Large-Scale Identification of Lysine Crotonylation Reveals Its Potential Role in Oral Squamous Cell Carcinoma.

Purpose: Lysine crotonylation, an emerging posttranslational modification, has been implicated in the regulation of diverse biological processes. However, its involvement in oral squamous cell carcinoma (OSCC) remains elusive. This study aims to reveal the global crotonylome in OSCC under hypoxic conditions and explore the potential regulatory mechanism of crotonylation in OSCC. Methods: Liquid-chromatography fractionation, affinity enrichment of crotonylated peptides, and high-resolution mass spectrometry were employed to detect differential crotonylation in CAL27 cells cultured under hypoxia. The obtained data were further subjected to bioinformatics analysis to uncover the involved biological processes and pathways of the dysregulated crotonylated proteins. A site-mutated plasmid was utilized to investigate the effect of crotonylation on Heat Shock Protein 90 Alpha Family Class B Member 1 (HAP90AB1) function. Results: A large-scale crotonylome analysis revealed 1563 crotonylated modification sites on 605 proteins in CAL27 cells under hypoxia. Bioinformatics analysis revealed a significant decrease in histone crotonylation levels, while up-regulated crotonylated proteins were mainly concentrated in non-histone proteins. Notably, glycolysis-related proteins exhibited prominent up-regulation among the identified crotonylated proteins, with HSP90AB1 displaying the most significant changes. Subsequent experimental findings confirmed that mutating lysine 265 of HSP90AB1 into a silent arginine impaired its function in promoting glycolysis. Conclusion: Our study provides insights into the crotonylation modification of proteins in OSCC under hypoxic conditions and elucidates the associated biological processes and pathways. Crotonylation of HSP90AB1 in hypoxic conditions may enhance the glycolysis regulation ability in OSCC, offering novel perspectives on the regulatory mechanism of crotonylation in hypoxic OSCC and potential therapeutic targets for OSCC treatment.

Development and Validation of a Prognostic Signature Based on the Lysine Crotonylation Regulators in Head and Neck Squamous Cell Carcinoma.

Background: Lysine crotonylation (Kcr) is a newly identified posttranslational modification type regulated by various enzymes and coenzymes, including lysine crotonyltransferase, lysine decrotonylase, and binding proteins. However, the role of Kcr regulators in head and neck squamous cell carcinoma (HNSCC) remains unknown. The aim of this study was to establish and validate a Kcr-related prognostic signature of HNSCC and to assess the clinical predictive value of this signature. Methods: The mRNA expression profiles and clinicopathological data from The Cancer Genome Atlas (TCGA) database were downloaded to explore the clinical significance and prognostic value of these regulators in HNSCC. The least absolute shrinkage and selection operator (LASSO) Cox regression model was used to generate the Kcr-related prognostic signature for HNSCC. Subsequently, the GSE65858 dataset from the Gene Expression Omnibus (GEO) database was used to validate the signature. The prognostic value of the signature was evaluated using the Kaplan-Meier survival, receiver operating characteristic (ROC) curve, and univariate and multivariate Cox regression analyses. Results: We established a nine-gene risk signature associated with the prognosis of HNSCC based on Kcr regulators. High-risk patients demonstrated significantly poorer overall survival (OS) than low-risk patients in the training (TCGA) and validation (GEO) datasets. Then, the time-dependent receiver operating characteristic (ROC) curve analysis showed that the nine-gene risk signature was more accurate for predicting the 5-year OS than other clinical parameters, including age, gender, T stage, N stage, and histologic grade in the TCGA and GEO datasets. Moreover, the Cox regression analysis showed that the constructed risk signature was an independent risk factor for HNSCC. Conclusion: Our study identified and validated a nine-gene signature for HNSCC based on Kcr regulators. These results might contribute to prognosis stratification and treatment escalation for HNSCC patients.

HSP90AB1 Promotes the Proliferation, Migration, and Glycolysis of Head and Neck Squamous Cell Carcinoma.

Background: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide. Heat shock protein 90 alpha family class B member 1 (HSP90AB1) is highly expressed in a variety of cancers and is associated with poor prognosis, however, its role in HNSCC is still poorly understood. This study aimed to explore the function HSP90AB1 played in HNSCC progression. Methods: The expression level of HSP90AB1 in HNSCC was analyzed by bioinformatics analysis and western blotting, and its relationship with clinicopathological parameters was analyzed by bioinformatics analysis and immunohistochemistry. Three stable HSP90AB1 knockdown HNSCC cell lines were constructed by lentiviral transfection. The effect of HSP90AB1 knockdown on the proliferation and migration of HNSCC cells was tested by CCK-8 assay, EdU incorporation assay, colony formation assay, nude mouse xenograft models, transwell migration assay, wound healing assay, and western blotting. The effect of HSP90AB1 knockdown on glycolysis in HNSCC cells was assessed by quantitative real-time PCR and related assay kits. Finally, the levels of Akt and phospho-Akt (Ser473) proteins after HSP90AB1 knockdown were detected by western blotting. Results: HSP90AB1 was highly expressed in HNSCC and associated with T grade, lymph node metastasis, and prognosis. Knockdown of HSP90AB1 inhibited the proliferation, migration, and glycolysis of HNSCC, and reduced the level of phospho-Akt. Conclusion: HSP90AB1 functions as an oncogene in HNSCC, and has the potential to become a prognostic factor and therapeutic target.

Cancer-associated fibroblasts secrete hypoxia-induced serglycin to promote head and neck squamous cell carcinoma tumor cell growth in vitro and in vivo by activating the Wnt/ß-catenin pathway.

BACKGROUND: The tumor microenvironment (TME) is known to play a prominent role in the pathology of head and neck squamous cell carcinoma (HNSCC). Cancer-associated fibroblasts (CAFs) have been reported to regulate tumor progression, and serglycin (SRGN), one of the paracrine cytokines of CAFs, has been reported to play an important role in various signaling pathways. Hypoxia is a distinct feature of the HNSCC TME. Here, we investigated the mechanism underlying CAF-secreted SRGN leading to HNSCC progression under hypoxia. METHODS: Immunohistochemical staining was used to detect SRGN expression in clinical HNSCC samples, after which its relation with patient survival was assessed. CAFs were isolated and SRGN expression and secretion by CAFs under normoxia and hypoxia were confirmed using qRT-PCR and ELISA assays, respectively. HNSCC sphere-forming abilities, stemness-related gene expression, and chemoresistance were assessed with or without SRGN treatment. A Wnt/ß-catenin pathway inhibitor (PNU-75,654) was used to block its activation, after which nuclear translocation of ß-catenin in the presence of SRGN with or without PNU-75,654 was evaluated. shRNAs were used to stably knock down SRGN expression in CAFs. HNSCC tumor cells with or without (SRGN silenced) CAFs were inoculated submucosally in nude mice after which tumor weights and sizes were determined to assess the effects of CAFs and SRGN on tumor growth. RESULTS: We found that SRGN was expressed in both HNSCC tumor and stroma cells, and that high SRGN expression in the stroma cells, but not in the tumor cells, was significantly related to a poor patient survival. After the extraction of CAFs and normal fibroblasts (NFs) from paired tumor samples and adjacent normal tissues, respectively, we found that the expression of CAF-specific genes, including fibroblast activation protein (FAP) and alpha-smooth muscle actin (α-SMA), was clearly upregulated compared to the expression in NFs. The hypoxia marker HIF-1α was found to be expressed in tumor stroma cells. Hypoxyprobe immunofluorescence staining confirmed stromal hypoxia in an orthotopic tongue cancer mouse model. Using qRT-PCR and ELISA we found that a hypoxic TME upregulated SRGN expression and secretion by CAFs. SRGN markedly enhanced the sphere-forming ability, stemness-related gene expression and chemoresistance of HNSCC tumor cells. SRGN activated the Wnt/ß-catenin pathway and promoted ß-catenin nuclear translocation. An in vivo study confirmed that CAFs can accelerate HNSCC tumor growth, and that this effect can be counteracted by SRGN silencing. CONCLUSIONS: Our data indicate that a hypoxic tumor stroma can lead to upregulation of SRGN expression. SRGN secreted by CAFs can promote ß-catenin nuclear translocation to activate downstream signaling pathways, leading to enhanced HNSCC cell stemness, chemoresistance and accelerated tumor growth.

Zhenqing recipe relieves diabetic nephropathy through the SIK1/SREBP-1c axis in type 2 diabetic rats.

BACKGROUND: Salt-inducible kinase 1 (SIK1) plays an important role in lipid metabolism, which inhibits lipid synthesis by directly phosphorylating multiple serine residue sites of sterol regulatory element-binding protein 1c (SREBP-1c). This study examined potential therapeutic effect of a Chinese herbal medicine Zhenqing Recipe (ZQR) and metformin on diabetic nephropathy and investigated whether the SIK1/SREBP-1c axis is involved. METHODS: The rat model of type 2 diabetes was developed by high-sucrose plus high fat diet for one month combined with low-dose Streptozocin intraperitoneal injection for three days, after which the presence of hyperglycemia and hyperlipidemia was examined to validate the model. The diabetic rats were then randomly allocated to diabetic groups treated by either ZQR or metformin, and normal rats receiving normal diet were included as a control group. Metabolic parameters, renal function, and renal triglyceride were examined and compared between groups. RESULTS: After a treatment of 12 weeks, ZQR and metformin significantly reduced serum glucose and triglyceride, inhibited diabetic nephropathy and improved renal function. The mRNA level of SIK1 was significantly lower in the diabetic rats than that in the control group, while the expression of SREBP-1c had an opposite pattern. However, after receiving ZQR or metformin treatment, the SIK1 level in diabetic rats increased and the SREBP-1c level was downregulated. Consistent results were observed at the protein level. CONCLUSION: The data suggested that similar to metformin, ZQR could alleviate diabetic nephropathy through SIK1/SREBP-1c axis.

A Novel Design of V-shaped Radial Forearm Free Flap Facilitates the Direct Closure of Donor Site Wound.

ABSTRACT: The radial forearm free flap (RFFF) is one of the commonly used flaps in the repair of head and neck soft tissue defects, especially small and medium-sized defects. The free skin grafts from abdomen are usually used to repair the RFFF donor site wound. This study aims to design a novel V-shaped RFFF, hoping that it might facilitate the direct closure of the forearm donor site wound. From August to December in 2019, 20 patients with oral cancers received radical surgeries, and V-shaped RFFFs were designed to repair the soft defects and the forearm donor site wound was directly closed. The patients were followed up for 6 months to assess the final outcome of repair. The results showed that the pre-designed V-shaped RFFF met the needs of soft tissue defect repair, with the size ranging from 24 cm2 to 30 cm2. Fifteen patients with tongue cancers and four with buccal cancers had satisfactory repair results, and only one patient with buccal cancer had mild limitation of mouth opening. There were 3 patients with a small area of ischemia. The mean postoperative hospital stay was 13.85 ±â€Š1.09 days. In 5 patients, wrist tilt motility decreased compared with that before surgery. The postoperative influence score of wrist exercise on daily life was 2.75 ±â€Š0.44 points. In conclusion, the V-shaped RFFF can meet the needs of small and medium-sized defect repair. This novel design can directly close the forearm donor site wound, which avoids surgical trauma to secondary donor site, and significantly reduces related complications.

Clinical analysis of 22 cases of lymphoepithelial carcinoma of parotid gland / 口腔疾病防治

Objective @# To investigate the diagnosis, treatment and prognosis of lymphoepithelial carcinoma of the parotid gland. @*Methods@#Data from 22 patients with parotid lymphoepithelial carcinoma from 2012 to 2019 were collected, and their clinical manifestations, imaging examinations, treatment methods and follow-up results were retrospectively analyzed.@*Results@# Among the 22 patients, 8 were males and 14 were females. The patients ranged from 26 to 61 years old, with a complaint duration ranging from 3 days to 18 years. One patient had multiple unilateral lumps in the parotid gland, and the other 21 patients had single unilateral lumps in the parotid gland. All patients underwent preoperative spiral CT examination. CT showed a soft tissue lump in the parotid tissue, the internal density shadow was not uniform, the CT value ranged from 26 to 81 Hu, and 15 patients presented elliptical lesions with clear boundaries. Seven patients presented nodular lesions, and the boundary was not clear. The diagnosis of all cases was ultimately based on pathological examination. Hematoxylin-eosin (HE) staining images showed active epithelial cell growth with atypia, mitotic figures could be seen, and abundant lymphocyte and plasma cell infiltration could be seen in the tumor stroma. All 22 patients received surgical treatment; 9 patients did not undergo cervical lymph node dissection. Twenty patients received adjuvant radiotherapy after surgery, and 10 of them received adjuvant chemotherapy at the same time. One patient only received chemotherapy after surgery, and one patient did not receive any other adjuvant therapy after surgery. All patients received follow-up visits. One patient died of liver metastasis 16 months after the operation, and the remaining patients survived without tumors for periods of 13 months to 8 years until the present. @*Conclusion @#Parotid lymphoepithelial carcinoma is a rare malignant tumor clinically. Pathology is still the gold standard for the diagnosis of lymphoepithelial carcinoma of the parotid gland. Radical resection of the tumor is the first choice of treatment. Selective neck lymph node dissection and postoperative adjuvant radiotherapy and chemotherapy can obtain better therapeutic effects according to clinical examination, imaging examination and neck conditions.

lncRNA-Z38 promotes occurrence and progression of gastric cancer though regulating TGF-β signaling pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨lncRNA-Z38对胃癌细胞恶性生物学行为的影响及其可能的调控机制。方法：构建lncRNA-Z38稳定过表达的胃癌细胞系（AGS、MKN74细胞），通过细胞成球实验检测过表达lncRNA-Z38对胃癌细胞肿瘤特征的影响。对过表达lncRNA-Z38的AGS细胞及对照细胞进行高通量测序，对差异表达基因进行KEGG富集分析，筛选出可能参与调控胃癌发生发展的信号通路，采用WB等技术验证lncRNA-Z38通过调控该信号通路参与提高胃癌细胞的干性。结果：成功构建lncRNA-Z38过表达细胞株，细胞成球实验提示lncRNA-Z38过表达细胞的成球能力显著高于对照细胞（P<0.05）。通过测序筛选出lncRNA-Z38过表达胃癌细胞中的1 999个差异表达基因，其中上调基因1 238个、下调基因761个；经KEGG富集分析得到其中变化最显著的通路为TGF-β信号通路（P<0.05）。WB实验结果提示，lncRNA-Z38高表达胃癌细胞中TGF-β信号通路组成基因编码蛋白ID3、BMP6显著上调（均P<0.05）、GDF-5、WNT8B显著下调（P<0.05）。结论：在胃癌中高度表达的lncRNA-Z38通过调控TGF-β信号通路提高胃癌细胞的干性。

A population-based competing risk survival analysis of patients with salivary duct carcinoma.

BACKGROUND: Salivary duct carcinoma (SDC) is a rare malignancy with high risk of local recurrence and distant metastases of the salivary gland. This study was designed to summarize the clinical and pathological features and to further evaluate them as potential prognostic factors for SDC in the salivary gland. METHODS: Clinical data of 266 patients diagnosed with SDC between 2004 and 2015 were collected from the Surveillance, Epidemiology, and End Results (SEER) database. The prognostic factors affecting overall survival (OS) and cancer-specific survival (CSS) were determined by Kaplan-Meier analyses and Cox proportional hazards model. The nomogram was established to predict OS and CSS for SDC. The predictive accuracy of the nomograms was measured by concordance index (C-index). RESULTS: The 3- and 5-year OS of SDC patients were 67.41% and 47.86%, while the 3- and 5-year CSS were 84.6% and 60.7%, respectively. The primary site, T stage and M stage were identified as independent prognostic factors for OS by the multivariate analysis, whereas N stage, M stage, the presence of multiple primary carcinomas and the treatment modalities were identified as independent prognostic factors for CSS. The C-index values of the prognostic nomogram based the risk factors affecting SDC OS and CSS were 0.703 (0.646-0.760) and 0.771 (0.691-0.851), respectively. CONCLUSIONS: SDC is an aggressive malignancy with a high proportion of advanced stage and lymph node metastases. Patients with increasing age, submandibular gland malignancy, advanced T stage, advanced N stage, advanced M stage, high lymph node ratio (LNR) and the presence of multiple primary carcinomas tend to have unfavorable outcomes. Radiotherapy or chemotherapy improve CSS remarkably. These factors will aid in effective therapeutic treatment modalities for SDC.

Co-expression of HIF-1 and TLR3 is associated with poor prognosis in oral squamous cell carcinoma.

This study investigates the prognostic impact of the expression of hypoxia inducible factor (HIF)-1α and Toll-like receptor (TLR) 3 detected by immunohistochemistry in oral squamous cell carcinoma (OSCC). The study also evaluates the treatment outcome by inhibition of the HIF-1α and TLR 3 pathway (nuclear factor [NF]-κB) in an OSCC transplantation model in nude mice. Statistical analysis of immunohistochemical results with clinical findings that included overall survival outcomes was performed for 90 OSCC patients. Forty nude mice were divided into four groups (control; inhibition of HIF-1α; inhibition of NF-κB; and inhibition of HIF-1α and NF-κB). Tumor weight and immunohistochemical results of each group were compared. The results show that co-detection of low HIF-1α/TLR3 expression is significantly correlated with a better prognosis for OSCC patients. Use of an inhibitor of the HIF-1 and TLR3 pathway in an OSCC transplantation model shows a good treatment outcome.

Hypoxia-Targeting Multifunctional Nanoparticles for Sensitized Chemotherapy and Phototherapy in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Chemotherapy in head and neck squamous cell carcinoma (HNSCC) has many systemic side effects, as well as hypoxia-induced chemoresistance. To reduce side effects and enhance chemosensitivity are urgently needed. METHODS: We synthesized a drug delivery system (named CECMa NPs) based on cisplatin (CDDP) and metformin (chemotherapeutic sensitizer), of which chlorin e6 (Ce6) and polyethylene glycol diamine (PEG) were synthesized as the shell, an anti-LDLR antibody (which can target to hypoxic tumor cells) was modified on the surface to achieve tumor targeting. RESULTS: The NPs possessed a great synergistic effect of chemotherapy and phototherapy. After laser stimulation, both CDDP and metformin can be released in situ to achieve anti-tumor effects. Meanwhile, PDT and PTT triggered by a laser have anticancer effects. Furthermore, compared with free cisplatin, CECMa exhibits less systemic toxicity with laser irradiation in the xenograft mouse tumor model. CONCLUSION: CECMa effectively destroyed the tumors via hypoxia targeting multimodal therapy both in vitro and in vivo, thereby providing a novel strategy for targeting head and neck squamous cell carcinoma.

The Management of Head and Neck Sarcoma.

OBJECTIVE: To describe the incidence, types, features, treatment and outcomes of head and neck sarcoma managed at a treatment center in eastern China. METHODS: Cases of head and neck soft tissue sarcoma and osteogenic sarcoma treated at the Stomatology Hospital of Nanjing University between 2008 and 2018 were retrospectively analyzed. Patient characteristics, site of lesion, main presenting symptoms, treatment, histology, local recurrence, development of metastatic disease, duration of follow-up and survival rates are described and compared. RESULTS: Sixty-three patients were diagnosed with head or neck sarcoma of which 42.9% had soft tissue sarcoma and 57.1% had osteogenic sarcoma. Of soft tissue sarcoma patients, the most frequently observed histologies were fibrosarcoma and malignant fibrous histiocytoma. Of 36 cases of osteogenic sarcoma, osteosarcoma, and fibrosarcoma of bone were most frequent. Mean latency period between initial symptoms and clinical presentation was 4.5 months. Radical resection was performed on 56 patients. For 33 patients, resection and radiotherapy were used and 10 patients received a triple combination of resection, chemotherapy and radiotherapy. Within the observation period, 17 patients died. CONCLUSIONS: Head and neck sarcomas, although rare, can represent a variety of pathological diagnoses. Surgery remains the main intervention although the data suggest chemotherapy, radical resection and irradiation as treatment. Outcomes are poor with high rates of local recurrence. Positive prognostic factors were tumor-free resection margins and choice of therapy. Due to the rarity of head and neck sarcoma, information remains limited and choice of treatment should be within the focus of clinical multi-center studies.

Anti-LDLR modified TPZ@Ce6-PEG complexes for tumor hypoxia-targeting chemo-/radio-/photodynamic/photothermal therapy.

Hypoxia, a state of low oxygen tension in solid tumors, is not only closely correlated with resistance to both radiotherapy and chemotherapy, but also associated with poor prognosis of tumors and regional lymph node status. Herein, based on the analysis of cell samples from tumor patients, low-density lipoprotein receptor (LDLR) was found to be overexpressed on the surface of hypoxic tumor cell membranes, and confirmed to be an effective hypoxia marker through specific binding with anti-LDLR antibody in solid tumors. In addition, using the special therapeutic microenvironment of hypoxia, tirapazamine (TPZ, which can be used as both a hypoxia-activated chemotherapy prodrug and radiotherapy sensitizer) was integrated with PEGylated photosensitizer chlorin e6 (Ce6-PEG) by self-assembly, and anti-LDLR was then modified on the surface to form tumor hypoxia-targeting multifunctional nanoparticles (CPTA). CPTA possesses a multimodal antitumor effect via a simultaneous photothermal therapy (PTT)/photodynamic therapy (PDT) effect generated by Ce6, and chemotherapy/radiotherapy actions sensitized by TPZ. It is noteworthy that tumor oxygen was consumed in the process of PDT and the hypoxia was subsequently exacerbated, which can greatly increase the TPZ-sensitized chemotherapy and lead to a synergistic antitumor effect. Both in vitro and in vivo experiments demonstrated that CPTA possesses an excellent therapeutic effect through PTT, PDT, and TPZ sensitized radiotherapy and chemotherapy. This hypoxic tumor targeting synergetic therapeutic strategy has great potential for future clinical transformation.

Rh type C-glycoprotein functions as a novel tumor suppressor gene by inhibiting tumorigenicity and metastasis in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC), a major histologic subtype of head and neck cancer, presents great mortality and morbidity worldwide. The aim of this study is to discover new potential biomarkers closely correlated with HNSCC progression. In this study, weighted gene co-expression network analysis was applied to construct a co-expression network, and the brown module was identified as the most correlated with HNSCC progression. Hub gene identification combined with survival analyses determined RHCG as a candidate biomarker for cancer progression and prognosis prediction. Further experimental results proved that RHCG was aberrantly downregulated in HNSCC tissues and cell lines. Moreover, decreased RHCG expression was shown to be associated with advanced stage and dismal prognosis in HNSCC patients. Functional assays revealed that RHCG could inhibit cell viability, clonogenicity, cell migration in vitro and suppress tumor formation in vivo. Further bioinformatics study demonstrated that DNA promoter hypermethylation of RHCG could lead to its downregulation and serve as potential prognostic maker in HNSCC. Our study reveals that RHCG acts as a tumor suppressor gene that plays a crucial role in inhibiting tumorigenicity and metastasis in HNSCC, which will shed light on the potential diagnostic and therapeutic strategies for HNSCC.

Metformin enhances gefitinib efficacy by interfering with interactions between tumor-associated macrophages and head and neck squamous cell carcinoma cells.

BACKGROUND: Tumor-associated macrophages (TAMs) play an important role in drug resistance in many tumors, including head and neck squamous cell carcinoma (HNSCC). However, how TAMs interact with HNSCC cells to induce drug resistance, especially under hypoxic conditions, is unclear. In this study, we investigated the mechanism of TAM-induced gefitinib resistance in HNSCC cells and sought for novel therapeutic strategies. METHODS: The effects of hypoxia-treated HNSCC cells on the migration and polarization of macrophages were analyzed. Recombinant cytokine proteins and neutralizing antibodies were used as controls. In addition, we assessed the cytotoxic effects of gefitinib on HNSCC cells treated with M2-type macrophage conditioned medium, and carried out a cytokine antibody array analysis, thereby revealing the key factor CCL15. The relationship between serum CCL15 expression levels and prognosis in HNSCC patients was analyzed. In addition, we performed bioinformatic analyses to pursue the mechanisms of CCL15-induced gefitinib resistance. Finally, metformin was used to evaluate the sensitizing effects of gefitinib treatment on HNSCC cells in vitro and in vivo. RESULTS: We found that HNSCC cells recruited macrophages by secreting VEGF and polarized the macrophages to the M2 phenotype through IL-6. Conversely, we found that M2-type TAMs promoted HNSCC cell resistance to gefitinib through paracrine CCL15 signaling. The serum CCL15 levels in HNSCC patients showed a significant correlation with patient prognosis. Furthermore, we found that M2-type TAMs could suppress the sensitivity of HNSCC cells to gefitinib through the CCL15-CCR1-NF-κB pathway. In addition, we found that metformin not only inhibited CCL15 expression in M2-type TAMs enhanced by hypoxia, but also suppressed CCR1 surface expression in HNSCC cells. Encouragingly, we found that metformin sensitized HNSCC cells to gefitinib treatment in vitro and in vivo. CONCLUSIONS: Based on our data we conclude that we have identified a novel interaction between M2-type TAMs and HNSCC cells that contributes to gefitinib resistance. We also found that metformin inhibited the cross-talk between macrophages and tumor cells, thereby eliciting therapeutic effects both in vitro and in vivo.

HOTTIP Functions as a Key Candidate Biomarker in Head and Neck Squamous Cell Carcinoma by Integrated Bioinformatic Analysis.

BACKGROUND: Accumulating evidence has demonstrated the pivotal role of long noncoding RNAs (lncRNAs) in competing endogenous RNA (ceRNA) networks for predicting survival and evaluating prognosis in cancer patients. However, the pathogenesis of head and neck squamous cell carcinoma (HNSCC) remains unclear, and prognostic biomarkers for HNSCC are still lacking. METHODS: A total of 546 RNA sequencing profiles of HNSCC patients with clinical outcome data were obtained from the Cancer Genome Atlas (TCGA) database, providing a large sample of RNA sequencing data. From these, 71 Long noncoding RNAs lncRNAs, 8 microRNAs (miRNAs), and 16 messenger RNAs (mRNAs) were identified to construct a HNSCC-specific ceRNA network (fold change >2, P < 0.05). Univariate and multivariate Cox proportional regression models were used to assess independent indicators of prognosis. Then the expression of lncRNAs harboring prognostic value was validated in human HNSCC cell lines and tumor samples from our cohort and another two datasets from GEO (Gene Expression Omnibus) databases. RESULTS: As a result, a 3-mRNA signature and 6-lncRNA signature were identified. The six-lncRNA signature exhibited the highest prognostic value. Notably, in the six lncRNAs, HOTTIP showed the greatest prognostic value and was significantly correlated with clinical stage and histological grade of HNSCC patients. Furthermore, it was proved that HOTTIP was upregulated in HNSCC cell lines and cancerous tissues compared with corresponding normal cell lines and normal tissues. Functional assessment analysis revealed that HOTTIP might play a key role in the oncogenesis and progression of HNSCC. CONCLUSION: The present study deepened our understanding of the ceRNA-related regulatory mechanism in the pathogenesis of HNSCC and identified candidate prognostic biomarkers for clinical outcome prediction in HNSCC. HOTTIP may function as a key candidate biomarker in HNSCC and serve as a prognostic marker for HNSCC patients.

A Novel Multimodal NIR-II Nanoprobe for the Detection of Metastatic Lymph Nodes and Targeting Chemo-Photothermal Therapy in Oral Squamous Cell Carcinoma.

Current surgical treatment for oral squamous cell carcinoma (OSCC) must be as precise as possible to fully resect tumors and preserve functional tissues. Thus, it is urgent to develop efficient fluorescent probes to clearly identify tumor delineation, as well as metastatic lymph nodes. Chemo-photothermal therapy combination attracted a growing attention to increase anti-tumor effect in various types of cancer, including OSCC. In the present study, we designed a multimodal NIR-II probe that involves combining photothermal therapy with chemotherapy, imaging OSCC tumors and detecting metastatic lymph nodes. Methods: In this study, we synthesized a novel near infrared (NIR)-II probe named TQTPA [4,4'-((6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazolo [3,4-g]quinoxaline-4,9-diyl)bis(thiophene-5,2-diyl))bis(N,N-diphenylaniline)] via the Suzuki reaction and prepared multimodal nanoparticles (NPs) loading TQTPA and cis-dichlorodiammine platinum (CDDP) (HT@CDDP) by hyaluronic acid. The characteristics of the NPs, including their photothermal and imaging capabilities were investigated in vitro and in vivo. Their anti-tumor efficacy was evaluated using orthotopic, tongue tumor-bearing, nude mice. Results: The NPs possessed good stability and water solubility and were pH/hyaluronidase sensitive. The good tissue penetration quality and active targeting ability enabled the NPs to draw the outline of orthotopic tongue tumors and metastatic lymph nodes as small as 1 mm in nude mice by IR-808 under NIR exposure. In vitro and in vivo experiments validated the biocompatibility and low systematic toxicity of the NPs. At the same time, the NPs acted as multimodal therapy agents, combining photothermal therapy with chemotherapy. Conclusion: With a good imaging capability and anti-tumor efficacy, our NPs successfully outlined orthotopic tongue tumors and metastatic lymph nodes as well as enabled chemo-photothermal therapy combination. Our study established a solid foundation for the application of new clinical diagnosis and treatment patterns in the future.

Metformin sensitizes hypoxia-induced gefitinib treatment resistance of HNSCC via cell cycle regulation and EMT reversal.

OBJECTIVES: The objectives of this study were to explore the mechanisms of metformin sensitization to hypoxia-induced gefitinib treatment in resistant head and neck squamous cell carcinoma (HNSCC) and evaluate the effects of this combined treatment strategy. METHODS: The effects of gefitinib treatment on HNSCC were measured under normoxic and hypoxic conditions. The relationship between hypoxia and cell cycle and epithelial-mesenchymal transition (EMT) in tumor cells were analyzed. Palbociclib and LY294002 were used in combination with gefitinib to evaluate the effects on HNSCC cell cytotoxicity during hypoxia. Finally, metformin was used to evaluate the sensitizing effects of gefitinib treatment on HNSCC in vivo and in vitro. RESULTS: Cell viability and apoptosis assays demonstrated a significant difference in HNSCC cells treated with gefitinib between the normoxia and hypoxia groups. Hypoxia induced the expression of cyclin D1, decreased the percentage of cells in G1, and promoted the EMT of tumor cells. Both palbociclib and LY294002 enhanced gefitinib-induced cytotoxicity of HNSCC cells under hypoxic conditions. Encouragingly, metformin sensitized HNSCC to gefitinib treatment in vivo and in vitro. CONCLUSION: Hypoxia promotes G1-S cell cycle progression and EMT in HNSCC, resulting in gefitinib treatment resistance. Metformin sensitizes HNSCC to gefitinib treatment, which might serve as a novel combined treatment strategy.

Antitumor effect of a Pt-loaded nanocomposite based on graphene quantum dots combats hypoxia-induced chemoresistance of oral squamous cell carcinoma.

BACKGROUND: Tumor microenvironment plays an important role in the chemoresistance of oral squamous cell carcinoma (OSCC). Hypoxia in the microenvironment is one of the important factors that contributes to OSCC chemoresistance; therefore overcoming hypoxia-mediated chemoresistance is one of the great challenges in clinical practice. METHODS: In this study, we developed a drug delivery system based on Pt-loaded, polyethylene glycol-modified graphene quantum dots via chemical oxidation and covalent reaction. RESULTS: Our results show that synthesized polyethylene glycol-graphene quantum dots-Pt (GPt) is about 5 nm in diameter. GPt sensitizes OSCC cells to its treatment in both normoxia and hypoxia conditions. Inductively coupled plasma-mass spectrometry assay shows that GPt enhances Pt accumulation in cells, which leads to a notable increase of S phase cell cycle arrest and apoptosis of OSCC cells in both normoxia and hypoxic conditions. Finally, compared with free cisplatin, GPt exhibits a strong inhibitory effect on the tumor growth with less systemic drug toxicity in an OSCC xenograft mouse tumor model. CONCLUSION: Taken together, our results show that GPt demonstrates superiority in combating hypoxia-induced chemoresistance. It might serve as a novel strategy for future microenvironment-targeted cancer therapy.

Prognostic factors of palatal mucoepidermoid carcinoma: a retrospective analysis based on a double-center study.

Mucoepidermoid carcinoma (MEC) of the palate is a common malignancy of minor salivary glands. This study was designed to identify the prognostic factors for MEC of the palate. The medical records of patients diagnosed with MEC of the palate who visited the Department of Oral and Maxillofacial Surgery at Nanjing Stomatological Hospital and the Department of Stomatology at Central Hospital of Xuzhou were retrospectively studied. The prognostic factors were determined using a Cox proportional hazards model. Furthermore, the expression of cancer stem cell (CSC) markers CD44, CD133, Nanog and Sox2 were detected in neoplastic samples of these patients by immunohistochemistry. As a result, both univariate analysis and multivariate analysis proved a high histological grade and an advanced tumor stage as negative prognostic factors for overall survival. By immunohistochemistry staining and survival analysis, a combination of CD44/CD133/SOX2 was found to have the strongest prognostic value for palatal MEC patients. In conclusion, the proposed nomogram which include histological grade and tumor stage along with cancer stem cell markers provides a more accurate long-term prediction for palatal MEC patients.