Activation of the mTOR Pathway by Oxaliplatin in the Treatment of Colorectal Cancer Liver Metastasis

Abstract

Background: Standard of care treatment for colorectal cancer liver metastasis consists of a cytotoxic chemotherapy in combination with a targeted agent. Clinical trials have guided the use of these combinatory therapies, but it remains unclear what the optimal combinations of cytotoxic chemotherapy with a targeted agent are.

Methods: Using a genomic based approach, gene expression profiling was obtained from tumor samples of patient with colorectal cancer liver metastasis who received an oxaliplatin based therapy. Early passaged colorectal cancer liver metastasis cell lines and patient derived xenografts of colorectal cancer liver metastasis were then treated with oxaliplatin and a mTOR inhibitor.

Results: Gene set enrichment analysis revealed that the mTOR pathway was activated in patients receiving oxaliplatin based therapy. Treatment of early passaged colorectal cancer lines and patient derived xenografts with oxaliplatin resulted in activation of the mTOR pathway. Combination therapy with oxaliplatin and a mTOR inhibitor resulted in a synergistic effect both in vitro and in vivo.

Conclusion: Our findings suggest a genomic based approach can be used to identify optimal combinations of cytotoxic chemotherapy with a targeted agent and that these observations can be validated both in vitro and in vivo using patient derived colorectal cancer cell lines and patient derived xenografts prior to clinical use.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Unsupervised hierarchical clustering on 39 colorectal cancer liver metastasis samples were performed with oxaliplatin treated samples boxed.

Unsupervised hierarchical clustering showed that oxaliplatin treatment did not influence the clustering of the 39 samples.

Fig 2. Activation of the mTOR pathway by oxaliplatin in vitro.

A. The level of phosphorylated p70 S6 kinase was increased in all five ATCC cell lines (HCT15, DLD-1, LoVo, HCT116, HT29 and Colo 205) by Day 3. B. The level of phosphorylated p70 S6 kinase was increased in all early passage colorectal cancer cell lines (CRC057, CRC119 and CRC240) by Day 3 or 4.

Fig 3. Oxaliplatin and Rapamycin Synergy Graphs were performed to determine the combination index (CI).

A. The growth of ATCC colorectal cell lines HCT15, DLD-1, LoVo, HCT116, HT29 and Colo 205 in the presence of oxaliplatin and rapamycin as single agents and in combination was analyzed by cytotoxicity assays (see materials and methods) to determine the dual-effect of the two agents on the cell lines. Mean Combination Index (CI) values (from three experiments) at 25%, 50%, 75% and 90% is plotted for each cell line. (CI < 1 denotes synergy between the two drugs). Oxaliplatin and rapamycin did show synergistic effect in DLD-1, LoVo, HT29 and Colo205 cell lines except HCT15 cell line. B. The growth of early passage colorectal cancer cell lines CRC057, CRC119 and CRC240 in the presence of oxaliplatin and rapamycin as single agents and in combination was analyzed by cytotoxicity assays (see materials and methods) to determine the dual-effect of the two agents on the cell lines. Mean Combination Index (CI) values (from three experiments) at 25%, 50%, 75% and 90% is plotted for each cell line. (CI < 1 denotes synergy between the two drugs). Oxaliplatin and rapamycin had synergistic effect in all three cell lines.

Fig 4. Activation of the mTOR pathway by oxaliplatin in vivo.

The level of phosphorylated p70 S6 kinase was increased in colorectal cancer PDXs by Day 2.

Fig 5. Synergy between oxaliplatin and everolimus in vivo.

Oxaliplatin and everolimus were shown to have synergic effect in CRC119 PDX based on tumor growth inhibition with the combination therapy compared to either oxaliplatin or everolimus alone. * Tumor sizes at Day 21, Control vs. Oxaliplatin + Everolimus: P