Fumagillin is the natural product isolated from fungus Aspergillus fumigatus, and is recognized as a potent anti-angiogenic compound. Substantial investigation has been focused on the anti-tumor activities of fumagillin and its analogues, some of which have been adopted in pre-clinical and clinical studies. However, investigation on the immunomodulating activities of this class of compounds is limited and results have been controversial. As there is intense interest in elucidating the interrelation between immune modulation and tumor development, novel immunopharmacological properties of chemotherapeutic agents have recently been explored for their therapeutic potentials in clinical applications. As a combination to both these research topics, fumagillin and its synthetic analogues were firstly investigated on different types of immune cells, such as T lymphocytes, dendritic cells and macrophages. F23, a fumagillin analogue with potent immunological activities, was further examined in three different murine cancer models, EL4 lymphoma, CT26 colon carcinoma and 4T1 mammary carcinoma, and their anti-tumor activities and intrinsic immunomodulatory effects were explored. Fumagillin and its analogues exert diversified functions in different types of immune cells. For example, they showed inhibitory effects on cell proliferation and cytokine production of T lymphocytes upon polyclonal stimulation, stimulatory effects on dendritic cells by inducing a highly-matured population, which contributed to induction of syngeneic and allogeneic lymphocyte proliferation and a preference to Th1 polarization, and multiple effects on macrophages based on phenotypic and cytokine analyses. Studies in murine cancer models showed that the fumagillin analogue F23 caused substantial inhibition of tumor development in three cancer models to different extents, with pronounced inhibitory effects on the expansion and functions of myeloid-derived suppressor cells (MDSCs), the signature cell population responsible for tumor progression and refractoriness to chemotherapeutic and immunotherapeutic agents, thereby suggesting the novel immunopharmacological properties of fumagillin and its analogues contributed to tumor suppression. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

In vertebrate epithelia, p120-catenin mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120s activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120s cadherin stabilizing- and RhoA-suppressing activity. Unexpectedly, removing p120s Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120s distinct activities toward E-cadherin and Rho are molecularly and functionally coupled, and this in turn enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.

published_or_final_version / Medicine / Master / Doctor of Medicine

published_or_final_version / Medicine / Master / Doctor of Medicine

Non-Hodgkin lymphomas often arise at sites of chronic inflammation, exposing them to oxidative stress, or increased levels of reactive oxygen species (ROS). Increases in ROS are associated with tumor initiation, promotion and progression. Chronic exposure to ROS may promote the transformation of lymphocytes to lymphoma. Anti-apoptotic proteins such as Bcl-2 are commonly overexpressed in lymphoid malignancies. The protective function of Bcl-2 is partially dependent on its ability to regulate the redox environment. Adaptation to oxidative stress via the upregulation of anti-oxidant defense enzymes or upregulation of anti-apoptotic proteins appear to, in part, confer resistance to chemotherapeutics through their ability to regulate the redox environment. This suggests that using an agent to disrupt redox homeostasis has potential as a therapeutic strategy to circumvent these resistance mechanisms. The following studies examine the use of the copper chelator drug, ATN-224, to modulate the redox environment and circumvent the upregulation of anti-oxidant defense enzymes and anti-apoptotic proteins. These studies demonstrate that ATN-224 inhibits the primary anti-oxidant defense enzyme SOD1 and the redox-driven proton pump CcOX (complex IV in the electron transport chain). This inhibits the ability of SOD1 and CcOX to regulate the cellular and/or mitochondrial redox environment, respectively. ATN-224 treatment increases oxidative stress and induces peroxynitrite-dependent cell death. Furthermore, ATN-224 induces the release of AIF from the mitochondria, resulting in caspase-independent cell death. Collectively, these findings suggest that modulating the redox environment with ATN-224 has therapeutic potential in the treatment of non-Hodgkin lymphoma.

Despite considerable progress in conventional cancer therapies, major challenges persist in the treatment of patients with advanced stage malignancies. Cancer immunotherapy (harnessing the immune system against tumors) has demonstrated limited success to date, partially due to the immunosuppressive environment generated by tumors. The mechanisms of cancer-induced immune suppression are multiple and include the promotion of immunosuppressive cells such as regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). MDSC expand in tumor-bearing hosts and play a central role in cancer immune evasion by inhibiting adaptive and innate immunity. Different approaches have been explored to negatively impact MDSC, each associated with specific pitfalls. In this study, we demonstrated that the anthracycline doxorubicin selectively eliminates MDSC in the spleen, blood and tumor beds. Furthermore, five days after doxorubicin treatment residual MDSC exhibited impaired suppressive function, which correlated with reduced reactive oxygen species (ROS) production, and down-regulation of arginase-1 and indoleamine 2,3-dioxygenase (IDO) expression. Of therapeutic relevance, the frequency of effector lymphocytes (CD4⁺ and CD8⁺ T cells) or natural killer cells (NK) to suppressive MDSC ratios was significantly increased following doxorubicin treatment of tumor-bearing mice. Importantly, the proportion of natural killer (NK) and cytotoxic T cells (CTL) expressing perforin and granzyme B and of CTL producing IFNγ was augmented following doxorubicin administration. The mechanism of doxorubicin-mediated elimination of MDSC was partly mediated by the increase of ROS production in MDSC at earlier time points after doxorubicin treatment. Consistently, MDSC isolated from gp91-/- mice were less sensitive to doxorubicin in vitro, and doxorubicin effects on MDSC in gp91-/- tumor-bearing mice were reduced. Of clinical significance, this drug efficiently combined with Th1 or Th17 lymphocytes to suppress tumor development and metastatic disease, resulting in better overall survival. MDSC isolated from patients with different types of cancer were also sensitive to doxorubicin-mediated cytotoxicity in vitro. Our results therefore indicate that doxorubicin may be used not only as a direct cytotoxic drug against tumor cells, but also as a potent immunomodulatory agent that selectively impairs MDSC-induced immunosuppression, thereby fostering the efficacy of T cell-based immunotherapy.

Cancer is a disease that is characterized by genetic mutations that occur leading to an increased propensity for abnormal cellular growth. Chromosome 1p36.3 is lost or aberrant in several tumor types, including neuroblastoma and melanoma. In order to understand the functional significance of chromosome 1p36.3 loss in tumors, our research has been focused on elucidating the function of cyclin dependent kinase 11 (CDK11), a gene that is located within this chromosomal region. CDK11 is a serine/threonine kinase and is a member of the p34cdc2-related family of kinases. CDK11 has multiple isoforms that are involved RNA processing, apoptosis and the cell cycle.CDK11 proteins are encoded by two different genes (Cdc2L1 and Cdc2L2), which are highly homologous to each other and are evolutionarily conserved. Our studies originated from investigating the regulation of the expression of CDK11 genes by isolating and characterizing the Cdc2L1 promoter region. We identified the basal promoter region and found that Cdc2L1 is regulated by transcription factors Ets-1, Skn-1 and E2F-1. Conversely, Cdc2L2 was shown to be regulated by TCF11/Nrf2, Ets-1 and CREB, displaying differential regulation of the two different CDK11 genes.CDK11p58 is generated during the G2/M phase of the cell cycle because of an internal ribosome entry site found within the full-length CDK11 transcript. The generation of CDK11p58 during the cell cycle had previously been shown to be a regulatory event; however the actual function of CDK11p58 remained unknown. Our research focused on determining potential interaction partners of CDK11p58 and elucidating the role of these interactions in the regulation of the cell cycle. Utilizing proteomic technology, we were able to find multiple potential interacting proteins with CDK11p58, including a RNA-dependent RNA helicase protein, DDX15. DDX15 is phosphorylated by CDK11p58 and both proteins are directly involved in cell cycle regulation. Additionally, CDK11p58 was found to associate with several cytoskeletal proteins, alpha- and beta-tubulin, lamin A, and vimentin, and heat shock proteins, Hsp70 and Hsp90. These investigations are only preliminary but provide a novel insight into the possible function of CDK11p58 during mitosis.

The adult mammalian brain hosts two regions of quiescent neural stem cells that continually generate new neurons throughout life. One of these regions, the subventricular zone (SVZ), primarily produces immature neuroblasts that migrate and integrate into the olfactory bulb during neurogenesis. Stem cells in the SVZ are of interest because of their potential utility in brain repair. Strikingly, stem cell location within this germinal region determines the type of neuronal progeny that a given stem cell generates, and stem cells retain this positional identity upon culture or transplantation. However, the molecular mechanisms that regulate fate specification and proliferation in neural stem cells remain largely unknown, and it is not yet clear whether the intrinsic properties of particular stem cells affect their response to oncogenic events or damage. My lab has shown that active sonic hedgehog (Shh) signaling occurs in the ventral SVZ and is necessary and sufficient for the regulation of neuronal fate in a specific group of ventral stem cells. The dorsal region of the SVZ does not have high shh pathway activation; despite expression of most pathway components, these cells appear to be resistant to stimulation by Shh ligand. I hypothesize that Shh pathway signaling is actively repressed in the dorsal SVZ through expression of the Gli3 transcription factor, and that this repression is necessary to generate dorsal neural progeny. My thesis seeks to determine which downstream components of the shh pathway control proliferation and fate specification in dorsal stem cells.

Perturbation of physiological processes stimulate a cascade of events resulting in adverse pathogical conditions, the most predominant being cancer. Colorectal cancer (CRC) is the third most common cancer in the United States and the third leading cause of cancer deaths among men and women. CRC is most commonly initiated by mutation of Adenomatous polyposis coli (APC), a tumor suppressor gene of the Wingless/Wnt pathway, which leads to constitutive activation of Wnt signaling responsible for the majority of CRC cases. Although significant progress has been made in understanding the sequential genetic events leading to cancer development, the precise genes and associated molecular events underlying tumor progression are still poorly understood. Our lab has previously reported a correlation between increased expression of claudin-1, a tight junction protein, in primary and metastatic tumor samples of CRC patients. Additionally, in adenomas of the APCMin mouse, an experimental model for CRC, and human cell lines harboring mutations in APC, claudin-1 expression is highly increased and mislocalized. Taken together, these studies suggest existence of a potential interplay between dysregulated claudin-1 expression and APC, thus supporting an essential role in the regulation of sporadic colonic neoplasia. In addition to genetic stimuli, CRC can be initiated by an inflammatory stimulus. Patients with inflammatory bowel disease, a chronic disorder of the gastrointestinal tract, are at high risk for developing CRC. This may be attributed, in part, to repeated injury and repair, which induces transformation of the colonic epithelium. As such, increased Claudin-1 expression is also reported to be associated with active IBD and colitis-associated cancer, yet its distinct role in inflammation has yet to be elucidated. This study investigates the specific contribution of increased claudin-1 expression to inflammation and CRC. These results identify a role for Claudin-1 in epithelial differentiation through a novel interaction with Notch signaling. Additionally, claudin-1 enhances tumorigenesis through upregulation of inflammation, IL-23/IL17 signaling, when combined with mutant APC. Once understood how overexpression claudin-1 contributes to tumor progression, we can better investigate its potential as a target for therapy.

Major Histocompatibility Complex (MHC) Class I molecules present peptides to CD8⁺ T cells and are essential for most adaptive immune responses. The first described-spliced tapasin (“isoform 1”) plays a critical role in MHC-I antigen presentation by facilitating peptide loading onto MHC-I molecules in the endoplasmic reticulum (ER). This thesis examines the expression, localization and function of two novel, alternatively-spliced isoforms of human tapasin that lack exon 7 (“isoform 2”) or both exons 6 and 7 (“isoform 3”). Isoform 1 contains a di-lysine ER-retention motif; the two novel isoforms encode different carboxy (C) termini that lack this motif. It was hypothesized that isoforms 2 and 3 would function in MHC-I cross-presentation of exogenous antigens in non-ER compartments. Isoform 2, like isoform 1, was found to be mainly ER-localized; however, both these isoforms were also found to co-localize in smaller amounts with the trans Golgi network and endo/lysosomes by confocal microscopy. Isoform 3 lacks a transmembrane domain and was found to be secreted from cells as well as being found within the ER. All isoforms were widely expressed at the RNA level in many tissues and cell types; however, mature dendritic cells (DCs) expressed the highest levels of all three isoforms, consistent with the high cross-presenting abilities of DCs. Both isoform 1 and 2 stabilized the transporters associated with antigen processing (TAP) in murine tapasin-/- cells, but isoform 3 did not due to its missing transmembrane domain. Isoform 1 and 2 mediated very similar effects on endogenous MHC-I presentation of self and viral peptides, on surface MHC-I thermostability, and on MHC-I maturation rates. Isoform 3 was found to decrease loading of exogenous peptides onto MHC-I. None of the isoforms influenced cross-presentation of the soluble antigen ovalbumin in a mouse dendritic cell line. This thesis also examines the effect of antigen presentation machinery (APM) re-expression in MHC-I-deficient tumor cell lines, B16F10 and CMT.64, which are deficient in TAP and tapasin. Virally-driven TAP1 and Tapasin expression increased MHC-I expression in the tumor cell lines, augmented tumor cell immunogenicity, and decreased tumor growth in vivo due to increased tumor cell elimination by the immune system.