Neither cetuximab nor MM-121 alone significantly reduced the tumor growth compared to PBS control. HNSCC cell lines. Combined treatment with cetuximab and MM-121 blocked EGFR and HER3 activities and inhibited PI3K/AKT and ERK signaling pathways and HNSCC cell growth more effectively than each antibody alone. HER3 knockdown reduced HER2 activation and re-sensitized cells to cetuximab. Cetuximab-resistant xenografts and PDX models revealed greater efficacy of dual EGFR and HER3 inhibition compared to single antibodies. In Xantocillin PDX tissue samples, cetuximab induced HER3 expression and MM-121 reduced AKT activity. Conclusion Clinically relevant PDX models demonstrate that dual targeting of EGFR and HER3 is usually superior to EGFR targeting alone in HNSCC. Our study illustrates the upregulation of HER3 by cetuximab as one mechanism underlying resistance to EGFR inhibition in HNSCC, supporting further clinical investigations using multiple targeting strategies in patients who have failed cetuximab-based therapy. Keywords: HNSCC, EGFR, HER3, PDX, Cetuximab Introduction Targeting epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinoma (HNSCC) is an attractive and rational strategy given that more than 90% of these tumors overexpress EGFR (1, 2). Cetuximab, a chimerized antibody against EGFR, remains the only Food and Drug Administration (FDA)-approved targeted agent for HNSCC since its approval in 2006. The addition of cetuximab to platinum and fluorouracil treatment resulted in improved overall survival of patients with recurrent/metastatic HNSCC, and this combination has been adopted as the current standard of care for this populace (3). Despite this success, the overall response rate to cetuximab as a single agent does not exceed 13%, with a response duration of less than 2C3 months (4). Moreover, intrinsic and acquired resistance during EGFR therapy inevitably occurs (5, 6). Several mechanisms have been recognized through which resistance to EGFR-targeted brokers occurs in HNSCC. EGFR gene mutation and compensatory signaling from HER3 and other EGFR (ErbB) family members have been suggested to be associated with sensitivity to cetuximab therapy in HNSCC (7, 8). HER3 (ErbB3) is usually a member of the human EGFR family which consists of four type 1 transmembrane tyrosine kinase receptors: HER1 (EGFR, ErbB1), HER2 (Neu, ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Upregulation of HER3 Xantocillin is commonly observed in numerous malignancies including breast, colorectal carcinoma, HNSCC, gastric, ovarian, prostate, and bladder cancers and correlates with poorer survival (9C13). Upon binding of HRG1, the physiological HER3 receptor ligand, HER3 dimerizes with other ErbB family members, preferentially HER2. Dimerization results in transphosphorylation of HER3 on tyrosine residues contained within the cytoplasmic tail of the protein (14C16). Phosphorylation of these sites creates SH2 docking sites for SH2-made up of proteins, and specifically PI3-kinase (17). HER3 is usually a potent activator of AKT as it possesses six tyrosine phosphorylation sites with YXXM motifs that serve as excellent binding sites of the p85 regulatory subunit of PI3K, resulting in subsequent activation of the downstream AKT pathway (18, 19). These six PI3K sites serve as a strong amplifier of HER3 signaling. Activation of this pathway further elicits several important biological processes including cell growth and survival (20). Ligand-independent HER2/HER3 conversation has also been reported in HER2-amplified cells (21). Since HER3 can dimerize with EGFR, HER2 even c-Met, it likely plays a central role in response to EGFR-targeted therapy. Identifying biomarkers that can predict the clinical activity of HER3 and EGFR-targeted therapy will be crucial in understanding the mechanism of resistance to anti-EGFR therapy in HNSCC. This study aimed to elucidate the role of HER3 in cetuximab resistance in HNSCC, to investigate whether adding anti-HER3 treatment to cetuximab-based regimens can improve the treatment of HNSCC in models more relevant to the medical center, and to understand the underlying anti-tumor mechanisms of anti-EGFR/HER3 methods. For Xantocillin the purpose, we resolved the role of HER3 in cetuximab resistance in three settings: HNSCC cell lines, a xenograft mouse model using a cetuximab resistant HNSCC cell collection, and multiple patient derived xenograft (PDX) mouse models using tissues from HNSCC patients. Materials PIK3R5 and Methods Cell lines and reagents Cetuximab was obtained from ImClone (New York, NY) and MM-121/SAR256212 was provided by Merrimack Pharmaceuticals (Cambridge, MA) and Sanofi (Bridgewater, NJ). Human HNSCC cell collection UMSCC1-P and its cetuximab resistant counterpart UMSCC1-C were provided by Dr. Paul Harari (University or college of Wisconsin School of Medicine and Public Health, Madison, WI). The genotypes of the two cell lines were confirmed using the STR method by the Emory Integrated Genomics Core. The genotype of these two cell lines is usually identical to the original UMSCC1 cell collection published previously by Zhao (22). All cell lines were managed in Dulbecco’s Modified Eagle’s Media (DMEM) with 5% FBS and 0.4 g/mL hydrocortisone at 37C, 10% CO2 (23). Colony formation assay Cells were.