Defining the clinical role of the mammalian target of rapamycin (mTOR) in lung cancer

Abstract

Lung cancer is the biggest cancer killer. Most patients present with advanced disease. These diseases are not amenable to surgery which currently provides the best chance of cure. New treatment approaches are urgently needed, and these will come from a greater understanding of the disease biology. Our group has identified growth factor signaling molecules, downstream of growth factor receptors, which are overexpressed in all SCLC tumours examined compared to lung epithelial cells. These include the growth controlling kinase the mammalian target of rapamycin (mTOR). Here the importance of mTOR in lung cancer was examined. We examined the expression of mTOR in NSCLC and SCLC patients. We demonstrated that total mTOR was overexpressed in early stage NSCLC patients and in SCLC patients. More specifically mTOR predicts poor outcome in early stage NSCLC patients. It appears to identify poor outcome in lymph node negative and stage IA patients where previously other markers have failed to consistently yield prognostic benefit. This information may help identify those at high risk of relapse. We demonstrate that using total protein immunohistochemistry may be more useful than using phospho-protein expression in assessing the contribution of a signaling protein to cancer biology in patient specimens. We show that in cell lines, formalin fixed cell line and in tumour biopsies phospho-mTOR signals varies in intensity with time from removing from media/blood supply. We suggest that no conclusions can be drawn from commonly used phospho-protein immunohistochemical studies and total protein studies may be more reliable. Previously our laboratory has shown that high concentrations of rapamycin, an mTOR inhibitor, block MEK/ERK signaling which is necessary for pro-survival/chemoresistance effects induced by FGF-2 in SCLC cells. We demonstrate that this phenomenon is also observed with the clinically relevant mTOR inhibitor- RAD001 and other growth factors/chemicals in other SCLC cell lines, but not in NSCLC cell lines. It is therefore possible that high concentrations of RAD001 might help to reduce FGF-2-induced chemoresistance in SCLC. Moreover, by impairing growth factor induced ERK signaling; RAD001 has an additional mechanism of blocking growth factor induced proliferation. This could be a potentially novel therapeutic method. We investigated the mechanism of how mTOR mediates the ability of high concentrations of rapalogues to suppress MEK/ERK signaling and FGF-2 triggered chemoresistance. Using siRNAi and a PP2A inhibitor to various components of the pathways involved, we have found that neither FKBP12 nor mTOR is directly involved whilst the phosphatase PP2A may aid the mTOR regulation of MEK/ERK signaling. We established, using combination analysis, that the H69 SCLC cell line is more sensitive to combined mTOR inhibitor/cytotoxic chemotherapy killing compared to the H510 SCLC cell line and the order of drug exposure may not be important. Collectively, our results show that mTOR is important in NSCLC as well as SCLC biology providing a possible biomarker for poor outcome and a suitable target for new therapies such as RAD001.