PKM2 promotes lymphatic metastasis of hypopharyngeal carcinoma via regulating epithelial-mesenchymal transition: an experimental research.

BACKGROUND: Patients with hypopharyngeal carcinoma (HPC) have a poor prognosis mainly because of lymphatic metastasis. This research aimed to determine the PKM2 role in lymphatic metastasis in HPC and the underlying molecular mechanism contributing to this phenomenon. METHODS: PKM2 in HPC was studied for its expression and its likelihood of overall survival using TCGA dataset. Western blotting, qRT-PCR, and IHC were employed to confirm PKM2 expression. Methods including gain- and loss-of-function were used to examine the PKM2 role in HPC metastasis in vitro and in vivo. In vitro and in vivo studies also confirmed lymphatic metastasis's mechanism. RESULTS: Prominent PKM2 overexpression was seen in patients with lymphatic metastasis of HPC, and there was an inherent relationship between a high PKM2 level and poor prognosis. In vitro research showed that knocking down PKM2 decreased tumor cell invasion, migration, and proliferation while promoting apoptosis and inhibiting epithelial-mesenchymal transition, but overexpressing PKM2 had the reverse effect. Animal studies suggested that PKM2 may facilitate tumor development and lymphatic metastasis. CONCLUSIONS: Our findings suggest that PKM2 may be a tumor's promoter gene of lymphatic metastasis, which may promote lymphatic metastasis of HPC by regulating epithelial-mesenchymal transition. PKM2 may be a biomarker of metastatic potential, ultimately providing a basis for exploring new therapeutic targets.

TRIM56: a promising prognostic immune biomarker for glioma revealed by pan-cancer and single-cell analysis.

Tripartite-motif 56 (TRIM56) is a member of the TRIM family, and was shown to be an interferon-inducible E3 ubiquitin ligase that can be overexpressed upon stimulation with double-stranded DNA to regulate stimulator of interferon genes (STING) to produce type I interferon and thus mediate innate immune responses. Its role in tumors remains unclear. In this study, we investigated the relationship between the expression of the TRIM56 gene and its prognostic value in pan-cancer, identifying TRIM56 expression as an adverse prognostic factor in glioma patients. Therefore, glioma was selected as the primary focus of our investigation. We explored the differential expression of TRIM56 in various glioma subtypes and verified its role as an independent prognostic factor in gliomas. Our research revealed that TRIM56 is associated with malignant biological behaviors in gliomas, such as proliferation, migration, and invasion. Additionally, it can mediate M2 polarization of macrophages in gliomas. The results were validated in vitro and in vivo. Furthermore, we utilized single-cell analysis to investigate the impact of TRIM56 expression on cell communication between glioma cells and non-tumor cells. We constructed a multi-gene signature based on cell markers of tumor cells with high TRIM56 expression to enhance the prediction of cancer patient prognosis. In conclusion, our study demonstrates that TRIM56 serves as a reliable immune-related prognostic biomarker in glioma.

Prognostic analysis of surgical treatment for T3 glottic laryngeal cancer based on different tumor extension patterns.

BACKGROUND AND OBJECTIVES: To investigate the prognostic impact of different tumor invasion patterns in the surgical treatment of T3 glottic laryngeal cancer. METHODS: We conducted a retrospective analysis of clinical data of 91 patients with T3 glottic laryngeal cancer. RESULTS: We found that the posterior invasion being significantly associated with involvement of the lamina of cricoid cartilage (P < 0.001), arytenoid cartilage (P = 0.001), and subglottic (P = 0.001). There was no statistical difference in survival outcomes between the total laryngectomy (TL) group and the partial laryngectomy (PL) group, but in the PL group, tumors with anterior invasion were associated with a better 5-year DFS than tumors with posterior invasion (HR: 4.681, 95% CI: 1.337-16.393, P = 0.016), and subglottic involvement was associated with worse LRRFS (HR: 3.931, 95% CI: 1.054-14.658, P = 0.041). At the same time, we found that involvement of the lamina of cricoid cartilage was an independent risk factor for postoperative laryngeal stenosis in PL patients (HR: 11.67, 95% CI: 1.89-71.98, P = 0.008). CONCLUSION: Selectively performed PL can also achieve favorable oncological outcomes comparable to those of TL. Posterior invasion and subglottic involvement are independent prognostic factors for recurrence after PL in T3 glottic laryngeal cancer, and involvement of the lamina of cricoid cartilage is associated with postoperative laryngeal stenosis. The tumor invasion pattern of patients with laryngeal cancer should be further subdivided to allow for selection of a more individualized treatment plan.

[Prognostic impact of different tumor invasion patterns in the surgical treatment of T3 glottic laryngeal cancer].

Objective:To investigate the prognostic impact of different tumor invasion patterns in the surgical treatment of T3 glottic laryngeal cancer. Methods:A retrospective analysis was conducted on the clinical data of 91 patients with T3 glottic laryngeal cancer. Results:Among the 91 patients, 58 cases （63.7%） had anterior invasion and 33 cases （36.3%） had posterior invasion. The posterior invasion was significantly correlated with invasions of the dorsal plate of cricoid cartilage （P<0.001）, arytenoid cartilage （P= 0.001）, and subglottic region（P = 0.001）. There was no statistical difference in survival outcomes between the total laryngectomy group and the partial laryngectomy group. But in the partial laryngectomy group, the 5-year disease-free survival（DFS） of patients with anterior invasive tumors was better than that of patients with posterior invasion tumors （HR: 4.681, 95%CI 1.337-16.393, P=0.016）, and subglottic invasion was associated with worse loco-regional recurrence-free survival（LRRFS）（HR: 3.931, 95%CI 1.054-14.658, P=0.041）. At the same time, we found that involvement of the dorsal plate of cricoid cartilage was an independent risk factor for postoperative laryngeal stenosis in partial laryngectomy patients （HR:11.67, 95%CI 1.89-71.98,P=0.008）. Conclusion:Compared with total laryngectomy, selected partial laryngectomy can also achieve favorable oncological outcomes. Posterior invasion and subglottic extension are independent prognostic factors for recurrence of partial laryngectomy in T3 glottic laryngeal cancer, and the involvement of the dorsal plate of cricoid cartilage is associated with postoperative laryngeal stenosis. The tumor invasion pattern of laryngeal cancer should be further subdivided in order to select a more individualized treatment plan.

[Clinical analysis of diversity of defect repair with supraclavicular island flap after head and neck tumor surgery].

Objective:To investigate the diversity and clinical effect of supraclavicular island flap in repairing the defect after head and neck tumor surgery. Methods:A retrospective analysis was performed on 30 patients who received the repair of head and neck defects with supraclavicular island flaps at Department of Otorhinolaryngology Head and Neck Surgery of the First Affiliated Hospital of Chongqing Medical University from January 2017 to March 2023. The sites and types of defects, intraoperative blood loss, time of flaps preparation, areas of flaps, survival of the flaps and other complications were recorded. Results:A total of 30 patients were enrolled, including 26 males and 4 females, aged 36-82 years. Among them, 22 patients with hypopharyngeal partial defect were repaired （19 patients with ipsilateral defect and 3 patients with contralateral defect）. In addition, 2 patients were repaired with contralateral pectoralis major musculocutaneous flap around the hypopharynx, the neck skin defect was repaired in 2 patients, the parotid skin defect was repaired in 2 patients, the temporal bone skin defect was repaired in 1 patient, and the cervical esophageal defect was repaired in 1 patient. The average blood loss during the operation was 8 ml, and the average time was 32 min. The flap areas ranged from 5.0 cm×4.0 cm to 20.0 cm×8.0 cm. 27 of 30 flaps survived（90.0%）, and pharyngeal fistula occurred in 6 patients after operation（4 flaps survived after local dressing）. One patient was complicated with venous thrombosis（the flap necrosis after local dressing）. Shoulder and neck functions（lift, internal rotation and abduction） were not significantly affected in 29 patients, and the function of 1 patient with shoulder infection was not affected after treatment. Conclusion:Supraclavicular island flap is a highly vascularized axial fascial flap. It is easy to make, thin, and soft in texture, and can be used to repair different sites and types of postoperative head and neck tumor defects with a low donor site complication rate. Good results in post-operative repair of head and neck tumors are worth promoting.

[Significance of neoadjuvant immunotherapy combined with chemotherapy in the treatment of larynx preservation in locally advanced hypopharyngeal squamous cell carcinoma].

Objective:To evaluate the clinical significance of neoadjuvant immunotherapy combined with chemotherapy in the treatment of larynx preservation in locally advanced hypopharyngeal squamous cell carcinoma. Methods:Patients with locally advanced HPSCC（cT3-T4aN0-N3M0） were eligible. All received 2 cycles of pembrolizumab combined with docetaxel and platinum neoadjuvant induction therapy. After two cycles, the efficacy was evaluated, followed by radical chemoradiotherapy or surgery and adjuvant chemoradiotherapy according to the efficacy. The primary endpoints were objective response rate（ORR） ，larynx-preservation（LP） rate at 3 months post-treatment and the adverse reactions during neoadjuvant therapy. Results:From December 2021 to December 2022, 10 patients with locally advanced HPSCC（cT3-T4aN0-N3M0） were enrolled. After 2 cycles of the neoadjuvant therapy, 2 patients achieved complete response（CR）, 7 patients achieved partial response（PR）, 1 patient was stable disease（SD）, objective response rate（ORR） was 90%, and disease control rate（DCR） was 100%. 5 patients received radical chemoradiotherapy, 5 patients received surgery and adjuvant chemoradiotherapy, four of them received partial laryngectomy and partial hypopharyngeal resection surgery, and one of them received total laryngectomy and partial hypopharyngeal resection surgery. All patients were able to withstand adverse reactions of neoadjuvant therapy and successfully completed the whole treatment of HPSCC without grade 3-4 treatment-related adverse reactions. There was no recurrence or metastasis during 3-18 months of follow-up. 1 patient died of severe pneumonia 3 months after the completion of radical chemoradiotherapy. At 3 months after treatment, the larynx-preservation rate was 80%. Conclusion:Neoadjuvant immunotherapy combined with chemotherapy has good short-term efficacy and the adverse reactions were tolerable. It can improve the larynx-preservation rate of patients with locally advanced HPSCC, thus improving the prognosis and quality of life of patients.

HSP90AA1 promotes lymphatic metastasis of hypopharyngeal squamous cell carcinoma by regulating epithelial-mesenchymal transition.

Background: Lymphatic metastasis (LM) emerges as an independent prognostic marker for hypopharyngeal squamous cell carcinoma (HSPSCC), chiefly contributing to treatment inefficacy. This study aimed to scrutinize the prognostic relevance of HSP90AA1 and its potential regulatory mechanism of concerning LM in HPSCC. Methods: In a preceding investigation, HSP90AA1, a differential gene, was discovered through transcriptome sequencing of HPSCC tissues, considering both the presence and absence of LM. Validation of HSP90AA1 expression was accomplished via qRT-PCR, western-blotting(WB), and immunohistochemistry(IHC), while its prognostic significance was assessed employing Kaplan-Meier survival analysis(KMSA), log-rank test(LR), and Cox's regression analysis(CRA). Bioinformatics techniques facilitated the prediction and analysis of its plausible mechanisms in LM, further substantiated by in vitro and in vivo experiments utilizing FaDu cell lines. Results: HSP90AA1 is substantially up-regulated in HPSCC with LM and is identified as an independent prognostic risk determinant. The down-regulation of HSP90AA1 can achieve inhibition of tumor cell proliferation, migration and invasion. Both in vivo experiments and Bioinformatics exploration hint at promoting LM by Epithelial-mesenchymal transition (EMT), regulated by HSP90AA1. Conclusions: HSP90AA1, by controlling EMT, can foster LM in HPSCC.This finding sets the foundation for delving into new therapeutic targets for HPSCC.

miR-19a mediates the mechanism by which SPHK2 regulates hypopharyngeal squamous cell carcinoma progression through the PI3K/AKT axis.

This study explored the expression of sphingosine kinase 2 (SPHK2) and microRNA miR-19a-3p (miR-19a-3p) in patients with Hypopharyngeal squamous cell carcinoma (HSCC) together with pathways affecting HSCC invasion and metastasis. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting (WB) were performed to assess the differential expression of SPHK2 and miR-19a-3p in patients with HSCC lymph node metastasis (LNM). Immunohistochemical (IHC) results were analyzed together with clinical information to evaluate their clinical significance. Subsequently, the functional effects of SPHK2 overexpression and knockdown on FaDu cells were evaluated in in vitro experiments. We performed in vivo experiments using nude mouse to assess the effects of SPHK2 knockdown on tumor formation, growth and LNM. Finally, we explored upstream and downstream signaling pathways associated with SPHK2 in HSCC. SPHK2 was significantly elevated in HSCC patients with LNM and survival was lower in patients with enhanced SPHK2 expression (P < 0.05). We also demonstrated that SPHK2 overexpression accelerated the proliferation, migration, and invasion. Using animal models, we further verified that SPHK2 deletion abrogated tumor growth and LNM. In terms of mechanism, we found that miR-19a-3p was significantly reduced in HSCC patients with LNM and was negatively associated with SPHK2. MiR-19a-3p and SPHK2 could regulate tumor proliferation and invasion through the PI3K/AKT axis. SPHK2 was found to contribute significantly to both LNM and HSCC patient prognosis and was shown to be an independent risk factor for LNM and staging in HSCC patients. The miR-19a-3p/SPHK2/PI3K/AKT axis was found to contribute to the development and outcome of HSCC.

Pedicled thoracoacromial artery compound flaps for circumferential hypopharyngeal reconstruction.

BACKGROUND: Total laryngeal and hypopharyngeal resection remained to be the mainly treatment option for advanced hypopharyngeal cancer, which resulted in complicated reconstructive challenge for circumferential hypopharyngeal defect. The pedicled thoracoacromial artery compound flaps included Thoracoacromial artery perforator (TAAP) flap and pectoralis major myocutaneous (PMMC) flap. This study is to evaluate the clinical application of the pedicled thoracoacromial artery compound flaps for circumferential hypopharyngeal reconstruction. METHODS: From May 2021 to April 2022, four hypopharyngeal cancer patients with circumferential hypopharyngeal defects were reconstructed by the pedicled thoracoacromial artery compound flaps. All patients were males. Patient age ranged from 35 to 62 years (average, 50 years). The Shoulder function were evaluated by SPADI. The average follow-up was 10.25 months (range from 4 to 18 months). RESULTS: All of the pedicled thoracoacromial artery compound flaps in our study survived. The defect length between tongue base and cervical esophagus ranged from 8 to 10 cm after total laryngeal and hypopharyngeal resection. The TAAP flap size ranged from 6 × 7 cm to 7 × 10 cm, and the PMMC flap size ranged from 6 × 7 cm to 9 × 12 cm. The pedicle length of TAAP and PMMC flaps varied, respectively, from 5 to 8 cm (mean 6.5 cm) and 7 cm to 11 cm (mean 8.75 cm). The mean time of TAAP and PMMC flaps harvest was, respectively, 82 min and 39 min. All patients were able to resume soft diet in the fourth week of postoperation, but one patient was operated by gastrostomy in the second month of postoperation because of pharyngeal cavity stenosis, and the patient successfully resumed oral soft diet by endoscopic balloon dilation after postoperation radiotherapy. All patients have resumed oral feeding at last. There were mild dysfunction for our patients according to SPADI during the mid-long follow-up. CONCLUSIONS: The pedicled thoracoacromial artery compound flaps have stable blood supply and provide adequate muscle coverage for greater protection during radiotherapy, and the microsurgical skills have no requirement. Therefore, the compound flaps provide a good choice for the reconstruction of circumferential hypopharyngeal defect, especially in the aged or patients with comorbidities who are not able to tolerate prolonged surgery.

Implantation of hydrogel-liposome nanoplatform inhibits glioblastoma relapse by inducing ferroptosis.

Glioblastoma is acknowledged as the most aggressive cerebral tumor in adults. However, the efficacy of current standard therapy is seriously undermined by drug resistance and suppressive immune microenvironment. Ferroptosis is a recently discovered form of iron-dependent cell death that may have excellent prospect as chemosensitizer. The utilization of ferropotosis inducer Erastin could significantly mediate chemotherapy sensitization of Temozolomide and exert anti-tumor effects in glioblastoma. In this study, a combination of hydrogel-liposome nanoplatform encapsulated with Temozolomide and ferroptosis inducer Erastin was constructed. The αvß3 integrin-binding peptide cyclic RGD was utilized to modify codelivery system to achieve glioblastoma targeting strategy. As biocompatible drug reservoirs, cross-linked GelMA (gelatin methacrylamide) hydrogel and cRGD-coated liposome realized the sustained release of internal contents. In the modified intracranial tumor resection model, GelMA-liposome system achieved slow release of Temozolomide and Erastin in situ for more than 14 d. The results indicated that nanoplatform (T+E@LPs-cRGD+GelMA) improved glioblastoma sensitivity to chemotherapeutic temozolomide and exerted satisfactory anti-tumor effects. It was demonstrated that the induction of ferroptosis could be utilized as a therapeutic strategy to overcome drug resistance. Furthermore, transcriptome sequencing was conducted to reveal the underlying mechanism that the nanoplatform (T+E@LPs-cRGD+GelMA) implicated in. It is suggested that GelMA-liposome system participated in the immune response and immunomodulation of glioblastoma via interferon/PD-L1 pathway. Collectively, this study proposed a potential combinatory therapeutic strategy for glioblastoma treatment.

ANXA6/TRPV2 axis promotes lymphatic metastasis in head and neck squamous cell carcinoma by inducing autophagy.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is highly aggressive with a significant tropism of lymph nodes, which restricts treatment options and negatively impacts patient outcomes. Although progress has been made in understanding the molecular mechanisms underlying lymphatic metastasis (LM), these mechanisms remain elusive. ANXA6 is a scaffold protein that participates in tumor pathogenesis and autophagy regulation; however, how ANXA6 affects autophagy and LM in HNSCC cells remains unknown. METHODS: RNA sequencing was performed on HNSCC clinical specimens with or without metastasis as well as on The Cancer Genome Atlas dataset to investigate ANXA6 expression and survival. Both in vitro and in vivo studies were performed to investigate the role of ANXA6 in the regulation of LM in HNSCC. The molecular mechanism by which ANXA6 interacts with TRPV2 was examined at the molecular level. RESULTS: ANXA6 expression was significantly upregulated in HNSCC patients with LM and higher expression was associated with poor prognosis. ANXA6 overexpression promoted the proliferation and mobility of FaDu and SCC15 cells in vitro; however, ANXA6 knockdown retarded LM in HNSCC in vivo. ANXA6 induced autophagy by inhibiting the AKT/mTOR signaling pathway in HNSCC, thereby regulating the metastatic capability of the disease. Furthermore, ANXA6 expression positively correlated with TRPV2 expression both in vitro and in vivo. Lastly, TRPV2 inhibition reversed ANXA6-induced autophagy and LM. CONCLUSIONS: These results indicate that the ANXA6/TRPV2 axis facilitates LM in HNSCC by stimulating autophagy. This study provides a theoretical basis for investigating the ANXA6/TRPV2 axis as a potential target for the treatment of HNSCC, as well as a biomarker for predicting LM.

CEACAM5 inhibits the lymphatic metastasis of head and neck squamous cell carcinoma by regulating epithelial-mesenchymal transition via inhibiting MDM2.

Lymph node (LN) metastasis affects both the management and prognosis of head and neck squamous cell carcinoma (HNSCC). Here, we explored the relationship between lymphatic metastasis and CEA family member 5 (CEACAM5), including its possible regulatory role in HNSCC. The levels of CEACAM5 in tissues from patients with HNSCC, with and without LN metastases, were assessed by transcriptome sequencing. The associations between CEACAM5 and the N stage of LN metastasis in HNSCC were predicted through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and a pan-cancer analysis of CEACAM5 expression in 33 common human tumors was conducted. CEACAM5 levels were analyzed in tumor and normal tissue specimens from HNSCC patients and the correlation between CEACAM5 levels and prognosis was evaluated. The influence of CEACAM5 on cell proliferation, invasion, migration, and apoptosis was investigated in HNSCC cell lines, as were the downstream regulatory mechanisms. A mouse model of LN metastasis was constructed. CEACAM5 levels were significantly higher in HNSCC tissue without LN metastasis than in that with LN metastasis. Similar findings were obtained for the clinical specimens. CEACAM5 levels were associated with better clinical prognosis. CEACAM5 was found to inhibit the proliferation and migration and promote the apoptosis of HNSCC cells. A mouse xenograft model showed that CEACAM5 inhibited LN metastasis. In conclusions, CEACAM5 inhibited epithelial-mesenchymal transition (EMT) in HNSCC by reducing murine double minute 2 (MDM2) expression and thereby suppressing LN metastasis. CEACAM5 has potential as both a prognostic marker and a therapeutic target in HNSCC.

RBM24 Mediates Lymph Node Metastasis and Epithelial-Mesenchymal Transition in Human Hypopharyngeal Squamous Cell Carcinoma by Regulating Twist1.

Objective: Despite the target RNA regulatory action of RBM24 (RNA Binding Motif 24), a protein implicated in multiple carcinomas, its role in HSCC remains unclear. Our study probed to understand the effect of RBM24 on HSCC. Materials and Methods: A combination of qRT-PCR, IHC, and western blot was employed to assess the HSCC tissue level of RBM24. The colony formation and CCK-8 assays were performed to estimate cellular proliferative potential, whereas the transwell assay was conducted to examine invasive and metastatic potential. The FaDu cell motility was assessed via the scratch-wound assay and EMT (epithelial-mesenchymal transition) by adopting qRT-PCR in conjunction with western blot and IF (immunofluorescence). The in-vivo effect of RBM24 on HSCC was investigated through modeling metastasis to the popliteal LNs (lymph nodes). Results: Among HSCC patients showing metastasis to LNs, prominent RBM24 downregulation was noted, with an intrinsic association between low RBM24 level and poor outcome. Knocking down RBM24 promoted cell multiplication, migration, and infiltration, while overexpression led to the opposite effects and inhibited the EMT. RBM24's suppressive action against the FaDu cell mobility and invasion was reversed by Twist1 overexpression. RBM24's suppressive actions against the tumor evolution and LN metastasis in HSCC in-vivo were also validated. Conclusion: As a carcinoma inhibitor gene, RBM24 regulates Twist1 to achieve LN metastasis and EMT suppression in HSCC.

Mechanisms of long non-coding RNAs in biological phenotypes and ferroptosis of glioma.

Glioma, one of the most common malignant tumors in the nervous system, is characterized by limited treatment, high mortality and poor prognosis. Numerous studies have shown that lncRNAs play an important role in the onset and progression of glioma by acting on various classical signaling pathways of tumors through signaling, trapping, guiding, scaffolding and other functions. LncRNAs contribute to the malignant progression of glioma via proliferation, apoptosis, epithelial-mesenchymal transformation, chemotherapy resistance, ferroptosis and other biological traits. In this paper, relevant lncRNA signaling pathways involved in glioma progression were systematically evaluated, with emphasis placed on the specific molecular mechanism of lncRNAs in the process of ferroptosis, in order to provide a theoretical basis for the application of lncRNAs in the anticancer treatment of glioma.

RAF1 promotes lymphatic metastasis of hypopharyngeal carcinoma via regulating LAGE1: an experimental research.

BACKGROUND: Lymphatic metastasis was an independent prognostic risk factor for hypopharyngeal carcinoma and was the main cause of treatment failure. The purpose of this study was to screen the differential genes and investigate the mechanism of lymphatic metastasis in hypopharyngeal carcinoma. METHODS: Transcriptome sequencing was performed on primary tumors of patients, and differential genes were screened by bioinformatics analysis. The expression of differential genes was verified by qRT-PCR, western-blotting and immunohistochemical, and prognostic value was analyzed by Kaplan-Meier and log-rank test and Cox's test. Next, FADU and SCC15 cell lines were used to demonstrate the function of differential genes both in vitro by EdU, Flow cytometry, Wound Healing and Transwell assays and in vivo by a foot-pad xenograft mice model. Proteomic sequencing was performed to screen relevant targets. In addition, in vitro and in vivo experiments were conducted to verify the mechanism of lymphatic metastasis. RESULTS: Results of transcriptome sequencing showed that RAF1 was a significantly differential gene in lymphatic metastasis and was an independent prognostic risk factor. In vitro experiments suggested that decreased expression of RAF1 could inhibit proliferation, migration and invasion of tumor cells and promote apoptosis. In vivo experiments indicated that RAF1 could promote tumor growth and lymphatic metastasis. Proteomic sequencing and subsequent experiments suggested that LAGE1 could promote development of tumor and lymphatic metastasis, and was regulated by RAF1. CONCLUSIONS: It suggests that RAF1 can promote lymphatic metastasis of hypopharyngeal carcinoma by regulating LAGE1, and provide a basis for the exploring of novel therapeutic target and ultimately provide new guidance for the establishment of intelligent diagnosis and precise treatment of hypopharyngeal carcinoma.

TBC1D14 inhibits autophagy to suppress lymph node metastasis in head and neck squamous cell carcinoma by downregulating macrophage erythroblast attacher.

Aims: This study aimed to identify the correlation and molecular mechanism between TBC1 domain family member 14 (TBC1D14) and lymph node metastasis (LNM) in head and neck squamous cell carcinoma (HNSCC). Methods: Whole transcriptome sequencing of HNSCC tissues with or without LNM was performed. TBC1D14 expression was quantified in HNSCC tissues. The role of TBC1D14 in HNSCC migration, invasion, autophagy, and LNM was investigated by wound healing, Transwell, western blotting, immunofluorescence, and transmission electron microscopy assays in vitro and in a mouse model in vivo. The correlation between autophagy and LNM was detected by wound healing and Transwell assays in vitro and western blotting in vivo. Mass spectrometry was used to identify the downstream target proteins. The correlation between TBC1D14 expression and macrophage erythroblast attacher (MAEA) expression was identified by qRT-PCR and western blotting assays in vitro and immunohistochemistry in vivo. The gain-of-function strategy was applied to further reveal the role of MAEA in the TBC1D14-induced autophagy of HNSCC cells. Results: TBC1D14 was a co-differentially expressed gene in the sequencing results, The Cancer Genome Atlas Data Portal, and Gene Expression Omnibus databases. TBC1D14 had a lower RNA and protein expression in HNSCC with LNM samples and was a favorable prognostic indicator. TBC1D14 inhibited the migration and invasion of HNSCC in vivo. Mechanistically, TBC1D14-induced autophagy suppression inhibited the migration and invasion of HNSCC. TBC1D14 expression negatively correlated with MAEA expression both in vitro and in vivo. Furthermore, MAEA overexpression could reverse TBC1D14-induced autophagy suppression. Conclusion: TBC1D14 is a novel LNM inhibitor in HNSCC and a favorable prognostic marker. TBC1D14 suppresses autophagy to inhibit LNM in HNSCC by downregulating MAEA expression. The results clarify the molecular mechanism of TBC1D14 in HNSCC.

RNA N6-methyladenosine reader IGF2BP2 promotes lymphatic metastasis and epithelial-mesenchymal transition of head and neck squamous carcinoma cells via stabilizing slug mRNA in an m6A-dependent manner.

BACKGROUND: Lymph node metastasis is the main cause of poor prognosis of head and neck squamous carcinoma (HNSCC) patients. N6-methyladenosine (m6A) RNA modification is an emerging epigenetic regulatory mechanism for gene expression, and as a novel m6A reader protein, IGF2BP2 has been implicated in tumor progression and metastasis. However, not much is currently known about the functional roles of IGF2BP2 in HNSCC, and whether IGF2BP2 regulates lymphatic metastasis through m6A modification in HNSCC remains to be determined. METHODS: The expression and overall survival (OS) probability of m6A-related regulators in HNSCC were analyzed with The Cancer Genome Atlas (TCGA) dataset and GEPIA website tool, respectively. The expression levels of IGF2BP2 were measured in HNSCC tissues and normal adjacent tissues. To study the effects of IGF2BP2 on HNSCC cell metastasis in vitro and in vivo, gain- and loss- of function methods were employed. RIP, MeRIP, luciferase reporter and mRNA stability assays were performed to explore the epigenetic mechanism of IGF2BP2 in HNSCC. RESULTS: We investigated 20 m6A-related regulators in HNSCC and discovered that only the overexpression of IGF2BP2 was associated with a poor OS probability and an independent prognostic factor for HNSCC patients. Additionally, we demonstrated that IGF2BP2 was overexpressed in HNSCC tissues, and significantly correlated to lymphatic metastasis and poor prognosis. Functional studies have shown that IGF2BP2 promotes both HNSCC cell migration as well as invasion via the epithelial-mesenchymal transition (EMT) process in vitro, and IGF2BP2 knockdown significantly inhibited lymphatic metastasis and lymphangiogenesis in vivo. Mechanistic investigations revealed that Slug, a key EMT-related transcriptional factor, is the direct target of IGF2BP2, and essential for IGF2BP2-regulated EMT and metastasis in HNSCC. Furthermore, we demonstrated that IGF2BP2 recognizes and binds the m6A site in the coding sequence (CDS) region of Slug and promotes its mRNA stability. CONCLUSIONS: Collectively, our study uncovers the oncogenic role and potential mechanism of IGF2BP2, which serves as a m6A reader, in controlling lymphatic metastasis and EMT in HNSCC, suggesting that IGF2BP2 may act as a therapeutic target and prognostic biomarker for HNSCC patients with metastasis.

Expression and Functional Relevance of ANXA1 in Hypopharyngeal Carcinoma with Lymph Node Metastasis.

PURPOSE: The purpose of this study is to investigate the expression and functional role of Annexin (ANXA1) in lymph node (LN) metastasis of hypopharyngeal carcinoma (HSCC). METHODS: Differentially expressed genes in tissue from HSCC with or without LN metastasis were obtained from a previous RNA sequencing experiment. The presence of LN metastasis is determined by pathological diagnosis after neck dissection. ANXA1 expression was detected by qRT-PCR and Western blotting. Immunohistochemistry was used to detect the expression of ANXA1 in 74 cases of HSCC and normal control tissues. We also evaluated the clinical significance of ANXA1 in HSCC. Differentially expressed genes related to ANXA1 were analyzed using bioinformatic tools, and potential mechanisms of action of ANXA1 were assessed using in vitro experiments. In these in vitro experiments, cell proliferation was detected by CCK8 staining, and colony formation, migration and invasion were assessed using Transwell assays, and apoptosis as well as cell cycle status were quantified by flow cytometry. RESULTS: ANXA1 was significantly downregulated in HSCC with LN metastasis. The survival rate of patients with low ANXA1 expression was significantly worse than that of patients with high ANXA1 expression (p<0.05). Silencing ANXA1 in cell culture experiments promoted the proliferation, migration and invasion of FaDu cells, inhibited apoptosis, and increased the proportion of cells in S phase. We furthermore found that the mRNA expression of ANXA1 was positively correlated with Yap1 expression (p<0.0001). Our in vitro experiments showed that ANXA1 regulates the expression of Yap1, and over-expression of Yap1 could reverse the effect of ANXA1 silencing on cancer cell progression. CONCLUSION: Our findings suggest that ANXA1 is a putative LN metastasis suppressor gene in tumor, which may suppress the LN metastasis of HSCC by regulating the expression of Yap1.

Extranodal Extension as an Independent Prognostic factor in Laryngeal Squamous Cell Carcinoma Patients.

The presence of Lymph node metastasis with extranodal extension (ENE) is considered to be an important adverse prognostic factor for survival in patients with head and neck cancer. The aim of this study was to determine the prognostic significance of ENE in patients with laryngeal squamous cell carcinoma (LSCC). Three hundred and fifty-five patients with LSCC who underwent surgical resection and neck dissection were included. The status of cervical lymph node was classified into three groups: pathological negative nodal (pN-), pathological positive nodal without ENE (ENE-), and pathological positive nodal with ENE (ENE+). A total of 85 of 355 (23.9%) LSCC were pathological nodal positive, and ENE was detected in 22/355 (6.2%) patients. ENE was associated with drinking (p=0.005), T stage (p=0.000), tumor location (p=0.000), and differentiation degree (p=0.000). The number of lymph node metastasis in ENE+ group was associated with almost twice compared to ENE- group (p=0.005). The 5-year overall survival rates for patients in the pN-, ENE-, and ENE+ groups were 86.4±2.6%, 75.9±6.3%, and 53.7±12.7%, respectively (p=0.000). After adjusting for confounding variables, ENE+ was associated with more than five times the hazard of death than pN- cases (p=0.000), and more than twice the hazard of death than ENE- cases (p=0.036). Compared to N2-3/ENE- cases, N2-3/ENE+ cases had the poorest survival rate (p=0.013). ENE+ was associated with worse outcomes compared to pN - or ENE- status. ENE is an independent prognostic factor in LSCC, and could be an indicator of the need for adjuvant treatment.

[Imaging of the parapharyngeal space tumors and selection of the treatment].

Objective:To analyze the imaging features of parapharyngeal space tumors, and to guide the choice of treatment. Method:A retrospective analysis of 75 cases' clinical data of parapharyngeal space tumor was carried out. All patients underwent enhanced CT and/or enhanced MRI, those tumors closely related to blood vessels and vascular tumors were examined by CTA or MRA, those closely related to peripheral nerves were examined by MRN. We evaluated the relationship between tumor and carotid sheath, parotid gland, skull base and hard palate. We choose the treatment according to the imaging features. Result:Imaging revealed 69 benign tumors and 6 malignant tumors. 8 benign tumors chose to follow up, 61 benign tumors and 6 malignant tumors underwent surgery. Among them, 17 cases of tumors were operated by the transoral approach assisted by endoscope, which were located in the medial of carotid sheath, with the main body protruding toward the oropharynx, below the level of the hard palate, and more than 5 mm away from the skull base; 31 cases were resected by the transcervical approach, and the tumor body protruded toward the neck（15 cases were lateral of the carotid sheath, and 16 cases were medial）; 16 cases were resected by the transparotid approach, and the tumor harbored a broad interconnecting margin with the parotid gland; there were 3 cases of other approaches（1 case of transnasal approach, 1 case of combined transnasal and transcervical approach, and 1 case of combined transauricular-cervical approach）. Postoperative complications occurred in 25 patients, including 9 cases of facial paralysis（6 cases of grade Ⅱ, 2 cases of grade Ⅲ, 1 case of grade Ⅳ）, 1 case of facial numbness, 5 cases of Hornor's syndrome; tongue extension deflection in 3 cases, 2 cases cough while drinking water, 1 case of postoperative hematoma formation, 1 case of oral errhysis, hearing loss in 1 case, shoulder shrugging and hand lifting weakness in 1 case, salivary fistula in 1 case, tracheotomy was performed in 1 case because of postoperative oropharyngeal swelling, swallowing obstruction in 1 case, gustatory sweating syndrome in 1 case and acute cerebral infarction was caused by thrombus detachment in 1 case. Conclusion:Imaging results are important basis for the treatment of parapharyngeal space tumors. The choice of surgical approach should be made based on imaging, including the relationship with the big vessels of the neck, the parotid gland, the hard palate, and the distance from the skull base, combined with the nature of the tumor.