Recent studies have elegantly shown that activated RAS could cause MAPK pathway activation MAPK signaling through direct activation of CRAF, or from the transactivation of BRAF CRAF heterodimers in the presence of vemurafenib, or perhaps through a variety of both elements. Certainly, introduction of an activated RAS mutant in to HT 29 cells generated sustained G ERK levels and resistance to vemurafenib. We found that inhibition of EGFR abrogated RAS activation, P CRAF induction, and P ERK re activation upon vemurafenib treatment in BRAF mutant CRC cells, suggesting that vemurafenib can produce sustained inhibition of mutant BRAF activity and suppression of ERK phosphorylation in the absence of EGFR mediated feedback signals. Particularly, we discovered that the Plastid sustained suppression of PERK achieved by combined RAF and EGFR inhibition leads to enhanced sensitivity in vitro and to tumefaction regressions in vivo. These studies suggest that BRAF mutant CRCs, like their melanoma counterparts, retain a strong dependence on MAPK signaling and that tumor responses are possible if the MAPK pathway is sufficiently inhibited in these cancers. Apparently, while EGFR seemed to mediate re activation of MAPK signaling in reaction to vemurafenib, we didn’t discover proof of increased EGFR activation by itself following vemurafenib treatment, as might be anticipated in a classical feedback loop. Indeed, R EGFR levels didn’t improve after treatment at any time point examined between 0 and 48 hours, despite the fact that MAPK activity appeared to recover since 3 6 hours after vemurafenib treatment. In fact, if any such thing, a slight decrease in R EGFR and full EGFR levels was seen at later timepoints. These results suggest that EGFR is lively in BRAF mutant CRC cells ahead of vemurafenib treatment, but that EGFR sends its signal to trigger CRAF and RAS only upon treatment. One possible order VX-661 explanation for this observation may contain Sprouty proteins, which are significant MAPK pathway feedback mediators that are transcribed in an ERK dependent manner. Sprouty proteins may prevent RTK mediated activation of RAS. Consistent with this hypothesis, we observed that Spouty4 levels decreased after-treatment with vemurafenib, and this decrease coincided with induction of P ERK and P CRAF. However, further studies are necessary to determine whether Sprouty proteins are involved with this p repression of EGFR dependent activation of downstream signaling. BRAF mutant CRC cell lines indicated higher levels of P and EGFR EGFR than BRAF mutant melanoma cell lines, and individual BRAF mutant CRCs demonstrated somewhat higher levels of P EGFR than BRAF mutant melanomas. These findings might explain why BRAF mutant CRCs are far more susceptible to EGFR mediated RAF inhibitor resistance through incomplete MAPK reduction. Apparently, while BRAF mutant cancer cells had internationally reduced levels of phosphorylated RTKs, BRAF mutant CRC cells showed high levels of a few phosphorylated RTKs.