Study On Simulataneous Targeting Of EGFR And MTOR Inhibits The Growth Of Colon Carcinoma Cells

Colorectal carcinoma is one of the most common cancers and the incidence of colon cancer is increasing year by year in the world. Studies of cancer signal pathways have resulted in the generation of novel therapeutic agents that target the signal pathways for treatment of colorectal carcinomas. Epidermal growth factor receptor (EGFR) of receptor tyrosine kinase family is highly expressed in colorectal carcinomas, leading to the activation of downstream kinase cell growth pathways and thereby cancer growth and progression. EGFR is currently targeted therapeutically at least by two approaches:small molecular inhibitors and neutralizing antibodies. Clinical trials of the antibodies have shown a response in approximately 10% of patients with metastatic colorectal carcinomas in association with EGFR gene amplification but not mutations. In contrast, small molecular inhibitors have shown no such a response in the patients. The mechanisms of the resistance to EGFR targeted therapeutics include the activation of redundant EGFR downstream kinase pathways in colorectal carcinoma. Current efforts have focused on development of combination therapies targeting the redundant cell growth pathways in colorectal carcinomas.EGFR can activate downstream phosphatidylinositol 3-kinase (PI3K) and Akt through interaction with intracellular adaptors, driving cancer progression and meta stasis. Mammalian target of rapamycin (mTOR), a serine and threonine kinase, is a recently identified downstream effector of the PI3K pathway that links EGFR with cell growth and proliferation. Recent study suggests that targeting of mTOR alone may not be sufficient enough to inhibit cancer grown because inhibition of mTor activates PI3K/Akt pathway. In this study, we show that simultaneous targeting of EGFR and mTor inhibits PI3K/Akt, Erk1/2 and mTOR pathways and thereby the growth of colorectal carcinoma cells.In the experiment, We used cell culture, cell proliferation assay, flow cytometry, Western blot and other experiments to study EGFR expression in human colon cancer tissues, the expression and activation of EGFR and mTOR pathway in human colon cancer cell lines. Erlotinib and Rapamycin inhibition cell growth, alone or in combination erlotinib and rapamycin on cell cycle and the pathway of the protein expression changes. This study investigated the combination of EGFR and mTOR inhibition colon cancer cell growth, and further enhance the molecular mechanism of combined treatment of colon cancer cells, provide the evidence for combination the targeting inhibitors of colon cancer in clinical trail.Test results of EGFR expression in human colon cancer tissues and cell lines:1. EGFR is expressed in human colon cancer tissues and corresponding normal tissues, and EGFR expression in tumor samples was no higher than the corresponding normal tissue. The results show that, EGFR is widely present protein in normal and tumor cell surface, and is highly expressed in colon tumors.2. The expression of phosphorylated Akt (phosphor-Akt), phosphorylated mTOR (phosphor-mTOR), phosphorylated rpS6 (phosphor-rpS6) in tumor tissue was significantly higher than the corresponding. Although the expressionthe of EGFR in colon tumor tissue was no significant difference in normal tissue, but the EGFR downstream of PI3K and mTOR signaling pathway in tumor tissue is fully activated. And phosphor-mTOR and phosphor-rpS6 in tumor tissues was positively correlated.3. Epidermal growth factor (EGFR) expressed in the HT-29, HCT-116, HCT-8, and DLD-1, and in both colon cancer cell lines it is activated4. EGF can activate the EGFR in colon cancer cells. And EGFR activation did not show significant positive correlation with EGF concentration and time. EGF can activate the EGFR and its downstream pathway in colon cancer cell line HT-29, HCT-8, DLD-1, and HCT-116.Following experimental results show Erlotinib and rapamycin can inhibition growth of colon cancer cells1. In KRAS wt colon cancer cell lines, EGFR inhibitor erlotinib targeted to inhibit EGF-mediated EGFR and its downstream Akt, mTor and Erk1/2 pathway activation. In KRAS mt colon cancer cell lines, mTOR pathway is sustained activation. Erlotinib in the KRAS mt colon cancer cell lines can inhibit EGF-mediated EGFR and downstream Akt, and Erk1/2 pathway activation, but can not inhibit the mTOR pathway activation.2. Erlotinib showed strong growth inhibition on colon cancer cell line and increasing intensity with the increase of drug concentration.3. Erlotinib inhibit cell growth and proliferation, block the cell proliferation cycle in KRAS mt and KRAS wt colon cancer cells, the tumor cell cycle arrest in G0/G1 phase, proliferative S phase to inhibit the growth of cells.4. Rapamycin has no significant inhibition on growth of KRAS wt and KRAS mt colon cancer cells. 5. Rapamycin completely inhibited the expression of phosphor-rpS6p in KRAS wt cells, only partially inhibited the expression of phosphor-rpS6p in KRAS mt cells, phosphor-Akt Expression did not reduce in the two cell lines, and tended to increase. So PI3K/Akt pathway can be activation of fed back.by inhibiting the mTOR pathwayFollowing experimental results show Erlotinib combine rapamycin can inhibition growth of colon cancer cells better1.Combination of Erlotinib and Rapamycin can significantly reduce the p-EGFR, p-Akt, p-rpS6 and p-ERK1/2 expression in KRAS wt cell lines and in KRAS mt cell lines. That combination of Rapamycin and Erlotinib can inhibite EGFR and mTOR pathway better.2.Combination of Erlotinib and Rapamycin inhibited the growth of colon cancer cells has increased dramatically than single drug.3.Combined Erlotinib and Rapamycin in KRAS wt and KRAS mt colon cancer cells may be better than monotherapy in cell cycle arresting. The role of combination therapy in KRAS wt cell lines was significantly better.The main innovation of this study1.Verifiy dual EGFR and mTOR signaling pathway activation in colon cancer tissues.2.Verifiy mTOR signaling pathway is sustained activation in the KRAS mt colon cancer cell lines; PI3K/Akt pathway can be activation of fed back.by inhibiting the mTOR pathway3.Ilustrates the molecular mechanism.the combination of Erlotinib and Rapamycin inhibited colon cancer cell growth better.Above all, EGFR pathway inhibition alone can not fully suppress the activation of mTOR pathway in KRAS mt colon cancer cells. mTOR inhibitors alone can not inhibited cell growth completely, PI3K/Akt pathway can be activation of fed back.by inhibiting the mTOR pathway. Then we combined inhibition of EGFR and mTOR pathways, to explore a new and effective methods for the molecular target of the treatment of colon cancer. Different methods results showed that combined inhibition can inhibit cell proliferation cycle better compared to Erlotinib alone. Therefore, the combination of Erlotinib and Rapamycin inhibition growth of colon cancer cells better, inhibition activation of EGFR and mTOR and downstream pathway, It is a new approach of colon cancer treatment.