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indicate that either treatment with PD98059 or silencing of Akt using shRNA in lung cancer xenografts significantly natural product libraries enhances the anti tumor efficacy of rapamycin in association with inhibition of Bad phosphorylation at S112 or S136 in tumor tissues. Importantly, PD98059 plus Akt shRNA block rapamycinstimulated Bad phosphorylation at both S112 and S136 sites in tumors, and more efficiently represses lung tumor growth than either PD98059 or Akt shRNA alone. Consistent with in vitro , PD98059 and Akt shRNA have no significant effect on S155 site phosphorylation of Bad in vivo. These findings suggest that blockage of rapamycin induced Bad phosphorylation at both S112 and S136 sites may not only sensitize cancer cells to rapamycin but also can overcome rapamycin resistance leading to increased anti tumor activity in vivo. To evaluate the role of apoptosis in tumor growth, a TUNEL assay was employed for measuring apoptosis in tumor tissues using Chromoblastomycosis a Tumor TACS In Situ Apoptosis Detection Kit. reveal that inhibition of Bad phosphorylation by PD98059 and Akt shRNA significantly enhances apoptosis in tumor tissues. Lung cancer, a major cigarette smoke related cancer, is the primary cause of cancer related mortality in the United States, accounting for more deaths than breast, prostate and pancreatic cancer combined. mTOR inhibitors, such as rapamycin and everolimus, have been evaluated as lung cancer therapeutics but with limited success. Previous reports indicate that rapalog activated Akt and MAPK ERK1/2 may contribute to the development of resistance to these agents. However, the downstream mediators of the survival consequence of rapamycin activated AKT and ERK1/2 remain unclear. Here we discovered that rapamycin, in addition to mTOR inhibition, potently stimulates Bad phosphorylation at S112 and S136 sites via activation of AKT and MAPK ERK1/2, which can lead to rapamycin resistance since increased Icotinib levels of Bad phosphorylation were observed in rapamycin resistant cells. Intriguingly, either blockage of Bad phosphorylation at S112 and S136 sites or expression of the nonphosphorylatable Bad mutant can reverse rapamycin resistance, suggesting that manipulation of Bad phosphorylation at these two sites should be an effective approach for overcoming rapamycin resistance. Because PKA is the physiological S155 Bad kinase and a previous study has demonstrated that rapamycin does not influence the activity of PKA, this helps explain why rapamycin has no effect on S155 Bad phosphorylation in vitro and in vivo. It is known that phosphorylation of Bad at one or more sites can inactivate the proapoptotic function of Bad. Therefore, we expect that rapamycin induced Bad phosphorylation at S112 or S136 will abolish the death promoting activity of Bad.