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Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines.

ABSTRACT: Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC₅₀ compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

SUBMITTER: Gomes INF

PROVIDER: S-EPMC8750399 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Head and neck cancer is the sixth most common malignancy around the world, and 90% of cases are squamous cell carcinomas. In this study, we performed a systematic investigation of the immunogenomic landscape to identify prognostic biomarkers for head and neck squamous cell carcinoma (HNSCC). We analyzed the expression profiles of immune-related genes (IRGs) and clinical characteristics by interrogating RNA-seq data from 527 HNSCC patients in the cancer genome atlas (TCGA) dataset, including 41 HPV+ and 486 HPV- samples. We found that differentially expressed immune genes were closely associated with patient prognosis in HNSCC by comparing the differences in gene expression between cancer and normal samples and performing survival analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to annotate the biological functions of the differentially expressed immunogenomic prognosis-related genes. Two additional cohorts from the Oncomine database were used for validation. 65, 56 differentially expressed IRGs was associated with clinical prognosis in total and HPV- samples, respectively. Furthermore, we extracted 10, 11 prognosis-related IRGs from 65, 56 differentially expressed IRGs, respectively. They were significantly correlated with clinical prognosis and used to construct the prognosis prediction models. The multivariable ROC curves (specifically, the AUC) were used to measure the accuracy of the prognostic models. These genes were mainly enriched in several gene ontology (GO) terms related to immunocyte migration and receptor and ligand activity. KEGG pathway analysis revealed enrichment of pathways related to cytokine-cytokine receptor interactions, which are primarily involved in biological processes. In addition, we identified 63 differentially expressed transcription factors (TFs) from 4784 differentially expressed genes, and 16 edges involving 18 nodes were formed in the regulatory network between differentially expressed TFs and the high-risk survival-associated IRGs. B cell and CD4 T cell infiltration levels were significantly negatively correlated with the expression of prognosis-related immune genes regardless of HPV status. In conclusion, this comprehensive analysis identified the prognostic IRGs as potential biomarkers, and the model generated in this study may enable an accurate prediction of survival.

Project description:Head and neck squamous cell carcinomas (HNSCC) arising from the oral cavity, pharynx, and larynx constitute the 6th most common human cancer. Human papillomavirus (HPV)-positive tumours are distinct from HPV-negative counterparts, with HPV status affording clear clinical utility, prognostic benefit and better treatment outcomes. In contrast to their HPV-positive counterparts, HPV-negative tumours are characterized by high mutational load and chromosomal aberrations, with varying copy number alteration (CNA) profiles. HNSCC are distinct tumours at the chromosomal, gene and expression levels, with additional insight gained from immune profiling. Based on mutational analyses, HNSCC are categorized as HPV-positive, HPV-negative CNA-silent, and HPV-negative CNA-high tumours. Furthermore, gene expression profiling segregates these tumours into atypical, classical, basal, and mesenchymal, with clear differences observed between tumours of the oral cavity, oropharynx, hypopharynx and larynx. Additional immune profiling further classifies tumours as either immune-active or immune-exhausted. The clinical utility and impact of these tumour molecular subtypes however remains to be determined. HNSCC harbor high levels of somatic mutations. They display loss at 3p and 18q and gain at 3q and 8q, with mutations in CDKN2A, TP53, CCND1, EGFR, PIK3CA, PTEN, NOTCH1, NSD1, FAT1, AJUBA and KMT2D. Important pathways include the p53 and RB pathways which are involved in cell cycle control and are frequently lost in HPV-negative tumours, the WNT-β-catenin pathway related to the mesenchymal subtype and smoking etiology, and the PI3K pathway which includes the most common genetic alteration in HPV-positive HNSCC. Understanding the mutational, genomic and transcriptomic landscape of HNSCC has leveraged better therapeutic approaches to manage this group of diseases, and it is hoped that additional insight into the molecular subtypes of HNSCC and its specific subsites will further drive improved strategies to stratify and treat patients with this debilitating disease.

Dataset Information

Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines.

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