Treatment Of Head And Neck Squamous Cell Carcinoma Research Articles

Black raspberry extract inhibits regulatory T-cell activity in a murine model of head and neck squamous cell carcinoma chemoprevention.

Head and neck squamous cell carcinomas (HNSCC) are one of the most diagnosed malignancies globally, with a 5-year survival rate of approximately 40% to 50%. Current therapies are limited to highly invasive surgery, aggressive radiation, and chemotherapies. Recent reports have demonstrated the potential phytochemical properties of black raspberries in inhibiting the progression of various cancers including HNSCCs. However, the effects of black raspberry extracts on immune cells of the tumor microenvironment, specifically regulatory T cells during HNSCC, have not been investigated. We used a mouse model of 4-nitroquinoline-1-oxide (4NQO) chemically induced HNSCC carcinogenesis to determine these effects. C57BL/6 mice were exposed to 4NQO for 16 weeks and regular water for 8 weeks. 4NQO-exposed mice were fed the AIN-76A control mouse diet or the AIN76 diet supplemented with black raspberry extract. At terminal sacrifice, tumor burdens and immune cell recruitment and activity were analyzed in the tumor microenvironment, draining lymph nodes, and spleens. Mice fed the BRB extract-supplemented diet displayed decreased tumor burden compared to mice provided the AIN-76A control diet. Black raspberry extract administration did not affect overall T-cell populations as well as Th1, Th2, or Th17 differentiation in spleens and tumor draining lymph nodes. However, dietary black raspberry extract administration inhibited regulatory T-cell recruitment to HNSCC tumor sites. This was associated with an increased cytotoxic immune response in the tumor microenvironment characterized by increased CD8+ T cells and enhanced Granzyme B production during BRB extract-mediated HNSCC chemoprevention. Interestingly, this enhanced CD8+ T-cell antitumoral response was localized at the tumor sites but not at spleens and draining lymph nodes. Furthermore, we found decreased levels of PD-L1 expression by myeloid populations in draining lymph nodes of black raspberry-administered carcinogen-induced mice. Taken together, our findings demonstrate that black raspberry extract inhibits regulatory T-cell recruitment and promotes cytotoxic CD8 T-cell activity at tumor sites during HNSCC chemoprevention. These results demonstrate the immunomodulatory potential of black raspberry extracts and support the use of black raspberry-derived phytochemicals as a complementary approach to HNSCC chemoprevention and treatment.

INVESTIGATION OF CIRCULAR RNAs AS BIOMARKERS IN HEAD AND NECK CANCER CELLS AND THE EFFECTS OF RADIOTHERAPY ON CIRCULAR RNAs

Objective: The head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide and the 5-year survival rate of patients is by 40-50 %. Radiotherapy is one of the important treatments for HNSCC. circRNAs, which are much more stabil than other RNAs because of their circular structure, have the potential to be a biomarker for all types of cancer. Therefore, the alterations of circRNAs expression in HNSCC cells and after irradiation was investigated. Methods: Pharyngeal cancer (Detroit-562) and healthy fibroblast (WI-38) cell lines were used. After appropriate irradiation dose for Detroit-562 cells was determined by colony formation assay, Detroit-562 cells were irradiated with 4 grays. The expression levels of 122 circRNA molecules in all groups were examined by RT-qPCR. The results were evaluated using CircNet and TCGA platforms. Results: In this study, 60 circRNAs were not expressed in any cell line, whereas 62 were found to be expressed in at least one of the groups, including irradiated Detroit-562, Detroit-562, and WI-38 cell lines. Among them, seven circRNAs were detected in all groups and expressed higher in Detroit-562 than two groups. Interestingly, these circRNAs were significantly decreased in irradiated group, exhibiting similar expression levels with healthy group. Bioinformatics analyses showed that the host genes of these circRNAs was also higher expressed in HNSCC tissue. Conclusions: Differential expression of circRNAs in cancers and changes with treatment revealed that they could be potential diagnostic and monitoring biomarkers in cancer. However, further studies need in this regard.

A novel necroptosis-related LncRNA signature for prediction of prognosis and therapeutic responses of head and neck squamous cell carcinoma.

Background: Long non-coding RNAs (lncRNAs) play an essential role in the occurrence and prognosis of tumors, and it has great potential as biomarkers of tumors. However, the roles of Necroptosis-related lncRNA (NRLs) in Head and neck squamous cell carcinoma (HNSCC) remain elusive. Methods: We comprehensively analyzed the gene expression and clinical information of 964 HNSCC in four cohorts. LASSO regression was utilized to construct a necroptosis-related lncRNA prognosis signature (NLPS). We used univariate and multivariate regression to assess the independent prognostic value of NLPS. Based on the optimal cut-off, patients were divided into high- and low-risk groups. In addition, the immune profile, multi-omics alteration, and pharmacological landscape of NLPS were further revealed. Results: A total of 21 NRLs associated with survival were identified by univariate regression in four cohorts. We constructed and validated a best prognostic model (NLPS). Compared to the low-risk group, patients in the high group demonstrated a more dismal prognosis. After adjusting for clinical features by multivariate analysis, NLPS still displayed independent prognostic value. Additionally, further analysis found that patients in the low-risk group showed more abundant immune cell infiltration and immunotherapy response. In contrast, patients in the high-risk group were more sensitive to multiple chemotherapeutic agents. Conclusion: As a promising tool, the establishment of NLPS provides guidance and assistance in the clinical management and personalized treatment of HNSCC.

Immunotherapeutic Strategies for Head and Neck Squamous Cell Carcinoma (HNSCC): Current Perspectives and Future Prospects.

Neoantigens are abnormal proteins produced by genetic mutations in somatic cells. Because tumour neoantigens are expressed only in tumour cells and have immunogenicity, they may represent specific targets for precision immunotherapy. With the reduction in sequencing cost, continuous advances in artificial intelligence technology and an increased understanding of tumour immunity, neoantigen vaccines and adoptive cell therapy (ACT) targeting neoantigens have become research hotspots. Approximately 900,000 patients worldwide are diagnosed with head and neck squamous cell carcinoma (HNSCC) each year. Due to its high mutagenicity and abundant lymphocyte infiltration, HNSCC naturally generates a variety of potential new antigen targets that may be used for HNSCC immunotherapies. Currently, the main immunotherapy for HNSCC is use of immune checkpoint inhibitors(ICIs). Neoantigen vaccines and adoptive cell therapy targeting neoantigens are extensions of immunotherapy for HNSCC, and a large number of early clinical trials are underway in combination with immune checkpoint inhibitors for the treatment of recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC). In this paper, we review recent neoantigen vaccine trials related to the treatment of HNSCC, introduce adoptive cell therapy targeting neoantigens, and propose a potential treatment for HNSCC. The clinical application of immune checkpoint inhibitor therapy and its combination with neoantigen vaccines in the treatment of HNSCC are summarized, and the prospect of using neoantigen to treat HNSCC is discussed and proposed.

An Investigation on Radiomics Feature Handling for HNSCC Staging Classification

The incidence of Head and Neck Squamous Cell Carcinoma (HNSCC) has been growing in the last few decades. Its diagnosis is usually performed through clinical evaluation and analyzing radiological images, then confirmed by histopathological examination, an invasive and time-consuming operation. The recent advances in the artificial intelligence field are leading to interesting results in the early diagnosis, personalized treatment and monitoring of HNSCC only by analyzing radiological images, without performing a tissue biopsy. The large amount of radiological images and the increasing interest in radiomics approaches can help to develop machine learning (ML) methods to support diagnosis. In this work, we propose an ML method based on the use of radiomics features, extracted from CT and PET images, to classify the disease in terms of pN-Stage, pT-Stage and Overall Stage. After the extraction of radiomics features, a selection step is performed to remove dataset redundancy. Finally, ML methods are employed to complete the classification task. Our pipeline is applied on the “Head-Neck-PET-CT” TCIA open-source dataset, considering a cohort of 201 patients from four different institutions. An AUC of 97%, 83% and 93% in terms of pN-Stage, pT-Stage and Overall Stage classification, respectively, is achieved. The obtained results are promising, showing the potential efficiency of the use of radiomics approaches in staging classification.

Identification of potent and novel inhibitors against RAC1: a Rho family GTPase.

Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most common form of cancer worldwide. It has high incidence and mortality rate making it one of the top causes of cancer related deaths. Tremendous efforts have being made towards treatment of HNSCC but still the overall survival rate hasn't improved much. Unregulated activation of Rho GTPase Ras-related C3 botulinum toxin substrate 1 or Rac1 has been reported in various tumor such as HNSCC, breast cancer, pancreatic cancer, etc. Rac1 is significant in activation and regulation of multiple signaling pathways and it's aberrant activation leads to uncontrolled proliferation, invasion and metastasis which contributes to the hallmarks of cancer. Therefore for treating proliferative disorders such as cancer, inhibition of Rac1 could be a viable approach. Rho GTPases were earlier considered "undruggable" due to their picomolar binding affinity for their guanine nucleotides. In addition presence of high micromolar concentrations of GDP (> 30μm) and GTP (> 300μm) in the cell, led to unsuccessful attempts in identification of potent or selective nucleotide competitive GTPase inhibitors. Therefore we identified small molecule inhibitors that target the GEF binding site of the Rho GTPase instead of nucleotide binding site by performing high throughput screening, molecular dynamics simulations, free energy calculations and protein-ligand interaction studies. As a result of this study, we identified four potential inhibitors against RAC1. This study provides a significant in-depth understanding of the Rho GTPases and can prove beneficial in the development of potential therapeutics against HNSCC.

E2F4 transcription factor is a prognostic biomarker related to immune infiltration of head and neck squamous cell carcinoma

To investigate the relationship between the transcription factor, E2F4, and head and neck squamous cell carcinoma (HNSCC), and to preliminarily explore the signaling pathways and immunological role of E2F4. The mRNA expression of E2F4 in HNSCC was evaluated by searching Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets. E2F4 protein expression was analyzed by immunohistochemistry using the CMU1h-ENT database. The association between E2F4 expression and tumor infiltration of immune cells was analyzed. Intracellular signaling by E2F4 was explored using KEGG and GO analysis. The correlation of E2F4 expression with clinical characteristics and its prognostic role were validated and analyzed in TCGA database. From the analysis of GEO and TCGA data, E2F4 expression was found to be up-regulated in HNSCC tumor tissues, and its level was associated with T, Grade, and M staging. Kaplan–Meier curve and Cox analyses indicated that the high expression of E2F4 was related to a poor prognosis. Thus, E2F4 was considered a potential prognostic factor for HNSCC. Immunohistochemical staining showed that E2F4 was mainly localized in the cell nucleus; it was highly expressed in HNSCC tissues, with a significant difference noted from that in pericancerous mucosa tissues. A correlation was observed between the differential expression of E2F4 and the immune infiltration of HNSCC. As revealed by KEGG and GO analysis, differential enrichment was found in the cell cycle, spliceosome, meiosis, microbial polysaccharide synthesis, and WNT signaling pathway, as well as in cyclic adenosine monophosphate, ERBB2, VEGF, GCNP and MYC pathways. E2F4 plays an important role in tumor progression and may be a critical biological prognostic factor for HNSCC. In addition, it functions in the nucleus as a transcription factor, regulates immune cells, and could be a promising molecular target for the diagnosis and treatment of HNSCC.

A Glycosyltransferase-Related Signature for Predicting Overall Survival in Head and Neck Squamous Cell Carcinoma.

Background: Here, we establish a prognostic signature based on glycosyltransferase-related genes (GTRGs) for head and neck squamous cell carcinoma (HNSCC) patients. Methods: The prognostic signature of GTRGs was constructed via univariate and multivariate Cox analyses after obtaining the expression patterns of GTRGs from the TCGA. A nomogram based on the signature and clinical parameters was established to predict the survival of each HNSCC patient. Potential mechanisms were explored through gene set enrichment analysis (GSEA) and immune cell infiltration, immune checkpoints, immunotherapy, and tumor mutational burden (TMB) analyses. The expression differences and prognostic efficacy of the signature were verified through the gene expression omnibus (GEO) and several online databases. Results: The prognostic signature was constructed based on five glycosyltransferases (PYGL, ALG3, EXT2, FUT2, and KDELC1) and validated in the GSE65858 dataset. The pathways enriched in the high- and low-risk groups were significantly different. The high-risk group had higher tumor purity; lower infiltration of immune cells, such as CD8+ T cells and Tregs; higher cancer-associated fibroblast (CAF) infiltration; lower immune function; and lower checkpoint expression. The signature can also be applied to distinguish whether patients benefit from immunotherapy. In addition, the high-risk group had a higher TMB and more gene mutations, including those in TP53, CSMD1, CDKN2A, and MUC17. Conclusion: We propose a prognostic signature based on glycosyltransferases for HNSCC patients that may provide potential targets and biomarkers for the precise treatment of HNSCC.

Fusobacterium nucleatum impairs DNA mismatch repair and stability in patients with squamous cell carcinoma of the head and neck.

Dysbiosis of the laryngeal microbiota has been demonstrated to the development of head and neck squamous cell carcinoma (HNSCC), but the association of Fusobacterium and Fusobacterium nucleatum (F. nucleatum) with DNA mismatch repair (MMR) and microsatellite instability (MSI) has not been investigated. The abundance of Fusobacterium and F. nucleatum, the status of deficient MMR (dMMR) and MSI, and MMR-related gene expression were analyzed in 171 HNSCC tissues, 61 paired para-tumor tissues, and 60 vocal cord polyp tissues. The molecular mechanism of F. nucleatum and MMR-related gene expression were investigated in two human HNSCC cell lines (Tu 686 and FD-LSC-1). Our results demonstrated that a high Fusobacterium abundance was detected in the HNSCC tissues and was exaggerated in the recurrent patients. We further found that a high Fusobacterium abundance was detected in the HNSCC tissues with dMMR and MSI. The Fusobacterium abundance was negatively correlated with the expression of MLH1, MSH2, and MSH6 in the HNSCC tissues. The Fusobacterium abundance was closely associated with the F. nucleatum abundance in the HNSCC tissues. F. nucleatum increased miR-205-5p expression to suppress MLH1, MSH2, and MSH6 expression via the TLR4- and MYD88-dependent innate immune signaling pathway, resulting in dMMR, DNA damage, and cell proliferation in HNSCC. F. nucleatum impacts HNSCC epigenetic changes in tissues with dMMR to promote DNA damage and cell proliferation by suppressing MMR-related gene expression via the TLR4/MYD88/miR-205-5p signaling pathway, which is valuable in the development of efficient strategies for HNSCC prevention and treatment. This study clearly indicates that Fusobacterium induced head and neck squamous cell carcinoma (HNSCC) aggressiveness to affect poor prognosis in HNSCC patients by epigenetic alteration of DNA mismatch repair (MMR) and microsatellite instability. Moreover, the research has shown that Fusobacterium nucleatum ( F. nucleatum ) impacts HNSCC epigenetic changes in tissues with deficient MMR to promote DNA damage and cell proliferation by suppressing MMRrelated gene expression via the TLR4/MYD88/miR-205-5p signaling pathway.

Target Definition in MR-Guided Adaptive Radiotherapy for Head and Neck Cancer.

Simple SummaryAdaptive radiotherapy for head and neck cancer has become more routine due to an increase in imaging quality and improvement in radiation techniques. With the availability of faster adaptive workflows, it is possible to adapt more easily to (daily) changes. MRI offers besides great anatomical imaging, also functional information about the tumor and surrounding tissue. The aim of this review is to provide current state of evidence about target definition on MRI for adaptive strategies in the treatment of head and neck cancer.In recent years, MRI-guided radiotherapy (MRgRT) has taken an increasingly important position in image-guided radiotherapy (IGRT). Magnetic resonance imaging (MRI) offers superior soft tissue contrast in anatomical imaging compared to computed tomography (CT), but also provides functional and dynamic information with selected sequences. Due to these benefits, in current clinical practice, MRI is already used for target delineation and response assessment in patients with head and neck squamous cell carcinoma (HNSCC). Because of the close proximity of target areas and radiosensitive organs at risk (OARs) during HNSCC treatment, MRgRT could provide a more accurate treatment in which OARs receive less radiation dose. With the introduction of several new radiotherapy techniques (i.e., adaptive MRgRT, proton therapy, adaptive cone beam computed tomography (CBCT) RT, (daily) adaptive radiotherapy ensures radiation dose is accurately delivered to the target areas. With the integration of a daily adaptive workflow, interfraction changes have become visible, which allows regular and fast adaptation of target areas. In proton therapy, adaptation is even more important in order to obtain high quality dosimetry, due to its susceptibility for density differences in relation to the range uncertainty of the protons. The question is which adaptations during radiotherapy treatment are oncology safe and at the same time provide better sparing of OARs. For an optimal use of all these new tools there is an urgent need for an update of the target definitions in case of adaptive treatment for HNSCC. This review will provide current state of evidence regarding adaptive target definition using MR during radiotherapy for HNSCC. Additionally, future perspectives for adaptive MR-guided radiotherapy will be discussed.

Abstract 6062: Radiosensitizing head and neck squamous cell carcinoma using isothiocyanates

Abstract Head and neck cancer kills over 400,000 people each year with the majority of cases being head and neck squamous cell carcinoma (HNSCC). Despite major advances in cancer treatment, radiation resistance contributes to increased morbidity and mortality of patients with HNSCC. Few FDA-approved radiosensitizers exist for the treatment of HNSCC and the majority are associated with severe adverse effects. Therefore, there is a clear need to examine novel treatment combinations to improve radiosensitivity and HNSCC outcomes. Isothiocyanates enhance response to cytokines, chemotherapy, and radiation in other in vitro and in vivo cancer models, including pancreatic and breast cancer. Isothiocyanates are antioxidants found in cruciferous vegetables such as watercress and broccoli. Phenethyl isothiocyanate (PEITC) was well tolerated by patients in chemopreventative clinical trials and was thus selected for this study. We hypothesize that PEITC radiosensitizes HNSCC cells through increasing DNA damage, apoptosis and reactive oxygen species (ROS). Non-human papilloma virus or P16 negative HNSCC are associated with increased radioresistance and as such human HNSCC cell lines HN5 and CAL27 were selected. Combination of PEITC and photon radiation (XRT) decreased cell survival by half as compared to cells treated with PEITC or XRT alone for both HN5 and CAL27 cells as determined by clonogenic assay (n≥3). PEITC enhanced XRT-induced apoptosis in HN5 and CAL27 cells as indicated by increased cleavage of effector caspases 3 and 7, which was reversed upon treatment with pan-caspase inhibitor, QVD-OPH. Preliminary comet assays identified that combination of PEITC with XRT significantly increased Tail DNA % relative to vehicle (p<0.001), PEITC alone (p<0.0001), or XRT alone (p<0.0001) in HN5 cells. Preliminary data in CAL27 cells identified significantly increased signal of y-H2Ax in cells treated with both PEITC and XRT was 2.2 fold higher than XRT alone (p<0.001) and 12.1 fold higher than PEITC alone (p<0.001) in CAL27 cells. PEITC increased XRT-induced ROS generation in CAL27 cells relative to independent treatment as determined by 2′,7′ dichlorodihydrofluorescein diacetate (DCFH-DA) staining. Taken together, these results support that PEITC synergistically enhances HNSCC cell response to XRT. The results of this study warrant further investigation into the potential of PEITC to improve radiation efficacy in pre-clinical HNSCC models with the long-term goal of testing this well-tolerated compound in clinical trials. Citation Format: Christina Ann Wicker, Samuel Thompson, Poornima Dubey, Vinita Takiar. Radiosensitizing head and neck squamous cell carcinoma using isothiocyanates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6062.

Abstract 3315: CRISPR-Cas9 screening reveals novel targets that govern HNSCC response to radiation therapy

Abstract Purpose: Although Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide, the high failure rates for non-HPV associated tumors and the absence of new effective therapies has caused the survival rate for HNSCC to remain stagnant for the past decade. As radiation is a frontline treatment for HNSCC, we sought to uncover novel actionable targets that affect cellular responses to radiation therapy. Experimental Procedures: We performed a kinome-wide CRISPR-Cas9 knockout screen in UM-SCC47, FaDu, Cal27, and Detroit562 HNSCC cell lines using a multi-fraction radiation regimen as a selective pressure. These four cell lines were chosen to enhance molecular diversity as they represent the classical, atypical, basal, and mesenchymal transcriptional subtypes of HNSCC. sgRNAs from irradiated and unirradiated controls underwent Next Generation Sequencing, and the results were analyzed via MAGeCK analysis. This approach provided a systematic method to identify sgRNAs that were enriched (loss of gene causes radioresistance) or depleted (loss of gene causes radiosensitivity) following HNSCC cell radiation treatment. Results: Our results indicate that Cal27 and Detroit562 cell lines share multiple targets that govern cell responses to radiation despite the different mutational backgrounds and transcriptional subtypes. In particular, sgRNAs targeting ATM and PRKDC were significantly depleted in Cal27 and Detroit562 cells (FDR<0.01, p<0.01). ATM and PRKDC are regulators of the DNA damage response, and loss of function of these genes is known to radiosensitize cells. Therefore, depletion of sgRNAs targeting these genes serves as internal validation of our screening methodology. sgRNAs targeting JAK1 were enriched in the Cal27 and Detroit562 cell lines (FDR<0.01, p<0.01), suggesting that loss of JAK1 caused decreased radiation-induced cell death. As a role for JAK1 in the cell response to DNA damage has not been described, we sought to validate and characterize the role of JAK1 in HNSCC cell radiation treatment using JAK1 knockout (KO) cell lines. Clonogenic assays revealed that JAK1 KO cells were radioresistant when treated with both single and multi-fraction radiation regimens, validating the results of the CRISPR screen. We observed cell signaling changes consistent with loss of JAK1 including decreased phospho-STAT3 and IRF9 signaling. To uncover pathways that may contribute to JAK1 KO cell radioresistance, we performed bulk RNA sequencing of JAK1 KO and control cell lines. Our analysis revealed that JAK1 KO cells may undergo an epithelial-mesenchymal transition among other signaling changes. Conclusion: The use of a CRISPR-Cas9 screen with radiation as a selective pressure revealed both known and novel targets that govern HNSCC cell line responses to radiation therapy. Loss of JAK1 causes radioresistance, and its role in HNSCC DNA damage response warrants further investigation. Citation Format: Vanessa M. Kelley, Marta Baro, Aanchal Katoch, Chatchai Phoomak, Hojin Lee, Joseph Contessa. CRISPR-Cas9 screening reveals novel targets that govern HNSCC response to radiation therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3315.

Abstract 88: ATAD3A enhances head and neck cancer malignancy via mitochondrial ERK1/2 signaling

Abstract Targeting mitochondria as a cancer therapeutic strategy has attracted more and more attention in the recent years. ATAD3A is identified as a nuclear-encoded mitochondrial enzyme that regulates mitochondrial dynamics, cholesterol metabolism, and signal transduction. However, its impact and underlying regulatory mechanisms in cancers remain to be determined. We report here that ATAD3A is highly expressed in head and neck squamous cell carcinoma (HNSCC), where it plays a critical role in favoring tumor progression. Loss of ATAD3A expression dramatically incudes tumor regression in orthotopic tongue tumor-bearing mice, whereas gain of ATAD3A expression produces the opposite effects. Mechanistically, ATAD3A binds to ERK1/2 at HNSCC cell mitochondria in the presence of VDAC1, and this interaction is essential for the activation of mitochondrial ERK1/2 signaling. Most importantly, the ATAD3A-ERK1/2 signaling axis enhances HNSCC development in a Ras-independent manner and, thus, tumor suppression is more effectively achieved when ATAD3A and Ras signaling pathways are co-inhibited. These novel findings highlight the importance of activation of ATAD3A-ERK1/2 axis in cancer progression and suggest that combined targeting of ATAD3A and Ras signaling could potentiate anticancer activity for HNSCC treatment. Citation Format: Liwei Lang, Reid Loveless, Zhaohui Qin, Peng Qiu, Chloe Shay, Yong Teng. ATAD3A enhances head and neck cancer malignancy via mitochondrial ERK1/2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 88.

A Novel Necroptosis-Associated IncRNAs Signature for Prognosis of Head and Neck Squamous Cell Carcinoma.

Purpose: The prognosis of head and neck squamous cell carcinoma (HNSCC) is poor. Necroptosis is a novel programmed form of necrotic cell death. The prognostic value of necroptosis-associated lncRNAs expression in HNSCC has not been explored. Methods: We downloaded mRNA expression data of HNSCC patients from TCGA databases. Prognostic lncRNAs were identified by univariate Cox regression. LASSO was used to establish a model with necroptosis-related lncRNAs. Kaplan-Meier analysis and ROC were applied to verify the model. Finally, functional studies including gene set enrichment analyses, immune microenvironment analysis, and anti-tumor compound IC50 prediction were performed. Results: We identified 1,117 necroptosis-related lncRNAs. The Cox regression showed 55 lncRNAs were associated with patient survival (p < 0.05). The risk model of 24- lncRNAs signature categorized patients into high and low risk groups. The patients in the low-risk group survived longer than the high-risk group (p < 0.001). Validation assays including ROC curve, nomogram and correction curves confirmed the prediction capability of the 24-lncRNA risk mode. Functional studies showed the two patient groups had distinct immunity conditions and IC50. Conclusion: The 24-lncRNA model has potential to guide treatment of HNSCC. Future clinical studies are needed to verify the model.

Melatonin inhibits EMT and PD-L1 expression through the ERK1/2/FOSL1 pathway and regulates anti-tumor immunity in HNSCC.

Melatonin is an endogenous hormone with various biological functions and possesses anti‐tumor properties in multiple malignancies. Immune evasion is one of the most important hallmarks of head and neck squamous cell carcinoma (HNSCC) and is closely related to tumor progression. However, as an immune modulator under physiological conditions, the roles of melatonin in tumor immunity in HNSCC remains unclear. In this study, we found that the endogenous melatonin levels in patients with HNSCC were lower than those in patients with benign tumors in head and neck. Importantly, lower melatonin levels were related to lymph node metastasis among patients with HNSCC. Moreover, melatonin significantly suppressed programmed death‐ligand 1 (PD‐L1) expression and inhibited epithelial–mesenchymal transition (EMT) of HNSCC through the ERK1/2/FOSL1 pathway in vitro and in vivo. In SCC7/C3H syngeneic mouse models, anti‐programmed death‐1 (PD‐1) antibody combined with melatonin significantly inhibited tumor growth and modulated anti‐tumor immunity by increasing CD8+ T cell infiltration and decreasing the regulatory T cell (Treg) proportion in the tumor microenvironment. Taken together, melatonin inhibited EMT and downregulated PD‐L1 expression in HNSCC through the ERK1/2/FOSL1 pathway and exerted synergistic effects with anti‐PD‐1 antibody in vivo, which could provide promising strategies for HNSCC treatment.

Immune checkpoint inhibitors in head and neck squamous cell carcinoma: A systematic review of phase-3 clinical trials.

BACKGROUNDThe outcomes of patients diagnosed with head and neck squamous cell carcinoma (HNSCC) who are not candidates for local salvage therapy and of those diagnosed with recurrent or metastatic disease are dismal. A relatively new systemic therapy option that emerged in recent years in the treatment of advanced HNSCC is immunotherapy using immune checkpoint inhibitors (ICIs). The safety profile and anti-tumor activity of these agents demonstrated in early phase clinical trials paved the way to the initiation of several promising phase-3 trials in the field.AIMTo evaluate the evidence on the effectiveness of ICIs in HNSCC, based on published phase-3 clinical trials.METHODSWe searched PubMed, Cochrane Library, Embase, and Scopus to identify published literature evaluating immunotherapy using ICIs in recurrent or metastatic HNSCC (R/M HNSCC) and locally advanced head and neck squamous cell carcinoma (LAHNSCC). We used a combination of standardized search terms and keywords including head and neck squamous cell carcinoma, recurrent, metastatic, locally advanced, immunotherapy, immune checkpoint inhibitors, monoclonal antibodies, programmed cell death protein-1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T- lymphocyte associated protein-4 (CTLA-4), and phase-3 clinical trial. A sensitive search filter was used to limit our results to randomized controlled trials.RESULTSFive phase-3 clinical trials have reported the data on the effectiveness of immunotherapy in HNSCC so far: Four in R/M HNSCC and one in LAHNSCC. In patients with R/M HNSCC, anti-PD-1 agents nivolumab and pembrolizumab demonstrated improved survival benefits in the second-line treatment setting compared to the standard of care (standard single-agent systemic therapy). While the net gain in overall survival (OS) with nivolumab was 2.4 mo [hazard ratio (HR) = 0.69, P = 0.01], that with pembrolizumab was 1.5 mo (HR = 0.80 nominal P = 0.0161). The anti-PD-L1 agent durvalumab with or without the anti-cytotoxic T- lymphocyte associated protein-4 agent tremelimumab did not result in any beneficial outcomes. In the first-line setting, in R/M HNSCC, pembrolizumab plus platinum-based chemotherapy resulted in significant improvement in survival with a net gain in OS of 2.3 mo (HR = 0.77, P = 0.0034) in the overall population and a net gain in OS of 4.2 mo in the PD-L1 positive (combined positive score > 20) population compared to standard of care (EXTREME regime). In patients with PD-L1 positive R/M HNSCC, monotherapy with pembrolizumab also demonstrated statistically significant improvement in survival compared to EXTREME. In LAHNSCC, immunotherapy using avelumab (an anti-PD-L1 agent) along with standard chemoradiation therapy did not result in improved outcomes compared to placebo plus chemoradiation therapy.CONCLUSIONAnti-PD-1 agents provide survival benefits in R/M HNSCC in the first and second-line settings, with acceptable toxicity profiles compared to standard therapy. There is no proven efficacy in the curative setting to date.

Functional Properties of Cancer Epithelium and Stroma-Derived Exosomes in Head and Neck Squamous Cell Carcinoma.

Stroma–cancer cell crosstalk involves a complex signaling network that contributes to tumor progression, including carcinogenesis, angiogenesis, migration, invasion, and therapy resistance in cancers. Exosomes, as extracellular membranous nanovesicles released by almost all types of cells, including tumor cells and stromal cells, play a critical role in signal delivery and material communication, in which the characteristics of their parent cells are reflected. The tumor or stroma-derived exosomes mediate cell–cell communication in the tumor microenvironment by transporting DNA, RNA, proteins, lipids, and metabolites. Recent studies on head and neck squamous cell carcinoma (HNSCC) have demonstrated that tumor-derived exosomes support various tumor biological behaviors, whereas the functional roles of stroma-derived exosomes remain largely unknown. Although these exosomes are emerging as promising targets in early diagnosis, prognostic prediction, and pharmaceutical carriers for antitumor therapy, there are still multiple hurdles to be overcome before they can be used in clinical applications. Herein, we systematically summarize the promotive roles of the epithelium and stroma-derived exosomes in HNSCC and highlight the potential clinical applications of exosomes in the treatment of HNSCC.

Characterization of the Immune Response to PD-1 Blockade during Chemoradiotherapy for Head and Neck Squamous Cell Carcinoma.

Simple SummaryBlockade of the PD-1/L1 interaction represents a breakthrough in the treatment for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC). Clinical and translational research suggests that this interaction may play a role in immune evasion during chemoradiotherapy. Using an immune-competent murine model of HNSCC, we demonstrate improved efficacy of PD-1 blockade with concurrent cisplatin-based chemoradiotherapy. Taking this approach into a clinical trial evaluating the anti-PD-1 agent, pembrolizumab, combined with chemoradiotherapy in HNSCC, we characterized the peripheral blood immune response to therapy. Our findings highlight that this combination is active in the murine model and circulating PD-1+ T-cell proportions were decreased during the clinical trial. However, additional findings from the clinical trial suggest a shift towards expression of other markers of immune exhaustion. As this treatment approach is being explored in large, randomized trials, these findings provide insight into potential pathways for treatment failure.Background: Chemoradiotherapy is a standard treatment for HNSCC. Blockade of the PD-1/L1-2 interaction may represent a target to overcome immune escape during this treatment. Methods: Utilizing a HNSCC mEERL C57BL/6 mouse model, we evaluated a PD-1 blockade alone or in combination with cisplatin-based chemoradiotherapy. Next, we evaluated peripheral blood mononuclear cells (PBMCs) with relative PD-1, TIM-3, and LAG-3 expression, and myeloid-derived suppressor-like (MDSC-like) populations from a clinical trial evaluating PD-1 blockade with chemoradiotherapy in HNSCC. Finally, we analyzed the effect of therapy on human T-cell clonality through T-cell Receptor (TCR) sequencing. Results: Anti-PD-1 monotherapy induced no response in the mEERL model; however, combination with chemoradiotherapy improved tumor clearance and survival. PBMCs from patients treated with this combination therapy demonstrate a decline in circulating T-cell populations with knockdown of PD-1 expressing CD3+CD4+ and CD3+CD8+ T cells during treatment. However, TIM-3, LAG-3 expressing T-cell and MDSC-like populations concordantly rose. During treatment, the TCR repertoire demonstrates overall clonal expansion, with both unique and previously reported T-cell clones. Conclusions: Our murine HNSCC model demonstrates efficacy of PD-1 blockade during chemoradiotherapy. However, while PD-1-expressing T cells decreased with this therapy, human PBMC findings also identified an increase in populations contributing to immune exhaustion. These findings further characterize PD-1 blockade during chemoradiotherapy for HNSCC and highlight potential competing mechanisms of immune evasion.

Design, synthesis and biological evaluation of new parbendazole derivatives for the treatment of HNSCC

Head and neck squamous cell carcinoma (HNSCC) is a lethal disease with a terrible prognosis, accounting for more than 900,000 new cases and 500,000 deaths each year, nevertheless, its pharmacotherapy is rather limited. Parbendazole was previously identified as a potential HNSCC therapy candidate in our research. Herein, we report the discovery of two series of parbendazole derivatives as tubulin inhibitors. Structure-activity relationship (SAR) analyses led to the discovery of compound 9q with the best pharmacological activities and pharmacokinetic properties. This compound exhibited reasonable inhibition activity on cell proliferation (HN6, CAL-27, Fadu) and tubulin polymerization, induced cell apoptosis, blocked cell cycle and suppressed cell migration and invasion. Compound 9q also displayed low toxicity and a favorable therapeutic effect on a xenograft tumor, indicating that it is a promising starting point for further research.

Sensory Neurotransmitter CGRP Modulates Oral Cancer Growth and Cancer-associated Immune Response

Head and neck squamous cell carcinoma (HNSCC) induces severe pain due in part to activation of primary afferent neurons by cancer-secreted mediators. Local neurotransmitter release (e.g., calcitonin gene-related peptide (CGRP)) from trigeminal neurons innervating the cancer has been linked to tumorigenesis. We hypothesize that CGRP exerts a dual effect on both cancer-associated pain and tumor progression, suggesting that CGRP may be a promising therapeutic target in HNSCC treatment. We used human tumor tissue and patient-reported outcomes to explore the relationship between CGRP+ sensory nerve innervation and cancer pain in patients. To determine CGRP receptor expression on tumor cells, immunohistochemistry and PCR were performed on human and mouse oral cancer cell lines. We used a syngeneic tongue tumor transplant mouse model of oral cancer and a global Calca knockout mouse (i.e. CGRP-KO) to investigate the impact of CGRP signaling on tumor growth and the associate immune response in vivo. We found prominent CGRP-immunoreactive sensory nerve presence innervating human HNSCC tumor tissue, which positively correlated to patient-reported pain (r2=0.357). Furthermore, human HNSCC cell lines expressed 3-fold more CGRP receptor, RAMP1, compared to a non-tumorigenic keratinocyte cell line. In tumor-bearing CGRP-KO mice, we found a significant reduction in tumor size at post-inoculation days 7 and 14 compared to wildtype. We also found a 4-fold increase in tumor infiltrating RAMP1-expressing CD4+ T cells, as well as a 5-fold increase cytotoxic CD8+ T cells and NK1.1+ NK cells in tumor tissue CGRP-KO mice compared to wildtype. This preliminary data suggests that CGRP signaling from sensory neurons may increase cancer associated pain and tumor progression. Further knowledge regarding the relationship between sensory neurons and cancer could allow for the repurposing clinically available nervous system drugs (e.g., anti-CGRP antibodies) for the treatment of cancer and cancer pain. Grant support from the Rita Allen Foundation.