These characters make HER3 an obligate heterodimerization partner for other HER receptors (16). simultaneously inhibiting the activation of HER3 and EGFR and consequently the downstream PI3K/AKT and ERK pathways and acquired resistance to cetuximab include mutations in the KRAS, BRAF and NRAS genes (9), a secondary mutation (S492R) in the extracellular domain name of EGFR receptor (9, 10), overexpression of the MET proto-oncogene (c-Met) (11), and in HNSCC, the expression of the in-frame deletion mutation of EGFR variant III (12). Recently, an increasing body of literature has suggested that resistance to anti-EGFR therapy occurs frequently through activation of option signaling pathways that bypass the original target (13, 14). Compensatory HER3 signaling and sustained PI3K/AKT Ziprasidone hydrochloride activation are associated with FLJ23184 sensitivity and resistance to anti-EGFR targeted therapies, especially in HNSCC (13-16). Unlike other HER receptors, HER3 has diminished intracellular kinase activity but has known ligands. These character types make HER3 an obligate heterodimerization partner for other HER receptors (16). HER3 contains six PI3K binding sites that are crucial for PI3K/AKT pathway activation (16). A preclinical study Ziprasidone hydrochloride reported an association between sensitivity to gefitinib and the overexpression of HER3 in HNSCC cell lines (17). Furthermore, after sustained exposure to gefitinib or erlotinib, cells showed upregulated HER3 and AKT phosphorylation, which correlated with HER3 translocation from your nucleus to the membrane (15). Increased expression of heregulin (HRG), a potent HER3 ligand, also provided a possible mechanism of cetuximab resistance in colorectal malignancy (18). There is a recent evidence reported that HER3 signaling plays an important role in acquired resistance to cetuximab, perhaps a more crucial one in comparison with MET in HNSCC and non-small cell lung malignancy (13). Direct targeting of HER3 by siRNA in cetuximab-resistant cells has been shown to restore cetuximab sensitivity (13). These data suggest an Ziprasidone hydrochloride opportunity to develop combinatorial strategies by using cetuximab and anti-HER3 agent in HNSCC. MM-121 (SAR256212) is usually a fully human antibody that directly binds to the extracellular domain name of HER3 (19, 20) and induces receptor downregulation resulting in the inhibition of downstream HER3-dependent pathways. As MM-121 has not previously been tested in HNSCC, we were interested in exploring its activity as a single agent and in combination with cetuximab in preclinical models of HNSCC. Overall, we found that HER3 was active in the majority of HNSCC cell lines, a combination of EGFR and HER3 inhibition provided improved antitumor activity relative to either inhibitor alone, and the combination effectively inhibited signaling through both ERK and PI3K/AKT pathways and in 2011, using the same STR profile (22). Colony formation assay Cells were plated in 6-well culture plates at the concentration of 200?per well. After 24h incubation, cells were treated with PBS, 2g/mL cetuximab, 20g/mL MM-121 or the cetuximab and MM-121combination (CM combination) for 9 days to form colonies as previously explained (25). The dose of cetuximab was chosen from our previous study (25) and the dose of MM-121 was chosen from an escalating serial doses which showed comparable pattern of synergistic effect in combination with cetuximab (data not shown). Medium was changed every three days. The colonies were then stained with 0.2% crystal violet with buffered formalin (Sigma). Colony figures were manually counted using Image J software. Cell figures 50 were considered as a colony. Cell proliferation assay The inhibition of cell proliferation by cetuximab and Ziprasidone hydrochloride MM-121 was analyzed by a cell proliferation assay as previously explained (26). Briefly, 2.5??105?cells were seeded in 60 mm dishes and incubated overnight. Cells were then treated with PBS, 62g/mL cetuximab, 125g/mL MM-121, and the combination for 72 Ziprasidone hydrochloride hours. The dose of MM-121 and cetuximab was chosen based on previous studies (19, 25) and our SRB assay (Sulforhodamine B cell proliferation assay) results (Supplementary Fig. S1). Cells were harvested by trypsinization and.