In the present study, we did not evaluate heregulin expression after acquired cetuximab resistance. analyzed. FaDuCR cells were resistant to cetuximab, whereas parental FaDu cells were susceptible to it. FaDuCR cells expressed consistently higher levels of phosphorylated Akt than FaDu cells despite cetuximab exposure. A comprehensive transcriptome analysis revealed that the HER3-ligand heregulin was upregulated in FaDuCR cells compared to FaDu cells. Heregulin knockdown in FaDuCR cells repressed HER3 and Akt phosphorylation and recovered cetuximab anticancer efficacy. In contrast, pan-HER family tyrosine kinase inhibitors such as afatinib decreased HER3 and Akt phosphorylation in FaDuCR cells and inhibited FaDuCR tumor growth. Two of the 28 HNSCC tumor samples presented aberrant heregulin expression comparable to that of FaDuCR cells and were resistant to cetuximab therapy. In HNSCC, heregulin-mediated HER3-Akt activation causes resistance to cetuximab but not to second-generation EGFR-tyrosine kinase inhibitors. Subpopulations with aberrant heregulin-expressing HNSCC might be resistant to cetuximab. encoding heregulin was among the 10 most highly upregulated genes. The quantitative PCR assay revealed approximately 13 higher heregulin expression GSK2636771 in FaDuCR cells than in FaDu cells (alters CRC and NSCLC cells harboring an EGFR-activating mutation in such a way that they are resistant to EGFR-targeted agents26,27. Heregulin might cause resistance to EGFR-targeted therapy in numerous types of cancer. Unlike CRC and NSCLC harboring an EGFR-activating mutation, HNSCC lacks biomarkers for optimizing EGFR-targeted therapy16. As high heregulin expression was detected in a subpopulation of HNSCC tumors, the correlation between heregulin expression and the clinical outcome of patients with HNSCC treated with cetuximab merits further investigation. Despite cetuximab exposure, cetuximab-resistant FaDuCR cells maintained Akt activation mediated by HER3 instead of EGFR. FaDuCR cells were more sensitive than parental FaDu cells to the Akt-specific inhibitor ipatasertib. Mutations in GSK2636771 PIK3CA can spontaneously induce the gene and its downstream Akt, causing cetuximab resistance28. Rabbit Polyclonal to ARHGEF11 PIK3CA mutations were detected in patients with CRC resistant to anti-EGFR antibody29. These results suggest that the Akt pathway plays a key role in cetuximab resistance. CRC frequently presents with Ras and Raf mutations. Consequently, the Ras/Raf/MAPK pathway contributes to anti-EGFR antibody resistance16. However, Ras/Raf mutations are uncommon in HNSCC. Therefore, HNSCC may acquire cetuximab resistance via aberrant Akt mediated by heregulin-dependent HER3 activation. Akt may also be a potential target for cancer therapy (and especially aberrant heregulin expression) in HNSCC. Unlike the anti-EGFR antibody cetuximab, the pan-HER family tyrosine kinase inhibitors afatinib and dacomitinib may prevent the proliferation of FaDuCR cells. This observation suggests that second-generation EGFR-TKIs could serve as an alternative therapy for patients with cetuximab-resistant and heregulin-expressing HNSCC. A previous study reported that heregulin causes HER3 to couple with HER2 in NSCLC harboring an EGFR-activating mutation. This mechanism induces resistance to first-generation EGFR-TKIs such as erlotinib but not to pan-HER family tyrosine kinase inhibitors such as afatinib25,26. In the present study, FaDuCR cells recovered the sensitivity to cetuximab in combination with anti-HER2 antibody pertuzumab. Therefore, the efficacy of pan-HER family tyrosine kinase inhibitor may be due to HER2 kinase inhibition. Heregulin evaluation by mRNA ISH could detect both cetuximab-resistant and optimal pan-HER family tyrosine kinase inhibitor subpopulations of HNSCC. Afatinib effectively inhibited heregulin expression in the present preclinical model. In contrast, a phase III clinical trial demonstrated significant but minimal improvement in PFS of patients with HNSCC compared GSK2636771 to that with methotrexate as a second-line treatment (HR?=?0.80; 95% CI?=?0.65C0.98)30. Subpopulations previously treated with anti-EGFR-targeted antibody were refractory to afatinib (HR?=?0.91; 95% CI?=?0.70C1.19). In the present study, we did not evaluate heregulin expression after acquired cetuximab resistance. However, 2 of the 28 GSK2636771 pre-cetuximab tumors presented aberrant heregulin expression resembling that in FaDuCR cells. The cetuximab resistance mechanism might be heterogeneous in HNSCC. Therefore, afatinib administration would have limited benefit in the treatment of heregulin-mediated, cetuximab-resistant tumors. The present study is based on a single cell line model and a limited number of clinical samples. To confirm the clinical relevance of the findings of the present study, we must analyze more samples from other HNSCC cohorts known GSK2636771 to have acquired resistance to cetuximab. However, HNSCC has limited treatment options compared to that of NSCLC. The molecular mechanisms of HNSCC have not been fully elucidated and their correlations with clinical outcome have not been established. Another report suggested that HNSCC preferentially expresses heregulin compared to other types of cancer31. In the present study, we found that.