Angiotensin-(1-7): A Peptide Hormone Inhibitor for the Treatment of Breast Cancer Targeting the Tumor Microenvironment
In 2009, 192,000 new cases of breast cancer were diagnosed in the United States and over 40,000 American women died from breast cancer, emphasizing the severity of the breast cancer epidemic. Recent advances in molecular profiling of breast tumors have identified three clinically-relevant subcategories of breast cancer: estrogen receptor (ER) positive, human epidermal growth factor receptor 2 (HER2) amplified, and triple negative (lacking the ER, the progesterone receptor (PR), and with normal expression of HER2) breast cancers. ER positive breast cancer is the most prevalent type of breast cancer accounting for over 70% of breast cancer cases while HER2-amplified breast cancer comprises an additional 20-25% of breast cancer cases. Drugs targeting estrogen biosynthesis, estrogen signaling, or HER2 amplification were developed and used for the treatment of breast cancer; however, drug resistance and adverse side effects have limited their use, emphasizing the need for new targeted drugs to treat breast cancer. 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 mm3 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-β (TGF-β), 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-(1-7) 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. Moreover, transfection of ZR-75-1 cells with siRNA to DUSP1 ablated the Ang-(1-7)-mediated reduction in PGES, demonstrating that Ang-(1-7) up-regulates the MAP kinase phosphatase DUSP1 to reduce ERK1/2 MAP kinase activities and PGES. A recently completed Phase I clinical trial on the use of Ang-(1-7) for the treatment of solid tumors demonstrated clinical benefit in 4 of 15 evaluable cancer patients. Since Ang-(1-7) reduced proliferation, inflammation and fibrosis in ZR-75-1 ER-positive and BT-474 HER2-amplified breast cancer cells and tumors, these results suggest that Ang-(1-7) may be an effective, first-in-class compound for the treatment of breast cancer by reducing tumor growth and targeting the reactive tumor microenvironment.
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