Abstract
BackgroundPrevious studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEK-inhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. However, rapid onset of resistance is a limit to combination therapies in KRAS mutated CRC.MethodsWe generated four different KRAS mutated CRC cell lines resistant to a combination of cetuximab (an anti-EGFR antibody) and refametinib (a selective MEK-inhibitor) after continuous exposure to increasing concentration of the drugs. We characterized these resistant cell lines by evaluating the expression and activation status of a panel of receptor tyrosine kinases (RTKs) and intracellular transducers by immunoblot and qRT-PCR. Oncomine comprehensive assay and microarray analysis were carried out to investigate new acquired mutations or transcriptomic adaptation, respectively, in the resistant cell lines. Immunofluorescence assay was used to show the localization of RTKs in resistant and parental clones.ResultsWe found that PI3K-AKT pathway activation acts as an escape mechanism in cell lines with acquired resistance to combined inhibition of EGFR and MEK. AKT pathway activation is coupled to the activation of multiple RTKs such as HER2, HER3 and IGF1R, though its pharmacological inhibition is not sufficient to revert the resistant phenotype. PI3K pathway activation is mediated by autocrine loops and by heterodimerization of multiple receptors.ConclusionsPI3K activation plays a central role in the acquired resistance to the combination of anti-EGFR and MEK-inhibitor in KRAS mutated colorectal cancer cell lines. PI3K activation is cooperatively achieved through the activation of multiple RTKs such as HER2, HER3 and IGF1R.
Highlights
Previous studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEKinhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines
In the present study we have investigated the mechanisms that eventually lead to resistance to the vertical suppression of MAPK pathway through combination of EGFR and MEK inhibition in a cellular model of primary resistance to anti-EGFR therapy constituted by KRAS mutated CRC cell lines
Previous studies have showed how, in KRAS-mutant CRC, PI3K activation is mediated by receptor tyrosine kinases (RTK) signaling, despite KRAS being capable of cross-activating PI3K [20, 28, 29], for this reason we investigated the status of multiple receptors
Summary
Previous studies showed that the combination of an anti-Epidermal growth factor (EGFR) and a MEKinhibitor is able to prevent the onset of resistance to anti-EGFR monoclonal antibodies in KRAS-wild type colorectal cancer (CRC), while the same combination reverts anti-EGFR primary resistance in KRAS mutated CRC cell lines. KRAS is able to directly activate PI3K signaling by binding to p110-PI3K subunit, there is increasing evidence that PI3K activation following MEK inhibition is correlated to RTK activity, paving the way to the use of RTK inhibitors in KRAS mutated CRC [20] With this respect, two different papers demonstrated that co-targeting of EGFR and MEK overcomes both acquired and primary resistance to anti-EGFR agents in CRC cellular models [21, 22]. In the present study we have investigated the mechanisms that eventually lead to resistance to the vertical suppression of MAPK pathway through combination of EGFR and MEK inhibition in a cellular model of primary resistance to anti-EGFR therapy constituted by KRAS mutated CRC cell lines
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