| The purpose of the studies described in this dissertation was to determine whether angiotensin-(1-7) [Ang-(1-7)], an endogenous seven amino-acid peptide hormone of the renin-angiotensin system, inhibits the growth of HER2-amplified and ER-positive orthotopic breast tumors and to identify the molecular mechanisms for the Ang-(1-7)-mediated reduction in tumor growth.;BT-474, which express the ER receptor and over-express HER2, and ZR-75-1 ER positive cells were injected into a mammary fat pad of athymic mice. Tumors were allowed to grow to a uniform size, 200 mm3 and 100 mm 3 for BT-474 and ZR-75-1, respectively, followed by treatment with Ang-(1-7) or saline for 18 days, to determine the effect of the drug on tumor growth. Ang-(1-7) administration reduced BT-474 and ZR-75-1 tumor volume by 68.2% and 77.0%, and tumor weight by 38.9% and 50%, respectively, indicating that the heptapeptide inhibits breast tumor growth. The Ang-(1-7)-mediated reduction in tumor growth was associated with a significant decrease in interstitial fibrosis, from 4.9 +/- 1.0%/field to 1.2 +/- 0.2%/field in BT474 tumors and from 23.3 +/- 2.4%/field to 8.3 +/- 0.8%/field in ZR-75-1 tumors, and a 66% reduction in collagen I deposition. Treatment with Ang-(1-7) also decreased perivascular fibrosis in BT-474 tumors, from 49.3 +/- 3.2% fibrosis/vessel compared to 13.4 +/- 2.2% fibrosis/vessel, demonstrating that the heptapeptide reduced both intratumoral and perivascular fibrosis. These results demonstrate that Ang-(1-7) inhibits both the growth and the tumor-associated fibrosis of HER2-amplified and ER positive breast cancer.;Fibroblasts were isolated from ZR-75-1 orthotopic breast tumors, to identify the molecular mechanisms for the anti-fibrotic response to Ang-(1-7) in breast cancer. Ang-(1-7) markedly attenuated the growth of tumor-associated fibroblasts, in association with a 36% reduction in the extracellular matrix protein fibronectin and a 44% decrease in active transforming growth factor-beta (TGF-beta), which plays a major role in the production of extracellular matrix proteins. The heptapeptide significantly increased the mitogen-activated protein (MAP) kinase phosphatase DUSP1 in tumor-associated fibroblasts, by 252 +/- 29% compared with untreated cells, with an associated 50% decrease in activities of the MAP kinases ERK1 and ERK2 [1.13 +/- 0.23 relative density units (RDU) in untreated cells compared 0.60 +/- 0.06 RDU in Ang-(1-7)-treated cells for pERK1, and 2.27 +/- 0.56 RDU in untreated cells compared to 0.84 +/- 0.08 RDU in Ang-(1-7)-treated cells for pERK2; n = 3--4; p < 0.05]. These results suggest that Ang-(1-7) increases DUSP1 and reduces MAP kinase activities to inhibit the proliferation of tumor-associated fibroblasts and the production of extracellular matrix proteins.;Ang-(1-7) significantly reduced cell proliferation in ZR-75-1 breast tumors, with a 61% reduction in Ki67 immunoreactivity. Moreover, Ang-(1-7) treatment markedly decreased ERK1/2 MAP kinase activities [for pERK1 from 0.51 +/- 0.13 RDU in tumors from mice treated with saline to 0.16 +/- 0.06 RDU in tumors treated with Ang-(1-7) and for pERK2 from 1.17 +/- 0.33 RDU following treatment with saline to 0.30 +/- 0.07 RDU following Ang-(1-7) treatment, p < 0.05; n = 5--6]. The decrease in phospho-ERK activities was accompanied by an increase in the MAP kinase phosphatase DUSP1 [from 0.23 +/- 0.03 RDU in tumors from mice treated with saline to 0.51 +/- 0.06 RDU in mice treated with Ang-(1-7), p < 0.05; n = 5--6]. Ang-(1-7) treatment of ZR-75-1 cells also increased DUSP1, by 2-fold compared to the amount of the immunoreactive protein in untreated cells, in agreement with a reduction in serum-stimulated ERK1/2 activities in cells treated with the heptapeptide, suggesting that Ang-(l-7) up-regulation of DUSP1 reduced phospho-ERK1/2. DUSP1 was reduced in ZR-75-1 cells, by transfection with siRNA to DUSP1, decreasing immunoreactivity by 60%. The reduction in DUSP1 prevented the Ang-(1-7)-mediated decrease in phospho-ERK1/ERK2, demonstrating that the heptapeptide up-regulates the MAP kinase phosphatase DUSP1 to reduce phospho-ERK1/ERK2 and subsequent effects of ERK signaling in ER positive breast cancer cells.;Ang-(1-7) administration also decreased cyclooxygenase 2 (COX-2) and prostaglandin E synthase (PGES) in ZR-75-1 orthotopic tumors, with no effect on prostacyclin synthase (PGIS), suggesting that the heptapeptide alters the ratio of proliferative to anti-proliferative prostaglandins. COX-2 and PGES were also reduced in ZR-75-1 cells treated with the heptapeptide. Co-treatment of ZR-75-1 cells with the serine/threonine phosphatase inhibitor okadaic acid and the tyrosine phosphatase inhibitor sodium vanadate prevented the Ang-(1-7)-mediated down-regulation of PGES, suggesting Ang-(1-7) up-regulates a phosphatase to reduce PGES expression. (Abstract shortened by UMI.)... |
