Head And Neck Squamous Cell Carcinoma Carcinogenesis Research Articles

Small Nucleolar RNAs in Head and Neck Squamous Cell Carcinomas.

Small nucleolar RNAs (snoRNAs), a distinct class of noncoding RNAs, encompass highly diverse structures and have a range of 60 to 300 nucleotides in length. About 90% of human snoRNAs are intronic and embedded within introns of their host gene transcripts. Most snoRNAs enriched in specific tissue correlate in abundance with their parental host genes. Advancements in high-throughput sequencing have facilitated the discovery of dysregulated snoRNA expression in numerous human malignancies including head and neck squamous cell carcinoma (HNSCC). Hundreds of differentially expressed snoRNAs have been identified in HNSCC tissues. Among 1,524 snoRNA genes in a 567 HNSCC cohort, 113 snoRNAs were found to be survival related. As for snoRNA's roles in HNSCC, based on the available evidence, dysregulated snoRNAs are closely associated with the carcinogenesis and development of HNSCC. Upregulated snoRNAs have been shown to augment the expression of other oncogenes or activate the Wnt/β-catenin signaling pathway, thereby promoting tumor cell viability, glycolysis, migration, and the epithelial-mesenchymal transition while inhibiting apoptosis in vitro. In vivo animal studies have further elucidated the functional roles of snoRNAs. Knockdown of host genes of these snoRNAs suppressed the Wnt/β-catenin signaling pathway and restrained tumor proliferation and aggressiveness in mice. The putative mechanisms underlying these observations are associated with the biological functions of snoRNAs, primarily involving microRNA-like functions through the generation of microRNA-like fragments and regulation of alternative splicing to yield diverse transcripts. While most of the snoRNAs are upregulated in HNSCC, 4 downregulated snoRNAs have been identified and annotated. SNORA36B (implicated in the regulation of DNA templates) and U3 (chr17, influencing cell proliferation) may serve as protective factors associated with prolonged overall survival. This review describes the viable structures of snoRNAs, endeavors to refine snoRNA sequencing technology, and summarizes snoRNAs' expression profile as well as their role in HNSCC progression for potential diagnostic and therapeutic strategies for HNSCC management.

Epithelial‑derived head and neck squamous tumourigenesis (Review).

Head and neck squamous cell carcinomas (HNSCCs), a heterogeneous group of cancers that arise from the mucosal epithelia cells in the head and neck areas, present great challenges in diagnosis, treatment and prognosis due to their complex aetiology and various clinical manifestations. Several factors, including smoking, alcohol consumption, oncogenic genes, growth factors, Epstein‑Barr virus and human papillomavirus infections can contribute to HNSCC development. The unpredictable tumour microenvironment adds to the complexity of managing HNSCC. Despite significant advances in therapies, the prediction of outcome after treatment for patients with HNSCC remains poor, and the 5‑year overall survival rate is low due to late diagnosis. Early detection greatly increases the chances of successful treatment. The present review aimed to bring together the latest findings related to the molecular mechanisms of HNSCC carcinogenesis and progression. Comprehensive genomic, transcriptomic, metabolomic, microbiome and proteomic analyses allow researchers to identify important biological markers such as genetic alterations, gene expression signatures and protein markers that drive HNSCC tumours. These biomarkers associated with the stages of initiation, progression and metastasis of cancer are useful in the management of patients with cancer in order to improve their life expectancy and quality of life.

Monoamine oxidase B inhibits epithelial-mesenchymal transition and trigger apoptosis via targeting ERK1/2 signaling pathway in head and neck squamous cell carcinoma.

Monoamine oxidase B (MAOB), a flavin monoamine oxidase, regulates biogenic and xenobiotic amine oxidative deaminization. We demonstrate MAOB expression in head and neck epithelium and its biological importance in head and neck squamous cell carcinoma (HNSCC) development. First, we found a possible MAOB downregulation in HNSCC using bioinformatic analysis. Second, we validated MAOB expression changes in vitro and assessed its tumorigenicity in HNSCC. Finally, preclinical xenograft models further confirmed our findings. Results proved that MAOB was significantly reduced in HNSCC tissues and cell lines. By comparing MAOB localization in patient specimens, we found that epithelial basal cells express MAOB and that it changes throughout HNSCC development. We observed that MAOB overexpression inhibited HNSCC cell malignancy via lentiviral transfection. We additionally discovered that selegiline partly counter-regulated MAOB overexpression-induced phenotypes in HNSCC cells. We found that MAOB is a potent biomarker and a unique and essential indication of HNSCC carcinogenesis.

Potential Roles of Activin in Head and Neck Squamous Cell Carcinoma Progression and Mortality.

Activin, a member of the TGF-β super family of cytokines, is involved in head and neck squamous cell carcinoma (HNSCC). This study examined the constituents of the activin axis in order to further elucidate the role of activin A in HNSCC progression. Immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR), MTT, and matrigel invasion assays, in addition to analysis of the tumor cancer genome atlas (TCGA), were employed. IHC in HNSCC and oral leukoplakia (OPL) lesions demonstrated increased expression of the inhibin subunit βA (INHBA) (p<0.0001), as well as activin receptor type IB (ACVR1B) (p<0.0032) compared to normal mucosa. TCGA analysis revealed increased INHBA expression was associated with lymph node positive tumors (p=0.024), decreased overall survival (p=0.0167), and decreased promoter methylation (p<0.0001). Concomitant up-regulated expression of gene pathways strongly correlated with INHBA expression demonstrated further deleterious effects on survival (p<0.0148). Activin may be an important component of early carcinogenesis in OPL and HNSCC with unfavorable effects on clinical end-points such as survival.

Overexpression of POFUT1 promotes malignant phenotype and mediates perineural invasion in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is one of the most aggressive neoplasms, which requires more effective prevention and treatment modalities. Previous studies found that protein O-fucosyltransferase 1 (POFUT1) upregulation promotes carcinogenesis, although the potential roles, underlying molecular mechanisms, and biological implications of POFUT1 in HNSCC were not investigated. In this study, in silico analyses referred POFUT1 as a potential oncogene in HNSCC. Further analysis of tumor and normal tissue samples as well as HNSCC cells with quantitative real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry showed significant overexpression of POFUT1 in HNSCC clinical tumor tissue specimens and cell lines compared to corresponding controls. In vitro investigations revealed that overexpression of POFUT1 promoted phenotypes associated with cancer aggressiveness and its knockdown in HNSCC cells suppressed those phenotypes. Further xenograft experiments demonstrated that POFUT1 is an oncogene in vivo for HNSCC. Immunohistochemical analysis with human clinical samples and cancer cell-dorsal root ganglion ex-vivo coculture model showed that deregulation of POFUT1 is involved in the perineural invasion of HNSCC cells. These results suggest POFUT1 expression as a potential prognostic marker for patients with head and neck cancer and highlight its potential as a target for HNSCC therapy, although more molecular clues are needed to better define the functions of POFUT1 related to HNSCC carcinogenesis.

Circadian Regulator-Mediated Molecular Subtypes Depict the Features of Tumor Microenvironment and Indicate Prognosis in Head and Neck Squamous Cell Carcinoma.

Circadian rhythm is involved in multiple biological activities and implicated in cancer development. However, the role of circadian rhythm in head and neck squamous cell carcinoma (HNSCC) has not been fully interpreted yet. Herein, the present study set out to explore the significance of circadian regulator genes (CRGs) in HNSCC. The molecular landscape and clinical significance of 13 CRGs in HNSCC were explored based on The Cancer Genome Atlas (TCGA). The biological functions of PER3, a key CRG, were validated by cellular experiments. The correlation of CRGs with microenvironment, pathway activities, and prognosis was determined by bioinformatic algorithms. A novel circadian score was introduced to evaluate the circadian modification pattern of each patient and further validated in an independent cohort from the Gene Expression Omnibus (GEO) dataset. CRGs presented high heterogeneity in HNSCC at both genomic and transcriptomic levels. Specifically, PER3 indicated a better prognosis and inhibited HNSCC cell proliferation. Moreover, HNSCC tissues displayed three circadian regulator patterns with distinct clinical outcomes, transcriptomic characteristics, and microenvironment features. Circadian score was an independent risk factor and exhibited excellent predictive efficiency in both the training cohort from the TCGA database and the validation cohort from the GEO database. CRGs played an indispensable role in HNSCC development. An in-depth exploration of circadian rhythm would improve the understanding of HNSCC carcinogenesis and confer novel insights for future clinical practices.

Inducible TgfbR1 and Pten deletion in a model of tongue carcinogenesis and chemoprevention.

Head and neck squamous cell carcinoma (HNSCC) is a significant public health problem, with a need for novel approaches to chemoprevention and treatment. Preclinical models that recapitulate molecular alterations that occur in clinical HNSCC patients are needed to better understand molecular and immune mechanisms of HNSCC carcinogenesis, chemoprevention, and efficacy of treatment. We optimized a mouse model of tongue carcinogenesis with discrete quantifiable tumors via conditional deletion of Tgfβr1 and Pten by intralingual injection of tamoxifen. We characterized the localized immune tumor microenvironment, metastasis, systemic immune responses, associated with tongue tumor development. We further determined the efficacy of tongue cancer chemoprevention using dietary administration of black raspberries (BRB). Three Intralingual injections of 500 µg tamoxifen to transgenic K14 Cre, floxed Tgfbr1, Pten (2cKO) knockout mice resulted in tongue tumors with histological and molecular profiles, and lymph node metastasis similar to clinical HNSCC tumors. Bcl2, Bcl-xl, Egfr, Ki-67, and Mmp9, were significantly upregulated in tongue tumors compared to surrounding epithelial tissue. CD4+ and CD8 + T cells in tumor-draining lymph nodes and tumors displayed increased surface CTLA-4 expression, suggestive of impaired T-cell activation and enhanced regulatory T-cell activity. BRB administration resulted in reduced tumor growth, enhanced T-cell infiltration to the tongue tumor microenvironment and robust antitumoral CD8+ cytotoxic T-cell activity characterized by greater granzyme B and perforin expression. Our results demonstrate that intralingual injection of tamoxifen in Tgfβr1/Pten 2cKO mice results in discrete quantifiable tumors suitable for chemoprevention and therapy of experimental HNSCC.

Expression and molecular regulation of non-coding RNAs in HPV-positive head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most prevalent malignancy worldwide. Accumulating evidence suggests that persistent HPV infection is closely related to a subset of HNSCC types, and the incidence of human papillomavirus (HPV)-positive HNSCC has been annually increasing in recent decades. Although the carcinogenesis of HPV-positive HNSCC has not been completely elucidated, it has been well confirmed that E6 and E7, the main viral oncoproteins are responsible for the maintenance of malignant transformation, promotion of cell proliferation, and increase in tumor invasion. Moreover, compared with HPV-negative HNSCC, HPV-positive HNSCC shows some special clinical-pathological features, which are possibly related to HPV infection and their specific regulatory mechanisms. Non-coding RNA (ncRNA) is a class of RNA lacking the protein-coding function and playing a critical regulatory role via multiple complex molecular mechanisms. NcRNA is an important regulatory pattern of epigenetic modification, which can exert significant effects on HPV-induced tumorigenesis and progression by deregulating downstream genes. However, the knowledge of ncRNAs is still limited, hence, a better understanding of ncRNAs could provide some insights for exploring the carcinogenesis mechanism and identifying valuable biomarkers in HPV-positive HNSCC. Therefore, in this review, we mainly focused on the expression profile of ncRNAs (including lncRNA, miRNA, and circRNA) and explored their regulatory role in HPV-positive HNSCC, aiming to clarify the regulatory mechanism of ncRNAs and identify valuable biomarkers for HPV-positive HNSCC.

Growth Regulated Oncogene-α Upregulates TNF-α and COX-2 and Activates NOD1/RIPK2 mediated-MAPK Pathway in Head and Neck Squamous Cell Carcinoma.

Purpose: The long-term prognosis and survival rate of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) are poor, although the identification of specific biomarkers that reveal its nature and aggressiveness has improved it. Growth-related oncogene alpha (Groα) and NOD1 (nucleotide-binding oligomerization domain 1) can be used as prognosis markers to identify subgroups of HNSCC patients with low survival rates and as potential therapeutic targets for HNSCC patients. However, the mechanism associated with the Groα-mediated NOD pathway in HNSCC progression remains unclear. Method: Overall survival analysis and multiple-gene comparison were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA). qRT-PCR and RT-PCR were used to analyze mRNA expression. Microarray, immunofluorescence staining or western blot analyses were carried out to detect protein expression. Results: Groα was significantly higher in the grade 4 HNSCC tumor tissues compared with that in grade 1-3 and healthy subjects. High expression of Groα, NOD1 and RIPK2 (receptor-interacting serine-threonine kinase 2) is correlated with survival rate in HNSCC patients. Treatment of SCC25 and OECM-1 cells with Groα increased the expression of NOD1 and RIPK2 in a concentration-dependent manner. The findings herein reveal the association of Groα, NOD1 and RIPK2 biomarkers with HNSCC carcinogenesis. Moreover, Groα is the major stimulus of inflammatory mediation and promotes TNF-α (tumor necrosis factor-α) and COX-2 (cyclooxygenase-2) expression in HNSCC. Groα induces TNF-α and COX-2 expression through regulation involving ERK (extracellular signal-regulated kinase)-, JNK (C-Jun N-terminal kinase)- and p38 MAPK (mitogen-activated protein kinase)-dependent signaling pathways. Conclusions: Our findings herein constitute the first evidence that Groα is important in HNSCC progression and metastasis via the NOD1-mediated MAPK pathway, suggesting a role for Groα and NOD1 in mediating metastasis and its potential as a therapeutic target.

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.

Abstract 4009: Interaction between NF-κB and NRF2 pathways lead to improved outcomes in HPV-associated head and neck cancer

Abstract HPV infection has been linked with various types of cancers. Current vaccines are likely to have limited impact in the prevention of head and neck squamous cell carcinoma (HNSCC) in several decades due to a high prevalence of this infection in older individuals. HPV-associated (HPV+) HNSCC is the most frequently diagnosed HPV related cancer and its incidence continues to rise. Despite this, little is known about the mechanism of HPV-driven carcinogenesis. Even though HPV+ HNSCC has a favorable prognosis, around 30% of patients show recurrence accompanied by severe side effects due to intensive radiotherapy. Hence, it is pertinent to develop de-intensification strategies to identify patients that are at low risk of recurrence. However, absence of molecular biomarkers greatly limits these studies. Data from two independent patient cohorts helped identify subtypes of HPV+ HNSCC based on presence or absence of NF-κB activating mutations. The subtype with NF-κB overactivity significantly correlated with better patient survival and the absence of an integrated HPV, suggesting that these tumors are driven by a distinct mechanism of oncogenesis, dependent on NF-κB overactivity and maintenance of episomal HPV. Most of these tumors had deletions of TRAF3 or CYLD genes, which are known NF-κB inhibitors. Another finding from this study was the presence of NRF2 downregulation in the subset of NF-κB overactive tumors. NRF2 is a major regulator of the cellular oxidative stress response. Its dysregulation has been known to help cancer cells survive and plays a role in radio resistance. We confirmed that baseline and inducible NRF2 transcriptional activity is downregulated in NF-κB overactive HPV+ HNSCC cells with TRAF3 or CYLD knockout. Interestingly, similar results were observed in these cells after overexpression of dominant negative CUL3 that stabilizes and activates NRF2, suggesting that stabilized NRF2 is inhibited in HPV+ HNSCC cells carrying constitutively active NF-κB. Clonogenic survival assay revealed that NF-κB active cells are more sensitive to radiation as compared to parental HPV+ HNSCC cells. In addition, we found lesser nuclear localization of NRF2 after treatment with small molecule NRF2 activator CDDO in CYLD/TRAF3 knockout cells. Confirmation of these findings post radiation may explain improved survival of patients with NF-κB overactive HPV+ HNSCC. Overall, our data suggests an inverse correlation between NF-κB and NRF2, with NF-κB potentially altering the oxidative stress response and conferring radio sensitivity to HPV+ HNSCC with TRAF3/CYLD mutations. Our aim is to uncover the unique mechanism of HPV carcinogenesis and finding novel therapeutic targets in HNSCC while improving quality of life in patients. Importantly, constitutively active NF-κB distinguishing the 2 subtypes of HPV+ HNSCC may serve as prognostic biomarkers to help clinicians with therapeutic decisions. Citation Format: Aditi Kothari, Travis Parke Schrank, Natalia Issaeva, Wendell Gray Yarbrough. Interaction between NF-κB and NRF2 pathways lead to improved outcomes in HPV-associated head and neck cancer [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 4009.

Human papilloma virus integration sites and genomic signatures in head and neck squamous cell carcinoma.

A prevalence of around 26% of human papillomavirus (HPV) in head and neck squamous cell carcinoma (HNSCC) has been previously reported. HPV induced oncogenesis mainly involving E6 and E7 viral oncoproteins. In some cases, HPV viral DNA has been detected to integrate with the host genome and possibly contributes to carcinogenesis by affecting the gene expression. We retrospectively assessed HPV integration sites and signatures in 80 HPV positive patients with HNSCC, by using a double capture‐HPV method followed by next‐generation Sequencing. We detected HPV16 in 90% of the analyzed cohort and confirmed five previously described mechanistic signatures of HPV integration [episomal (EPI), integrated in a truncated form revealing two HPV‐chromosomal junctions colinear (2J‐COL) or nonlinear (2J‐NL), multiple hybrid junctions clustering in a single chromosomal region (MJ‐CL) or scattered over different chromosomal regions (MJ‐SC) of the human genome]. Our results suggested that HPV remained episomal in 38.8% of the cases or was integrated/mixed in the remaining 61.2% of patients with HNSCC. We showed a lack of association of HPV genomic signatures to tumour and patient characteristics, as well as patient survival. Similar to other HPV associated cancers, low HPV copy number was associated with worse prognosis. We identified 267 HPV‐human junctions scattered on most chromosomes. Remarkably, we observed four recurrent integration regions: PDL1/PDL2/PLGRKT (8.2%), MYC/PVT1 (6.1%), MACROD2 (4.1%) and KLF5/KLF12 regions (4.1%). We detected the overexpression of PDL1 and MYC upon integration by gene expression analysis. In conclusion, we identified recurrent targeting of several cancer genes such as PDL1 and MYC upon HPV integration, suggesting a role of altered gene expression by HPV integration during HNSCC carcinogenesis.

Frequency of PIK3CA mutations in different subsites of head and neck squamous cell carcinoma in southern Thailand.

BackgroundPhosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations have been reported in many cancers, including head and neck squamous cell carcinoma (HNSCC). The frequency of these mutations varies among tumor locations and might be relevant to treatment outcomes among HNSCC. In this study, we examined the frequency of PIK3CA mutations in the different subsites of HNSCC.MethodsNinety-six fresh biopsy specimens were investigated for mutations in PIK3CA exons 4, 9, and 20 using allele-specific real-time polymerase chain reaction. Patient characteristics and survival were analyzed and compared between specimens with or without PIK3CA mutations.ResultsThe study included primary tumors originating from the oral cavity (n = 63), hypopharynx (n = 23), and oropharynx (n = 10). We identified mutations in 10.4% of patients (10 of 96 specimens). The overall mutational frequency was 17.4% (4/23) and 9.5% (6/63) in the hypopharynx and oral cavity, respectively. No patients with oropharyngeal carcinoma had mutations. Among the 10 mutant specimens, five were missense mutations (exon 9 [E545K] in two samples and exon 20 [H1047R] in three samples) and five were silent mutations in exon 20 (T1025T). Mutations were not found in exon 4. Among 84 patients with available clinical data, we found no significant differences in clinical characteristics and survival based on the presence or absence of PIK-3CA mutations.ConclusionsThe results indicate that PIK3CA mutations are involved in HNSCC carcinogenesis, and the hypopharynx should be considered a primary site of interest for future studies, particularly in Southeast Asian populations.

Age-specific oncogenic pathways in head and neck squamous cell carcinoma-are elderly a different subcategory?

In recent clinical practice, an increasing number of elderly patients suffering from head and neck squamous cell carcinoma (HNSCC) of unknown pathophysiology is observed. The majority of HNSCC patients can roughly be divided into three subcategories. First, a small group of young patients who present with variants of genomic aberrations and inheritable diseases like Fanconi anaemia. Second, an increasing population of HPV-related HNSCCs that are regarded as genomic stable tumours with a more favourable prognosis. Though HPV-related tumours used to be more common among younger males, a notable rise in the elderly population is observed. The third subcategory, that of HPV-negative tumours, has been shown to be more heterogeneous with involvement of a variety of oncogenic pathways related to lifestyle factors like smoking and alcohol consumption, often seen in middle-aged males. Some of these pathways could be related to age, such as TP53 alterations, EGFR activation, apoptotic pathway alterations and field cancerization. In this narrative review, we provide an overview of established and newly discovered age-specific pathophysiological mechanisms underlying HNSCC. We propose a fourth subcategory of patients with a suspected different pathophysiology: elderly (HPV-negative) HNSCC patients without a history of tobacco and alcohol consumption. In this subcategory, carcinogenesis seems to be a multi-step process based on genomic instability, immunosenescence, cell cycle disruption and telomere shortening. To conclude, we discuss suggestions for future research to fill the knowledge gap about age-dependent HNSCC carcinogenesis.

MiRNAs as Biomarkers for Diagnosing and Predicting Survival of Head and Neck Squamous Cell Carcinoma Patients.

Simple SummaryHead and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide. It arises from the epithelium of the upper aerodigestive tract. Increasing evidence suggests that there is a significant role of microRNAs in HNSCC formation and progression. The aim of this study was to explore and compare the expression of HNSCC related miRNAs in tumor vs neighboring healthy tissue of HNSCC patients with tumors located in either the oral cavity, oropharynx, or larynx. Our results demonstrated that expression of these miRNAs was significantly different not only between healthy and tumor tissues, but also among tumor locations. Further analysis indicated that microRNA expression could be used to distinguish between tumor and healthy tissues, and prognose the overall survival of patients.Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth most common cancer worldwide. These tumors originate from epithelial cells of the upper aerodigestive tract. HNSCC tumors in different regions can have significantly different molecular characteristics. While many microRNAs (miRNAs) have been found to be involved in the regulation of the carcinogenesis and pathogenesis of HNSCC, new HNSCC related miRNAs are still being discovered. The aim of this study was to explore potential miRNA biomarkers that can be used to diagnose HNSCC and prognose survival of HNSCC patients. For this purpose, we chose a panel of 12 miRNAs: miR-146a-5p, miR-449a, miR-126-5p, miR-34a-5p, miR-34b-5p, miR-34c-5p, miR-217-5p, miR-378c, miR-6510-3p, miR-96-5p, miR-149-5p, and miR-133a-5p. Expression of these miRNAs was measured in tumor tissue and neighboring healthy tissue collected from patients diagnosed with HNSCC (n = 79) in either the oral cavity, oropharynx, or larynx. We observed a pattern of differentially expressed miRNAs at each of these cancer locations. Our study showed that some of these miRNAs, separately or in combination, could serve as biomarkers distinguishing between healthy and tumor tissue, and their expression correlated with patients’ overall survival.

The mRNA expression levels of calpains and their activity in malignant and dysplastic epithelium of the upper respiratory tract

Background. The calpain proteolytic system plays an important role in the development of cancer. Detection of early cancer in the upper respiratory tract is often challenging, as symptoms are largely non-specific, and most cases are diagnosed at an advanced stage. Aim . To identify candidate markers of transition from premalignant lesions to invasive carcinoma, we studied mRNA expression levels of CAPN1 and CAPN2 and the total activity of calpains in the tumor tissues of patients with head and neck squamous cell carcinoma (HNSCC) and in the epithelial dysplasia-affected tissues of patients with chronic diseases of the upper respiratory tract. Materials and methods. The study included 32 patients with HNSCC (Т1-3N0-1М0) and 12 patients with chronic diseases of the upper respiratory system associated with epithelial dysplasia. The expression levels of CAPN1 and CAPN2 were assessed using real-time polymerase chain reaction (PCR). The calpain activity was determined by hydrolysis of the fluorogenic Suc-LLVY-AMC oligopeptide. Results. The mRNA expression levels of CAPN1 and CAPN2 were respectively 3 and 4 times higher in the tumor tissue of patients with HNSCC than in the tissue of patients with endothelial dysplasia in the upper respiratory tract. The level of calpain activity was 4.4 times higher in patients with HNSCC than in patients with epithelial dysplasia of different severity. Conclusion. The elevated mRNA expression levels of CAPN1 and CAPN2 and their activity in the tumor tissues of patients with HNSCC compared to patients with chronic respiratory diseases associated with epithelial dysplasia are likely to characterize a high potential for transition from precancerous lesion to cancer. To clarify the role of calpains in the carcinogenesis of HNSCC, further studies of intact tissues using animal models are required.

Abstract 2367: Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma

Abstract MicroRNAs (miR) are evolutionarily highly conserved non-coding RNAs that play roles in fundamental cellular functions by post-translational gene regulation. In cancer pathogenesis, miRs exert both anti-tumorigenic and pro-tumorigenic effects by virtue of miR-specific and context-dependent mechanisms. The role of chromosomal changes and mutation status on miR expression in cancer remains unknown. Here we provide evidence that chromosome arm 1p loss and P53 mutations are associated with lower levels of miR-34a, particularly in squamous cancers such as head and neck squamous cell carcinoma (HNSCC). miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET, and modulates cell proliferation. miR-34a expression is inversely correlated with MET in tumor tissue. We found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Moreover, ectopic expression of miR-34a reduces HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes and proteins involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Our work demonstrates a role of miR-34a in maintaining tumor immunity, as overexpression of miR-34a resulted in a decease in percentage of anti-inflammatory (M2-like) TAMs and PDL1+TAMs, which are reported to have pro-tumorigenic activity. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells, myeloid cells, and regulatory T cells. Our results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity, and could potentially represent a new therapeutic approach for HNSCC. Citation Format: Xun Wu, Yi-Shing Lisa Cheng, Methew Matthen, Angela Yoon, Gary Schwartz, Shashi Bala, Alison M. Taylor, Fatemeh Momen-Heravi. Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2367.

Potential Therapeutic Significance of Laminin in Head and Neck Squamous Carcinomas.

Simple SummaryHead and neck cancers (HNC) account for approximately 500,000 new cases of tumors annually worldwide and are represented by upper aerodigestive tract malignant neoplasms, which particularly arise in oral cavity, larynx, and pharynx tissues. Thus, due to the biological diversity between the upper aerodigestive organs, and to the heterogeneity of risk factors associated with their malignant transformation, HNC behavior, and prognosis seem to strongly vary according to the tumor site. However, despite to the heterogeneity which characterizes head and neck tumors, squamous cell carcinomas (SCC) represent the predominant histopathologic HNC subtype. In this sense, it has been reported that SCC tumor biology is strongly associated with deregulations within the extracellular matrix compartment. Accordingly, it has been shown that laminin plays a remarkable role in the regulation of crucial events associated with head and neck squamous cell carcinomas (HNSCC) progression, which opens the possibility that laminin may represent a convergence point in HNSCC natural history.Head and neck squamous cell carcinomas (HNSCC) are among the most common and lethal tumors worldwide, occurring mostly in oral cavity, pharynx, and larynx tissues. The squamous epithelia homeostasis is supported by the extracellular matrix (ECM), and alterations in this compartment are crucial for cancer development and progression. Laminin is a fundamental component of ECM, where it represents one of the main components of basement membrane (BM), and data supporting its contribution to HNSCC genesis and progression has been vastly explored in oral cavity squamous cell carcinoma. Laminin subtypes 111 (LN-111) and 332 (LN-332) are the main isoforms associated with malignant transformation, contributing to proliferation, adhesion, migration, invasion, and metastasis, due to its involvement in the regulation of several pathways associated with HNSCC carcinogenesis, including the activation of the EGFR/MAPK signaling pathway. Therefore, it draws attention to the possibility that laminin may represent a convergence point in HNSCC natural history, and an attractive potential therapeutic target for these tumors.

Integrative Analysis of DNA Methylation Data and Transcriptome Data Identified a DNA Methylation-Dysregulated Four-LncRNA Signature for Predicting Prognosis in Head and Neck Squamous Cell Carcinoma.

Increasing evidence has demonstrated the crosstalk between DNA epigenetic alterations and aberrant expression of long non-coding RNAs (lncRNAs) during carcinogenesis. However, epigenetically dysregulated lncRNAs and their functional and clinical roles in Head and Neck Squamous Cell Carcinoma (HNSCC) are still not explored. In this study, we performed an integrative analysis of DNA methylation data and transcriptome data and identified a DNA methylation-dysregulated four-lncRNA signature (DNAMeFourLncSig) from 596 DNA methylation-dysregulated lncRNAs using a machine-learning-based feature selection method, which classified the patients of the discovery cohort into two risk groups with significantly different survival including overall survival, disease-specific survival, and progression-free survival. Then the DNAMeFourLncSig was implemented to another two HNSCC patient cohorts and showed similar prognostic values in both. Results from multivariable Cox regression analysis revealed that the DNAMeFourLncSig might be an independent prognostic factor. Furthermore, the DNAMeFourLncSig was substantially correlated with the complete response rate of chemotherapy and may predict chemotherapy response. Functional in silico analysis found that DNAMeFourLncSig-related mRNAs were mainly enriched in cell differentiation, tissue development and immune-related pathways. Overall, our study will improve our understanding of underlying transcriptional and epigenetic mechanisms in HNSCC carcinogenesis and provided a new potential biomarker for the prognosis of patients with HNSCC.

Targeting mTOR-CCL20 Signaling May Improve Response to Docetaxel in Head and Neck Squamous Cell Carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) usually show a dismal prognosis. It is this worthwhile to develop new, effective therapeutic regimens for these patients, such as molecular targeted therapy, which is promising as an alternative or combination treatment for HNSCC. The mammalian target of rapamycin (mTOR) pathway, which plays an important role in the carcinogenesis of HNSCC, is the most frequently activated, and is thus worthy of further investigation. In this study, two human HNSCC cell lines, FaDu and SAS, were evaluated for cell growth with trypan blue staining and tumor growth using an orthotopic xenograft model. The immunohistochemical expression of mTOR in the subcutaneous xenograft model and the inhibitory effects of docetaxel on the growth and state of activation of the PI3K/mTOR pathway were also evaluated and examined by colony formation and Western blot, respectively. Cell proliferation and migration were measured by water-soluble tetrazolium salt (WST-1) and OrisTM cell migration assay, respectively. Furthermore, the effects of rapamycin and BEZ235, a phosphatidylinositol 3-kinases (PI3K) and mTOR inhibitor in combination with docetaxel or CCL20 were evaluated in the FaDu and SAS cells. The results showed that the expression of mTOR was significantly higher in the SAS and FaDu xenograft models than in the control. Docetaxel treatment significantly suppressed HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. Additionally, when administered in a dose-dependent fashion, mTOR inhibitors inhibited the growth and migration of the HNSCC cells. This combination was synergistic with docetaxel, resulting in almost complete cell growth and migration arrest. In conclusion, docetaxel significantly inhibited HNSCC cell proliferation and migration in vitro via the PI3K/mTOR/CCL-20 signaling pathway. The synergistic and additive activity of mTOR inhibitors combined with docetaxel shows potential as a new treatment strategy for HNSCC.