Partial epithelial mesenchymal transition (p-EMT) was found to play a potential role in the initial stage of metastasis in human head and neck squamous cell carcinoma (HNSCC). Some long noncoding RNAs (lncRNAs) have been reported to function as promoters or inhibitors of cancer metastasis. This study aimed to identify p-EMT-related lncRNAs in HNSCC. Differentially expressed lncRNAs (DE-lncRNAs) and mRNAs (DEGs) in HNSCC obtained from The Cancer Genome Atlas (TCGA) were screened out by using the “edgeR” package. DE-lncRNAs in the Oral squamous cell carcinoma (OSCC) lncRNA microarray dataset GSE84805 were screened out by using the “limma” package. Slug-related lncRNAs were determined by Pearson correlation analysis (|Pearson correlation coefficient| ≥ 0.4, p < 0.01) based on TCGA. Survival analysis were performed for the overlapping DE-lncRNAs by using the “Survival” package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to predict the potential functions of MYOSLID. RT-qPCR and In Site Hybridization (ISH) were used to explore the MYOSLID expression and its clinical significance in HNSCC specimens. Immunohistochemical staining, siRNA, wound healing assay, transwell assay, and western blot were used to explore the biological function and potential molecular mechanisms. MYOSLID was identified as a Slug-related lncRNA and with prognostic value among the 9 overlapping DE-lncRNAs. GO and KEGG analyses revealed that MYOSLID was closely related to important biological processes and pathways that regulate cancer metastasis. The results of univariate and multivariate Cox regression analysis based on TCGA and HNSCC tissue microarray data suggested MYOSLID was an independent prognostic factor. MYOSLID expression in HNSCC was closely correlated with Slug, PDPN and LAMB3. The knockdown of MYOSLID in OSCC cell line significantly inhibited cell migration and invasion compared to those in the control cells. In addition, the knockdown of MYOSLID significantly reduced Slug, PDPN and LAMB3 expression levels. However, the knockdown of MYOSLID had no effect on the expression levels of the EMT biomarkers E-cadherin and Vimentin. Our study revealed that MYOSLID expression was closely related to the p-EMT program in HNSCC, and it might be a new predictive biomarker for aggressive HNSCC.

: Oral squamous cell carcinoma (OSCC) represents over 90% of oral cancer incidence, while its mechanisms of tumorigenesis remain poorly characterized. In this study, we applied RNA-seq and microRNA-seq methodologies in four pairs of cancer and adjacent normal tissues to profile the contribution of miRNAs to tumorigenesis-altered functional pathways by constructing a comprehensive miRNA-mediated mRNA regulatory network. There were 213 differentially expressed (DE) miRNAs and 2172 DE mRNAs with the involvement of negative miRNA-mRNA interactions identified by at least two pairs of cancerous tissues. GO analysis revealed that the upregulated microRNAs significantly contributed to a global down-regulation of a number of transcription factors (TFs) in OSCC. Among the negative regulatory networks between the selected miRNAs (133) and TFs (167), circadian rhythm genes (RORA, RORB, RORC, and CLOCK) simultaneously regulated by multiple microRNAs were of particular interest. For instance, RORA transcript was predicted to be targeted by 25 co-upregulated miRNAs, of which, miR-503-5p, miR-450b-5p, miR-27a-3p, miR-181a-5p and miR-183-5p were further validated to directly target RORA, resulting in a stronger effect on RORA suppression together. In addition, we showed that the mRNA and protein expression levels of RORα were significantly decreased in most OSCC samples, associated with advanced clinical stage and poor prognosis. RORα significantly suppressed the proliferation of OSCC cells in vitro and in vivo. Attenuated RORα decreased p53 protein expression and suppressed p53 phosphorylation activity. Altogether, our results strongly suggest the importance of the role of miRNAs in regulating the activity of circadian rhythm-related TFs network during OSCC tumorigenesis, and provide further clues to understand the clinical link between circadian rhythm and cancer therapy.

X-box-binding protein 1 (XBP1) contributes to various types of cancer including breast, bladder cancer and esophageal squamous cell carcinoma. The aim of the study was to examine the metastatic role of XBP1 in oral squamous cell carcinoma (OSCC), and identify possible downstream molecules. Immunohistochemical staining was conducted on tissue microarrays comprising 96 OSCC cases to determine the expression level of XBP1 and analyze its association with metastasis, clinicopathological characteristics and survival prognosis. Compared with the adjacent normal tissues of OSCC, the expression of XBP1 was significantly increased in the tumor center and front area, and lymph nodes metastases (P<0.05). A relatively high XBP1 expression was associated with histological grades (P<0.05), advanced clinical stages (P<0.05), unfavorable 5-year survival (P=0.027). Suppressed XBP1 expression caused a significant reduction of cell invasion capability (P<0.05). AXL and the downstream molecules, such as PI3K, MMP1, MMP3, and uPA were significantly suppressed when XBP1 expression was inhibited in OSCC cells. Once XBP1 was activated by Thapsigargin, AXL expression was restored. Moreover, aberrant AXL expression was associated with XBP1 overexpression in OSCC tissues (P<0.05). In conclusion, XBP1 is a potential target that is relevant to suppressing cell invasion and is associated with patient prognosis in OSCC.

Purpose: Neoadjuvant anti–PD-1 immunotherapy combined with chemotherapy has shown promising pathologic responses in various cancers, including oral squamous cell carcinoma (OSCC). However, the pathologic response of lymph node (LN) metastases remains poorly understood. This study aims to systematically evaluate the pathologic response rates (pRR) of LN metastases in patients with OSCC and identify potential targets to improve therapeutic outcomes. Patients and Methods: We assessed the pRRs of LN metastases and matched primary tumors (PT) in patients with OSCC enrolled in a randomized, two-arm, phase II clinical trial (NCT04649476). Single-cell and spatial transcriptomics and multiplex IHC were performed to analyze the tumor microenvironment and identify potential therapeutic targets in LN metastases. A neoadjuvant orthotopic OSCC mouse model was established to evaluate the therapeutic potential of these targets. Results: We observed significant heterogeneity in pathologic regression of LN metastases, with lower pRRs compared with PTs. pRRs in LN metastases were correlated with overall and disease-free survival in patients with OSCC. We identified an abundance of macrophages in LN metastases exhibiting an unfolded protein response and activated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling. These macrophages contributed to an extracellular matrix–enriched microenvironment through interactions with fibroblasts, which hindered T cell–mediated cytotoxicity. Pharmacologic inhibition of the PERK pathway significantly enhanced anti–PD-1 therapy in LN metastases and PTs in the mouse model. Conclusions: Our study confirms that the pathologic response of LN metastases in patients with OSCC undergoing neoadjuvant immunotherapy or immunochemotherapy is inferior to that of PTs. It suggests that targeting the PERK pathway in macrophages could be a potential strategy to enhance treatment outcomes.

Neoadjuvant immunotherapy is a first-line treatment for recurrent and metastatic head and neck squamous cell carcinoma (HNSCC). However, only a fraction of patients with advanced HNSCC benefit from immunotherapy. Identifying accurate and accessible biomarkers is essential for optimal patient selection. In this study, we integrated single-cell RNA sequencing and T-cell receptor sequencing to comprehensively characterize the tumor immune microenvironment (TIME) of HNSCC biopsies prior to a phase II neoadjuvant immunotherapy clinical trial. Tumor-specific MHC-II (tsMHC-II) was identified as a superior predictor of response to neoadjuvant immunotherapy in HNSCC compared with PD-L1. Mechanistically, tsMHC-II ignited a hot TIME and enhanced the effect of PD-1 blockade by recruiting T cells through the induction of chemokines, particularly CCL5. Moreover, tsMHC-II triggered a Th1 response and activated CD4+ and CD8+ T-cell expansion, suppressing HNSCC growth in a CD4+ T-cell–dependent manner. Simultaneously, tsMHC-II facilitated an increase in PD-1+CD4+ T cells and a modest elevation in tumor PD-L1, thereby enhancing sensitivity to anti–PD-1 therapy. This study highlights that tsMHC-II, by generating an inflamed TIME, is crucial in enhancing the effectiveness of neoadjuvant immunotherapy in HNSCC. Significance: scRNA-seq analysis identifies tumor-specific MHC-II as a predictor for neoadjuvant immunotherapy response in HNSCC and provides insights into the complex networks that ignite a hot tumor immune microenvironment and regulate T-cell infiltration.