Browsing by Author "Carson, Daniel D"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    A two-pronged approach towards the development of novel therapeutics for advanced endometrial cancer
    (2015-11-24) Engel, Brian Joseph; Carson, Daniel D; Jacot, Jeffrey G; McCrea, Pierre D; Stern, Michael; Wagner, Daniel S
    Endometrial cancer is the fourth most common cancer among women. The standard of care involves hysterectomy with adjuvant radiation and chemotherapy for advanced disease. Despite these efforts, treatment of advanced and metastatic disease is not very effective. This body of work describes a two-pronged approach to address lack of treatments for advanced endometrial cancer. The first approach was an in-depth study of the mechanism and physiological effect of mucin 1 (MUC1)-driven epidermal growth factor receptor (EGFR) expression and signaling. MUC1 is a large, heavily glycosylated transmembrane protein that functions to lubricate surfaces, provides protection from external insult and plays an important role in embryo implantation. EGFR is a receptor tyrosine kinase that influences cellular proliferation, migration and apoptosis. MUC1 increases EGFR gene expression, mRNA levels, protein levels and signaling in endometrial cancer cell lines. Consequently, MUC1 expression is associated with increased EGF-dependent cellular proliferation, survival and resistance to EGFR inhibitors. In addition, MUC1 and EGFR co-expression is associated with increased cellular proliferation in endometrial tumors. The second approach involved the development and characterization of an advanced three dimensional (3D) hyaluronic acid (HA)-based culture model that is compatible with existing high throughput drug screening methodologies. This system incorporates three layers: an acellular cushion layer; an encapsulated cancer cell layer for growth in 3D; and a collagen-containing layer that supports the growth of stromal cells on top of the hydrogel (2.5D). The robustness of this system was evaluated by incorporating endometrial or prostate cancer cells with associated stromal cells. Both culture systems provided high cancer and stromal cell viability and facilitated paracrine interactions. The response to cytotoxic drugs from cells cultured in 3D HA better matched clinical data than cells grown in 2D and 3D-alginate. These studies provide mechanistic evidence for regulation that occurs in advanced endometrial cancer, as well as an improved platform to screen for effective therapeutics. The 3D culture system could be leveraged to evaluate novel therapeutics for the treatment of advanced endometrial cancer which may include MUC1 and EGF-directed therapies.
  • Thumbnail Image
    Item
    MUC16/CA125 Regulation by the Proinflammatory Cytokines TNF α and IFNγ and the PPARγ Agonist Rosiglitazone in Breast and Ovarian Cancers
    (2016-04-22) Morgado, Micaela; Bartel, Bonnie; Carson, Daniel D
    MUC16 is a high molecular weight transmembrane mucin (TM) carrying the CA125 epitope, a well-known molecular marker for human cancers. TMs are restricted to the apical surface of normal epithelia. TMs not only are over-expressed, but also lose polarized distribution in cancers. Similar to other TMs, MUC16 is overexpressed in a variety of epithelial cancers. In this work the regulation of MUC16 by the proinflammatory cytokines, TNFα and IFNγ, in addition to the PPARγ agonist rosiglitazone, a drug used in the treatment of type 2 diabetes, was explored. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα or IFNγ when used alone; however, combined treatment with both cytokines resulted in a moderate (3-fold or less) to large (>10-fold) stimulation of MUC16 mRNA and protein expression in a variety of cancer cell types indicating that this may be a general response. Human cancer tissue microarray analysis indicated that MUC16 expression directly correlates with TNFα and IFNγ staining intensities in certain cancers. NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. The 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Rosiglitazone exerts a dual role in the regulation of MUC16 since at low pharmacologically relevant concentrations it further stimulates MUC16 in combination with cytokines, but at high concentrations inhibits cytokine stimulated MUC16. This regulation also is PPARγ dependent. In this work methods were developed to manipulate MUC16 expression that can provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.
  • Thumbnail Image
    Item
    Role of SULF1 in the regulation of metastatic prostate cancer progression
    (2020-04-24) Brasil da Costa, Fabio Henrique; Carson, Daniel D; Warmflash, Aryeh
    The establishment of new prostate cancer (PCa) metastases in the bone marrow accompanies a classical stromal reaction orchestrated primarily by the bidirectional engagement of cancer cells and fibroblasts. As result, tumor-associated macrophages (TAMs) are recruited to the tumor and display a fluid spectrum of behaviors that may promote or suppress tumor progression. These stromal and immune reactions, although initially in place to constrain tumor growth, have been shown to ultimately benefit cancer cells, for example, due to increased bioavailability of growth factors and matrix remodeling. Also, deposition of the heparan sulfate (HS) proteoglycan perlecan (HSPG2) and secretion of HS-modifying enzymes are increased in the matrix as result of the Cancer-CAF-TAM trialogue. In this work, to investigate PCa bone metastases, we developed a novel, bone marrow mimetic hydrogel containing collagen type I, hyaluronic acid, and HS-decorated domain 1 of perlecan (COL1-HA-PlnDm1). Our goal was to use this model to study the prevalence and role of growth factor-releasing enzymes, like sulfatases 1 and 2 (SULF1/SULF2) and heparanase. We first demonstrated that PCa cells, bone marrow fibroblasts, and Mφs have excellent viability, show in vivo-like morphology, and exhibit classical phenotypic markers in COL1-HA-PlnDm1 hydrogels. Furthermore, we found that SULF1 is the major HS modifier and is predominantly expressed by activated fibroblasts both in vivo and in vitro. Then, we discovered that TAMs are highly prevalent in PCa bone marrow metastases, as indicated by CD163 and CD206 expression, and Mφs in 3D displayed similar phenotype. Additionally, alternatively activated macrophages induced upregulation of SULF1 and HSPG2 in fibroblasts in vitro. Finally, our 3D triculture data shows that when SULF1 is knocked out from fibroblasts, PCa cell proliferation and cluster volume are significantly increased in response to treatment with Wnt3a, even in the presence of Mφs. We conclude that 1) our 3D hydrogel model is a physiologically relevant in vitro system to discover fundamental aspects of HS signaling in the tumor microenvironment; and 2) the dynamics of SULF1 expression and activity suggest a tumor suppressing role in the context of Wnt3a signaling in PCa. Further studies are necessary to determine SULF1’s value as a prognostic marker or a candidate target for new therapies.
  • Thumbnail Image
    Item
    Toward a better understanding of human polarized epithelial biology: Mucin 4 in endometrium and polarity complexes in salivary epithelium
    (2017-04-20) Chapela, Patricia Jensen; Carson, Daniel D
    The epithelium is one of four major tissues in the human body. It covers every surface and cavity. Epithelial cells have an intracellular organization they use to differentiate the top, or apical surface, of the cell from the basal surface at the bottom. The cell localizes transmembrane proteins to specific regions for particular functions. Loss of this polarization cause cells to function abnormally as can occur in cancer. Therefore, understanding the basic function and organization of epithelial cells will aid in many areas of human health from diagnosis of disease to tissue regeneration. This work focuses on two separate projects; mucin expression in multiple cell lines originating from human female reproductive tissues, and cellular apicobasal polarity in salivary epithelial cells. For the first focus, cells localize mucins to their apical surface to protect and lubricate the external cell surface. Over the course of my studies in multiple epithelial cell lines, I found mucin 4 (MUC4) stead-state levels of transcripts and proteins increase when cells are exposed to the pro-inflammatory cytokines, interferon and tumor necrosis factor . In healthy endometrium, MUC4 was not present at levels comparable to mucin 1 or mucin 16; however, it was detectable in endometrial cancer. In light of the response to pro-inflammatory cytokines, it is not surprising to have higher quantities of MUC4 in endometrial tumor tissue. Furthermore, in cancer, cells tend to lose their polarity allowing proteins to interact that are typically separated. In endometrial cancer, this loss of polarity allows MUC4 to stabilize a growth receptor, ERBB2, normally relegated to the basal membrane. The second project involved a close examination of salivary epithelial tissue revealing ductal cells maintain their polarity with different proteins than the highly secretory acinar cells. Specifically, ductal cells have higher levels of a key polarity protein, SCRIB, and the mRNA of INADL, a component of the apical polarity complex. However, while these proteins may not be abundant in acinar cells, acinar epithelial cells do establish and maintain polarity. Acinar cells localize tight junction proteins in different structures than ductal cells due in part to their different functions. This project focused on identifying the difference between these two cell types in the salivary gland and the culture conditions necessary to polarize cultured primary cells. The ability to differentiate cultured cells into both epithelial cell types is vital to the generation of a functional salivary gland. These projects encompassed two different questions involving the phenotypes of different epithelial cells in different tissues. From the first project, observed responses to cytokines suggested a potential role for MUC4 in inflammatory conditions. In the second project, the ultimate goal of developing an autologous salivary gland for implantation in xerostomia patients requires determining the growth factors and basement membrane cues fundamental to developing a properly organized and functioning salivary gland. The complete work detailed here gives insight into the function of epithelial cells in these different contexts.