Establishment of head and neck squamous cell carcinoma mouse models for cetuximab resistance and sensitivity.

Aim: Acquired resistance to the targeted agent cetuximab poses a significant challenge in finding effective anti-cancer treatments for head and neck squamous cell carcinoma (HNSCC). To accurately study novel combination treatments, suitable preclinical mouse models for cetuximab resistance are key yet currently limited. This study aimed to optimize an acquired cetuximab-resistant mouse model, with preservation of the innate immunity, ensuring intact antibody-dependent cellular cytotoxicity (ADCC) functionality. Methods: Cetuximab-sensitive and acquired-resistant HNSCC cell lines, generated in vitro, were subcutaneously engrafted in Rag2 knock-out (KO), BALB/c Nude and CB17 Scid mice with/without Matrigel or Geltrex. Once tumor growth was established, mice were intraperitoneally injected twice a week with cetuximab for a maximum of 3 weeks. In addition, immunohistochemistry was used to evaluate the tumor and its microenvironment. Results: Despite several adjustments in cell number, cell lines and the addition of Matrigel, Rag2 KO and BALB/C Nude mice proved to be unsuitable for xenografting our HNSCC cell lines. Durable tumor growth of resistant SC263-R cells could be induced in CB17 Scid mice. However, these cells had lost their resistance phenotype in vivo. Immunohistochemistry revealed a high infiltration of macrophages in cetuximab-treated SC263-R tumors. FaDu-S and FaDu-R cells successfully engrafted into CB17 Scid mice and maintained their sensitivity/resistance to cetuximab. Conclusion: We have established in vivo HNSCC mouse models with intact ADCC functionality for cetuximab resistance and sensitivity using the FaDu-R and FaDu-S cell lines, respectively. These models serve as valuable tools for investigating cetuximab resistance mechanisms and exploring novel drug combination strategies.

Co-Targeting the EGFR and PI3K/Akt Pathway to Overcome Therapeutic Resistance in Head and Neck Squamous Cell Carcinoma: What about Autophagy?

Resistance to EGFR-targeted therapy is a major obstacle on the road to effective treatment options for head and neck cancers. During the search for underlying mechanisms and regulators of this resistance, there were several indications that EGFR-targeted therapy resistance is (partially) mediated by aberrant signaling of the PI3K/Akt pathway. Genomic alterations in and/or overexpression of major components of the PI3K/Akt pathway are common in HNSCC tumors. Therefore, downstream effectors of the PI3K/Akt pathway serve as promising targets in the search for novel therapeutic strategies overcoming resistance to EGFR inhibitors. As both the EGFR/Ras/Raf/MAPK and the PI3K/Akt pathway are involved in autophagy, combinations of EGFR and PI3K/Akt pathway inhibitors can induce an autophagic response in tumor cells. This activation of autophagy can be seen as a "double-edge sword", depending on the cellular context. Autophagy is largely known as a cytoprotective mechanism, but it can also be a mechanism of programmed (autophagic) cell death. The activation of autophagy during anti-cancer treatment is, therefore, not necessarily a bad sign. However, in HNSCC, the role of therapy-induced autophagy as an anti-tumor mechanism is still largely unclear. Further research is warranted to understand the potential of combination treatments targeting both the EGFR and PI3K/Akt pathway.

The prognostic impact of the immune signature in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of tumors that retain their poor prognosis despite recent advances in their standard of care. As the involvement of the immune system against HNSCC development is well-recognized, characterization of the immune signature and the complex interplay between HNSCC and the immune system could lead to the identification of novel therapeutic targets that are required now more than ever. In this study, we investigated RNA sequencing data of 530 HNSCC patients from The Cancer Genome Atlas (TCGA) for which the immune composition (CIBERSORT) was defined by the relative fractions of 10 immune-cell types and expression data of 45 immune checkpoint ligands were quantified. This initial investigation was followed by immunohistochemical (IHC) staining for a curated selection of immune cell types and checkpoint ligands markers in tissue samples of 50 advanced stage HNSCC patients. The outcome of both analyses was correlated with clinicopathological parameters and patient overall survival. Our results indicated that HNSCC tumors are in close contact with both cytotoxic and immunosuppressive immune cells. TCGA data showed prognostic relevance of dendritic cells, M2 macrophages and neutrophils, while IHC analysis associated T cells and natural killer cells with better/worse prognostic outcome. HNSCC tumors in our TCGA cohort showed differential RNA over- and underexpression of 28 immune inhibitory and activating checkpoint ligands compared to healthy tissue. Of these, CD73, CD276 and CD155 gene expression were negative prognostic factors, while CD40L, CEACAM1 and Gal-9 expression were associated with significantly better outcomes. Our IHC analyses confirmed the relevance of CD155 and CD276 protein expression, and in addition PD-L1 expression, as independent negative prognostic factors, while HLA-E overexpression was associated with better outcomes. Lastly, the co-presence of both (i) CD155 positive cells with intratumoral NK cells; and (ii) PD-L1 expression with regulatory T cell infiltration may hold prognostic value for these cohorts. Based on our data, we propose that CD155 and CD276 are promising novel targets for HNSCC, possibly in combination with the current standard of care or novel immunotherapies to come.

Microenvironment-driven intratumoral heterogeneity in head and neck cancers: clinical challenges and opportunities for precision medicine.

Squamous cell carcinoma of the head and neck (SCCHN) is among the most prevalent cancer types worldwide. Despite multimodal therapeutic approaches that include surgical resection, radiation therapy or concurrent chemoradiation, targeted therapy and immunotherapy, SCCHN is still associated with a poor prognosis for patients with locally advanced or recurrent/metastatic (R/M) diseases. Although next-generation sequencing data from thousands of SCCHN patients have provided a comprehensive landscape of the somatic genomic alterations in this disease, genomic-based precision medicine is not implemented yet in routine clinical use since no satisfactory genetic biomarker has been identified for diagnosis, patient outcome prediction and selection of tailored therapeutic options. The lack of significant improvement in SCCHN patient survival over the last decades stresses the need for reliable predictive biomarkers and new therapeutic strategies for personalized clinical management of SCCHN patients. Targeting the SCCHN-associated microenvironment or the interaction of the latter with cancer cells may represent such paradigm shift in the development of new strategies to treat SCCHN patients, as exemplified by the recent implementation of immune checkpoint inhibitors to improve clinical outcomes by increasing anti-tumor immune responses in SCCHN patients. Several clinical trials are in progress in SCCHN patients to evaluate the activity of monoclonal antibodies and small-molecule inhibitors targeting the tumor microenvironment (TME) at different treatment settings, including combinations with adjuvant surgery, radiation therapy and chemotherapy. This review describes the current knowledge about the influence of the TME on intratumoral heterogeneity and clinical relapse in human SCCHN patients. More precisely, the role of hypoxia as well as the presence of non-cancer cells (e.g. cancer-associated fibroblasts and immune cells) on therapy response of SCCHN cells is highlighted. We also discuss relevant (pre)clinical models that may help integrate the microenvironment-tumor cell interplay in translational research studies for SCCHN. Finally, this review explores potential therapeutic strategies that may exploit the crosstalk between TME and SCCHN cells in order to implement fundamental changes in the tumor treatment paradigm of patients with locally advanced or R/M SCCHN.

Recent insights in the PI3K/Akt pathway as a promising therapeutic target in combination with EGFR-targeting agents to treat head and neck squamous cell carcinoma.

Resistance to therapies targeting the epidermal growth factor receptor (EGFR), such as cetuximab, remains a major roadblock in the search for effective therapeutic strategies in head and neck squamous cell carcinoma (HNSCC). Due to its close interaction with the EGFR pathway, redundant or compensatory activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been proposed as a major driver of resistance to EGFR inhibitors. Understanding the role of each of the main proteins involved in this pathway is utterly important to develop rational combination strategies able to circumvent resistance. Therefore, the current work reviewed the role of PI3K/Akt pathway proteins, including Ras, PI3K, tumor suppressor phosphatase and tensing homolog, Akt and mammalian target of rapamycin in resistance to anti-EGFR treatment in HNSCC. In addition, we summarize PI3K/Akt pathway inhibitors that are currently under (pre)clinical investigation with focus on overcoming resistance to EGFR inhibitors. In conclusion, genomic alterations in and/or overexpression of one or more of these proteins are common in both human papillomavirus (HPV)-positive and HPV-negative HNSCC tumors. Therefore, downstream effectors of the PI3K/Akt pathway serve as promising drug targets in the search for novel therapeutic strategies that are able to overcome resistance to anti-EGFR treatment. Co-targeting EGFR and the PI3K/Akt pathway can lead to synergistic drug interactions, possibly restoring sensitivity to EGFR inhibitors and hereby improving clinical efficacy. Better understanding of the predictive value of PI3K/Akt pathway alterations is needed to allow the identification of patient populations that might benefit most from these combination strategies.

The Role of Akt in Acquired Cetuximab Resistant Head and Neck Squamous Cell Carcinoma: An In Vitro Study on a Novel Combination Strategy.

The epidermal growth factor receptor (EGFR) is a therapeutic target in head and neck squamous cell carcinoma (HNSCC). Resistance to EGFR-targeted therapies, such as cetuximab, poses a challenging problem. This study aims to characterize acquired cetuximab resistance mechanisms in HNSCC cell lines by protein phosphorylation profiling. Through this, promising combination treatments can be identified to possibly overcome acquired cetuximab resistance in HNSCC. Protein phosphorylation profiling showed increased phosphorylation of Akt1/2/3 after cetuximab treatment in acquired cetuximab resistant cells compared to cetuximab sensitive cells, which was confirmed by western blotting. Based on this protein phosphorylation profile, a novel combination treatment with cetuximab and the Akt1/2/3 inhibitor MK2206 was designed. Synergy between cetuximab and MK2206 was observed in two cetuximab sensitive HNSCC cell lines and one acquired cetuximab resistant variant in simultaneous treatment schedules. In conclusion, this study demonstrates that increased Akt1/2/3 phosphorylation seems to be characteristic for acquired cetuximab resistance in HNSCC cell lines. Our results also show an additive to synergistic interaction between cetuximab and MK2206 in simultaneous treatment schedules. These data support the hypothesis that the combination of cetuximab with PI3K/Akt pathway inhibition might be a promising novel therapeutic strategy to overcome acquired cetuximab resistance in HNSCC patients.

The Right Partner in Crime: Unlocking the Potential of the Anti-EGFR Antibody Cetuximab via Combination With Natural Killer Cell Chartering Immunotherapeutic Strategies.

Cetuximab has an established role in the treatment of patients with recurrent/metastatic colorectal cancer and head and neck squamous cell cancer (HNSCC). However, the long-term effectiveness of cetuximab has been limited by the development of acquired resistance, leading to tumor relapse. By contrast, immunotherapies can elicit long-term tumor regression, but the overall response rates are much more limited. In addition to epidermal growth factor (EGFR) inhibition, cetuximab can activate natural killer (NK) cells to induce antibody-dependent cellular cytotoxicity (ADCC). In view of the above, there is an unmet need for the majority of patients that are treated with both monotherapy cetuximab and immunotherapy. Accumulated evidence from (pre-)clinical studies suggests that targeted therapies can have synergistic antitumor effects through combination with immunotherapy. However, further optimizations, aimed towards illuminating the multifaceted interplay, are required to avoid toxicity and to achieve better therapeutic effectiveness. The current review summarizes existing (pre-)clinical evidence to provide a rationale supporting the use of combined cetuximab and immunotherapy approaches in patients with different types of cancer.

Cetuximab-induced natural killer cell cytotoxicity in head and neck squamous cell carcinoma cell lines: investigation of the role of cetuximab sensitivity and HPV status.

BACKGROUND: The epidermal growth factor receptor (EGFR) is overexpressed by 80-90% of squamous cell carcinoma of head and neck (HNSCC). In addition to inhibiting EGFR signal transduction, cetuximab, a monoclonal antibody targeting EGFR can also bind to fragment crystallisable domain of immunoglobulins G1 present on natural killer (NK), causing antibody-dependent cellular cytotoxicity (ADCC). However, presence of cetuximab resistance limits effective clinical management of HNSCC. METHODS: In this study, differences in induction of ADCC were investigated in a panel of ten HNSCC cell lines. Tumour cells were co-cultured with NK cells and monitored using the xCELLigence RTCA. RESULTS: While ADCC was not influenced by HPV status, hypoxia and cetuximab resistance did affect ADCC differentially. Intrinsic cetuximab-resistant cell lines showed an increased ADCC induction, whereas exposure to hypoxia reduced ADCC. Baseline EGFR expression was not correlated with ADCC. In contrast, EGFR internalisation following cetuximab treatment was positively correlated with ADCC. CONCLUSION: These findings support the possibility that resistance against cetuximab can be overcome by NK cell-based immune reactions. As such, it provides an incentive to combine cetuximab with immunotherapeutic approaches, thereby possibly enhancing the anti-tumoural immune responses and achieving greater clinical effectiveness of EGFR-targeting agents.