The PI3K/Akt/mTOR and Ras/Raf/ERK (MAPK) signaling pathways are constitutively activated and serve critical functions in the pathogenesis of colorectal cancer (CRC). mTOR signaling is involved in cell growth and metabolism; rapamycin inhibits the kinase activity of mTORC1 but leads to feedback activation of upstream PI3K/Akt signaling, resulting in cell survival and chemoresistance. MAPK signaling is involved in cell proliferation and differentiation; sorafenib is a multikinase inhibitor that inhibits Raf kinase. Recent studies suggest that inhibition of mTORC1 with rapamycin leads to feedback induction of MAPK signaling through a S6K-PI3K-Ras feedback loop. Therefore, we investigated whether dual targeting of mTOR and MAPK signaling pathways, using rapamycin and sorafenib, respectively, has therapeutic benefits in CRC.

Methods: HCT116 human CRC cells (mutant K-Ras; wild-type B-Raf) were treated with either rapamycin (50 nM) or sorafenib (5 µM) alone or in combination. Effects on cell proliferation (measured by cell counting), apoptosis (measured by detecting the level of histone- associated DNA fragments in mono- and oligonucleosomes), cell cycle progression (measured by fluorescence-activated cell sorting), migration (measured by wound healing assay) and invasion through matrigel (measured by modified Boyden chamber assay) were analyzed.

Results: (i) pAktSer473 and pERKThr202/Tyr204 are overexpressed together in stage IV CRC tissues and matched liver metastasis tissues from the same patient (Spearman's correlation=0.77; p<0.0001). (ii) Inhibition of mTORC1 using rapamycin or targeted shRNA-mediated inhibition of mTORC1 proteins, mTOR and Raptor, leads to strong feedback activation of MAPK signaling. (iii) Combined treatment with rapamycin and sorafenib prevents feedback induction of both PI3K/Akt and MAPK signaling pathways. (iv) Rapamycin or sorafenib significantly decreased cell proliferation, migration and invasion compared to the control group; combinatorial treatment significantly potentiated proliferation, migration and invasion compared to single agent treatment. (v) Sorafenib, but not rapamycin, increased apoptosis compared to the control group; combination treatment significantly increased induction of apoptosis compared to monotherapy. (vi) Single agent treatment with rapamycin or sorafenib inhibited G1 -S phase cell cycle progression; combinatorial treatment increased growth arrest compared to rapamycin, but not sorafenib monotherapy.

Conclusions: K-Ras mutant CRCs, which display clinical resistance to anti-EGFR therapy, are sensitive to the anti-tumorigenic effect of sorafenib. Furthermore, combinatorial inhibition of mTOR and MAPK signaling pathways, using rapamycin and sorafenib, respectively, significantly potentiates attenuation of growth and progression compared to monotherapy in K-Ras mutant CRCs. This effect may be due to reduced feedback activation of PI3K/Akt and MAPK signaling. CRC is a heterogeneous disease with multiple aberrant signaling pathways driving the oncogenic behavior of tumors; simultaneous inhibition of parallel signaling pathways may be more effective than single pathway inhibition in CRC therapies.

Citation Information: Clin Cancer Res 2010;16(14 Suppl):A41.