Identification Of Candidate Predictive Biomarker Of Response To Rapalogs Therapy In Human Cancer

The mammalian target of rapamycin (mTOR) regulates multiple processes, including cell growth, apoptosis, metabolism, mRNA translation, angiogenensis, cell-cycle progression, and has been recognized as a master switch in cell proliferation. The mTOR-mediated pathway is commonly deregulated in cancer and has been validated as an important target for chemotherapy.Rapamycin is the first discovered mTOR inhibitor and its analogs temsirolimus and everolimus have been approved for the treatment of advanced renal cancer in May 2007 and April 2009 respectively. Rapamycin and its analogs temsirolimus, everolimus and deforolimus are being tested in a wide spectrum of clinical human cancers. Although preclinical pharmacological results of rapalogs are encouraging, the objective response rates with rapalogs in clinical trials were modest and variable. Identification of biomarkers capable of predicting response to rapalogs is of increasing interest.We analyzed pairwise Pearson correlation coefficients (r) between rapalogs activity (GI50) and gene expression profile for each NCI-60 cell line. p27/KIP1 showed the highest positive correlation among 9,706 gene probes tested. At cellular levels, breast cancer MCF-7, T47D and BT-474 cells, expressing high levels of p27/KIP1, were sensitive to rapalogs, whereas the cells expressed low levels of p27/KIP1, such as MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells, exhibited resistance to rapalogs. Consistently, down-regulation of p27/KIP1 by siRNA conferred MCF-7 and BT-474 cells insensitive to rapalogs. Moreover, a significant positive correlation between p27/KIP1 gene expression and rapamycin anti-tumor activity was also observed in mice bearing different human cancer cell xenografts. Mechanistic study indicated this correlation is likely determined by the basal level of p27/KIP1 regardless of the redistribution of p27/KIP1 upon rapalogs treatment. Moreover, in MCF-7 cells, p27/KIP1 pulled-down by an anti-CDK4 antibody significantly decreased after rapamycin treatment, indicating a significant decrease in the association of p27/KIP1 with CDK4. Meanwhile, p27/KIP1 immunoprecipitated by anti-CDK2 antibody increased under the same condition, indicating enhanced association between p27/KIP1 and CDK2. This phenomenon failed to be observed in rapamycin-resistant MDA-MB-231 cells, and the association of p27/KIP1 with CDKs is weaker than that in MCF-7 cells. The removal of p27/KIP1 from cyclinE-CDK2 is an essential step for S-phase entry. Thus, we propose p27/KIP1 may provide a putative threshold to block G1/S transition. The p27/KIP1 levels in sensitive cells are beyond the threshold and cells are arrested in G1-phase or vice versa.In conclusion, p27/KIP1 expression level is positively correlated with the anticancer activity of rapalogs in vitro and in vivo. We propose p27/KIP1 expression level may be also a candidate predictive biomarker for patient selection for rapalogs-based therapy.During computing pairwise Pearson correlation coefficients (r) between rapamycin activity and genes expression patterns in NCI-60 cells to search for the predictors of rapalogs response based on gene expression profile, we found the expression of CXCL12 in addition to p27/KIP1 was also significantly correlated with rapamycin activity. CXCL12 was initially cloned by Japanese scientists, which is involed in the metastasis of gastric carcinoma and determined to be an independent prognostic factor. Gastric cancer is the second leading cause of cancer-related death worldwide. Tumor metastasis is a leading cause of death in advanced gastric cancer paitents. Thus, therapeutic strategy for gastric cancer metastasis, as well as the early diagnosis and treatment, are very important. CXCL12/CXCR4 axis, a common pair of chemokine/chemokine receptor cancer cells, plays an important role in metastasis of gastric carcinoma. Recently, spioradic studies reported that rapamycin is also able to inhibit gastric cancer cell migration. Therefore, Gastric cancer cells were selected as models to study the role of mTOR signal pathway in the cells migration mediated by CXCL12/CXCR4 axis.We found that stimulation of gastric cancer cells with CXCL12 activated PI3K/Akt/mTOR signaling cascade, which is consistent with results obtained with other cell lines. On the other hand, pretreatment with CXCR4 antagonist AMD-3100, PI3K inhibitor PI-103 or mTOR inhibitor rapamycin abrogated enhanced phosphorylated Akt and/or phosphorylated S6K1 induced by CXCL12. These results indicated that CXCL12 interacted with and atcivated CXCR4 receptor, which in turn activated PI3K kinase and its downstream signaling including phosphorylation of Akt and mTOR. More over, stimulation of CHO-K1 cells stably expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of PIP3, which provided direct evidence activation of PIP3 by CXCL12.The classⅠPI3K are divided into class 1A (α,β,δ) and class 1B (γ) according to their different catalytic subunits, which are activated by different extra-or intra-cellular signaling molecules. Down-regulation of p110beta subunit by transient transfection of siRNA targeting p110βbut not p110αblocked increase in phosphorylated Akt and phosphorylated S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor TGX-221 blocked activation of PI3K/Akt/mTOR signaling cascade by CXCL12 in MKN-45 cells. Moreover, CXCR4 immunoprecipitated by anti-p110beta antibody increased after CXCL12 stimulation, indicating a significant increase in the association of p110βand CXCR4. Thus, these results demonstrated that CXCR4 interacted with p110(3 and actived the downstream signaling.As we demonstrated that PI3K/Akt/mTOR pathway was important for the downstream signaling, we proposed that inhibitors targeting PI3K/mTOR could inhibit cell migration induced by CXCL12. In a transwell model, the chemotactic responses of MKN-45 cells to CXCL12 were significantly blocked by PI-103 and rapamycin. However, their activities were less potent in FBS-stimulated cell migration. Further studies inhibition of migration by rapamycin or PI-103 might be related to prevention of F-actin reorganization and downregulation of proteins involved in cell motility, such as Rho A. Furthermore, rapamycin also inhibited the secretion of CXCL12 and the expression of its receptor CXCR4, which may form a positive feedfact to further abolishing upstream signaling leading to cell migration.Finally, we found MKN-45 and MKN-28 cells, expressing high levels of CXCL12, were sensitive to rapamycin in its activity inhibiting migration, whereas SGC-7901 cells expressing low levels of CXCL12, exhibited resistance to rapamycin. Together with the fact that gastric cells expressing high levels of CXCL12 are more sensitive to rapamycin-induced antiproliferation, our results indicated that mTOR inhibitor may be used for therapy of metastic gastric cancer expressing high levels of CXCL12.In summary, we have shown that mTOR pathway play an important role in cell migration mediated by CXCL12/CXCR4 axis and mTOR pathway inhibitors could inhibit CXCL12-stimulated cells migration. On the other hand, cells that express high levels of CXCL12 mRNA were more sensitive to rapamycin in its activity in inhibiting both cell migration and proliferation. We proposed that drugs targeting mTOR pathway may be used for the therapy metastic gastric cancer expressing high levels of CXCL12.