These results strongly suggest that the EGFR is transactivated by neurotensin in HCT116 cells and that this transactivation is involved in mediating the Akt phosphorylation stimulated by neuro tensin. Since the PI3K/Akt pathway is important in sev eral regulations besides cell proliferation, such as promoting cell survival by enhancing resistance to apop tosis, the EGFR mediated activation by neuro tensin may have significant roles in the malignant phenotype in these cells. It is unclear why neurotensin activates different path ways in the different the cell lines. It is known that HCT116 and Panc 1 cells both harbour a KRAS muta tion, while HT29 cells have a mutant BRAF. Further more, HT29 and HCT116 cells harbour mutations in the catalytic a polypeptide of phosphoinositide 3 kinase, and HT29 cells also have mutated p53.
While it is known that mutations in KRAS, BRAF and PIK3CA may determine the responsiveness to targeted therapies such as EGFR, MEK or mTOR inhibitors, the consequences of these mutations for neurotensin signal ling in the different cell lines are not obvious. Whereas we found that neurotensin treatment stimulated Akt phosphorylation in the three cell lines examined, an ear lier report using NTSR1 transfected AV12 cells found that neurotensin inhibited basal and EGF or insulin sti mulated Akt phosphorylation, which was ascribed to a negative regulation mediated through Gq. It has been found that classical PKC isoforms mediate feed back inhibition of EGFR transactivation by Gq coupled receptor agonists.
The degree of EGFR induced transactivation involvement in signalling by neurotensin may thus depend on the strength of PKC mediated feed back inhibition in different cells. In this context, it is of interest that HCT116 cells have a higher expression of the classical isoform PKCbII than HT29 cells. Interestingly, while the results showed that EGFR acti vation was required for neurotensin stimulated phos phorylation of Akt, we did not obtain complete inhibition of this effect by pretreatment with neither GM6001, cetuximab or gefitinib. Contrary to this, Akt phosphorylation induced by direct activation of the EGFR by TGFa or EGF was completely suppressed by gefitinib or cetuximab. Also, neurotensin stimulated Shc phosphorylation was completely suppressed. One possi ble explanation is that neurotensin also might induce release of ligands that activate ErbB3 or ErbB4 recep tors.
The HCT116 cells have been found to release sev eral ligands that activate the ErbB receptor family. The lack of complete inhibition induced by GM6001 pretreatment could imply that Brefeldin_A EGFR transacti vation could also be induced independently of ligand shedding by an intracellular calcium mediated mechan ism, possibly involving Pyk2 or Src. Alternatively, neurotensin might induce transactivation of the insulin like growth factor 1 receptor, as observed in human colonic epithelial cells.