Abstract B34: Angiotensin-(1-7) attenuates the proliferation of cancer-associated fibroblasts in triple negative breast cancer
Authors: Arter Alison L. , Gallagher Patricia E. , Tallant E. Ann 
Published: August 2014

The interaction between cancer cells and the surrounding microenvironment transforms the stroma into an abnormal phenotype, altering normal function, tissue architecture, cellular morphology, and extracellular matrix-cell interactions that directly contribute to formation of neoplasia. About 80% of reactive stroma associated with breast carcinoma is comprised of activated myofibroblasts or cancer-associated fibroblasts which secrete extracellular matrix proteins resulting in the desmoplasia that is associated with advanced breast tumor progression. Cancer-associated fibroblasts, transformed by cytokines such as transforming growth factor-β (TGF-β), secrete tumor promoting growth factors and extracellular matrix components, to promote tumor initiation, growth, and metastases. Inhibition of cancer-associated fibroblast proliferation and function may be an effective therapeutic strategy, since these cells play a vital role in carcinogenesis. Angiotensin-(1-7) [Ang-(1-7)] is an endogenous, seven amino acid peptide hormone of the renin-angiotensin system with anti-proliferative and anti-fibrotic properties. We showed that Ang-(1-7) inhibited the growth of MDA-MB-231 human triple negative breast tumors growing in the mammary fat pads of athymic mice with a concomitant 3-fold decrease in interstitial tumor fibrosis (n=9-11, p&lt;0.0001). Cancer-associated fibroblasts were isolated from orthotopic breast tumors, to assess the effect of Ang-(1-7) on fibroblast proliferation. The isolated tumoral fibroblasts stained positive for fibronectin, vimentin, collagen I, and β-smooth muscle actin, confirming that the isolated cells were activated myofibroblasts. Ang-(1-7) significantly reduced the growth of cancer-associated fibroblasts isolated from orthotopic triple negative breast tumors (n=4-5, p&lt;0.05). The decrease in cancer-associated fibroblast proliferation was accompanied by attenuation of the activation of the growth-promoting mitogen-activated protein kinases (MAPK) ERK1 and ERK2 (n=3, p&lt;0.001) as quantified by Western blot hybridization. In contrast, incubation of the heptapeptide hormone resulted in a 2.5-fold increase in the MAPK phosphatase DUSP1 (dual specificity phosphatase 1) in cancer-associated fibroblasts (n=3, p&lt;0.05). These studies suggest that Ang-(1-7) promotes the dephosphorylation of MAPK by up-regulating DUSP1 to reduce this proliferative signaling pathway in cancer-associated fibroblasts and prevent tumor growth. TGF-β is a potent stimulator of fibroblast activation and plays an important role in promoting fibrosis, including activation of the MAP kinase pathway to stimulate fibroblast proliferation. TGF-β activates tumor-promoting cancer-associated fibroblasts by phosphorylation of Smad2/3 which translocates to the nucleus to induce pro-fibrotic gene expression. Stimulation of the Smad signaling pathway by TGF-β results in extracellular matrix protein synthesis and deposition as well as its own production, creating an autocrine cycle of fibroblast activation and extracellular matrix deposition. TGF-β was quantified by Western blot hybridization in protein homogenates from myofibroblasts isolated from orthotopic breast tumors. TGF-β stimulation of cancer-associated fibroblasts significantly increased phospho-Smad2/3 which was reduced approximately 60% by treatment with Ang-(1-7) (n=3, p&lt;0.001), indicating that the heptapeptide inhibits both the growth of stromal fibroblasts as well as the signaling pathways that participate in extracellular matrix protein production. These findings suggest that Ang-(1-7) may serve as a first-in-class chemotherapeutic agent for triple negative breast cancer in part through a reduction in the proliferation and function of cancer-associated fibroblasts in the tumor microenvironment.

Citation Format: Alison L. Arter, Patricia E. Gallagher, E. Ann Tallant. Angiotensin-(1-7) attenuates the proliferation of cancer-associated fibroblasts in triple negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B34.