The USP7-STAT3-granzyme-Par-1 axis regulates allergic inflammation by promoting differentiation of IL-5-producing Th2 cells.

Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.

Salivary gland-type cancers: cross-organ demographics of a rare cancer.

BACKGROUND: Salivary gland-type cancers (SGTCs) are histologically heterogeneous and can affect organs other than the salivary glands. Some tumors outside the salivary glands are diagnosed on their unique histological characteristics. Comprehensive cross-organ studies on SGTCs are limited. METHODS: We retrospectively analyzed the data of patients with salivary duct carcinoma (SDC), adenoid cystic carcinoma (AdCC), mucoepidermoid carcinoma (MEC), epithelial-myoepithelial carcinoma (EMC), acinic cell carcinoma (AcCC), and polymorphous adenocarcinoma (PAC) who visited our institution between 2009 and 2019. The primary tumor sites were classified into four categories; major salivary glands, head/neck (H/N) excluding (exc) major salivary glands (MSG) regions, broncho-pulmonary regions, and "others". H/N exc MSG was further divided into three subcategories, nasal/paranasal sinus, oral and pharynx/larynx. RESULTS: We identified 173 patients with SGTCs, with SDC, AdCC, MEC, EMC, AcCC, and PAC accounting for 20%, 42%, 27%, 3%, 8%, and 1% of the cases, respectively. The most frequent primary site was the major salivary glands (64%), followed by H/N exc MSG regions (27%), broncho-pulmonary regions, and "others", thus non-salivary gland origins accounted for 9% of all cases. Patients with SDC, MEC, AcCC, or SGTC of the major salivary glands and broncho-pulmonary regions were more frequently treated by surgery. The overall survival time of the patients with MEC was significantly better than that of patients with SDC or EMC. CONCLUSIONS: This cross-organ study highlights the clinical significance of SGTCs, underscoring the need for developing novel therapies for this rare disease entity.

Factors contributing to the diagnosis and onset prediction of perennial allergic rhinitis in high-risk children: A sub-analysis of the CHIBA study.

BACKGROUND: This study aimed to clarify the diagnostic and predictive factors for perennial allergic rhinitis (PAR) onset in children by analyzing the results of the Chiba High-risk Birth Cohort for Allergy study, which examined newborns with a family history of allergies. METHODS: Overall, 306 pregnant women were recruited. Their newborns were examined by otolaryngologists and pediatric allergists at 1, 2, and 5 years of age. Participants with clinical and laboratory data available at all consultation points were considered eligible. RESULTS: Among 187 eligible participants, the prevalence rates of PAR were 2.1%, 4.3%, and 24.1% at 1, 2, and 5 years of age, respectively. AR-specific nasal local findings and eosinophils in nasal smear were observed in a substantial number of patients with PAR at 1 and 2 years of age. Factors present up to 2 years of age that were associated with PAR onset at 5 years of age, in descending order, were as follows: sensitization to house dust mites (HDM), nasal eosinophilia, and sensitization to cat dander. In 44 cases with HDM sensitization, nasal eosinophilia up to 2 years of age achieved a sensitivity of 76.0% and a specificity of 73.7% for predicting PAR onset at 5 years. CONCLUSIONS: Rhinitis findings and nasal eosinophilia are useful auxiliary diagnostic items for pediatric PAR. Sensitization to HDM and nasal eosinophilia were the most influential factors associated with future PAR onset. A combination of these factors may facilitate the prediction of PAR onset.

Tumorigenic activation around HPV integrated sites in head and neck squamous cell carcinoma.

Human papillomavirus (HPV) is causally involved in the development of head and neck squamous cell carcinoma (HNSCC). The integration of HPV drives tumorigenesis through expression of oncogenic viral genes as well as genomic alterations in surrounding regions. To elucidate involvement of epigenetic dysregulation in tumorigenesis, we here performed integrated analyses of the epigenome, transcriptome and interactome using ChIP-seq, RNA-seq and Hi-C and 4C-seq for HPV(+) HNSCCs. We analyzed clinical HNSCC using The Cancer Genome Atlas data and found that genes neighboring HPV integration sites were significantly upregulated and were correlated with oncogenic phenotypes in HPV(+) HNSCCs. While we found four HPV integration sites in HPV(+) HNSCC cell line UPCI-SCC-090 through target enrichment sequencing, 4C-seq revealed 0.5 to 40 Mb of HPV-interacting regions (HPVIRs) where host genomic regions interacted with integrated HPV genomes. While 9% of the HPVIRs were amplified and activated epigenetically forming super-enhancers, the remaining non-amplified regions were found to show a significant increase in H3K27ac levels and an upregulation of genes associated with GO terms, for example, Signaling by WNT and Cell Cycle. Among those genes, ITPR3 was significantly upregulated, involving UPCI-SCC-090-specific super-enhancer formation around the ITPR3 promoter and in the 80-kb-downstream region. The knockdown of ITPR3 by siRNA or CRISPR deletions of the distant enhancer region led to a significant suppression of cell proliferation. The epigenetic activation of HPVIRs was also confirmed in other cell lines, UM-SCC-47 and UM-SCC-104. These data indicate that epigenetic activation in HPVIRs contributes, at least partially, to genesis of HPV(+) HNSCC.

Single-cell immunoprofiling after immunotherapy for allergic rhinitis reveals functional suppression of pathogenic TH2 cells and clonal conversion.

BACKGROUND: Allergic rhinitis is a growing problem worldwide. Currently the only treatment that can modify the disease is antigen-specific immunotherapy, but its mechanism of action is not fully understood. OBJECTIVE: We comprehensively investigated the role and changes of antigen-specific T cells before and after sublingual immunotherapy (SLIT) for Japanese cedar pollinosis. METHODS: We cultured peripheral blood mononuclear cells obtained both before and 1 year after initiating SLIT and used a combination of single-cell RNA sequencing and repertoire sequencing. To investigate biomarkers, we used cells from patients participating a phase 2/3 trial of SLIT tablets for Japanese cedar pollinosis and cells from outpatients with good and poor response. RESULTS: Antigen-stimulated culturing after SLIT led to clonal expansion of TH2 and regulatory T cells, and most of these CD4+ T cells retained their CDR3 regions before and after treatment, indicating antigen-specific clonal responses and differentiation resulting from SLIT. However, SLIT reduced the number of clonal functional TH2 cells but increased the trans-type TH2 cell population that expresses musculin (MSC), TGF-ß, and IL-2. Trajectory analysis suggested that SLIT induced clonal differentiation of the trans-type TH2 cells differentiated into regulatory T cells. Using real-time PCR, we found that the MSC levels increased in the active SLIT group and those with good response after 1 year of treatment. CONCLUSION: The combination of single-cell RNA sequencing and repertoire analysis helped reveal part of the underlying mechanism: SLIT promotes the expression of MSC on pathogenic TH2 cells and suppresses their function. MSC may be a potential biomarker of SLIT for allergic rhinitis.

Differences in the expression of multidrug resistance proteins in chronic rhinosinusitis according to endotype.

BACKGROUND: Chronic rhinosinusitis is a common disease of the nasal cavity and is classified into two major endotypes, which are neutrophilic and eosinophilic. Some patients with neutrophilic and eosinophilic chronic rhinosinusitis are refractory to treatment, and the mechanism of drug resistance is not completely understood. METHODS: Nasal polyp samples were collected from patients with non-eosinophilic chronic rhinosinusitis (nECRS) and eosinophilic chronic rhinosinusitis (ECRS). Transcriptomic and proteomic analyses were performed simultaneously. Gene Ontology (GO) analysis was conducted to extract genes involved in drug resistance. Then, GO analysis results were validated via real-time polymerase chain reaction and immunohistochemistry analysis. RESULTS: The nasal polyps of patients with ECRS were enriched with 110 factors in the genes and 112 in the proteins, unlike in those of patients with nECRS. GO analysis on the combined results of both showed that the factors involved in extracellular transportation were enriched. Our analysis focused on multidrug resistance protein 1-5 (MRP1-5). Real-time polymerase chain reaction revealed that the MRP4 expression was significantly upregulated in ECRS polyps. Immunohistochemical staining showed that the MRP3 and MRP4 expressions significantly increased in nECRS and ECRS, respectively. MRP3 and MRP4 expressions were positively correlated with the number of neutrophil and eosinophil infiltrates in polyps and associated with the tendency to relapse in patients with ECRS. CONCLUSIONS: MRP is associated with treatment resistance and is expressed in nasal polyps. The expression pattern had different features based on chronic rhinosinusitis endotype. Therefore, drug resistance factors can be associated with therapeutic outcomes.

Genome-Wide Super-Enhancer-Based Analysis: Identification of Prognostic Genes in Oral Squamous Cell Carcinoma.

Advanced-stage oral squamous cell carcinoma (OSCC) patients are treated with combination therapies, such as surgery, radiation, chemotherapy, and immunotherapy. However, OSCC cells acquire resistance to these treatments, resulting in local recurrence and distant metastasis. The identification of genes involved in drug resistance is essential for improving the treatment of this disease. In this study, we applied chromatin immunoprecipitation sequencing (ChIP-Seq) to profile active enhancers. For that purpose, we used OSCC cell lines that had been exposed to cetuximab for a prolonged period. In total, 64 chromosomal loci were identified as active super-enhancers (SE) according to active enhancer marker histone H3 lysine 27 acetylation (H3K27ac) ChIP-Seq. In addition, a total of 131 genes were located in SE regions, and 34 genes were upregulated in OSCC tissues by TCGA-OSCC analysis. Moreover, high expression of four genes (C9orf89; p = 0.035, CENPA; p = 0.020, PISD; p = 0.0051, and TRAF2; p = 0.0075) closely predicted a poorer prognosis for OSCC patients according to log-rank tests. Increased expression of the four genes (mRNA Z-score ≥ 0) frequently co-occurred in TCGA-OSCC analyses. The high and low expression groups of the four genes showed significant differences in prognosis, suggesting that there are clear differences in the pathways based on the underlying gene expression profiles. These data indicate that potential stratified therapeutic strategies could be used to overcome resistance to drugs (including cetuximab) and further improve responses in drug-sensitive patients.

Identification of Tumor-Suppressive miR-30e-3p Targets: Involvement of SERPINE1 in the Molecular Pathogenesis of Head and Neck Squamous Cell Carcinoma.

Recently, our studies revealed that some passenger strands of microRNAs (miRNAs) were closely involved in cancer pathogenesis. Analysis of miRNA expression signatures showed that the expression of miR-30e-3p (the passenger strand of pre-miR-30e) was significantly downregulated in cancer tissues. In this study, we focused on miR-30e-3p (the passenger strand of pre-miR-30e). We addressed target genes controlled by miR-30e-3p that were closely associated with the molecular pathogenesis of head and neck squamous cell carcinoma (HNSCC). Ectopic expression assays demonstrated that the expression of miR-30e-3p attenuated cancer cell malignant phenotypes (e.g., cell proliferation, migration, and invasive abilities). Our analysis of miR-30e-3p targets revealed that 11 genes (ADA, CPNE8, C14orf126, ERGIC2, HMGA2, PLS3, PSMD10, RALB, SERPINE1, SFXN1, and TMEM87B) were expressed at high levels in HNSCC patients. Moreover, they significantly predicted the short survival of HNSCC patients based on 5-year overall survival rates (p < 0.05) in The Cancer Genome Atlas (TCGA). Among these targets, SERPINE1 was found to be an independent prognostic factor for patient survival (multivariate Cox regression; hazard ratio = 1.6078, p < 0.05). Aberrant expression of SERPINE1 was observed in HNSCC clinical samples by immunohistochemical analysis. Functional assays by targeting SERPINE1 expression revealed that the malignant phenotypes (e.g., proliferation, migration, and invasion abilities) of HNSCC cells were suppressed by the silencing of SERPINE1 expression. Our miRNA-based approach will accelerate our understanding of the molecular pathogenesis of HNSCC.

Identification of Tumor Suppressive Genes Regulated by miR-31-5p and miR-31-3p in Head and Neck Squamous Cell Carcinoma.

We identified the microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC) tissues by RNA sequencing, in which 168 miRNAs were significantly upregulated, including both strands of the miR-31 duplex (miR-31-5p and miR-31-3p). The aims of this study were to identify networks of tumor suppressor genes regulated by miR-31-5p and miR-31-3p in HNSCC cells. Our functional assays showed that inhibition of miR-31-5p and miR-31-3p attenuated cancer cell malignant phenotypes (cell proliferation, migration, and invasion), suggesting that they had oncogenic potential in HNSCC cells. Our in silico analysis revealed 146 genes regulated by miR-31 in HNSCC cells. Among these targets, the low expression of seven genes (miR-31-5p targets: CACNB2 and IL34; miR-31-3p targets: CGNL1, CNTN3, GAS7, HOPX, and PBX1) was closely associated with poor prognosis in HNSCC. According to multivariate Cox regression analyses, the expression levels of five of those genes (CACNB2: p = 0.0189; IL34: p = 0.0425; CGNL1: p = 0.0014; CNTN3: p = 0.0304; and GAS7: p = 0.0412) were independent prognostic factors in patients with HNSCC. Our miRNA signature and miRNA-based approach will provide new insights into the molecular pathogenesis of HNSCC.

Molecular Pathogenesis of the Coronin Family: CORO2A Facilitates Migration and Invasion Abilities in Oral Squamous Cell Carcinoma.

In humans, the coronin family is composed of seven proteins containing WD-repeat domains that regulate actin-based cellular processes. Some members of the coronin family are closely associated with cancer cell migration and invasion. The Cancer Genome Atlas (TCGA) analysis revealed that CORO1C, CORO2A, and CORO7 were significantly upregulated in oral squamous cell carcinoma (OSCC) tissues (p < 0.05). Moreover, the high expression of CORO2A was significantly predictive of the 5-year survival rate of patients with OSCC (p = 0.0203). Overexpression of CORO2A was detected in OSCC clinical specimens by immunostaining. siRNA-mediated knockdown of CORO2A suppressed cancer cell migration and invasion abilities. Furthermore, we investigated the involvement of microRNAs (miRNAs) in the molecular mechanism underlying CORO2A overexpression in OSCC cells. TCGA analysis confirmed that tumor-suppressive miR-125b-5p and miR-140-5p were significantly downregulated in OSCC tissues. Notably, these miRNAs bound directly to the 3'-UTR of CORO2A and controlled CORO2A expression in OSCC cells. In summary, we found that aberrant expression of CORO2A facilitates the malignant transformation of OSCC cells, and that downregulation of tumor-suppressive miRNAs is involved in CORO2A overexpression. Elucidation of the interaction between genes and miRNAs will help reveal the molecular pathogenesis of OSCC.

Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma.

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.

Stratification of HPV-associated and HPV-negative oropharyngeal squamous cell carcinomas based on DNA methylation epigenotypes.

While the incidence of oropharyngeal squamous cell carcinoma (OPSCC) has been increasing in these two decades, primarily due to human papillomavirus (HPV), stratification of OPSCC into molecular subgroups showing different clinicopathological features has not been fully investigated. We performed DNA methylome analysis using Infinium 450k for 170 OPSCC cases, including 89 cases in our cohort and 81 cases reported by The Cancer Genome Atlas, together with targeted exon sequencing analysis. We stratified OPSCC by hierarchical clustering analysis using methylome data. Methylation levels of classifier markers were validated quantitatively using pyrosequencing, and area under the curve (AUC) values of receiver operating characteristics (ROC) curves were calculated. OPSCC was stratified into four epigenotypes: HPV(+) high-methylation (OP1), HPV(+) intermediate-methylation (OP2), HPV(-) intermediate-methylation (OP3) and HPV(-) low-methylation (OP4). Ten methylation marker genes were generated: five to classify HPV(+) cases into OP1 and OP2, and five to classify HPV(-) cases into OP3 and OP4. AUC values of ROC curves were 0.969 and 0.952 for the two marker panels, respectively. While significantly higher TP53 mutation and CCND1 copy number gains were observed in HPV(-) than in HPV(+) groups (p < 0.01), no significant difference of genomic aberrations was observed between OP1 and OP2, or OP3 and OP4. The four epigenotypes showed significantly different prognosis (p = 0.0006), distinguishing the most favorable OPSCC subgroup (OP1) among generally favorable HPV(+) cases, and the most unfavorable OPSCC subgroup (OP3) among generally unfavorable HPV(-) cases. HPV(+) and HPV(-) OPSCC are further divided into distinct DNA methylation epigenotypes, showing significantly different prognosis.

Establishment of epigenetic markers to predict irradiation efficacy against oropharyngeal cancer.

Irradiation, or chemoradiotherapy, is a curative treatment for oropharyngeal squamous cell carcinoma (OPSCC). Its invasiveness, however, can often negate its efficacy. Therefore, developing methods to predict which patients would benefit from irradiation is urgent. Promoter DNA hypermethylation was recently reported to correlate with favorable OPSCC prognosis. It is still unclear, however, whether there is an association between promoter DNA methylation and response to irradiation. In this study, we analyzed DNA methylation in the specimens from 40 OPSCC patients who had undergone irradiation, using the Infinium assay. Our results showed significant correlation between high levels of promoter DNA methylation and better response to treatment (P < 0.01). We used the 10 most differentially-methylated genes between responders and non-responders to develop a panel of predictive markers for efficacy. Our panel had high sensitivity, specificity and accuracy (92%, 93% and 93%, respectively). We conducted pyrosequencing to quantitatively validate the methylation levels of 8 of the 10 marker genes (ROBO1, ULK4P3, MYOD1, LBX1, CACNA1A, IRX4, DPYSL3 and ELAVL2) obtained by Infinium. The validation by pyrosequencing showed that these 8 genes had a high prediction performance for the training set of 40 specimens and for a validation set of 35 OPSCC specimens, showing 96% sensitivity, 89% specificity and 94% accuracy. Methylation of these markers correlated significantly with better progression-free and overall survival rates, regardless of human papillomavirus status. These results indicate that increased DNA methylation is associated with better responses to irradiation therapy and that DNA methylation can help establish efficacy prediction markers in OPSCC.

Efficacy of Desloratadine and Levocetirizine in Patients with Cedar Pollen-Induced Allergic Rhinitis: A Randomized, Double-Blind Study.

BACKGROUND: No comparative study of antihistamines that differ in structural system has been conducted in allergic rhinitis. OBJECTIVE: This was a randomized, double-blind, crossover comparative study to verify the efficacy of antihistamines that differ in structural system. METHODS: A total of 50 patients with moderate or more severe Japanese cedar pollen-induced allergic rhinitis were randomized to receive either placebo, desloratadine 5 mg (a tricyclic), or levocetirizine 5 mg (a piperazine). One dose of the study drug was orally administered at 9 pm on the day before a pollen exposure test, which was performed for 3 h (9 a.m. to 12 p.m.) to assess symptoms in an environmental challenge chamber (ECC). Nasal and ocular symptoms were compared at an airborne pollen level of 8,000 grains/m3. The primary endpoint was mean total nasal symptom score (TNSS) from 120 to 180 min in the ECC. Subjects with a difference of ≥1 in TNSS between 2 drugs were extracted to the relevant drug-responsive group. RESULTS: The difference in TNSS from placebo was -2.42 (p < 0.0001) with levocetirizine and -1.66 (p < 0.01) with desloratadine, showing that both drugs were significantly more effective than placebo in controlling symptoms, but with no statistically significant difference between the 2 drugs. There were 12 subjects in the desloratadine-responsive group and 24 subjects in the levocetirizine-responsive group, with no contributor to response was detected. CONCLUSION: Levocetirizine tended to control nasal symptoms more effectively than desloratadine. However, the response to each antihistamine varied among individuals and the predictors to the response are unknown. CLINICAL TRIAL REGISTRATION NUMBER: UMIN ID: UMIN000029653.

Sublingual administration of liposomes enclosing alpha-galactosylceramide as an effective adjuvant of allergen immunotherapy in a murine model of allergic rhinitis.

BACKGROUND: Sublingual immunotherapy (SLIT) is an established efficacious approach for the treatment of allergic rhinitis (AR). However, SLIT requires a long administration period to establish stable and adequate responses. This study investigated the efficacy of the sublingual administration of an allergen with liposomes enclosing α-GalCer (α-GC-liposome) as a potential adjuvant in mice with AR. METHODS: Mice with AR induced by OVA received the sublingual administration of OVA, α-GC-liposomes, or OVA plus α-GC-liposomes for 7 days. After nasal re-challenge with OVA, nasal symptoms were evaluated. The serum levels of OVA-specific Ig, the cytokine production of CD4+ T cells in the cultures of cervical lymph node (CLN) cells, and the gene expression of CLNs were analyzed. RESULTS: Although IL-4, IL-5 and IL-13 production from CD4+ T cells in CLN cells was significantly inhibited by the sublingual administration of OVA alone in mice with AR induced by OVA, their nasal symptoms were not significantly diminished. However, the combined sublingual administration of α-GC-liposomes and OVA completely suppressed nasal symptoms, downregulated Th2 and Th17 type cytokine production in CD4+ T cells as well as Th2 and Th17 gene expressions, and upregulated Th1 type cytokine production as well as Th1 gene expressions in CLN cells. Additionally, the serum levels of specific IgG2a were promoted, and specific IgE and IgG1 were inhibited. CONCLUSIONS: Our findings suggest that the sublingual administration of an allergen with α-GC-liposomes as an adjuvant might increase the therapeutic efficacy and effectiveness of this treatment method.

Involvement of aberrantly expressed microRNAs in the pathogenesis of head and neck squamous cell carcinoma.

MicroRNAs (miRNAs) are small noncoding RNAs that act as fine-tuners of the post-transcriptional control of protein-coding or noncoding RNAs by repressing translation or cleaving RNA transcripts in a sequence-dependent manner in cells. Accumulating evidence have been indicated that aberrantly expressed miRNAs are deeply involved in human pathogenesis, including cancers. Surprisingly, these small, single-stranded RNAs (18-23 nucleotides) have been shown to function as antitumor or oncogenic RNAs in several types of cancer cells. A single miRNA has regulating hundreds or thousands of different mRNAs, and individual mRNA has been regulated by multiple different miRNAs in normal cells. Therefore, tightly controlled RNA networks can be disrupted by dysregulated of miRNAs in cancer cells. Investigation of novel miRNA-mediated RNA networks in cancer cells could provide new insights in the field of cancer research. In this review, we focus on head and neck squamous cell carcinoma (HNSCC) and discuss current findings of the involvement of aberrantly expressed miRNAs in the pathogenesis of HNSCC.

The high expression of FOXA1 is correlated with a favourable prognosis in salivary duct carcinomas: a study of 142 cases.

AIMS: Salivary duct carcinoma (SDC) is an uncommon, aggressive tumour that, histologically, resembles high-grade mammary ductal carcinoma, and is characterised by the expression of androgen receptor (AR). The androgen signalling pathway, a potential therapeutic target, can be regulated by FOXA1. This study aimed to evaluate the clinicopathological implications of FOXA1 in SDC. METHODS AND RESULTS: We examined the relationship between the immunoexpression of FOXA1 and FOXA1 mutations and clinicopathological factors, including the biomarker status and clinical outcome, in 142 SDCs. FOXA1 was expressed in 128 SDCs (90.1%); the immunoexpression was heterogeneous. SDCs with a higher FOXA1 labelling index (LI) (≥20%) more frequently showed less advanced tumors on T classification (P = 0.002). FOXA1 LI was correlated positively with the AR expression value (r = 0.430, P < 0.001). PI3K and p-mTOR positivity, and intact-PTEN, were associated with a higher FOXA1 LI. Twenty-two of 121 SDCs (18.2%) harboured FOXA1 gene mutations at the flanking regions in and around the forkhead DNA binding domain; however, the given gene mutation and the expression of FOXA1 were not significantly correlated. A multivariate analysis revealed that SDCs with a higher FOXA1 LI were associated with longer overall survival and progression-free survival (P = 0.029 and 0.016, respectively). CONCLUSIONS: In SDC, FOXA1, which may biologically interact with the AR and PI3K signalling pathways, is a putative biomarker that may be associated with a favourable prognosis. Further studies are needed to apply the findings to the development of targeted personalised therapy for patients with SDC.

Regulation of ITGA3 by the anti-tumor miR-199 family inhibits cancer cell migration and invasion in head and neck cancer.

For patients with head and neck squamous cell carcinoma (HNSCC), survival rates have not improved due to local recurrence and distant metastasis. Current targeted molecular therapies do not substantially benefit HNSCC patients. Therefore, it is necessary to use advanced genomic approaches to elucidate the molecular mechanisms underlying the aggressiveness of HNSCC cells. Analysis of our microRNA (miRNA) expression signature by RNA sequencing showed that the miR-199 family (miR-199a-5p, miR-199a-3p, miR-199b-5p and miR-199b-3p) was significantly reduced in cancer tissues. Ectopic expression of mature miRNA demonstrated that all members of the miR-199 family inhibited cancer cell migration and invasion by HNSCC cell lines (SAS and HSC3). These findings suggested that both passenger strands and guide strands of miRNA are involved in cancer pathogenesis. In silico database and genome-wide gene expression analyses revealed that the gene coding for integrin α3 (ITGA3) was regulated by all members of the miR-199 family in HNSCC cells. Knockdown of ITGA3 significantly inhibited cancer cell migration and invasion by HNSCC cells. Moreover, overexpression of ITGA3 was confirmed in HNSCC specimens, and high expression of ITGA3 predicted poorer survival of the patients (P = 0.0048). Our data revealed that both strands of pre-miR-199a (miR-199a-5p and miR-199a-3p) and pre-miR-199b (miR-199b-5p and miR-199b-3p) acted as anti-tumor miRNA in HNSCC cells. Importantly, the involvement of passenger strand miRNA in the regulation of cellular processes is a novel concept in RNA research. Novel miRNA-based approaches for HNSCC can be used to identify potential targets for the development of new therapeutic strategies.