Nitric Oxide in Physiologic Concentrations Targets the Translational Machinery to Increase the Proliferation of Human Breast Cancer Cells: Involvement of Mammalian Target of Rapamycin/eIF4E Pathway

Abstract

Nitric oxide (NO) in nanomolar (nmol/L) concentrations is consistently detected in tumor microenvironment and has been found to promote tumorigenesis. The mechanism by which NO enhances tumor progression is largely unknown. In this study, we investigated the possible mechanisms and identified cellular targets by which NO increases proliferation of human breast cancer cell lines MDA-MB-231 and MCF-7. DETA-NONOate, a long acting NO donor, with a half-life of 20 h, was used. We found that NO (nmol/L) dramatically increased total protein synthesis in MDA-MB-231 and MCF-7 and also increased cell proliferation. NO specifically increased the translation of cyclin D1 and ornithine decarboxylase (ODC) without altering their mRNA levels or half-lives. Critical components in the translational machinery, such as phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets, phosphorylated eukaryotic translation initiation factor and p70 S6 kinase, were up-regulated following NO treatment, and inhibition of mTOR with rapamycin attenuated NO induced increase of cyclin D1 and ODC. Activation of translational machinery was mediated by NO-induced up-regulation of the Raf/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase/ERK (Raf/MEK/ERK) and phosphatidylinositol 3-kinase (PI-3 kinase)/Akt signaling pathways. Up-regulation of the Raf/MEK/ ERK and PI-3 kinase/Akt pathways by NO was found to be mediated by activation of Ras, which was cyclic guanosine 3 ¶,5 ¶-monophosphate independent. Furthermore, inactivation of Ras by farnesyl transferase inhibitor or K-Ras small interfering RNA attenuated NO-induced increase in proliferation signaling and cyclin D1 and ODC translation, further confirming the involvement of Ras activation during NOinduced cell proliferation. [Cancer Res 2007;67(1):289-99]

Effect of rapamycin, an mTOR inhibitor, on radiation sensitivity of lung cancer cells having different p53 gene status

“…7). The activation of mTOR can increase the proliferation rate through the increased translation of cell cycle effectors such as cyclin D1 (41). In thyroid cancer cells, RAD001 treatment reduced cyclin D1 and cyclin D3 protein levels, confirming that mTOR activation contributes to cell proliferation (42).…”

The chemical biology of nitric oxide: Implications in cellular signaling

“…At sustained NO levels between 10-30 nM, phosphorylation of ERK occurs through a cGMP dependent mechanism in MCF7 and endothelial cells. At 30-60 nM NO Akt was phosphorylated 40,42,43 . When NO reaches a threshold concentration of about 100 nM, HIF-1α is stabilized 40 .…”

Increased NOS2 predicts poor survival in estrogen receptor–negative breast cancer patients

“…Aberrant expression of NOS2 has been observed in many types of solid tumors, including breast and colon cancer, and also in melanoma (12)(13)(14)(15)(16). NO can both cause DNA damage and protect from cytotoxicity and either inhibit or stimulate cell proliferation and migration as well as apoptosis (17)(18)(19)(20)(21). The effect of NO will be dependent on the microenvironment, including tissue oxygen tension and local superoxide concentrations.…”