Browsing by Author "Morgado, Micaela"

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    A 3D inﾠvitro model of patient-derived prostate cancer xenograft for controlled interrogation of inﾠvivo tumor-stromal interactions
    (Elsevier, 2016) Fong, Eliza L.S.; Wan, Xinhai; Yang, Jun; Morgado, Micaela; Mikos, Antonios G.; Harrington, Daniel Anton; Navone, Nora M.; Farach-Carson, Mary C.
    Patient-derived xenograft (PDX) models better represent human cancer than traditional cell lines. However, the complex in vivo environment makes it challenging to employ PDX models to investigate tumor-stromal interactions, such as those that mediate prostate cancer (PCa) bone metastasis. Thus, we engineered a defined three-dimensional (3D) hydrogel system capable of supporting the co-culture of PCa PDX cells and osteoblastic cells to recapitulate the PCa-osteoblast unit within the bone metastatic microenvironment in vitro. Our 3D model not only maintained cell viability but also preserved the typical osteogenic phenotype of PCa PDX cells. Additionally, co-culture cellularity was maintained over that of either cell type cultured alone, suggesting that the PCa-osteoblast cross-talk supports PCa progression in bone, as is hypothesized to occur in patients with prostatic bone metastasis. Strikingly, osteoblastic cells co-cultured with PCa PDX tumoroids organized around the tumoroids, closely mimicking the architecture of PCa metastases in bone. Finally, tumor-stromal signaling mediated by the fibroblast growth factor axis tightly paralleled that in the in vivo counterpart. Together, these findings indicate that this 3D PCa PDX model recapitulates important pathological properties of PCa bone metastasis, and validate the use of this model for controlled and systematic interrogation of complex in vivo tumor-stromal interactions.
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    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.
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    p62/SQSTM1 is required for cell survival of apoptosis-resistant bone metastatic prostate cancer cell lines
    (Wiley, 2014) Chang, Megan A.; Morgado, Micaela; Warren, Curtis R.; Hinton, Cimona V.; Farach-Carson, Mary C.; Delk, Nikki A.
    BACKGROUND: Bone marrow stromal cell (BMSC) paracrine factor(s) can induce apoptosis in bone metastatic prostate cancer (PCa) cell lines. However, the PCa cells that escape BMSC-induced apoptosis can upregulate cytoprotective autophagy. METHODS: C4-2, C4-2B, MDA PCa 2a, MDA PCa 2b, VCaP, PC3, or DU145 PCa cell lines were grown in BMSC conditioned medium and analyzed for mRNA and/or protein accumulation of p62 (also known as sequestome-1/SQSTM1), Microtubule-associated protein 1 light chain 3B (LC3B), or lysosomal-associated membrane protein 1 (LAMP1) using quantitative polymerase chain reaction (QPCR), Western blot, or immunofluorescence. Small interfering RNA (siRNA) was used to determine if p62 is necessary PCa cell survival. RESULTS: BMSC paracrine signaling upregulated p62 mRNA and protein in a subset of the PCa cell lines. The PCa cell lines that were insensitive to BMSC-induced apoptosis and autophagy induction had elevated basal p62 mRNA and protein. In the BMSC-insensitive PCa cell lines, siRNA knockdown of p62 was cytotoxic and immunostaining showed peri-nuclear clustering of autolysosomes. However, in the BMSC-sensitive PCa cell lines, p62 siRNA knockdown was not appreciably cytotoxic and did not affect autolysosome subcellular localization. CONCLUSIONS: A pattern emerges wherein the BMSC-sensitive PCa cell lines are known to be osteoblastic and express the androgen receptor, while the BMSC-insensitive PCa cell lines are characteristically osteolytic and do not express the androgen receptor. Furthermore, BMSC-insensitive PCa may have evolved a dependency on p62 for cell survival that could be exploited to target and kill these apoptosis-resistant PCa cells in the bone.
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    Tumor necrosis factor-α and interferon-γ stimulate MUC16 (CA125) expression in breast, endometrial and ovarian cancers through NFκB
    (Impact Journals, LLC, 2016) Morgado, Micaela; Sutton, Margie N.; Simmons, Mary; Warren, Curtis R.; Lu, Zhen; Constantinou, Pamela E.; Liu, Jinsong; Francis, Lewis L.W.; Conlan, R.Steven; Bast, Robert C.Jr.; Carson, Daniel D.
    Transmembrane mucins (TMs) are restricted to the apical surface of normal epithelia. In cancer, TMs not only are over-expressed, but also lose polarized distribution. MUC16/CA125 is a high molecular weight TM carrying the CA125 epitope, a well-known molecular marker for human cancers. MUC16 mRNA and protein expression was mildly stimulated by low concentrations of TNFα (2.5 ng/ml) or IFNγ (20 IU/ml) 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. We show that NFκB is an important mediator of cytokine stimulation of MUC16 since siRNA-mediated knockdown of NFκB/p65 greatly reduced cytokine responsiveness. Finally, we demonstrate that the 250 bp proximal promoter region of MUC16 contains an NFκB binding site that accounts for a large portion of the TNFα response. Developing methods to manipulate MUC16 expression could provide new approaches to treating cancers whose growth or metastasis is characterized by elevated levels of TMs, including MUC16.