Thesis (M. Phil.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 153-173). Also available online.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes bibliographical references and vita.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 107-111). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Mary C. Farach-Carson, Dept. of Biological Sciences. Includes bibliographical references.

Thesis--Cornell University. / Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Pathology / Master / Master of Philosophy

A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). These TICs are believed to display unique survival mechanisms, and account for failure in therapeutic treatments. It is also believed that, effective treatments against the diseases can only be developed through targeting and eliminating these TICs. We previously identified TIC populations derived from hepatocellular carcinoma (HCC) that are characterized by membrane expression of CD133. As findings from our previous studies were mostly based on HCC cell lines, here, we first identified rare CD133+ subpopulations in freshly resected HCC specimens, but not their non-tumor counterparts. We also found increased CD133 expression to be associated with advanced disease stages, increased recurrence rate and poorer overall survival in HCC patients. Next, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133+ liver TICs isolated from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133+ cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133+ liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis and initiate tumors. In further support of these observations, IL-8 repression in CD133+ liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be also preferentially expressed in CD133+ liver TICs. Exogenous NTS treatment resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, key components of the MAPK signaling pathway. Enhanced IL-8 secretion by CD133+ TICs can in turn activate an IL-8 positive feedback loop through MAPK signaling. Subsequent studies from CD133 sorted cells found only CD133+ TICs, but not CD133- cells were able to response to exogenous NTS / IL-8 stimulations with concomitant up-regulation of CD133, suggested that the preferential expression of NTS / IL-8 signaling cascade was also important in CD133+ TICs self-renewal and maintenance. Further to its role as a liver TIC marker, CD133 also plays functional roles in conferring TICs properties via regulating NTS / IL-8 / CXCL1 / MAPK signaling. These results suggested that CD133+ liver TICs promote angiogenesis, tumorigenesis and selfrenewal through NTS-induced activation of the IL-8 signaling cascade. In conclusion, our findings had identified rare expressions of CD133 in clinical HCC specimens and hence its prognostic values. We also show for the first time the functional roles of CD133 in conferring tumorigenic potential to liver TICs. The characterization of underlying molecular signaling in CD133+ liver TICs in this study should provide not only a better understanding of the mechanisms regulating this specific population of cells but also novel insights that could allow the development of more effective therapeutic treatments of this disease. / published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Acute myeloid leukemia (AML) is a group of heterogeneous diseases characterized by an abnormal increase in myeloblasts in circulation and/or bone marrow. Internal tandem duplication (ITD) of the fms-like tyrosine kinase 3 (FLT3) gene occurs in about 30% of AML and is associated with an inferior prognosis. Tyrosine kinase domain (TKD) mutations occur in about 5% with uncertain prognostic significance. Intensive chemotherapy and allogeneic hematopoietic stem cell transplantation (HSCT) are the mainstays of treatment. However these approaches have reached a deadlock with a cure rate of 30-40%. Targeting FLT3 in AML with multi-tyrosine-kinase inhibitors has been evaluated in Phase II/III clinical trials. Despite an initial clearance of myeloblasts, the leukemia invariably progresses despite continuous treatment. The mechanisms of drug resistance and leukemia progression, hence the effective therapeutic strategies are currently unknown, limiting its clinical application. These issues were addressed in the present study. In the first part, 13 patients with chemo-refractory or relapsed FLT3-ITD+ AML received sorafenib 200-400 mg twice daily of whom 12 patients achieved clearance or near clearance of bone marrow blasts after a median of 27 days (range 21-84 days). There was evidence of myeloid differentiation of the leukemia blasts at remission. Leukemia progression occurred in 9 patients after a median of 72 days (range 54-287 days) and in 4 out of 6 patients it was dominated by clones carrying double FLT3-ITD and -TKD mutations. Microarray studies comparing myeloblasts before sorafenib treatment (sorafenib naïve) and at subsequent progression (sorafenib resistant) demonstrated up-regulation of 64 genes including ALDH1A1, JAK3 and TESC whose functions were unknown in AML. Transplantation of sorafenib naïve and resistant myeloblasts into NOD/SCID mice recapitulated their clinical behavior when the animals were treated with sorafenib. Both ITD and TKD mutations at D835 were identified in leukemia initiating cells (LICs) from sorafenib naïve samples. These results suggested that sorafenib have selected more aggressive sorafenib-resistant subclones carrying both FLT3-ITD and D835 mutations. In the second part, the gene encoding tescalcin (TESC), that was up-regulated at sorafenib resistance and was known to activate a sodium/hydrogen exchange (NHE1), was evaluated to examine its link with TKI resistance. TESC was highly expressed in FLT3-ITD+ AML cell lines MOLM-13 and MV4-11 and its knock-down by siRNA lowered intracellular pH and induced apoptosis. The results were recapitulated by treatment with a NHE1 inhibitor, 5-(N,N-Hexamethylene)amiloride (HMA). Induction of sorafenib resistance in MOLM-13 cell line (MOLM-13-RE) significantly increased its sensitivity to HMA. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD+ AML cells significantly reduced leukemia initiation in NOD/SCID mouse xenotransplantation. Normal CD34+ cells engraftment was not affected. HMA treatment significantly enhanced suppression of FLT3 signaling by sorafenib even in sorafenib resistant cell lines. These observations provided novel information about the pathogenetic role of TESC-NHE1-pHi in sorafenib resistance in AML. In conclusion, the information derived from the present study has provided mechanistic insights to the emergence of drug resistance during sorafenib treatment and important guide for future therapeutic strategies targeting FLT3-ITD+ AML. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy