Solid tumors typically require angiogenesis, the development of new blood vessels, for growth and metastasis. Vascular endothelial growth factor (VEGF) induces angiogenesis by activating receptors on host endothelial cells. One such receptor, Flt-1, occurs as either a membrane bound or a secreted form (sFlt-1) that can inhibit angiogenic signaling. Previous studies have shown that variation in mRNA expression of VEGF and its receptors KDR, sFlt-1 and Flt-1 occurs in pathological angiogenesis, i.e. metastatic tumorigenesis. We hypothesize that the ratio of sFlt-1:Flt-1 mRNA will be altered in the presence of solid tumors. The objective of this study was to evaluate the expression of sFlt-1 and Flt-1 mRNAs in a mouse metastatic tumor model using CT26.CL25 cells. CT26.CL25 cells are VEGF-producing murine colon carcinoma cells transfected with the lacZ gene, which expresses B-galactosidase activity. These cells, injected intravenously, form tumor nodules in the lung. A pilot study revealed development of lung nodules in mice nine days after intravenous injection with 105 cells. In a second study, twenty-five 10- week-old female Balb/c mice were injected intravenously, via tail vein, with 2 x 105cells, and fifteen with vehicle control. Lung nodules developed in all mice injected with cells. Tissues were harvested by routine necropsy and either formalin-fixed for routine histology/histochemistry or stored for quantitative RT-PCR (QPCR) analysis of gene expression. Under microscopic evaluation, sections of lungs stained with Hematoxylin & Eosin (H&E) revealed nodules composed of polygonal neoplastic cells. cDNA from lungs (14 tumor-bearing, 10 controls) and cultured CT26.CL25 cells was analyzed by QPCR using primers and TaqMan probes directed against sFlt-1, Flt-1, KDR, VEGFA, PlGF (Placental Growth Factor), Angiotensin Converting Enzyme (ACE), 18S ribosomal RNA and neoR (neomycin phosphotransferase). We observed an increased sFlt-1:Flt-1 ratio in tumor-bearing versus control lungs, suggesting that tumor-derived signals may influence sFlt-1 and Flt-1 expression differentially. Additionally, there was increased expression of Flt-1, sFlt-1 and KDR in tumors versus controls, but not in VEGF expression in tumors versus controls. Interestingly, expression of PlGF was increased in tumors versus controls, suggesting its role as an enhancer of tumor progression in the presence of other angiogenic factors. Together, these findings indicate that solid tumor angiogenesis results from an intricate balance of various angiogenic factors. / Master of Science

Thesis (M.A.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Evidence that dietary fats influence carcinogenesis comes from both epidemiological and experimental data. Previous experimental studies suggest that dietary fat acts as a promoter in chemically induced carcinogenesis and this effect depends on the degree of saturation and concentration of dietary fat. [TRUNCATED] / 2999-01-01

NM23-H1 represents the first identified metastasis suppressor, exhibiting reduced expression in breast carcinoma and melanoma, and an ability to inhibit metastatic growth without significant impact on the transformed phenotype. Although its molecular mechanism of action is not fully understood, NM23-H1 possesses at least three enzymatic activities that may mediate metastasis suppressor function. It catalyzes nucleoside diphosphate kinase (NDPK) activity, as well as protein histidine kinase and 3’-5’ exonuclease activities. As 3’-5’ exonucleases are generally required for maintenance of genomic integrity, this activity represents a plausible mediator to underlie the metastasis suppressor function of NM23-H1 protein. To investigate the relevant activity of NM23-H1 in metastasis suppression, we constructed a panel of NM23-H1 mutant variants with selective enzymatic lesions. Previous studies have identified some key amino acid residues important for the enzymatic characteristics of NM23-H1. However, none of them are selective for disrupting the 3’-5’ exonuclease activity. In this study, we show that a substitution of Glu5 to alanine results in a dramatic, selective loss in 3’-5’ exonuclease property without significant affecting other enzymatic activities. To measure the extent to which the exonuclease function opposes mutation and metastasis, NM23-deficient and metastatic cell lines with forced expression of NM23-H1 variants are analyzed in nude mice. In spontaneous metastasis models, NM23-H1 mutants deficient in 3‘-5’ exonuclease activity significantly disrupt the capacity of metastasis suppression of wild-type protein, indicating that the 3’-5’ exonuclease activity of NM23-H1 is necessary for the spontaneous metastasis-suppressing effects. As 3'-5' exonucleases are generally associated with DNA repair process, we have also studied the contributions of yeast NM23 homologue YNK1 to genomic integrity in Saccharomyces cerevisiae. Consistent with an antimutator function, ablation of YNK1 significantly results in increased mutation rates following exposure to UV irradiation and the alkylating agent methyl methanesulfonate (MMS). The impaired DNA-damage response of ynk1Δ cells suggests a role of human homologue NM23 in DNA repair. More evidence is being collected in our laboratory to demonstrate a role for NM23-H1 in maintaining genomic integrity. Collectively, our findings of DNA repair activity of NM23-H1 will contribute to the understandings of the mechanisms in metastasis suppression and new drug discoveries.

The majority of cancer related deaths occur due to the invasive growth of metastatic lesions. In the early stages of metastasis, circulating cell interact with the endothelial cells to establish at a distant site. In inflammation endothelial activation results in induction of adhesion molecules on the endothelium that participate in the homing of leukocytes. Because of the interactions of metastatic cells with the endothelium, the question was whether some of the characteristic molecules of endothelial activation were induced during metastasis. In vivo pulmonary metastatic models were used to characterize the expression profile of endothelial activation. Immunohistochemistry identified VCAM-1 to be induced on the pulmonary endothelium following tumour cell arrest. VCAM-1 upregulation was not observed prior to tumour cells arrest or within the first hours. In contrast, tumour cell arrest appeared to be required for endothelial activation, arguing against a mechanism analogous to leukocyte homing. The upregulation of VCAM-1 upon tumour cell arrest corresponded with the initiation of platelet clot formation around the tumour cell and recruitment of leukocytes to the site, both previously shown to be essential for metastasis. Disruption of both phenomena, either through genetic or pharmacological manipulation, demonstrated that in contrast to the recruited leukocytes, platelets were involved in inducing endothelial activation. Another protein investigated was VAP-1. In contrast to VCAM-1, central to VAP-1 adhesive function is its enzymatic activity. Blocking the functions of either molecule highlighted their role in facilitating the recruitment of the leukocyte population to the tumour cell. Disruption of which led to a significant attenuation of metastasis. While VCAM-1 and VAP-1 function appears critical in the early steps of metastasis, their inhibition had no effect at later stages of pulmonary colonization.

Metastasis is the most fearsome aspect of breast cancer (BC) a common disease in women, because it drives mortality. Although BC can invade almost any organ, it is most often found to invade the bone (31-79%), the brain (3-12%), the liver (8-18%) and the lung (11-13%). The site of distant metastasis is often associated with cause of death and length of survival. This dissertation examines whether the presence of select lifestyle and clinical factors can predict metastatic spread to the lung and/or the liver for a particular woman with advanced breast cancer. A systematic review of the literature identified tobacco use as a risk factor for lung metastasis in women with BC and suggested that obesity, hormone replacement therapy prior to BC diagnosis, hormonal therapy post diagnosis, and post-mastectomy radiation therapy may have an impact on this association. The review also uncovered that liver disease (i.e. hepatic steatosis, chronic hepatitis B infection, cirrhosis) is associated with the occurrence of liver metastasis in patients with colorectal cancer and that hyperglycemic and oxidative stress conditions as well as alcohol consumption were found to be associated with liver metastasis in colorectal or BC patients. We conducted a retrospective hospital-based case-control study of the association of select lifestyle and clinical factors with metastases detected in the lung and the liver among women diagnosed with stages II-IV BC and seen at the Columbia University Medical Center from 2008 to 2013. Select relevant clinical variables were extracted from the hospital patient charts and lifestyle factors from patients’ responses to a questionnaire developed for the purposes of this research. We examined whether smoking and / or post-mastectomy radiation therapy to the breast and/or the chest area were associated with an increased risk of 1st site lung metastasis in our sample of women with metastatic BC. We found that lifestyle factors such as smoking history or BMI at diagnosis did not affect the likelihood of 1st site lung metastasis in our sample of women. We also investigated whether a history of alcohol intake or chronic liver disease was associated with risk of developing a 1st site liver metastasis. Our analyses suggested that lifestyle factors such as alcohol intake or obesity might not affect the likelihood of 1st site liver metastasis in women with metastatic BC. We also report that a history of chronic liver disease significantly increased the odds of 1st site liver metastasis. Given our findings around adjuvant post mastectomy radiation therapy and chronic liver disease, we suggest collecting adjuvant treatment or relevant comorbid information in larger cohort studies. A better understanding of the relationship between these factors and the sites of metastasis has the potential to increase our understanding of the metastatic process. If we can find ways to identify women at high risk of metastatic disease, or develop preventive or therapeutic measures against lung or liver metastasis, we can hope to reduce mortality from metastases.

The metastasis suppressor gene NM23-H1 has been shown to possess three enzymatic activities including nucleoside diphosphate kinase, histidine-dependent protein kinase and 3’-5’ exonuclease activity. While these properties have been demonstrated in vitro using recombinant proteins, the contribution of these activities to suppression of metastatic dissemination is unknown. Site-directed mutagenesis studies were used to identify amino acid residues which are required for proper function of each enzymatic activity associated with H1, providing a platform for studying the importance of each function on an individual basis. To assess the relevance of these activities to melanoma progression, a panel of mutants harboring selective lesions disrupting the enzymatic activities of H1 were overexpressed using stable transfection in two melanoma cell lines, WM793 (isolated from a vertical growth phase human melanoma), and the metastatic derivative cell line 1205LU. In vitro correlates of metastasis measuring motility and invasion were used in an attempt to identify the mechanism mediating H1-dependent motility suppression of cancer cells. Surprisingly, all mutants studied retained full motility suppression in this setting, suggesting that the enzymatic functions associated with H1 are not required for inhibiting cell migration. Instead, gene expression analyses conducted on the panel of stable transfectants indicate that differences in steady-state mRNA levels of genes involved in mitogen-activated protein kinase (MAPK) signaling showed significant correlations with H1 expression and motility suppression. RNAi studies have confirmed that H1-dependent modulation of the expression of two genes in particular, BRAP and IQGAP2, contribute to the observed phenotype, suggesting a novel mechanism used by NM23 to control cellular migration in human melanoma.

Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. While some progress has been made in the understanding of metastasis, the detailed molecular mechanisms that define the various stages of the process remain elusive. A major rate limiting step in metastasis is the loss of function of metastasis suppressor genes which block a cascade of crucial steps including the loss of adhesion of primary tumor cells, intravasation into the blood and lymphatics with subsequent extravasation at distant sites, and the formation of new colonies. Our examination of gene expression profiles from a breast cancer model system consisting of cell lines with the same genetic lineage representing the benign, carcinoma in situ and the metastatic stages led to the identification of a candidate metastasis suppressor gene, serum deprivation response (SDPR). We observed that stable SDPR over-expression in highly metastatic breast cancer model cell lines significantly suppressed metastatic nodule formation in NOD/SCID mice. Furthermore, meta-analysis of pre-existing gene expression data suggests that the loss of SDPR expression significantly correlated with relapse of breast cancer in patients who underwent therapy. We found that the mechanism of SDPR function involves activation of the p53 pathway and inhibition of ERK and NF-κB signaling pathways. SDPR increased the apoptotic population, hindered growth in 3D cell culture and impaired migration. Moreover, SDPR was suppressed by promoter DNA methylation in metastatic cell line models and its expression was restored by 5-aza-2'-deoxycytidine treatment. Together, our results reveal that SDPR is a novel metastasis suppressor gene with potential value as a target for future therapeutic applications.

Metastasis suppressors regulate multiple steps during the process of dissemination of tumor cells from primary sites to distant organs, while they do not affect the growth of the primary tumor. Previously, we identified NDRG1 (N-myc downstream regulated gene 1) as a tumor metastasis suppressor gene and found that it is negatively involved in metastatic progression of prostate and breast cancers. To elucidate the molecular mechanism of NDRG1 function, we used the yeast two-hybrid system to identify proteins interacting with NDRG1. In the first part of this project, we demonstrate that NDRG1, interacts with the Wnt receptor, LRP6, followed by blocking of the Wnt signaling, and therefore, orchestrates a cellular network that impairs the metastatic progression of tumor cells in vitro and in animal model. We also found that restoring NDRG1 expression by a small molecule compound significantly suppressed the capability of otherwise highly metastatic tumor cells to thrive in circulation and distant organs in animal models. In addition, our analysis of clinical cohorts data indicate that Wnt+/NDRG-/LRP+ signature has a strong predictable value for recurrence-free survival of cancer patients. Collectively, we have identified NDRG1 as a negative master regulator of Wnt signaling during the metastatic progression, and therefore revealed a novel control mechanism of Wnt signaling in tumor progression. Previously, we identified the metastasis promoting transcription factor, ATF3, as a downstream target of NDRG1. Further analysis revealed that the KAI1 promoter contained a consensus binding motif of ATF3, suggesting a possibility that NDRG1 suppresses metastasis through inhibition of ATF3 expression followed by activation of KAI1 gene. In the second part of this project, we examine a possible link between two metastasis suppressor genes, NDRG1 and KAI1, through ATF3. We demonstrated that ectopic expression of NDRG1 was able to augment endogenous KAI1gene expression in prostate cancer cell lines, while silencing NDRG1 accompanied with significant decrease in KAI1 expression in vitro and in vivo. In addition, our results of ChIP analysis indicate that ATF3 indeed bound to the promoter of KAI1 gene. Importantly, our promoter-based analysis revealed that ATF3 modulated KAI1 transcription through cooperation with other endogenous transcription factor as co-activator (ATF3-JunB) or co-repressor (ATF3-NFêB). Moreover, loss of KAI1 expression significantly abrogated NDRG1-mediated metastatic suppression in vitro as well as in a spontaneous metastasis animal model, indicating that KA11 is a functional down-stream target of NDRG1 pathway. Our result of immunohistochemical analysis showed that loss of NDRG1 and KAI1 occurs in parallel as prostate cancer progresses. We also found that a combined expression status of these two genes serves as a strong independent prognostic marker to predict metastasis-free survival of prostate cancer patients. Taken together, our result revealed a novel regulatory network of two metastasis suppressor genes, NDRG1 and KAI1, which together concerted metastasis-suppressive activities through intrinsic transcriptional cascade.

No description available.

Anterior Gradient-2 protein (AGR2) has recently been linked to the onset of several pathologies including asthma and inflammatory bowel disease. Most interestingly, it has been discovered to influence the transformation of cells and metastatic growth essential to cancer development, and has subsequently been linked to the development of resistance to anti-cancer therapeutics. AGR2 protein is overexpressed in a diverse range of human cancer types, and has been detected secreted into the extracellular milieu. Thus, AGR2 protein represents a compelling pro-oncogenic signalling intermediate in tumour emergence and endurance. This thesis presents an interdisciplinary approach including structural biology, cell biology and synthetic biology, and clinical studies to shed more light on the role of AGR2 in cancer development. Synthetic cell based reagents were developed to define the dominant pathways that are reprogrammed in a cell as a result of AGR2 synthesis. A cell panel was engineered incorporating the AGR2 (and mutants thereof) allele into the AGR2-null A375 cell line. These tools were then coupled to quantitative proteomics (SILAC) to unravel the mechanism whereby introduction of AGR2 alters cell phenotype, allowing identification of dominant pathways affected by AGR2 signalling. Using pathway analysis tools, the dominant pathway suppressed by wt-AGR2 expression highlighted the p53-signalling axis. DNA damage induced p53 stabilisation and p21 induction by cisplatin treatment confirmed the influence of AGR2 gene expression. Further data analysis identified the outlying protein expression changes identified by SILAC was the anti-viral cell cycle regulator TSG101 (tumour susceptibility gene 101), and confirmed by immunoblotting. Transfection and silencing studies of TSG101 confirmed that TSG101 attenuates p53 function. These data provide a mechanism to explain the most dominant pathways reprogrammed by AGR2 expression, incorporating ER stress response, proliferation markers and p53 pathway attenuation. Further advances were made in analysis of the function, regulation, and drugability of AGR2 protein. Assays were devised to define the subunit structure of AGR2 as a dimer unit; subsequent functional studies defined intrinsically disordered motifs that regulate stability of the dimer. A two-site sandwich microtiter assay (2SMTA) was designed to screen for self-peptides and mutations that regulate oligomer stability. These assays were used to identify the first biochemical property of AGR2 being that the dimer unit is required for maximal binding to the AAA+ protein, and well characterised AGR2 interactor, Reptin. In addition, based on this dimeric structure, a novel solution based dimerisation assay was developed to identify natural products that are able to disrupt the dimer suggesting that AGR2 itself can be targeted in principle with small molecules for therapeutic purposes.