Epigenetic regulation of VENTXP1 suppresses tumor proliferation via miR-205-5p/ANKRD2/NF-kB signaling in head and neck squamous cell carcinoma.

An increasing number of studies have shown that long noncoding RNAs (lncRNAs) play important roles in tumor development and progression. However, their involvement in head and neck squamous cell carcinoma (HNSCC) remains largely unknown. Epigenetic regulation is one major mechanism utilized by cancer cells to control lncRNA expression. We identified that lncRNA VENTXP1 was epigenetically silenced in multiple cancer types, and its lower expression was correlated with poorer survival in HNSCC patients. Through in silico analysis and experimental validation, we identified miR-205-5p and its direct interacting partner of VENTXP1, which regulates HNSCC cell proliferation and tumorigenicity. Using RNA-seq and differential gene expression analysis, we further identified ANKRD2 as a miR-205-5p target, which plays an essential role in modulating NF-kB signaling. These findings suggest that VENTXP1 inhibits tumor growth via suppressing miR-205-5p/ANKRD2-mediated NF-kB signaling in HNSCC. Thus, pharmaceutical targeting of DNA methylation to restore VENTXP1 expression might constitute a therapeutic strategy for HNSCC.

Long non-coding RNA ANRIL promotes tumorgenesis through regulation of FGFR1 expression by sponging miR-125a-3p in head and neck squamous cell carcinoma.

ANRIL (CDKN2B antisense RNA 1, CDKN2B-AS1) is involved in the progression of various cancers. However, its role in head and neck squamous cell carcinoma (HNSCC) remains unclear. In this study, we found that ANRIL expression was upregulated in HNSCC and correlated with tumor progression. Further functional analysis showed that knockdown of ANRIL significantly inhibited proliferation in vivo and in vitro. ANRIL functioned as a ceRNA (competing endogenous RNAs) for miR-125a-3p and upregulated FGFR1 (fibroblast growth factor receptor-1), which could promote tumor growth. Moreover, we confirmed that ANRIL promoted HNSCC activity via FGFR1 with a FGFR1 inhibitor in vivo and in vitro. Thus, it could be concluded that ANRIL promoted the progression of HNSCC via miR-125a-3p/FGFR1/MAPK signaling, which might provide a new target for the diagnosis and treatment of HNSCC.

STT3-dependent PD-L1 accumulation on cancer stem cells promotes immune evasion.

Enriched PD-L1 expression in cancer stem-like cells (CSCs) contributes to CSC immune evasion. However, the mechanisms underlying PD-L1 enrichment in CSCs remain unclear. Here, we demonstrate that epithelial-mesenchymal transition (EMT) enriches PD-L1 in CSCs by the EMT/ß-catenin/STT3/PD-L1 signaling axis, in which EMT transcriptionally induces N-glycosyltransferase STT3 through ß-catenin, and subsequent STT3-dependent PD-L1 N-glycosylation stabilizes and upregulates PD-L1. The axis is also utilized by the general cancer cell population, but it has much more profound effect on CSCs as EMT induces more STT3 in CSCs than in non-CSCs. We further identify a non-canonical mesenchymal-epithelial transition (MET) activity of etoposide, which suppresses the EMT/ß-catenin/STT3/PD-L1 axis through TOP2B degradation-dependent nuclear ß-catenin reduction, leading to PD-L1 downregulation of CSCs and non-CSCs and sensitization of cancer cells to anti-Tim-3 therapy. Together, our results link MET to PD-L1 stabilization through glycosylation regulation and reveal it as a potential strategy to enhance cancer immunotherapy efficacy.

[Safety and efficacy of ultrasound hyperthermia combined with chemotherapy and radical operation for advanced oral squamous cell carcinoma].

PURPOSE: To explore the safety and efficacy of ultrasound hyperthermia combined with chemotherapy and radical operation for advanced oral squamous cell carcinoma (OSCC). METHODS: Nine patients who had definite pathological diagnosis were enrolled in this clinical trail from 2015 to 2016. All patients underwent ultrasound hyperthermia combined with chemotherapy before radical operation. The treatment regime was as follows: Docetaxel and cisplatin (75mg/m2) used on the first day, fluorouracil (750 mg/m2) infused from 1st to 5th day. All patients received 2 cycles of thermo-chemotherapy, the therapeutic temperature was set to be 40-42 degrees centigrade, ultrasound hyperthermia was performed for 40 minutes every other day for 5 times. The therapeutic outcomes were evaluated by observation of clinical tumor regression. RESULTS: Of the 9 patients, complete response（CR）was seen in 1 case, partial response (PR) was seen in 3 cases, stable disease（SD）was seen in 5 cases. Complications were not severe and tolerable. CONCLUSIONS: Ultrasound hyperthermia combined with chemotherapy (TPF) may improve the therapeutic effect in advanced OSCC without obvious adverse reactions, and the toxicity and side effects are well tolerated.

Long-term results of a randomized phase III trial of TPF induction chemotherapy followed by surgery and radiation in locally advanced oral squamous cell carcinoma.

Previously, we conducted a randomized phase III trial of TPF (docetaxel, cisplatin, and 5-fluorouracil) induction chemotherapy in surgically managed locally advanced oral squamous cell carcinoma (OSCC) and found no improvement in overall survival. This study reports long-term follow-up results from our initial trial. All patients had clinical stage III or IVA locally advanced OSCC. In the experimental group, patients received two cycles of TPF induction chemotherapy (75mg/m2 docetaxel d1, 75mg/m2 cisplatin d1, and 750mg/m2/day 5-fluorouracil d1-5) followed by radical surgery and post-operative radiotherapy; in the control group, patients received upfront radical surgery and post-operative radiotherapy. The primary endpoint was overall survival. Among 256 enrolled patients with a median follow-up of 70 months, estimated 5-year overall survival, disease-free survival, locoregional recurrence-free survival, and distant metastasis-free survival rates were 61.1%, 52.7%, 55.2%, and 60.4%, respectively. There were no significant differences in survival rates between experimental and control groups. However, patients with favorable pathologic responses had improved outcomes compared to those with unfavorable pathologic responses and to those in the control group. Although TPF induction chemotherapy did not improve long-term survival compared to surgery upfront in patients with stage III and IVA OSCC, a favorable pathologic response after induction chemotherapy may be used as a major endpoint and prognosticator in future studies. Furthermore, the negative results observed in this trial may be represent type II error from an underpowered study. Future larger scale phase III trials are warranted to investigate whether a significant benefit exists for TPF induction chemotherapy in surgically managed OSCC.

Randomized phase III trial of induction chemotherapy with docetaxel, cisplatin, and fluorouracil followed by surgery versus up-front surgery in locally advanced resectable oral squamous cell carcinoma.

PURPOSE: To evaluate induction chemotherapy with docetaxel, cisplatin, and fluorouracil (TPF) followed by surgery and postoperative radiotherapy versus up-front surgery and postoperative radiotherapy in patients with locally advanced resectable oral squamous cell carcinoma (OSCC). PATIENTS AND METHODS: A prospective open-label phase III trial was conducted. Eligibility criteria included untreated stage III or IVA locally advanced resectable OSCC. Patients received two cycles of TPF induction chemotherapy (docetaxel 75 mg/m(2) on day 1, cisplatin 75 mg/m(2) on day 1, and fluorouracil 750 mg/m(2) on days 1 to 5) followed by radical surgery and postoperative radiotherapy (54 to 66 Gy) versus up-front radical surgery and postoperative radiotherapy. The primary end point was overall survival (OS). Secondary end points included local control and safety. RESULTS: Of the 256 patients enrolled onto this trial, 222 completed the full treatment protocol. There were no unexpected toxicities, and induction chemotherapy did not increase perioperative morbidity. The clinical response rate to induction chemotherapy was 80.6%. After a median follow-up of 30 months, there was no significant difference in OS (hazard ratio [HR], 0.977; 95% CI, 0.634 to 1.507; P = .918) or disease-free survival (HR, 0.974; 95% CI, 0.654 to 1.45; P = .897) between patients treated with and without TPF induction. Patients in the induction chemotherapy arm with a clinical response or favorable pathologic response (≤ 10% viable tumor cells) had superior OS and locoregional and distant control. CONCLUSION: Our study failed to demonstrate that TPF induction chemotherapy improves survival compared with up-front surgery in patients with resectable stage III or IVA OSCC.

Contactin 1 (CNTN1) expression associates with regional lymph node metastasis and is a novel predictor of prognosis in patients with oral squamous cell carcinoma.

The contactin 1 (CNTN1) gene exerts oncogene­like activities and its expression has been linked to several human malignancies. In this study, a possible association between CNTN1 expression and clinicopathological parameters and clinical outcomes in patients with oral squamous cell carcinoma (OSCC) was examined. CNTN1 protein expression was evaluated by immunohistochemistry in OSCC tissues of 45 patients. For the immunohistochemical assessment of CNTN1 expression, the cytoplasmic staining labeling index was analyzed using a semiquantitative score. The association between CNTN1 protein levels and clinicopathological factors was analyzed using the Mann-Whitney U test for categorical variables and the Kruskal-Wallis test for continuous variables. The effects of CNTN1 expression on overall and disease-free survival were assessed by using univariate survival analysis. The transcript levels of CNTN1 were detected in OSCC cell lines. In addition, specific siRNA against CNTN1 was applied to investigate the effect exerted by CNTN1 ablation on OSCC cell lines by proliferation and invasion assays in vitro. During follow-up, 16 patients (35.56%) had succumbed to OSCC; the median follow-up of patients was 5.0 years (range, 0.2-8.3). A high expression of CNTN1 was markedly associated with the regional lymph node metastasis of patients with OSCC (P=0.006). CNTN1 expression was significantly associated with overall survival of patients with OSCC (P=0.032; log-rank test) and disease-free survival of patients with OSCC (P=0.038; log-rank test). In addition, CNTN1 ablation notably suppressed the invasion potential of OSCC cell lines, but there was no significant change in the proliferation of OSCC cell lines by CNTN1 knockdown in vitro. The study supports CNTN1 as a novel predictor of regional lymph node metastasis in patients with OSCC and a prognostic marker for OSCC in patients.

Expression of podoplanin in salivary gland adenoid cystic carcinoma and its association with distant metastasis and clinical outcomes.

Distant metastasis is a common cause of mortality in patients with salivary gland adenoid cystic carcinoma (SACC). However, presently, the development of distant metastasis is unable to be predicted in clinical practice. Recent studies have shown that overexpression of podoplanin is associated with metastasis and survival in patients with several cancer types. The purpose of the present study was to determine whether podoplanin is overexpressed in SACC and whether such overexpression is associated with distant metastasis and survival. Podoplanin expression was determined using immunohistochemistry (IHC) in tumors from 40 SACC patients. The expression status was analyzed in regards to patient clinicopathological parameters and survival rates. Overexpression of podoplanin was detected in 13 (32.5%) of the 40 tumors. Overexpression was significantly associated with disease-free survival (P=0.025) and distant metastasis (P=0.015), although it was not associated with recurrence and overall survival. In conclusion, podoplanin is overexpressed in a subset of SACCs and may be a biomarker predicting distant metastasis in patients with SACC.

Primary oral mucosal melanoma: advocate a wait-and-see policy in the clinically N0 patient.

PURPOSE: Oral mucosal melanoma (OMM) is a rare disease associated with a very poor prognosis. Because well-established treatment protocols for OMM are in short supply, prognostic information regarding recent treatment modalities for this disease were sought. PATIENTS AND METHODS: A retrospective chart review was performed of 61 patients who were treated for OMM from 1998 through 2005. The clinical features and treatment modalities were identified and correlated with the outcomes. RESULTS: There were 41 male and 20 female patients (ratio, 2.1:1) with a mean age of 54.1 years. The mean follow-up was 31.9 months, and the overall 2-year and 5-year survival rates were 51.1% and 30.3%, respectively. According to the seventh edition of the American Joint Committee on Cancer staging system, there were 31 patients (50.8%) with stage III tumors. A more advanced stage and a tumor of at least 2 cm were associated with worse survival (P < .001 and P = .036, respectively). Elective lymph node dissection and biochemotherapy were not associated with a higher total survival rate (P = .53 and P = .76, respectively). CONCLUSIONS: OMM has a male predilection. The seventh edition of the American Joint Committee on Cancer stage and tumor size are effective prognostic parameters for patients with OMM. The American Joint Committee on Cancer staging system provides useful information for predicting the ultimate outcome and should be used as the primary staging system. Elective node dissection and adjuvant biochemotherapy offer no additional advantage in increasing the patient survival rate. A wait-and-see policy is advocated for patients with clinical stage N0 cancer.

Establishment and characterization of a rabbit oral squamous cell carcinoma cell line as a model for in vivo studies.

The incidence of oral squamous cell carcinoma (SCC) is increasing but the long-term survival rate remains low. An animal model would therefore be helpful for evaluation of new treatment modalities for oral SCC. Hamster is small animal, therefore, the cancer of hamster cheek pouch is not optimal for tumor imaging. The VX2 cell line has been used in many carcinoma-related studies, including oral SCC research, but it is derived from cutaneous tissue and not mucosa. We chemically induced tongue squamous cell carcinoma in rabbits and subsequently established a rabbit squamous cell line. The cells grew in multiple layers without contact inhibition for 60 passages over 2 years and were positive for cytokeratin (CK). Electron microscopy revealed that cells were polygonal with rich microvilli on the surface, and there were desmosomes between cells and bundles of tonofibril beside the cell membrane. The chromosome number ranged from 71 to 272, with a modal value of 145 (12.4%). The cells were transplantable into nude mice subcutaneously or rabbit submucosally and produced carcinomas in all the animals. The cell line should be a useful tool for the study of the biological characteristics of oral SCC, especially tongue SCC.

Neck dissection and post-operative chemotherapy with dimethyl triazeno imidazole carboxamide and cisplatin protocol are useful for oral mucosal melanoma.

BACKGROUND: Oral mucosal melanoma (OMM) is a clinically rare disease with poor prognosis. Various treatment methods have been investigated with the aim of improving the prognosis. This study aimed to analyze the data of a single institution in the management of OMM. METHODS: A total of 78 consecutive OMM patients were included in this retrospective study. The intraoral lesion was treated either by cryotherapy, surgery or both; the neck was treated by neck dissection or observation; post-operative chemotherapy with dimethyl triazeno imidazole carboxamide and cisplatin was performed in some patients. The Kaplan-Meier method was used for statistical analysis. RESULTS: Among the 78 patients, there were 50 males and 28 females with an average age of 53.8 years (ranging from 27 to 85 years). The most common sites of OMM were the hard palate and gingiva. The main cause of death in OMM was distant metastasis. No significant difference was found between the intraoral/cervical lesion recurrence/post-operative distant metastasis and the intraoral lesion site/biopsy method/treatment method. The metastasis rate of cervical lymph node was high in the OMM patients, even in the patients with clinically negative necks. Cervical lesion recurrence was correlated with N stage and intraoral lesion recurrence. The survival period was longer in the patients with T3 staging, clinical stage III disease, with post-operative chemotherapy and without post-operative distant metastasis when compared to those patients with T4a staging, clinical stage IV disease, without post-operative chemotherapy and with post-operative distant metastasis. CONCLUSIONS: Radical surgery including wide intraoral resection and neck dissection is recommended for OMM patients. Post-operative chemotherapy may also be beneficial for both primary and recurrent OMM patients.

[Inhibition effects of tumor necrosis factor-alpha gene transduced tumor drainage node of lymphocytes from tongue cancer on SCID mice transplanted tumor established with human tongue carcinoma cell lines].

OBJECTIVE: To observe the in vivo inhibition effects of tumor necrosis factor-alpha (TNF-alpha) gene transduced tumor drainage node of lymphocytes (DNL) from tongue cancer on SCID mice transplanted tumor. METHODS: 15 human tongue carcinoma models were established in SCID mice by subcutaneously injection of squamous cell carcinoma line Tca8113. TNF-alpha gene introduced DNL, combined with low dose Pinyancin (PYC), were locally injected into tumor site. The inhibition rate was determined by the weights at the 8th week after tumor dissection and fresh specimens were prepared and subject to histopathologic examination under transmission electron microscope, and in situ TUNEL was used to detect apoptosis. RESULTS: The TNF/DNL and rIL-2 group, and the TNF/DNL and rIL-2 and PYC group both exerted a strong inhibition effect on the implanted tumor. Treated tumors of the TNF/DNL and rIL-2 and PYC group were significantly reduced in comparison with those of the TNF/DNL and rIL-2 group (P < 0.05). The apoptosis of tumor in the TNF/DNL and rIL -2 group was evidenced based on transmission electron microscope and TUNEL analysis, and the apoptosis index was higher than that of control group (P < 0.05). CONCLUSION: Local injection of DNL modified with TNF-alpha gene, combined with low dose PYC, exert a synergistic antitumor effect. Apoptosis may be an important mechanism of squamous cell carcinoma killed by TNF/DNL.

[Evaluation of fluorodeoxyglucose-position-emission tomography-computer tomography imaging on head and neck squamous cell carcinomas including primary tumors and lymph node metastases].

OBJECTIVE: To evaluate (18)F-fluorodeoxyglucose-position-emission tomography-computer tomography imaging ((18)F-FDG-PET-CT) on head and neck squamous cell carcinoma(HNSCCA) and lymph node metastasis. METHODS: (18)F-FDG-PET-CT imaging of 20 patients with HNSCCA was evaluated retrospectively. RESULTS: All the primary tumors were correctly diagnosed by (18)F-PET-CT imaging and SUV(avg) of the primary tumors was (6.22 +/- 2.20). All the sensitivity, specificity, positive predictive value and the negative predictive value were 100%. In detecting lymph node metastasis, the sensitivity was 51%, specificity 97.7%, false positive rate 2.3%, false negative rate 49%, positive predictive value 82%, and negative predictive value 91.2%. CONCLUSIONS: (18)F-FDG-PET-CT imaging was valuable in detecting HNSCCA and lymph node metastasis. SUV was helpful for differential diagnosis between benign or malignant tumors but it needs further study to determine the cutoff SUV for differentiating lymph node metastasis.

Expression of Glut-1 in primary and recurrent head and neck squamous cell carcinomas, and compared with 2-[18F]fluoro-2-deoxy-D-glucose accumulation in positron emission tomography.

The aim of the study was to investigate the pattern of glucose transporter-1 (Glut-1) expression in primary and recurrent head and neck squamous cell carcinomas (HNSCCAs) and the relation between Glut-1 expression and 2-[18F]fluoro-2-deoxy-D-glucose - positron emission tomography (FDG-PET). Standardised uptake values (SUVs) were used to evaluate FDG uptake by the tumour. Sections were stained immunohistochemically for Glut-1, which showed that high SUVs were seen in all HNSCCAs, and patients with higher T stage tumours or less well-differentiated tumours showed significantly higher SUVs than those with lower stage tumours or better-differentiated tumours (P=0.001 and 0.04, respectively). Glut-1 immunostaining was present in all cases. The Glut-1 staining index in primary HNSCCAs was significantly lower than that in recurrent HNSCCAs (P=0.03), and the index of better-differentiated tumours lower than that of poorly-differentiated tumours (P=0.02). However, there was no significant correlation between SUV(mean) and the Glut-1 staining index. In conclusion, our data suggest that high FDG uptakes were seen with overexpression of Glut-1 in primary and recurrent HNSCCAs. SUV(mean) was related to tumour T stage and grade of differentiation, which indicated that SUV was helpful in evaluating tumours. The expression of Glut-1 in recurrent HNSCCAs was higher than that in primary HNSCCAs, and in poorly-differentiated HNSCCAs higher than in better-differentiated HNSCCAs, which indicated that Glut-1 may have a useful role as a predictor for poor prognosis in HNSCCAs. However, there was no significant correlation between FDG accumulation and Glut-1 expression.

[Genetic linkage analysis and mutation detection in Chinese families with basal cell nevus syndrome].

OBJECTIVE: To study the molecular genetic etiology of a Chinese pedigree with basal cell nevus syndrome. METHODS: The proband and his affected mother and a unaffected individual in the pedigree were chosen and peripheral blood was collected from them for DNA. Direct sequencing was performed to detect the mutations of PTCH gene. In order to further confirm the results of sequence analysis, all available family members were analyzed with genetic linkage analysis using 3 highly polymorphic microsatellite DNA markers in the region of 9q22.3-q31. RESULTS: No mutations of PTCH gene was detected in the proband's mother, one synonymous mutation was detected in the proband. Linkage analysis showed that the Lod scores of the 3 markers were: D9S283, Z = -2.11 (theta = 0.00); D9S1690, Z = -2.95 (theta = 0.00); D9S1677, Z = -5.94 (theta = 0.00). CONCLUSIONS: In this pedigree, mutation of PTCH gene is not related to the underlying pathogenesis of the syndrome.

[Experimental study on ultrasound heating affecting the sensitivity of oral carcinoma to chemotherapy drugs].

OBJECTIVE: To explore the influence of chemosensitivity of Tca8113 cells by modified MTT assay after the animal model of Tca8113 were treated by the ultrasound hyperthermia. METHODS: The MTT assay of the BALB/C nu/nu mice model of Tca8113 cells treated by the ultrasound hyperthermia in vivo was performed. RESULTS: The chemosensitivity to the 9 kinds of drugs demonstrated no significant differences between the Tca8113 cells in the control group, the 39 degrees C-treated group and the groups treated from 41 degrees C to 44 degrees C. But significant differences between the 40 degrees C-treated group and the 41 degrees C or 42 degrees C-treated group existed. In the heating-time grades test, there were no significant differences in the chemosensitivity to the 9 kinds of drugs between these three pairs of group (the control group and the 15 min-treated group, the 30 min-treated and the 45 min-treated group, the 60 min-treated and the 75 min-treated group). But there were significant differences between the 30 min-treated or the 45 min-treated group and the 60 min-treated or the 75 min-treated group. CONCLUSION: Ultrasound hyperthermia performed in 42 degrees C for 30-45 min can improve the chemosensitivity of Tca8113 cells to some drugs significantly, which confirms the rationality of synchronous combination of hyperthermia and chemotherapy in the chemosensitivity point of view for the first time.

[A study on the mechanism of inducing apoptosis of Tca8113 cells by means of ultrasound hyperthermia].

PURPOSE: To investigate the mechanism of apoptosis in Tca8113 cells induced by ultrasound hyperthermia by detecting changes in related index. METHODS: Tca8113 cells were treated in vitro by ultrasound hyperthermia in different heating temperatures (38 degrees C to 44 degrees C,10 minutes) and heating times (42 degrees C,10 to 60 minutes), and then the dynamic changes of early apoptosis and secondary necrosis treated by 42 degrees C for 10 minutes were assayed to detect deltapsim and Caspase-3 levels of different groups by flow cytometry (FCM). The means of each group were compared by ANOVA with SAS6.12 software package. RESULTS: After heated by ultrasound in 42 degrees C for 10 minutes, the early apoptosis of Tca8113 was detected. The apoptosis index reached its highest level at the 6th to 8th hour, then decreased rapidly and maintained in a lower level after 12 hours. The level of secondary necrosis increased with the level of early apoptosis, but kept in a higher level until the 10th hour, the level of secondary necrosis correlated with that of the early apoptosis (r = 0.7909, P = 0.0064). The fraction of cells with low mitochondria membrane potential and increased activity of Caspase-3 were detected either in the heating-temperature grads group or in the heating-time grads group, which showed significant relationship between thetwo apoptosis related index (r = 0.89189, P = 0.0029 in the heating-temperature grads group; r = 0.9679, P = 0.0003 in the heating-time grads group). CONCLUSIONS: Tca8113 cells developed apoptosis after heated by ultrasound hyperthermia along with deltapsim level decreasing and Caspase-3 activity increasing. Ultrasound hyperthermia induces apoptosis of Tca8113 cells by the mitochondrum-Caspase pathway.