Browsing by Subject "ErbB2"
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Item Inhibitory actions of Ah receptor agonists and indole-containing compounds in breast cancer cell lines and mouse models(Texas A&M University, 2005-08-29) Walker, Kelcey Manae BeckerThe aryl hydrocarbon receptor (AhR) binds synthetic and chemoprotective phytochemicals, and research in this laboratory has developed selective AhR modulators (SAhRMs) for treatment of breast cancer. Activation of the AhR through agonists such as TCDD inhibits hormone activation of several E2-responsive genes in breast cancer cell lines. In this study, inhibition of E2-induced proliferation and gene expression by TCDD has been investigated in the uterus of wildtype, ERKO and AhRKO mice. Cyclin D1, DNA polymerase ?, and VEGF mRNA levels are induced by E2 through ER? in the uterus as determined by in situ hybridization studies. TCDD down-regulated E2-induced cyclin D1 and DNA polymerase ? expression, but not E2-induced VEGF expression, in wild-type mice, but not AhRKO mice, confirming the role of the AhR. Furthermore, protein synthesis was not necessary for induction of cyclin D1 or DNA polymerase ?gene expression by E2 or inhibition of these responses by TCDD. Therefore, AhR-ER? crosstalk directly regulates the expression of genes involved in cell proliferation in vivo. AhR agonists induce down-regulation of ErbB family receptors in multiple tissues/organs suggesting possible inhibitory interactions with chemotherapeutic potential. Recently, it has been reported that the SAhRM 1,1??,2,2??-tetramethyldiindolylmethane inhibited DMBA-induced mammary tumor growth in rats and also inhibited MAPK and PI3-K pathways in human breast cancer cells. BT-474 and MDA-MB-453 cell lines are ErbB2-overexpressing breast cancer cells that express functional AhR and exhibit constitutive activation of MAPK and PI3-K pathways. Therefore, 1,1??,2,2??-tetramethyldiindolylmethane-induced inhibition of ErbB2 signaling was investigated in these cells lines and in the MMTV-c-neu mouse mammary tumor model, which overexpresses ErbB2 in the mammary gland. The growth of ErbB2 overexpressing cell lines and mammary tumors was inhibited by 1,1??,2,2??-tetramethyldiindolylmethane; however, modulation of MAPK or PI3-K pathways and cell cycle proteins nor induction of apoptosis by 1,1',2,2'-tetramethyldiindolylmethane was observed in the ErbB2overexpressing cell lines. Current studies are investigating mitochondrial effects of 1,1??,2,2??-tetramethyldiindolylmethane in the ErbB2-overexpressing cell lines, as well as continuing studies on gene expression profiles in the mammary glands of MMTV-c-neu mice to better understand and identify critical genes that are responsible for ErbB2-mediated transformation and growth of cancer cells/tumors.Item Integrated roles of mechanics, motility, and disease progression in cancer(2010-12) Baker, Erin Lynnette; Bonnecaze, R. T. (Roger T.); Zaman, Muhammad H. (Muhammad Hamid); Schmidt, Christine E.; Dunn, Andrew K.; Liechti, Kenneth M.The broad objective of this research is to examine the relationship between the cellular micromechanical environment and disease progression in cancer. The mechanical stiffness of cancerous tissue is a key feature that distinguishes it from normal tissue and thus facilitates its detection clinically. While numerous inroads have been achieved toward elucidating molecular mechanisms that underlie diseases such as cancer, quantitative characterization of associated cellular mechanical properties and biophysical attributes remains largely incomplete. To this end, the present research provides insight into the following questions: (1) What is the effect of extracellular matrix (ECM) stiffness and architecture on internal cancer cell rheology and cytoskeletal organization? (2) What are the integrated effects of ECM stiffness and cell metastatic potential on the intracellular rheology and morphology of breast cancer cells? (3) What are the integrated effects of ECM stiffness, ECM architecture, and cell metastatic potential on the motility of breast cancer cells? To examine these phenomena, the present research utilizes a multidisciplinary engineering approach that integrates experimental rheology, theoretical mechanics, confocal microscopy, computational algorithms, and experimental cell biology. Briefly, genetically altered cancer-mimicking cells are cultured within synthetic ECMs of varying mechanical stiffness and structure, where they are then observed using time-lapsed confocal microscopy. Image analyses and computational algorithms are then employed to extract measures of cell migration speed and intracellular stiffness via particle-tracking microrheology techniques. Major results show that ECM stiffness elicits an intracellular mechanical response only within the framework of physiologically relevant matrix environments and that a key cell-matrix attachment protein (the integrin) plays an essential role in this phenomenon. Additional results indicate that a well-known breast cancer-associated biomarker (ErbB2) is responsible for sensitizing mammary cells to ECM stiffness. Finally, results also show that a switch in ECM architecture significantly hinders the migratory capacity of ErbB2-associated cells, which may explain why the ErbB2 biomarker is detected with much higher frequency in early stage breast cancer than in later stage invasive and metastatic cancers. In total, these findings inform the fields of mechanobiology and cancer biology by systematically linking cell rheology, cell motility, matrix mechanics, and disease progression in cancer.Item Mechanistic Investigation of Tolfenamic Acid, Betulinic Acid, and Aspirin in Anti-Cancer Therapy(2012-10-19) Liu, XinyiTolfenamic acid (TA), betulinic acid (BA) and acetylsalicylic acid (aspirin) are anticancer drugs, and Sp1, Sp3, Sp4 transcription factors and growth factor 2 (EGFR2, HER2 / ErbB2) are important molecular markers in cancer cells. In this study the molecular mechanisms by which these anticancer drugs target downregulation of Sp1, Sp3, Sp4 and ErbB2 are investigated in breast cancer cells. TA inhibits growth of ErbB2-overexpressing BT474 and SKBR3 breast cancer cells by inhibiting ErbB2 expression. In cells treated with TA, ErbB2 mRNA expression and promoter activity were decreased, and this was due to decreased mRNA stability in BT474 cells. In both cell lines, TA also decreased expression of the YY1 and AP-2 transcription factors that are required for basal ErbB2 expression. These effects were accompanied by decreased ErbB2-dependent kinase activities, induction of p27, and decreased expression of cyclin D1. In addition, TA also inhibited tumor growth in BT474 cells orthotopic mouse model. However, Sp proteins were not the major target of TA in these breast cancer cells and this contrasts to results in pancreatic cancer cells where TA decreased expression of Sp proteins. BA inhibits growth of ErbB2 ?overexpressing BT474 and MDA-MB-453 and induced apoptosis in these cells. BA induced proteasome-independent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, Sp4 and survivin, an Sp-regulated gene, and BA also decreased expression of ErbB2, ErbB2-regulated kinases and also YY1, a transcription factor that regulates ErbB2 expression in these cells. Knockdown of Sp1, Sp3, Sp4 and their combination by RNA interference was accompanied by decreased expression of ErbB2, YY1 and luciferase activity in cells transfected with a construct containing the GC-rich YY1 promoter linked to a luciferase reporter gene. BA-dependent repression of Sp1, Sp3, Sp4 and Sp regulated genes was due in part to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a) which constitutively inhibits ZBTB10 expression. The effects of BA on the miR-27a:zBTB10-Sp transcription factor axis were inhibited in cells cotreated with the cannabinoid 1 (CB1) and CB2 receptor antagonists AM251 and AM630 respectively. However, in vitro binding studies with ? 10 mM BA and a radiolabeled cannabinoid did not indicate direct competitive binding of BA to the CB1 and CB2 receptors, suggesting a possible role for other CB-like G protein-coupled receptors. Aspirin inhibits growth, induces apoptosis and decreases cell migration in BT474 and MDA-MB-453 breast cancer cells that overexpress the ErbB2 oncogene. Aspirin also downregulated ErbB2 expression in these cells and this was accompanied by inhibition of downstream kinases including phospho-Akt (p-Akt) and phospho-mitogen-activated protein kinase (p-MAPK). Aspirin also decreased expression of survivin, vascular endothelial growth factor (VEGF) and YY1 which regulates ErbB2 expression in these cell lines. Aspirin also downregulates Sp1, Sp3 and Sp4, and further investigation of the underlying mechanism of action showed that aspirin-induced downregulation of Sp transcription factors and Sp-regulated genes could be inhibited in part by proteasome inhibitior lactacystin and phosphatase inhibitors including sodium orthovanadate (SOV). These results were consistant with the induction of several phosphatases by aspirin in BT474 and MDA-MB-453 cells and these include mitogen-activated protein kinase phosphatase-5 (MKP-5) and MKP-1. Aspirin-induced downregulation of Sp1, Sp3 and Sp4 are reversed in cells transfected with an oligonucleotide (siMKP5) that knocks down MKP5 by RNA interference whereas siMKP-1 did not block Sp protein downregulation demonstrating for the first time a linkage between a drug-induced phosphatase (MKP-5) and Sp downregulation. These results suggest that TA, BA and aspirin represent novel and promising new anticancer drugs for cancer treatment by targeting Sp proteins and ErbB2 oncogene.