrated that c-Met activity was necessary for MUC1 and EGFR to promote migration and invasion. In addition, we wanted to identify the role of EGFR membrane localization in membrane identity and tumor initiation. We established several EGFR localization mutants to compare to wild-type basolateral EGFR and we performed proof-of-concept experiments to show that these mutants will be useful in future studies. Finally, we studied the effect of MUC1 and EGF loss on tissue architecture and function in the lactating mammary gland. EGF is the primary ligand for EGFR during lactation, and MUC1 is highly expressed during this period of mammary development. In addition, it has been shown that EGFR and MUC1 interact at the apical cell surface of lactating mammary ducts, yet there is no link between lactation and tumor formation. We hypothesized that MUC1 and EGFR interaction may have a role in maintaining tissue architecture and lactation function in the mouse mammary gland. We found instead that the loss of MUC1 and EGF had no noticeable effect on lactation and did not result in tissue defects. These studies further clarified the relationship between MUC1 and EGFR in several different contexts, showing a role for their interaction in metastatic progression, and showing that their ablation has no effect in the lactating mammary gland. Future studies will elucidate the role of MUC1 and EGFR interaction in tumor initiation, and we have taken several steps in our studies toward that goal.

Pleckstrin homology (PH) domains are structurally conserved domains, which generally bind to phosphatidylinositol phosphate (PtdInsP) lipids. They are present in a variety of proteins, including those that are upregulated in cancer growth and metastasis, and represent a crucial component of intracellular signaling cascades and membrane translocation. Thus, they may be considered as attractive targets for cancer drug therapy. AKT (protein kinase B), a pleckstrin homology lipid binding domain and a serine/threonine kinase-containing protein, is a key component of the phophatidylinositol-3-kinase (PI3K)/AKT cell survival signaling pathway which is activated in a variety of cancers, including prostate, pancreatic, and skin cancers. In this study, I report the finding of a novel inhibitor of AKT; PH-427. I describe its effects on binding to the PH domain of AKT thus preventing its binding to PtdIns3-P at the plasma membrane and subsequent activation. In vivo testing of the drug led to reduction of tumor size and numbers in a mouse pancreatic cancer model. Additional testing of PH-427 on squamous cell carcinomas revealed that the drug is able to reduce tumor burden and multiplicity in vivo when topically applied. Thus, we demonstrate proof-of-principle in targeting PH domains as a viable cancer drug therapy option. The effects of PH-427 raised the intriguing possibility that targeting PH domains may have beneficial effects in other signaling pathways with PH domain-containing proteins. Guanine exchange factors (GEFs) contain a Dbl homology (DH) domain and a PH domain and have been shown to be involved in the process of metastasis. More specifically, RacGEFs activate Rac1 GTPase by facilitating the exchange of GDP to GTP. Over-expression of certain GEFs has been shown to contribute to increased malignancy in a variety of cancers. T-lymphoma invasion and metastasis-inducing protein-1 (Tiam1) is a highly conserved GEF and contains an N-terminal pleckstrin homology domain (nPH) and a DH/C-terminal PH domain (cPH). Tiam1 has been found to be over-expressed in several cancers, including breast, colon and prostate cancers. In this study, I describe the identification, development, experimental testing, and potential mechanism of action of novel small molecule inhibitors targeting the RacGEF Tiam1 to inhibit prostate cancer bone metastasis.

Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamines are organic cations shown to control gene expression at the transcriptional, post-transcriptional, and translational level. The activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is associated with normal and neoplastic growth. A single nucleotide polymorphism (SNP, rs2302615, SNP +316 nucleotides 3' of the transcriptional start site) in the ODC1 gene has been found to be both functional and prognostic for risk of colorectal carcinogenesis. A comprehensive investigation of genetic variability in ODC1 gene was performed. We confirmed frequencies of 12 SNPs occurring in participants of a clinical cancer prevention trial. We identified haplotypes accounting for over 90% of the genetic diversity in the ODC1 gene. Mechanistically, we addressed two of them, which account for more than half of the participants in the clinical trial. Two ODC1 intron 1 SNPs, rs2302616 (SNP +263 nucleotides 3' of the transcriptional start site) and rs2302615, were found to be associated with disease processes. Both of them predicted metachronous adenoma and response to agents targeting the polyamine pathway in participants of the clinical trial. The rs2302616 functionally modulate a DNA G-quadruplex structure and predicted the ODC1 rate-limiting product putrescine by genotype. Both SNPs cooperate to modulate ODC1 transcriptional activity involving both a G-quadruplex structure and Sp1 binding site at rs2302616, and rs2302615 flanked MYC-binding E-boxes. Haplotype analysis, using both these SNPs, might provide better discrimination of both disease prognosis and treatment prediction in cancer chemoprevention clinical trials.

Obesity rates have risen steeply in recent decades. This is accompanied with increased prevalence of several obesity-related disorders including type 2 diabetes. Genetic, environmental, and lifestyle factors (increased caloric intake and/or decreased physical activity) predispose individuals to type 2 diabetes by decreasing the body's responsiveness to the pancreatic hormone insulin, a physiological phenomenon commonly referred to as insulin resistance. Insulin resistance occurs due to induction of inflammation characterized by increased secretion of pro-inflammatory cytokines from enlarged adipose tissue, endoplasmic reticulum stress, and oxidative stress associated with excess blood glucose. There is a strong correlation between insulin resistance and decreased circulating levels of adiponectin (APN), a hormone implicated in promoting insulin-like activities. Further, inflammation negatively affects both insulin and adiponectin levels. It is recognized that folding of APN 18mer-subunits (insulin-sensitizing oligomer) is hindered in obese and type 2 diabetic individuals. Likewise, formation of normal hexameric insulin complex is compromised in type 2 diabetes. Insulin biogenesis, packaging, and assembly are impaired and unable to compensate for high blood glucose levels. As insulin and APN are key metabolic hormones essential for proper glucose regulation, maintaining their correct folding and assembly is required for conserving overall metabolic homeostasis. This dissertation centers on investigating proper assembly pathways of APN and insulin isoforms to form the higher order complexes necessary for their function. The interaction between APN oligomers was studied in the presence and absence of zinc, previously shown to inhibit formation of disulfide bonds in APN. We observed that zinc protects APN from collapse under acidic conditions and likely stabilizes oligomers through high affinity histidine coordination. The interaction between oligomers was further assessed by analyzing conformational differences between oligomers through tryptophan fluorescence. Reduced oligomers were observed to have significant structural differences compared to oxidized oligomers indicated by changes in fluorescent intensities. The capacity of APN chaperone DSBA-L to promote assembly was also evaluated although no significant changes were observed. In addition, the interaction between zinc and insulin was assessed where we observed that in the presence of zinc, insulin is significantly protected from reduction and precipitation. Zinc formed large complexes with insulin under reducing environments to induce high structural stability of insulin oligomers. We then utilized the strong conformational stability of insulin to develop a novel insulin analog with properties to slowly release insulin in circulation and more quickly in the presence of high glucose concentrations. Insulin modification is at preliminary stages and requires further experimentation. Together, these results indicate that zinc plays a significant role in multimerizing properties to provide high stability towards APN and insulin structures. Zinc enhances multimerization of oligomers to both promote activity of APN and protect insulin from reduction and premature breakdown to monomers. Through this study we better identified the folding pathway of APN and elucidated the strong intermolecular forces involved in oligomer association. In addition, the multimerization pattern of insulin to large conformation complexes is observed to mediate protection under reducing conditions. This has implications in the development of new therapeutic options to promote insulin-sensitization and insulin activity to regulate plasma glucose levels. In addition, we propose the development of a novel insulin-analog to mimic physiological insulin secretion, currently unavailable in the market.

Growing evidence indicates that serine proteases known as kallikreins are associated with malignancy and may have potential diagnostic/prognostic applications in cancer. Kallikreins are the largest group of serine proteases. Kallikrein enzymes are often involved in proteolytic cascades through their function in degradation of extracellular matrix proteins and promotion of angiogenesis. Kallikrein 6 (KLK6) is a member of the family of fifteen highly conserved secreted trypsin- or chemotrypsin-like serine proteases. Over-expression of KLK6 has been observed in different pathophysiological states such as neurodegenerative diseases, inflammation and various cancers, including colorectal cancer. In Chapter 3 we elucidated the miRNA-based mechanism of regulation of invasion in metastatic colorectal cancer over-expressing KLK6. We developed HCT116 colon stable isogenic cell lines with knockdown of KLK6 expression using short-hairpin interference RNA (shKLK6 clones). The shKLK6 clones had decreased expression and secretion of KLK6 protein with a minimal effect on cell growth and viability in cell culture. SCID mice injected with shKLK6-3 clone 3 cells exhibited a statistically significant increase in the survival rates (P=0.005), decrease in the incidence of distant metastases and a shift in the location of the metastatic foci closer to the cell's injection site. Levels of KLK6 protein secreted into the bloodstream were significantly lower in animals injected with shKLK6-3 clone 3 compared to HCT116 control clone 1 (P < 0.04). Through bioinformatics analyses we identified and validated three miRNAs, which are important in post-translational modification of bioactive proteins, proliferation, migration and p38 MAPK signaling pathway. In Chapter 4 we developed Caco-2 colon stable isogenic cell lines with expressing enzymatically active or mutant KLK6 protein (Caco-2 stable clones). We employed these cell lines to investigate the importance of KLK6 enzymatic activity of initiation of cell invasion using in vitro and in vivo models.

The integrity of the genome relies on the maintenance of chromosomes, the structural embodiment of the genetic material. Disruption of chromosome replication can lead to extensive genomic rearrangements, spanning kilobase (Kb) to megabase (Mb) regions. Some chromosome rearrangements are inherently dynamic, beginning as a single unstable rearrangement from which multiple rearrangements emerge. The rare formation and transient behavior of unstable chromosomes renders their study challenging. Here I characterize the genetic ontogeny of unstable chromosomes in a budding yeast model, from initial replication error to unstable chromosome formation to their resolution. I find that the initial error often arises in or near the telomere and frequently forms unstable chromosomes that later resolve to an internal "collection site" in the middle of the chromosome. The initial telomere-proximal unstable chromosome is increased in cells mutant for telomerase, the Tel1 checkpoint kinase and even the Rad9 checkpoint protein, with no known telomere-specific function. Defects in Tel1 and the Rrm3 DNA helicase, or the Tel1-MRX complex and 9-1-1 checkpoint clamp, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. I performed a candidate genetic screen of instability in telomere maintenance and DNA damage response (DDR) proteins to characterize the interplay of pathways regulating senescence and genomic instability. Collectively, my results suggest that unstable chromosomes form in or near damaged telomeres, independently of end degradation (Exo1-independent), by either nonhomologous end joining (partially Lig4-dependent) or by faulty template switch during replication (Lig4- and Rad52-independent). The telomere-proximal unstable chromosomes then rearrange further to the middle of the chromosome. These results implicate telomere replication errors as a common source of widespread genomic changes and make substantial progress to our understanding of the initiation and fate of unstable chromosomes in the eukaryotic genome.

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease. HTLV-1 Related Endogenous Sequence (HRES-1), a human endogenous retrovirus, produces 2 retroviral-like Gag capsid proteins (p8 and p15) that share significant sequence homology to the U1-subunit of the small ribonucleoprotein complex (U1sn-RNP), an autoantigen of lupus. The central hypothesis is that molecular mimicry between HRES-1 and U1sn-RNP serves as a priming event in SLE via the production of cross-reactive autoantibodies. Anti-HRES-1/U1sn-RNP serological responses in subjects with SLE and comparison populations were characterized. An overlapping peptide set mapping the HRES-1 p8 and p15 proteins was used. SLE subjects produce IgG to several regions of HRES-1. Healthy subjects or those with RA, HIV-1 infection, or HTLV-1-infection produced no significant anti-HRES-1 IgG. Anti-HRES-1 antibodies deposited in the kidneys of patients with SLE glomerulonephritis were identified. Our data suggests that HRES-1 plays a role in SLE by means of a molecular mimicry mechanism with U1sn-RNP.

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