published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

Retinoblastoma binding protein 6 (RBBP6) is a nuclear protein, previously implicated in the regulation of cell cycle and apoptosis. It is a multi-domain protein containing a Zinc finger, a RING finger, an Rb binding domain, a p53 binding domain and a novel N-terminal protein domain, the so called, Domain With No Name or DWNN. The RBBP6 gene encodes three isoforms of this particular protein. A common feature of all three isoforms of RBBP6 is the presence of the N-terminal DWNN domain. RBBP6 isoform 3 is comprised of the DWNN domain only. The DWNN itself has a ubiquitin-like fold, sharing 22% similarity with ubiquitin. It is likely that DWNN regulates intracellular levels of the two tumour suppressors, Rb and p53 through the ubiquitin-proteasome pathway and as such, DWNN may therefore play a role in the deregulation of cell cycle control in cancer cells. A mouse homologue, P2P-R of the gene has been implicated in mitotic apoptosis. The expression of DWNN, RBBP6 and their roles in the cell cycle, apoptosis and human cancer were investigated. RT-PCR and real-time PCR were used to determine the gene expression of DWNN and RBBP6 variants in human cancer cells. An anti-human DWNN antibody was characterized using both Western Blotting analysis and MALDI-TOF mass spectroscopy to determine whether the antibody specifically recognizes DWNN and RBBP6 isoforms, or if it recognizes other proteins. Western blotting was also used to determine the nature of the DWNN in human cell lines. A DWNN probe and the characterized anti-human antibody were used to localize DWNN and RBBP6 gene products at the mRNA and protein levels using ISH/FISH and Immunohistochemistry respectively. Cell labelling was also performed using this antibody to localize RBBP6 products in human cell lines. RNA interference and over-expression of DWNN and RBBP6 gene products was carried out to further investigate the role of RBBP6 products in the cell cycle, apoptosis and carcinogenesis. Cloned RT-PCR products of RBBP6 binding domains, the RING finger domain, pRb-binding and p53-binding domains in human cancers cell lines (Hek 293T, MCF7, HeLa and HepG2 cells) showed no mutations, but MCF-7 cells showed the lowest expression of the RBBP6. Real-time PCR and Western blotting analysis confirmed that MCF-7 cells express very little DWNN (RBBP6 isoform 3) and RBBP6 gene products when compared to Hek 293T, HeLa and HepG2 cells. It was also shown that the anti-human DWNN antibody recognizes the DWNN domain (RBBP6 isoform 3) and the larger RBBP6 isoforms. Using 2D gel electrophoresis and MALDI-TOF spectrometry, it was also found that DWNN is associated with other proteins namely, Recoverin and a hypothetical protein XP_002342450. This result suggested that DWNN may be a ubiquitin-like protein, which may be specific to these proteins in human cells. FISH and IHC demonstrated that the DWNN domain and its relatives are down-regulated in human cancers at both mRNA and protein levels, respectively. In contrast, however, cell staining showed that the expression of the DWNN gene products was high during the G2/Mitosis transition. Knocking-down the DWNN domain or over-expressing it did not sensitise the Hek 293T cells to Camptothecin (CPT)-induced apoptosis but rather slowed down cell growth. These results strongly suggest that the DWNN gene is likely to be involved in cell cycle control. Up-regulation in mitotic cells and down-regulation in cancers also implies that RBBP6 gene products may additionally be involved in cell cycle arrest. Moreover, down-regulation in human cancers particularly indicates that the loss of its function which correlates with loss of cell cycle control in this disease may be involved in the pathogenesis of cancer. This was confirmed by up-regulation of the DWNN in arsenic trioxide induced cell cycle arrested cells specifically at G2/M phase where a p53-dependent cell cycle arrest ensued. It is thus proposed that the DWNN may be implicated both as a p53 stabilizer and additionally as a G2/M progression regulator.

The aim of this thesis is to evaluate the role of the extracellular environment in regulating breast cancer cell morphological and invasive characteristics. In vitro experiments of breast cancer cell lines in three dimensional matrices, which afford control over variables of interest while maintaining physiological relevance, were utilized for this purpose. We evaluated the sensitivity of cell morphology to the dimensionality, biochemistry, and mechanical properties of the extracellular environment as well as the reciprocal effects cells display when remodeling the extracellular environment during invasion. Chapter 1 introduces background material on breast cancer development, classification systems, and in vitro methods of research. Chapter 2 describes protocols for cell care and experiments used in these studies. In chapter 3, we explore the role of fibrillar collagen I environments in breast cancer cell invasion. This was motivated by previous research that has associated high breast tissue density with breast cancer risk and poor prognosis as well as tissue stiffness with cancer cell aggressiveness. Breast cancer cells were found to regain an invasive phenotype in sterically constrained environments when the extracellular matrix included a fibrillar component. In chapter 4, the relationship between cell morphology and invasive behavior in various dimensional contexts was assessed. Anecdotal evidence has shown stellate morphology may be associated with epithelial to mesenchymal transition and invasive capacity in cancer cells. Differences in the dimensionality and biochemistry of the environment resulted in changes to cell aggregate morphology. Although morphology did not predict invasive capacity as measured by spheroid invasion in collagen I, invasion was found to correlate with cancer-related gene expression profiling, suggesting the ability of cancer cells to utilize more than one mode of invasion. Chapter 5 explores to what degree the presence of invasive cells can give rise to invasive behavior from noninvasive cells. Segregation of cell subtypes during co-culture spheroid formation was found to be altered in the presence of BME. When implanted into collagen gels, invasive cell lines that generate structural changes to the extracellular matrix on their own were able to confer invasive behavior to otherwise noninvasive cell lines in some cases. Chapter 6 summarizes these findings and suggests further studies. Appendix 1 lists useful abbreviations. In Appendices 2 and 3, codes for ImageJ and Matlab-based analyses are recorded. Through this work, we see how cell morphology and invasive capacity are influenced by the extracellular environment. Cells that can interact with components of the extracellular matrix through matrix-specific integrins show a range of capacities for remodeling the extracellular environment, which in turn plays a role in invasive capacity. We anticipate that enhanced understanding of the role of the extracellular environment in regulating cell morphology and invasive behavior will lead to advances in the study of cell locomotion as well as in cancer research, diagnosis, and treatment.

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Medicine / Master / Master of Philosophy

published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

Tumor heterogeneity has long been observed and recognized as a challenge to cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses proposed to explain such a phenomenon. A specific cancer stem cell marker has not been determined for non-small cell lung cancers (NSCLC), preventing the definitive evaluation of whether the biology of NSCLC is governed by the CSC model. This study aimed to analyze the expression of candidate CSC markers and using the identified putative marker, to isolate CSC and determine the applicability of the CSC model in NSCLC. The expression or activities of four putative stem cell markers, CD24, CD44, CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours. Markers with increased expression after treatment were considered potential CSC markers and used for isolating tumor cell subpopulations from the untreated cell lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further characterized by functional and phenotypic studies. Flow cytometry showed amongst the 4 markers, only ALDH1 expression was significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise, higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells formed tumor in SCID mice which were serially transplantable to 2nd and 3rd recipients, while no tumor was formed from ALDH- cells with even ten times the number of cells. Also, expression analysis revealed higher expression of the pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view of previous studies reporting CD44 as a CSC marker in lung cancer, double marker selection of putative CSC was performed to compare ALDH1+CD44+ and ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation possessed higher self-renewal ability and chemo-resistance. Cell migration and invasion assays showed differences between the ALDH1+CD44+ and ALDH1- CD44+ subpopulations. The significance of these observations require further investigation. In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem cells as evidenced by enhanced self-renewal and differentiation abilities in ALDH1+ subpopulation. Furthermore, this study observed the presence of at least two potential stem cell subpopulations in NSCLC cells with differential selfrenewal, chemotherapy resistance and cell mobility properties. Further investigations are required to validate these observations and to investigate the underlying mechanisms. Better understanding of these issues would help to solve the challenges brought by tumor heterogeneity in lung cancer therapy. / published_or_final_version / Pathology / Master / Master of Philosophy

Angiogenesis is one of the essential hallmarks of cancer, typically breast cancer. Signaling from VEGFR-2 is necessary for the execution of VEGF-induced proliferation, migration, and tube formation of cultured endothelial cells in vitro and the onset of angiogenesis on tumors in vivo. Ellagic acid is a naturally existing small molecular polyphenol widely found in fruits and vegetables. It was reported that ellagic aicd interfered with some angiogenesis-dependent pathologies. Yet the mechanisms involved were not fully understood. Thus we analyzed its anti-angiogenesis effects and mechanisms on human breast cancer utilizing in vitro and in vivo methodologies. Besides, the in silico analysis was carried out to further analyze the structure-based interaction between ellagic aicd and VEGFR-2. The influences of ellagic aicd on VEGF-induced endothelial cells were studied by proliferation, tube formation and migration in vitro experiments. Kinase activity assay and western blotting were utilized to explore the effects of ellagic aicd on VEGFR-2 induced signaling pathway. Organ-based chick aortic ring model, in vivo Chorioallantoic membrane model and in vivo breast cancer xenografts were built to determine the anti-angiogenesis effects of ellagic aicd. Besides, software LigandFit algorithm in Discovery Studio 2.1 (Accelrys Inc., San Diego, CA) was applied to further understand the structure-based interaction between ellagic aicd and VEGFR-2. We found that ellagic aicd impeded a series of VEGF-induced angiogenesis processes including proliferation, migration and tube formation of endothelial cells. Besides, it directly inhibited VEGFR-2 tyrosine kinase activity and its downstream signaling pathways including MAPK and PI3K/Akt on endothelial cells. Ellagic aicd also obviously inhibited sprouts formation from chicken aorta and neo-vessel formation in chick chorioallantoic membrane. The growth and the P-VEGFR2 expression in breast tumors treated with ellagic aicd were also significantly suppressed. In the molecular docking simulation experiment, the structure-based interaction of VEGFR-2 with ellagic acid was found to be stable conformation by hydrogen bonds within residues Lys866 and Glu883 as well as by π–π interactions within residue Phe1045 at ATP binding pocket of VEGFR-2 catalytic domain. Taken together, ellagic aicd could exert anti-angiogenesis effects via VEGFR-2 signaling pathway in breast cancer. / published_or_final_version / Chinese Medicine / Master / Master of Philosophy

Esophageal cancer is the third most common digestive tract malignancy. Along with surgery, genotoxic drugs (e.g. cisplatin) and radiotherapy are the mainstays of treatment for this disease. Environmental factors and environmental stress-induced responses contribute to esophageal tumorigenesis and chemoresistance. Studying key molecules in stress-induced signal pathway can help unravel the underlying mechanisms and discover rational therapeutic targets. Cyclin D1 is DNA damage response protein. Genotoxic stress induces rapid cyclin D1 degradation and the molecules mediating this response are cell-type dependent. The first part of this study investigated the changes of cyclin D1 expression in response to genotoxic stress in immortalized esophageal epithelial cells, which are experimental models commonly used to study the early events of cancer development. The results showed that cyclin D1 underwent rapid proteasomal degradation before p53-induced p21 accumulation, which substantiates that cyclin D1 plays a role in eliciting cell cycle arrest very early in the DNA damage response. FBXO31 and FBX4, two F-box proteins previously reported to mediate cyclin D1 degradation, were found to be accumulated and unchanged, respectively, after ionizing irradiation in immortalized esophageal epithelial cells and esophageal squamous cell carcinoma (ESCC) cell lines. Yet, knockdown of FBXO31 did not rescue rapid cyclin D1 degradation upon UV or ionizing irradiation. This led to the hypothesis that accumulation of FBXO31 may have novel functions beyond mediating cyclin D1 degradation in cells responding to genotoxic stress. The second part of this study explored the function of FBXO31 in genotoxic stress response. The accumulation of FBXO31 in cancer cells after exposure to various genotoxic stresses was found to coincide with p38 deactivation, giving the clue that FBXO31 may negatively regulate this important pathway. Further studies revealed that knockdown of FBXO31 resulted in sustained activation of stress-activated MAPKs (SAPKs) p38 and JNK, as well as increase in UV-induced cell apoptosis, whereas overexpression of FBXO31 had opposite effects. The inhibitory role of FBXO31 on SAPK activation and apoptosis was confirmed by shRNA rescue experiments. Consistent with the observed anti-apoptotic effect, soft agar, colony formation and in vivo xenograft experiments showed that FBXO31 had oncogenic function in ESCC. Moreover, in vitro and in vivo results showed that knockdown of FBXO31 could sensitize ESCC cells to cisplatin treatment. The mechanism underlying the inhibition of SAPKs by FBXO31 was investigated in the third part of this study. Co-immunoprecipitation results showed that FBXO31 could interact with MKK6 (a p38 activator), but not p38, JNK1, or other MAP2Ks. FBXO31 was found to be co-localized with MKK6 in the cytoplasm. Mapping of interaction domains of FBXO31 revealed that aa 115-240 and aa 351-475 were responsible for binding to MKK6. Further study found that binding of FBXO31 to MKK6 could facilitate the K48-linked polyubiquitination and degradation of MKK6. Taken together, the results of this study showed that FBXO31 accumulation upon genotoxic stress can promote the degradation of MKK6 via K48-linked ubiquitination, thereby inhibiting SAPK activation and protecting cancer cells from genotoxic stress-induced apoptosis. FBXO31 may be a potentially useful therapeutic target to overcome chemoresistance in cancer therapy. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy