Current data suggest that the process of melanoma tumorigenesis is enhanced by aberrant constitutive expression of the inducible form of nitric oxide synthase (iNOS), an enzyme catalyzing the generation of nitric oxide (NO), normally in response to cytokines. The presence of reactive NO in melanoma is indicated by detection of nitrotyrosine (NT), which is an end-product of the chemical reaction of NO with proteins; NT formation is known to alter intracellular signaling, especially when the target is a tyrosine kinase substrate. Production of low levels (pM) of NO by human tumors is proposed to be responsible for increased angiogenesis, growth, invasion, genomic instability and resistance to apoptosis. Our analysis of human melanoma specimens from patients with advanced (Stage III) disease indicates that patients whose tumors express iNOS are more likely to die within 2 years of neoadjuvant biohemotherapy than patients without iNOS (p<0.001).
Specific Aim 1 comes from the clinic to the laboratory to further test the significance of iNOS and NT in tissue specimens as prognostic markers for primary and metastatic melanoma patients. The translational goals of Specific Aim 1 are validation of iNOS and NT as new prognostic markers for survival in melanoma patients and to determine whether either marker provides independent prognostic value. Significant results will then be submitted to American Joint Commission on Cancer, Committee on Melanoma for inclusion in future staging revisions, as the first molecular marker for melanoma to be validated.
Specific Aim 2 tests the hypothesis that iNOS is controlled by specific, identifiable gene transcriptional regulators including constitutively active NFKB. NFKB activation is known to regulate basal as well as cytokine-induced iNOS in normal human cells, however its role in regulation of melanoma iNOS is unknown.
Specific Aim 3 builds on further upstream mechanisms of iNOS activation and tests the association and functional role of mutated B-raf, which is reported to drive MAPK independently of Ras in melanoma cells. A novel molecular heteroduplex analysis will be employed to detect mutated B-raf in paraffin-embedded tumor specimens, and study of its association with iNOS and constitutive NFKappaB. The regulation of mutated B-raf activity is also proposed in Specific Aim 4 as a means to regulate iNOS and, by extension, melanoma growth.
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