mutated ((stimulated Akt phosphorylation and increased plating efficiency. that the expression of together with the enhanced expression of amphiregulin (AREG) can identify HNSCC patients who are less likely to benefit from combination treatment with the anti-EGFR antibody cetuximab and docetaxel. Although mutations in occur in HNSCC at a rather low frequency, amplification of the wild-type gene (gene increases PI3K activity in HNSCC cells, which leads to growth factor-independent colony formation.18 It is known that a mutation leads to constitutive K-RAS activity N-(p-Coumaroyl) Serotonin supplier that is associated with the stimulated autocrine production of the EGFR ligand AREG19 and resistance to EGFR-TK inhibitors in NSCLC. However, it is not known whether mutations lead to the activation of the PI3K/Akt and MAPK/ERK pathways, the specific role of each pathway in clonogenicity needs to be investigated in both mutation or mutation results in constitutive K-RAS activity, as demonstrated by a pull-down assay using the GST-tagged Raf1-Ras-binding domain (Raf1-RBD) protein (Fig.?1A). Interestingly, although SAS and UT5R cells are < 0.001). Similarly, for the HNSCC cell lines, the DTs of the SAS (24.01 1.96 h) and UT5R (27.61 2.34 h) cells were significantly shorter than that of either the UT5 (39.68 8.55 h) or UT15 (48.08 3.04 h) cells (< 0.001) (Fig. S1A). The DT for FaDu cells (29.46 1.90 h) was significantly longer than that of the SAS cells (< 0.001) but was not significantly longer than that of the UT5R cells (= 0.087) (Fig. S1A). Cells with a short DT (A549, H460, SAS, and UT5R) presented a significant increase in clonogenic activity, as shown by plating efficiency (PE) (Fig. S1B). sequencing was performed to analyze whether the increased clonogenic activity in the NSCLC (A549 and H460) and HNSCC cells (SAS and UT5R) was due to a potential mutation in the gene. The data for the mutational status of (summarized in Table S1) indicate that the gene was mutated only in the A549 (G12S) and H460 (Q61H) cells and not in the HNSCC SAS and UT5R cells presenting a short DT and high PE. On the basis of these results, it can be assumed that the level of K-RAS activity rather than its mutational status correlates with clonogenic activity (Fig. S1B). As an additional proof for the role of K-RAS in clonogenic activity, the HNSCC FaDu cells were transiently transfected with a plasmid expressing mutated < 0.001). The HTB-182 cells, with a very low expression of EGFR (Fig. S2), N-(p-Coumaroyl) Serotonin supplier did not response to erlotinib (Fig.?2A), and erlotinib (1 M) had no effect on clonogenic activity in the HNSCC cells SAS and UT5R, which present high wild-type K-RAS activity, even at the higher concentration of 2.5 M. In contrast, the clonogenic activity of HNSCC cells presenting low levels of K-RAS activity (UT5, UT15, and FaDu) was completely blocked (Fig.?2B). Figure?2. K-RAS activity is associated with erlotinib resistance and accompanied with increased autocrine production of AREG. (A and B) The effect of erlotinib on clonogenic activity was determined using a clonogenic assay. The data points shown … Previously, we showed that mutation is associated with an enhanced autocrine production of the EGFR ligand AREG.19,20 As the < 0.001). Based on the possible role of K-RAS activity in the response to N-(p-Coumaroyl) Serotonin supplier erlotinib, the influence of this activity on erlotinib resistance in in FaDu cells led to the enhanced phosphorylation of Akt at S473 (Fig.?1D). Similarly, as DDX16 indicated by the data presented in Figure S3, a N-(p-Coumaroyl) Serotonin supplier 24 h treatment of the erlotinib-resistant < 0.05) (Fig.?4B). Most interestingly, the clonogenic activity of FaDu cells (in.