Doctor of Philosophy, The Ohio State University, 2016, Molecular, Cellular and Developmental Biology

Breast cancer is the second leading cause of cancer related death women worldwide despite significant advances in detection, diagnosis and treatment. The high mortality rates with accompanying therapeutic resistance and relapse over the years can be predominantly attributed to the immense heterogeneity of breast cancer. Importantly, the tumor microenvironment, which is comprised of stromal cells including cancer-associated fibroblasts (CAFs) contributes significantly to mammary tumor heterogeneity. Specifically, fibroblasts constitute the bulk of the tumor stroma and is now acknowledged to play an important role in regulating tumor progression. However, exact signaling pathways governing the cross-talk between the tumor cells and fibroblasts remain to be elucidated. In this study, we examined the fibroblast-specific roles of the proto-oncogene Ets2 and the tumor suppressor Pten in ErbB2-driven tumorigenesis. In the first part of this study, we show that deletion of fibroblast-specific Ets2 in the ErbB2 tumor model delayed the progression of these tumors by inhibiting tumor angiogenesis. We then independently examined the function of tumor suppressor Pten within the stromal fibroblasts. Previous studies demonstrate that loss of fibroblast-specific Pten promoted ErbB2-driven tumorigenesis. To identify how PTEN null fibroblasts induce the malignant transformation of associated ErbB2+ epithelia, we evaluated both phenotypic and gene expression changes within pre-neoplastic epithelium of mice with and without stromal PTEN. (i) Fibroblast-specific PTEN deletion causes an expansion of mammary epithelial stem cell (MaSC)-enriched subpopulations and alters their stem cell activity. Specifically, MaSC-enriched basal/myoepithelial cells from stromal PTEN null mice exhibited decreased levels of NOTCH3 receptor, a key player in the maintenance of stem cell fate. Interestingly, we discovered that PTEN null fibroblasts have low JAGGED-1 expression levels, a transmembrane li (open full item for complete abstract)

Committee: Michael Ostrowski (Advisor) Subjects: Cellular Biology; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2007, Animal Science

Failure to terminate the inflammatory response results in chronic inflammation that may lead to disease or cancer, especially in epithelial cells. We explored mammary epithelial cells as a model to identify mechanisms of anti-inflammatory activity in epithelia alone in the absence of immune cells. Bacterial endotoxin (ET) added to SCp2 mammary secretory epithelial cells: (1) induced both interleukin-6 (IL-6) secretion and nitric oxide (NO) production, but with unexpected delay in expression of mRNA for iNOS compared to IL-6; and (2) NFκB activation by 1 h after ET application (post-ET) that was transient for NFκB/p65 but persisted for NFκB/p50. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in ET-induced IL-6 and iNOS regulation via NFκB activation. Serum supplementation but not soluble extracellular matrix (EHS) enhanced ET-induced IL-6 mRNA expression and protein secretion without affecting iNOS mRNA expression or NO production, confirming the different modes of regulation of IL-6 and iNOS expressions. Culturing SCp2 cells on a confluent monolayer of SCg6 mouse mammary myoepithelial cells increased IL-6 secretion dramatically even in the absence of ET, with ET treatment further increasing IL-6 secretion but having little effect on induction of NO production over that for SCp2 cells alone; showing importance of microenvironment and cell-cell interaction in regulation of inflammation and likely its link to cancer in epithelia. ET-induced inflammation in SCp2 cells was used to screen and identify anti-inflammatory fractions of methanol extracts of wild Lebanese Centaurea ainetensis, used in Lebanese folk medicine to treat inflammatory diseases. A partially purified solid phase (SPE columns) methanolic elution fraction of C. ainetensis followed by methanol gradient elution on reverse phase HPLC chromatography (RP-HPLC) strongly inhibited ET-induc (open full item for complete abstract)

Committee: Floyd Schanbacher (Advisor); Charles Brooks (Other); James DeWille (Other); Joy Pate (Other) Subjects:

Doctor of Philosophy, The Ohio State University, 2004, Veterinary Biosciences

CCAAT/Enhancer binding proteins (C/EBPs) are members of the leucine zipper family of transcription factors and play key roles in cell growth, differentiation and apoptosis. C/EBP family member, C/EBPδ, plays an important role in the G0 growth arrest of mouse mammary epithelial cells in vitro and in mouse mammary gland involution in vivo. The role of C/EBPδ in human mammary epithelial cells has not yet been established. Serial Analysis of Gene Expression (SAGE) analysis of human breast tumors, however, has recently demonstrated that C/EBPδ is one of a subset of genes that is down-regulated in breast cancer. These results suggest that C/EBPδ may play an important role in human mammary epithelial cell growth control. The goal of this dissertation project was three-fold. First, characterize the expression of C/EBPδ and determine its role in G0 growth arrest in normal and tumorigenic human mammary epithelial cells. Secondly, investigate upstream signaling regulators of the C/EBPδ promoter. And lastly, investigate signaling events important for the initiation and maintenance of G0 growth arrest in human mammary epithelial cells. In summary, this dissertation has revealed that activation of the JAK/STAT3 signaling pathway is required for the induction of C/EBPδ and plays a key role in the induction of G0 growth arrest of human mammary epithelial cells. This growth inhibitory signaling pathway is increasingly disrupted in mammary epithelial cells as cells progress from normal to neoplastic. Activation of the STAT3 signaling pathway in normal mammary epithelial cells and the initiation and maintenance of G0 growth arrest is the result of transcriptional up-regulation and secretion of growth inhibitory cytokines (LIF, IL-6, OSM) into the media, establishing an autocrine regulatory mechanism.

Committee: Jim DeWille (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, The Ohio State University, 2002, Molecular, Cellular, and Developmental Biology

Work from this dissertation has demonstrated that C/EBPbeta is the predominant C/EBPbeta isoform expressed in the normal mouse mammary gland and mouse mammary tumors in vivo. Specific analysis of the C/EBPbeta isoforms demonstrated that LIP protein levels are directly influenced by the protein isolation procedure, indicating that LIP levels are modulated by protein isolation induced proteolysis. C/EBPbeta LIP was not detected in the normal mammary gland, but was detected in mammary tumors. Analysis of the affect of C/EBPbeta LIP overexpression on mammary epithelial cell proliferation and differentiation demonstrated that LIP did not have a significant effect on proliferation, but abrogated differentiation by an extracellular matrix-independent mechanism. This work has lead to the model that disruption of the differentiation program by LIP allows mammary epithelial cells to undergo continued proliferation, which may result in breast cancer. In addition, we demonstrated for the first time that C/EBPdelta mRNA exhibits a relatively short half-life in G0 growth arrested mouse and human mammary epithelial cells. Results of oligo/RNase H cleavage analysis and RACE-PAT revealed a short C/EBPdelta mRNA half-life in addition to demonstrating that the C/EBPdelta mRNA poly(A) tail is relatively short. The poly(A) tail length is not modulated during C/EBPdelta mRNA degradation, which suggests a deadenylation-independent pathway. The C/EBPdelta protein also exhibited a short half-life in G0 growth arrested mouse and human mammary epithelial cells. Results of ubiquitination inhibitor studies demonstrated that C/EBPdelta protein is degraded in an ubiquitin-dependent manner, exclusively in the nucleus during G0 growth arrest. Finally, replacement of the C/EBPdelta 3' untranslated region (UTR) with the bovine growth hormone 3' UTR increased C/EBPdelta mRNA half-life, implying a role for the 3' UTR in the regulation of C/EBPdelta mRNA stability. Analysis of the C/EBPdelta 3' UTR iden (open full item for complete abstract)

Committee: James DeWille (Advisor) Subjects: Biology, Molecular

Doctor of Philosophy, Case Western Reserve University, 2009, Genetics

Breast cancer is the most common cancer among women, affecting approximately one million women worldwide. Understanding the cellular and microenvironmental changes that contribute to breast cancer initiation and progression will allow the most effective treatment regimens to be discovered. The studies described here began with normal human mammary epithelial cells (HMECs) obtained from reduction mammoplasties. The stepwise addition of specific genetic alterations created a transformation model that mimics the progression of breast cancer. Constitutive cyclin D1/cyclin-dependent kinase (CDK) activity promotes the hyperphosphorylation and inactivation of the RB family of tumor suppressor proteins. To study the role of wild-type p53 and its ability to be reactivated in breast cancers, a cyclin D1/CDK fusion protein (D1/CDK) was expressed in immortalized HMECs allowing the cells to grow anchorage-independently, a hallmark of transformation. Importantly, the D1/CDK-mediated transformation occurred even in the presence of wild-type p53, providing a model to study p53 reactivation by the therapeutic compound Nutlin-3, an HDM2 antagonist. Interestingly, p53 could be activated by treatment with Nutlin-3, leading to a p53-dependent, but RB-independent growth arrest through the stable down-regulation of critical cell cycle proteins. Since a significant percentage of breast cancers overexpress cyclin D1 and have constitutive CDK activity, this study provides important information regarding the efficacy of p53 reactivation in such tumors. Further work to reveal tumor suppressive mechanisms which are overcome during tumorigenesis examined the effect of the cytokine Oncostatin M (OSM) on normal and transformed HMECs, uncovering a regulatory switch that can suppress or promote growth. In HMECs, OSM induced a STAT3-dependent, p53-independent growth arrest. However, the overexpression of c-MYC not only prevented OSM-induced growth arrest but promoted transformation. Therefore, OSM in (open full item for complete abstract)

Committee: George Stark PhD (Advisor); Helen Salz PhD (Committee Chair); Clark Distelhorst MD (Committee Member); Mark Jackson PhD (Advisor); Joseph Nadeau PhD (Committee Member); Zhenghe Wang PhD (Committee Member) Subjects: Genetics; Molecular Biology