Angiotensin-(1-7) [Ang-(1-7)] is an endogenous seven-amino acid peptide hormone with antiproliferative properties. Our previous studies showed that Ang-(1-7) inhibits the growth of human lung cancer cells in vitro and reduces the size of human lung tumor xenografts in vivo. In the current study, s.c. injection of Ang-(1-7) not only caused a significant reduction in human A549 lung tumor growth but also markedly decreased vessel density, suggesting that the heptapeptide inhibits angiogenesis to reduce tumor size. A decrease in human endothelial cell tubule formation in Matrigel was observed following a 16 h incubation with Ang-(1-7), with a maximal reduction at a 10 nmol/L concentration. Ang-(1-7) had similar antiangiogenic effects in the chick chorioallantoic membrane, causing a >50% decrease in neovascularization. The Ang-(1-7)-induced reduction in both endothelial cell tubule formation and vessel formation in the chick was completely blocked by the specific Ang-(1-7) receptor antagonist [d-proline7]-Ang-(1-7), suggesting that these biological actions are mediated by an AT(1-7) receptor. Ang-(1-7) significantly reduced vascular endothelial growth factor-A protein and mRNA in tumors from mice treated with the heptapeptide compared with saline controls as well as in the parent A549 human lung cancer cells in culture. These results suggest that Ang-(1-7) may attenuate tumor angiogenesis by reducing vascular endothelial growth factor-A, a primary proangiogenic protein. Taken together, this study shows that Ang-(1-7) exhibits significant antiangiogenic activity and may be a novel therapeutic agent for lung cancer treatment targeting a specific AT(1-7) receptor.

Angiotensin-(1-7) ] is an endogenous peptide of the renin-angiotensin system with vasodilator and antiproliferative properties. Our previous studies showed that Ang-(1-7) reduced serum-stimulated growth of human lung cancer cells in vitro through activation of a unique AT (1-7) receptor. The current study investigates the effect of Ang-

Lung cancer is a leading cause of death in both men and women, with over 1,000,000 new cases diagnosed worldwide annually and a 5-year survival rate of only 14%, a figure that has improved little in the past thirty years. This poor prognosis suggests a need for novel approaches for the treatment and prevention of lung cancer. The renin-angiotensin system is an established, primary regulator of blood pressure, homeostasis, and natriuresis; however, compelling evidence indicates that the angiotensin peptides also play a role in cell proliferation and inflammation. Angiotensin II is a vasoconstrictor, a mitogen, and an angiogenic factor, while angiotensin-(1-7) has vasodilator, anti-proliferative, and anti-angiogenic properties. This review focuses on studies examining the renin-angiotensin system in pulmonary cancers and whether clinical intervention of this pathway may serve as an effective chemotherapeutic and/or chemopreventive modality for lung cancer.

BackgroundHuman mitochondrial peptide deformylase (PDF) has been proposed as a novel cancer therapeutic target. However, very little is known about its expression and regulation in human tissues. The purpose of this study was to characterize the expression pattern of PDF in cancerous tissues and to identify mechanisms that regulate its expression.MethodsThe mRNA expression levels of PDF and methionine aminopeptidase 1D (MAP1D), an enzyme involved in a related pathway with PDF, were determined using tissue panels containing cDNA from patients with various types of cancer (breast, colon, kidney, liver, lung, ovarian, prostate, or thyroid) and human cell lines. Protein levels of PDF were also determined in 2 colon cancer patients via western blotting. Colon cancer cells were treated with inhibitors of ERK, Akt, and mTOR signaling pathways and the resulting effects on PDF and MAP1D mRNA levels were determined by qPCR for colon and lung cancer cell lines. Finally, the effects of a PDF inhibitor, actinonin, on the proliferation of breast, colon, and prostate cell lines were determined using the CyQUANT assay.ResultsPDF and MAP1D mRNA levels were elevated in cancer cell lines compared to non-cancer lines. PDF mRNA levels were significantly increased in breast, colon, and lung cancer samples while MAP1D mRNA levels were increased in just colon cancers. The expression of PDF and MAP1D varied with stage in these cancers. Further, PDF protein expression was elevated in colon cancer tissue samples. Inhibition of the MEK/ERK, but not PI3K or mTOR, pathway reduced the expression of PDF and MAP1D in both colon and lung cancer cell lines. Further, inhibition of PDF with actinonin resulted in greater reduction of breast, colon, and prostate cancer cell proliferation than non-cancer cell lines.ConclusionsThis is the first report showing that PDF is over-expressed in breast, colon, and lung cancers, and the first evidence that the MEK/ERK pathway plays a role in regulating the expression of PDF and MAP1D. The over-expression of PDF in several cancers and the inhibition of cancer cell growth by a PDF inhibitor suggest this enzyme may act as an oncogene to promote cancer cell proliferation.

Angiogenesis inhibition is an important therapeutic strategy for advanced stage prostate cancer. Previous work from our laboratory showed that sustained stimulation of Rap1 by 8-pCPT-2'-O-Me-cAMP (8CPT) via activation of Epac, a Rap1 GEF, or by expression of a constitutively active Rap1 mutant (cRap1) suppresses endothelial cell chemotaxis and subsequent angiogenesis. When we tested this model in the context of a prostate tumor xenograft, we found that 8CPT had no significant effect on prostate tumor growth alone. However, in cells harboring cRap1, 8CPT dramatically inhibited not only prostate tumor growth but also VEGF expression and angiogenesis within the tumor microenvironment. Subsequent analysis of the mechanism revealed that, in prostate tumor epithelial cells, 8CPT acted via stimulation of PKA rather than Epac/Rap1. PKA antagonizes Rap1 and hypoxic induction of 1α protein expression, VEGF production and, ultimately, angiogenesis. Together these findings provide evidence for a novel interplay between Rap1, Epac, and PKA that regulates tumor-stromal induction of angiogenesis.

MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf Kinase Inhibitory Protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.

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The ability of cancer cells to survive and grow at primary and metastatic sites is influenced to a large degree by the surrounding cells in the tumor environment. In this process, tumor cells secrete signals that recruit specific cell types to their immediate vicinity and induce these cells to produce factors critical to tumor development, while the surrounding normal tissue, or stroma, detects and responds to developing lesions. While the clinical importance of this complex dialogue between the tumor and the surrounding tissue has long been appreciated, its molecular basis is not well understood. In particular, studies attempting to characterize interactions between tumors and the surrounding tissue have been confounded largely for technical reasons relating to the difficulty of precisely identifying and separating the developing tumor cells from normal cells nearby, and of confidently assigning the factors identified in later analyses to the proper tissue of origin. Recent developments in high-throughput sequencing technology offer an opportunity to overcome these difficulties and precisely identify factors associated with the stromal response to developing tumors on a genomic scale. Using a novel sequencing strategy applied to a xenograft model of triple-negative breast cancer, we are able to unambiguously assign 93.3% of mapped reads to tumor or stromal transcriptomes, with a misassignment rate approaching 0.01%. These methods have allowed us to directly compare transcript levels in stroma containing developing tumors to those levels observed in naive stroma, and to subsequently identify a set of candidate factors likely involved in the stromal response to developing lesions. We then extended this method to contrast stromal responses to near-isogenic tumors with differing metastatic potential and invasive phenotype conferred by expression of the RKIP metastasis suppressor, thereby identifying candidate factors likely to be specifically relevant to metastatic colonization. Surprisingly, we observed significant up-regulation of genes associated with macrophage chemotaxis and macrophage infiltration in the invasive stroma, which was not present in stroma surrounding tumors expressing RKIP. These findings were confirmed by immunohistochemistry using the original cell line as well as parallel experiments in a syngeneic model, suggesting that by utilizing RNA sequencing technology in an innovative way we can increase our understanding of the interplay between tumors and their local environment. Our model allows us to contrast simultaneous high-precision gene expression estimates in tumors and stroma to infer instances of crosstalk. In the invasive microenvironment, we observed coordinated suppression of genes involved in negatively regulating endothelial cell proliferation accompanied by elevated expression of genes whose products bind growth factors in both tumors and stroma. Finally, we extended these analyses to identify candidate paracrine interactions that are likely to be suppressed in noninvasive contexts involving stromal ALK and MDK which may directly contribute to observed differences in cell growth, migration and angiogenesis. Citation Format: Casey A. Frankenberger, Russell O. Bainer, Jyotsana Menon, Claudia Chavarria, Katelyn Michelini, Pàll Melsted, Yoav Gilad, Marsha R. Rosner. Using species-specific RNAseq reveals stromal reprogramming in triple-negative breast cancer xenografts. [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 B25.