One of the earlier mandatory steps in the carcinogenesis pathway is the tumor promotion event which allows for the clonal expansion of neoplastic cells. The main focus of this project is to define the potential autocrine/paracrine mechanisms regulating the growth of human tumors and utilize these as rational targets for the early detection and intervention of malignant disease. Recently, we have demonstrated that the newly identified hypotensive peptides, adrenomedullin (AM) and its gene related partner, proadrenomedullin N-terminal peptide (PAMP), play an important role in the proliferative process associated with carcinogenesis, embryogenesis, and wound repair. AM has been shown to function as a mitogen, apoptosis survival factor and an angiogenic factor, all of these characteristics playing a critical part in the in vivo proliferative process. Our prior studies have shown that a variety of human tumor cell lines and tumors express AM and the AM-receptor (AM-R), implicating these ligand/receptor partners in a possible autocrine growth mechanism regulating neoplasm proliferation. Prior studies from our group and others have shown that a variety of human cancer cell lines and tumors express AM and the AM-receptor (AM-R) and that these products are upregulated during hypoxic insult. Multiple reports have now been published which demonstrate patients with elevated tumor AM expression (breast, prostate and colon CA) have a worsen clinical prognosis. Recently, we have shown that over-expressing AM in breast cancer cells (MCF-7 and T47D) dramatically increases their tumorigenicity capabilities. When comparing stable transfectants of AM over-expressors to negative control cells, the enhanced AM producing clones were resistant to apoptosis induced by serum-deprivation, had increased angiogenic activity in vitro and in vivo and formed tumors in nude mice while the parent (or empty plasmid) controls were incapable of mediating similar physiological events. We have also shown that MMP-2 rapidly degrades AM (<5 minute half-life) and that the AM binding protein, complement factor H, completely blocks this enzymatic mediated destruction. Hence, tumor cells producing both AM and complement factor H would have a selective growth advantage by virtue of their ability to circumvent MMP-2 activity and extending the biological half-life of AM. As a side note to our MMP-2/AM studies, we found that during the enzymatic processing of AM (a hypotensive peptide, AM1-52) by MMP-2 one of the resulting fragmentary products (AM11-22) is a potent vasoconstrictor which may play a regulatory role in modulating blood pressure. Employ invention report filed 7/24/2002. In collaboration with Drs Laurent Ozbun and Sonia Jakowlew and using a yeast-2-hybridization strategy, we have demonstrated that AM binds to microtubule-associated proteins (MAP1A and RANBP9) and proteins associated with cell cycle regulation (CDC2-related protein kinase 5 and TLH29). Interestingly, we now have demonstrated that PAMP may be as important as AM in regulating the carcinogenesis pathway. In collaboration with Dr. William Stetler-Stevenson (Pathology Branch, NCI) and using the direct in vivo angiogenic assay (DIVAA), we have found that PAMP is 1,000,000 times more potent on a molar basis than bFGF, VEGF or AM in inducing neovascularization. PAMP produces measurable vessel outcropping at 1fM while bFGF, VEGF and AM are indistinguishable from negative controls at concentrations <1nM. Employ invention report filed 7/24/2002.In conclusion, our collective studies offer a rational approach to identify potential biological targets for the early detection and intervention of malignant disease.

National Institute of Health (NIH)
Division of Clinical Sciences - NCI (NCI)
Intramural Research (Z01)
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Julian, Miguel; Cacho, Monica; Garcia, Mario A et al. (2005) Adrenomedullin: a new target for the design of small molecule modulators with promising pharmacological activities. Eur J Med Chem 40:737-50
Cuesta, Natalia; Martinez, Alfredo; Cuttitta, Frank et al. (2005) Identification of adrenomedullin in avian type II pneumocytes: increased expression after exposure to air pollutants. J Histochem Cytochem 53:773-80
Zudaire, Enrique; Cuesta, Natalia; Martinez, Alfredo et al. (2005) Characterization of adrenomedullin in birds. Gen Comp Endocrinol 143:10-20
Martinez, Alfredo; Zudaire, Enrique; Julian, Miguel et al. (2005) Gastrin-releasing peptide (GRP) induces angiogenesis and the specific GRP blocker 77427 inhibits tumor growth in vitro and in vivo. Oncogene 24:4106-13
Martinez, Alfredo; Zudaire, Enrique; Portal-Nunez, Sergio et al. (2004) Proadrenomedullin NH2-terminal 20 peptide is a potent angiogenic factor, and its inhibition results in reduction of tumor growth. Cancer Res 64:6489-94
Martinez, Alfredo; Oh, Hae-Ryong; Unsworth, Edward J et al. (2004) Matrix metalloproteinase-2 cleavage of adrenomedullin produces a vasoconstrictor out of a vasodilator. Biochem J 383:413-8
Martinez, Alfredo; Julian, Miguel; Bregonzio, Claudia et al. (2004) Identification of vasoactive nonpeptidic positive and negative modulators of adrenomedullin using a neutralizing antibody-based screening strategy. Endocrinology 145:3858-65
Zudaire, Enrique; Martinez, Alfredo; Ozbun, Laurent L et al. (2004) Characterization of adrenomedullin in non-human primates. Biochem Biophys Res Commun 321:859-69
Ajona, Daniel; Castano, Zafira; Garayoa, Mercedes et al. (2004) Expression of complement factor H by lung cancer cells: effects on the activation of the alternative pathway of complement. Cancer Res 64:6310-8
Macpherson, Gordon R; Ng, Sylvia S W; Forbes, Siiri L et al. (2003) Anti-angiogenic activity of human endostatin is HIF-1-independent in vitro and sensitive to timing of treatment in a human saphenous vein assay. Mol Cancer Ther 2:845-54

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