The efficacy and safety of dalpiciclib, a cyclin-dependent kinase 4/6 inhibitor, in patients with advanced head and neck mucosal melanoma harboring CDK4 amplification.

BACKGROUND: Mucosal melanoma (MM) is a rare but devastating subtype of melanoma. Our previous studies have demonstrated robust anti-tumor effects of cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors in head and neck MM (HNMM) patient-derived xenograft models with CDK4 amplification. Herein, we aimed to investigate the efficacy and safety of dalpiciclib (SHR6390), a CDK4/6 inhibitor, in HNMM patients harboring CDK4 amplification. METHODS: The anti-tumor efficacy of dalpiciclib was assessed by HNMM patient-derived xenograft (PDX) models and patient-derived tumor cells (PDC) in vivo and in vitro. Immunohistochemical analyses and western blot were then performed to assess the markers of cell proliferation and CDK4/6 signaling pathway. For the clinical trial, advanced recurrent and/or metastatic HNMM patients with CDK4 amplification were treated with dalpiciclib 125 mg once daily for 21 consecutive days in 28-day cycles. The primary endpoint was disease control rate (DCR). Secondary endpoints included safety, objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: Dalpiciclib profoundly suppressed growth of HNMM-PDX and PDC with CDK4 amplification, whereas it showed relatively weak suppression in those with CDK4 wild type compared with vehicle. And dalpiciclib resulted in a remarkable reduction in the expression levels of Ki-67 and phosphorylated Rb compared with control group. In the clinical trial, a total of 17 patients were enrolled, and 16 patients were evaluable. The ORR was 6.3%, and the DCR was 81.3%. The estimated median PFS was 9.9 months (95% CI, 4.8-NA), and the median OS was not reached. The rate of OS at 12 months and 24 months was 68.8% (95% CI, 0.494-0.957) and 51.6% (95% CI, 0.307-0.866), respectively. The most frequent adverse events were neutrophil count decrease, white blood cell count decrease, and fatigue. CONCLUSIONS: Dalpiciclib was well-tolerated and displayed a durable benefit for HNMM patients with CDK4 amplification in this study. Further studies on CDK4 inhibitors and its combination strategy for MM are worth further exploration. TRIAL REGISTRATION: ChiCTR2000031608.

A multicenter randomized phase II trial of hyperthermia combined with TPF induction chemotherapy compared with TPF induction chemotherapy in locally advanced resectable oral squamous cell carcinoma.

BACKGROUND: Hyperthermia has been reported to cause cancer stage regression, thus providing surgical opportunities in patients with unresectable tumors and improving the quality of life of patients by preserving certain organs. METHODS: A prospective open-label phase II trial was conducted to evaluate the efficacy of hyperthermia combined with induction chemotherapy in patients with locally advanced resectable oral squamous cell carcinoma (OSCC). Patients received hyperthermia combined with two cycles of 5-fluorouracil, cisplatin, and docetaxel (TPF) induction chemotherapy regimens or TPF induction chemotherapy alone, followed by radical surgery with postoperative radiotherapy. The primary endpoint was the clinical response rate of the induction chemotherapy. The secondary endpoints were overall survival (OS), disease-free survival (DFS), and toxicity. RESULTS: A total of 120 patients were enrolled, and 115 patients were included in the clinical response analysis. The clinical response rate was significantly higher in the experimental arm than in the control arm (65.45% vs. 40.00%, p = 0.0088). There were no unexpected toxicities, and hyperthermia and induction chemotherapy did not increase the perioperative morbidity rate. Moreover, there was a significant improvement in DFS, but no significant difference in OS between the two arms. In the subgroup analysis, increased OS and DFS rates were associated with patients with favorable clinical response after induction chemotherapy in the total population, experimental arm, and control arm. CONCLUSIONS: Our study demonstrates that hyperthermia combined with induction chemotherapy is associated with a high response rate and provides a new treatment option for patients with resectable stage III or IVA OSCC.

Altered expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 during nimotuzumab therapy correlates with responses and prognosis of oral squamous cell carcinoma patients.

BACKGROUND: Since the inhibitory immune checkpoint receptors have been described to benefit the OSCC patients clinically, it is unknown that whether their expression in tumor immune microenvironment (TME) can determine the clinical outcome in response to nimotuzumab therapy. METHODS: We examined the expression patterns of immune checkpoint receptors (including TIM-3, LAG-3, PD-L1, and CTLA-4) and an immunoregulatory enzyme called IDO in 36 OSCC patients during nimotuzumab therapy by immunohistochemistry. Then, we divided the recruited patients into clinical responders and non-responders according to computed tomography (CT) scan and analyzed the relationship between the immunological parameters and clinical outcome. RESULTS: We observed that nimotuzumab therapy significantly increased the expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 in the TME, and the expression of LAG-3 and PD-L1 before nimotuzumab therapy was inversely correlated with the overall survival. In clinical non-responders, we found the expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 was significantly increased during nimotuzumab therapy, and the expression of TIM-3, LAG-3, IDO, PD-L1, and CTLA-4 before nimotuzumab therapy was negatively correlated with the overall survival. However, in clinical responders, neither of those showed significant. CONCLUSIONS: It suggests that these immune checkpoint receptors and IDO could be considered as biomarkers to reflect immune status in the tumor microenvironment during nimotuzumab therapy. Blockade of these immune checkpoint receptors might enhance nimotuzumab-based cancer immunotherapy, thus potentially improving clinical outcomes of OSCC patients.

Stabilization of Slug by NF-κB is Essential for TNF-α -Induced Migration and Epithelial-Mesenchymal Transition in Head and Neck Squamous Cell Carcinoma Cells.

BACKGROUND/AIMS: Slug protein, a transcription factor for the induction of epithelial-mesenchymal transition (EMT) and cancer cell invasion and metastasis, is frequently upregulated in human epithelial cancers. However, mutation of this gene in cancer is rare, and the mechanism of its dysregulation remains unknown, especially in head and neck squamous cell carcinoma (HNSCC). METHODS: We examined the role of TNF-α in the stabilization of Slug by immunoprecipitation-westernblot analysis. Migration of HNSCC cells with or without knockdown of Slug gene expression was assayed by a wound healing assay. Immunohistochemical staining analysis was used to measurement Slug levels in both normal and HNSCC tumor tissues. RESULTS: The inflammatory cytokine TNF-α stabilized Slug protein by inhibiting its ubiquitination through the NF-κB pathway. Inhibition of NF-κB or knockdown of p65 abrogated the TNF-α-induced stabilization of Slug. Knockdown of Slug expression inhibited cancer cell migration and EMT characteristics induced by TNF-α. Moreover, increased levels of Slug were found to correlate with lymph node metastasis and predict poor prognosis in patients with HNSCC. CONCLUSIONS: NF-κB-mediated stabilization of Slug underlies the inflammation-induced EMT and metastasis in HNSCC, which may serve as a therapeutic target for metastatic HNSCC.

Ultrasound hyperthermia enhances chemo-sensitivity in oral squamous cell carcinoma by TRIF-mediated pathway.

BACKGROUND: Hyperthermia is currently used as an alternative to surgery or in combination with chemotherapy and/or radiation therapy in the treatment of oral squamous cell carcinoma. However, little has been known about the change in chemo-sensitivity after hyperthermia and the mechanism underlie it in oral squamous cell carcinoma. METHODS: The aim of this study was to explore the influence of chemo-sensitivity of CAL-27 and SCC-4 cells by histoculture drug response assay after the animal model treated by the ultrasound hyperthermia. Then, we conducted a microarray between xenograft after hyperthermia at 42°C for 45 minutes and that with no treatment. We further confirmed the expression of TRIF in hyperthermia by immunohistochemistry, RT-PCR and Western blot. RESULTS: The chemo-sensitivity to five kinds of drugs demonstrated ultrasound hyperthermia performed in 42°C for 45 minutes would reach the highest inhibition rate in CAL-27 and SCC-4 cells. The microarray dataset revealed that 847 mRNA were upregulated and 1031 were downregulated. GO and pathway analyses indicated that they play an important role in translational initiation, nucleoplasm, and poly (A) RNA binding in the hyperthermia process. We further confirmed the expression of TRIF was downregulated in hyperthermia along with inactivation of NF-κB pathway. CONCLUSIONS: The experiments confirms the rationality of synchronous combination of hyperthermia and chemotherapy and may provide a better treatment that the use of sensitivity testing in such cases may lead to individualized, more effective therapy.

The clinical outcome of pembrolizumab for patients with recurrent or metastatic squamous cell carcinoma of the head and neck: a single center, real world study in China.

Background: The KEYNOTE-048 and KEYNOTE-040 study have demonstrated the efficacy of pembrolizumab in recurrent or metastatic squamous cell carcinoma of the head and neck (R/M HNSCC), we conducted this real-world study to investigate the efficacy of pembrolizumab in patients with R/M HNSCC. Methods: This is a single-center retrospective study conducted in the Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Shanghai, China). Between December 2020 and December 2022, a total of 77 patients with R/M HNSCC were included into analysis. The primary endpoint of the study was overall survival (OS), and the secondary endpoints were progression-free survival (PFS), overall response rate (ORR)and toxicity.Efficacy was assessed according to RECIST version 1.1.SPSS 27.0 and GraphPad Prism 8.0 software were utilized to perform the statistical analysis. Results: By the cut-off date (February 28, 2023), the median OS,PFS and ORR were 15.97 months,8.53 months and 48.9% in patients treated with the pembrolizumab regimen in the first line therapy. Among these patients, 17 patients received pembrolizumab with cetuximab,and 18 received pembrolizumab with chemotherapy.We observed no significant differences between two groups neither in median OS (13.9 vs 19.4 months, P=0.3582) nor PFS (unreached vs 8.233 months, P= 0.2807). In the ≥2nd line therapy (n=30), the median OS, PFS and ORR were 5.7 months, 2.58 months and 20% respectively. Combined positive score (CPS) was eligible from 54 patients. For first line therapy, the median OS and PFS were 14.6 and 8.53 months in patients with CPS ≥1, and median OS and PFS were 14.6 and 12.33 months in patients with CPS ≥20. The immune-related adverse events (irAEs) were occurred in the 31 patients (31/77, 40.26%), and the most common potential irAEs were hypothyroidism (25.97%), and pneumonitis (7.79%). Conclusion: Our real-world results indicated that pembrolizumab regimen is a promising treatment in patients with R/M HNSCC.

The association between body mass index and efficacy of pembrolizumab as second-line therapy in patients with recurrent/metastatic head and neck squamous cell carcinoma.

BACKGROUND: Recent evidence suggested a potential correlation between BMI and the efficacy of immune checkpoint inhibitors in cancer patients. This study aimed to evaluate the prognostic value of the body mass index (BMI) in recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) patients treat with pembrolizumab. METHODS: The current retrospective cohort study enrolled 49 R/M HNSCC patients underwent at least one cycle of pembrolizumab as second-line treatment from June 2018 to October 2020. Survival analysis of immunotherapy prognosis and risk factor analysis of age, gender, BMI, ECOG-PS, CPS, rT-stage, tumor site, and tube feeding. RESULTS: Among the 49 patients, the BMI at the time of immunotherapy ranged from 14.5 to 32.0 kg/m2 . The Kaplan-Meier analysis showed that the BMI was significantly correlated with overall survival time (OS, p = 0.0007) and progression-free survival time (PFS, p = 0.0012). BMI, gender, prior treatment, serum albumin level, ECOG-PS, CPS and rT-stage were analyzed in multivariate Cox regression model analysis after adjusted for potential confounding clinical variables. Patients with underweight (OS:HR = 6.862, 95% CI:1.566-30.064, p = 0.011; PFS:HR = 5.672, 95% CI:1.364-23.586, p = 0.017);ECOG≥2 (OS:HR = 0.250, 95% CI:0.086-0.731, p = 0.011;PFS:HR = 0.284, 95% CI:0.101-0.805, p = 0.018); CPS <1(OS: HR = 4.34, 95% CI:1.271-15.464, p = 0.019; PFS:HR = 3.859, 95% CI:1.180-12.618, p = 0.025) and rT4-stage(OS:HR = 4.380, 95% CI:1.452-13.209, p = 0.009;PFS: HR = 3.799, 95% CI:1.240-11.638, p = 0.019) suffered higher risk of mortality. CONCLUSIONS: The BMI at the time of clinical diagnosis was showed to be an independent predictive factor for R/M HNSCC patients receiving pembrolizumab. Compared with normal weight patients, underweight patients have worse clinical prognosis.

A human mucosal melanoma organoid platform for modeling tumor heterogeneity and exploring immunotherapy combination options.

Mucosal melanoma (MM), an aggressive rare subtype of melanoma, is distinct from cutaneous melanoma and has poor prognoses. We addressed the lack of cell models for MM by establishing 30 organoids of human oral MM (OMM), which retained major histopathological and functional features of parental tumors. Organoid groups derived from chronologically or intratumorally distinct lesions within the same individual displayed heterogeneous genetics, expression profiles, and drug responses, indicating rapid tumor evolution and poor clinical response. Furthermore, transcriptome analysis revealed receptor tyrosine kinases (RTKs) signaling, particularly NGFR, a nerve growth factor receptor, was significantly up-regulated in OMMs and organoids from patients resistant to anti-programmed cell death protein 1 (anti-PD-1) therapy. Combining anti-PD-1 with anlotinib (a phase 2 multitarget RTK inhibitor for OMM) or NGFR knockdown enhanced the effective activity of immune cells in organoid-immune cell coculture systems. Together, our study suggested that OMM organoids serve as faithful models for exploring tumor evolution and immunotherapy combination strategies.

A pilot study of camrelizumab with docetaxel and cisplatin for the first line treatment in recurrent/metastatic oral squamous cell carcinoma.

Pembrolizumab with cisplatin and 5-fluorouracil showed survival benefit but relatively high occurrence of treatment-related adverse events (TRAEs) for recurrent/metastatic oral squamous cell carcinoma (R/M OSCC). A more tolerable regime is needed. This trial enrolled 20 R/M OSCC patients with previously untreated and PD-L1 positive. Patients were administered camrelizumab with docetaxel and cisplatin every 3 weeks for six cycles, followed by camrelizumab monotherapy every 3 weeks until disease progression or intolerable toxicity. The primary endpoint was occurrence of grade ≥ 3 TRAEs, secondary endpoints included overall survival (OS), progression-free survival (PFS), and overall response rate (ORR). 45% patients experienced grade ≥ 3 TRAEs, which the most common were anemia (15%), stomatitis (15%), and neutropenia (10%). The most common potential immune-related adverse events were reactive cutaneous capillary endothelial proliferation (RCCEP; 60%), hypothyroidism (35%), and pneumonitis (15%). No treatment-related deaths occurred. The median OS, PFS, and ORR was 14.4 months, 5.35 months, and 40.0% respectively. The study also found RCCEP occurrence, lower FOXP3+ cells, and higher density of intratumor tertiary lymphoid structure were associated with improved efficacy. Our data suggest that camrelizumab with docetaxel/cisplatin as first-line therapy was well tolerable and had potentially favorite efficacy in PD-L1-positive patients with R/M OSCC.

Candidate therapeutic agents in a newly established triple wild-type mucosal melanoma cell line.

BACKGROUND: Mucosal melanoma has characteristically distinct genetic features and typically poor prognosis. The lack of representative mucosal melanoma models, especially cell lines, has hindered translational research on this melanoma subtype. In this study, we aimed to establish and provide the biological properties, genomic features and the pharmacological profiles of a mucosal melanoma cell line that would contribute to the understanding and treatment optimization of molecularly-defined mucosal melanoma subtype. METHODS: The sample was collected from a 67-year-old mucosal melanoma patient and processed into pieces for the establishment of cell line and patient-derived xenograft (PDX) model. The proliferation and tumorigenic property of cancer cells from different passages were evaluated, and whole-genome sequencing (WGS) was performed on the original tumor, PDX, established cell line, and the matched blood to confirm the establishment and define the genomic features of this cell line. AmpliconArchitect was conducted to depict the architecture of amplified regions detected by WGS. High-throughput drug screening (HTDS) assay including a total of 103 therapeutic agents was implemented on the established cell line, and selected candidate agents were validated in the corresponding PDX model. RESULTS: A mucosal melanoma cell line, MM9H-1, was established which exhibited robust proliferation and tumorigenicity after more than 100 serial passages. Genomic analysis of MM9H-1, corresponding PDX, and the original tumor showed genetic fidelity across genomes, and MM9H-1 was defined as a triple wild-type (TWT) melanoma subtype lacking well-characterized "driver mutations". Instead, the amplification of several oncogenes, telomerase reverse transcriptase (TERT), v-Raf murine sarcoma viral oncogene homolog B1 (BRAF), melanocyte Inducing transcription factor (MITF) and INO80 complex ATPase subunit (INO80), via large-scale genomic rearrangement potentially contributed to oncogenesis of MM9H-1. Moreover, HTDS identified proteasome inhibitors, especially bortezomib, as promising therapeutic candidates for MM9H-1, which was verified in the corresponding PDX model in vivo. CONCLUSIONS: We established and characterized a new mucosal melanoma cell line, MM9H-1, and defined this cell line as a TWT melanoma subtype lacking well-characterized "driver mutations". The MM9H-1 cell line could be adopted as a unique model for the preclinical investigation of mucosal melanoma.

The baseline oral microbiota predicts the response of locally advanced oral squamous cell carcinoma patients to induction chemotherapy: A prospective longitudinal study.

PURPOSE: Among oral squamous cell carcinoma (OSCC) patients who receive docetaxel, cisplatin, and 5-fluorouracil (TPF) induction chemotherapy, those with a favorable pathological response tend to obtain satisfactory clinical outcomes, while the total population exhibit no survival benefit. Thus, there is an urgent need to improve the therapeutic effect of TPF by applying personalized treatment according to distinct biomarkers. METHODS AND MATERIALS: In the present study, we collected oral rinse samples from 44 OSCC patients enrolled in our prospective multicenter random phase II trial before TPF induction chemotherapy to conduct 16S rRNA gene sequencing and metagenomic analysis. Patients were administrated with two cycles of TPF induction chemotherapy (75 mg/m2 cisplatin and 75 mg/m2 docetaxel on day 1 and 750 mg/m2 fluorouracil from the first to the fifth day), and then divided into responsive and nonresponsive groups according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. RESULTS: In the 16S rRNA gene sequence analysis, Fusobacterium and Mycoplasma were more enriched in the nonresponsive group, while Slackia was more enriched in the responder group at the genus level. In the metagenomic shotgun sequencing analysis, Fusobacterium nucleatum was more enriched in the nonresponsive group. Functional analysis showed that the platinum drug resistance pathway and microRNAs in cancer and RNA degradation pathways were remarkably associated with patient sensitivity to induction chemotherapy. CONCLUSIONS: Our data suggest that the oral microbiome may play an important role in the OSCC patient sensitivity to TPF induction chemotherapy and offer novel potential biomarkers for predicting the response to TPF induction chemotherapy.

A novel intronic circular RNA, circGNG7, inhibits head and neck squamous cell carcinoma progression by blocking the phosphorylation of heat shock protein 27 at Ser78 and Ser82.

BACKGROUND: There is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC). METHODS: We conducted whole-transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression. RESULTS: In this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27-JNK/P38 mitogen-activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27-JNK/P38 MAPK signaling and promoted tumor progression. CONCLUSIONS: Our results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27-JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue in the oromaxillofacial head and neck region: A retrospective analysis of 105 patients.

BACKGROUND: Extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) in the oromaxillofacial head and neck region is rare, with limited data available. This retrospective study explored the clinical features, stage, treatment, and prognosis of this disease. METHODS: Overall, 105 patients with MALT lymphomas in the oromaxillofacial head and neck region were included in this retrospective analysis. SPSS 22.0 software package was used for data analysis and a two-tailed P value of ≤.05 was considered statistically significant. Primary endpoints of the study were the complete response (CR) rate, overall survival (OS), and progression-free survival (PFS). RESULTS: About 52% of the patients had long-term xerostomia, autoimmune diseases, or chronic parotitis and 81% had diseases involving the large salivary glands. Ann Arbor staging of the patients was as follows: stages I/II, 73 patients and stages III/IV, 32 patients. In the 97 patients followed up, CR rate after initial treatment was 80%. Tumor progression was observed in 12 patients and 14 patients died. There was a significant difference between the rate of CR in localized (87%) and disseminated (67%) lymphoma patients (P = .02). The 5- and 10-year PFS of the localized lymphoma patients were both 91%, whereas those of the disseminated lymphoma patients were 83% and 65%, respectively (P = .03). The 5-year PFS rates of the chemotherapy and non-chemotherapy groups in the disseminated lymphoma patients were 85% and 73% (P = .04). Meanwhile, the 5-year PFS rates of the rituximab and non-rituximab groups in the disseminated lymphoma patients were 100% and 70% (P = .03). In multivariate analysis, MALT Lymphoma International Prognostic Index (MALT-IPI) was an independent prognostic factor affecting OS, whereas Ann Arbor staging affected PFS. CONCLUSIONS: This study suggests that the outcome after initial treatment of MALT lymphomas in the oromaxillofacial head and neck region is satisfactory and that this disease progresses slowly. The CR rate and PFS of localized lymphoma patients are better than those of disseminated lymphoma patients. Systemic treatment (chemotherapy or rituximab) may improve PFS in disseminated disease patients. MALT-IPI and Ann Arbor staging are independent prognostic factors.

EREG-driven oncogenesis of Head and Neck Squamous Cell Carcinoma exhibits higher sensitivity to Erlotinib therapy.

Rationale: The oncogenesis of head and neck squamous cell carcinoma (HNSCC) is believed to result from oncogene activation and tumor suppressor inactivation. Here, we identified a new oncogenic role for the EREG gene in HNSCC. Methods: The TCGA database and immunohistochemistry assay were used to analyze expression of EREG in HNSCC tissues. Immunoblotting was performed to identify the EGFR-mediated pathways altered by EREG. The role of EREG in oncogenesis was investigated in vivo and in vitro. Results: Upregulated EREG expression predicted a poor prognosis and triggered HNSCC oncogenic transformation by activating the epidermal growth factor receptor (EGFR) signaling pathway. We also demonstrated the direct association of EREG with EGFR and that this binding required EGFR domains I and III and the N57 residue of EREG. Moreover, EREG overexpression was shown to promote HNSCC oncogenesis by inducing C-Myc expression, and the pharmacological inhibition of C-Myc rescued EREG-promoted HNSCC oncogenesis. Unlike other EGFR ligands, EREG could mimic EGFR mutations by sustaining the activation of the EGFR-Erk pathway, and high EREG expression was positively associated with the response to treatment with the EGFR inhibitor erlotinib. Furthermore, knockdown of EREG decreased sensitivity to erlotinib treatment in vitro and in vivo. Conclusions: These results identify the EREG-EGFR-C-Myc pathway as a crucial axis that drives HNSCC oncogenesis and show that EREG expression could be a predictive functional marker of sensitivity to erlotinib therapy in HNSCC.

TLR4 activation leads to anti-EGFR therapy resistance in head and neck squamous cell carcinoma.

Epidermal growth factor receptor (EGFR) is highly expressed in head and neck squamous cell carcinoma (HNSCC) and related to cancer progression. The resistance to anti-EGFR therapy remains a major clinical problem in HNSCC. In this study, we found that TOLL-like receptor 4 (TLR4) was highly expressed in 50% of EGFR overexpressed HNSCC biopsies, which correlated to worse prognosis in patients. In HNSCC cell lines, activation of TLR4 reversed cetuximab-induced the inhibition of proliferation, migration and invasion. LPS induced of TLR4 signaling was potentiated under cetuximab treatment, showing increased activation of downstream NF-κB and MAPK pathways. Accordingly, cetuximab treatment also increased expression of TNF-α, COX2, and other molecules involved in TLR4 induced tumor inflammation. Mechanistically, we found inhibition of EGFR by cetuximab led to decreased phosphorylation of Src and sequentially Src-medicated activation of Cbl-b. This inhibited Cbl-b-mediated degradation of the key TLR4 adaptor protein MyD88 and activated TLR4 signaling. TLR4 or MyD88 overexpressed CAL27 and SCC4 cells grew faster and were more resistant to cetuximab and gefitinib both in vitro and in vivo. Out study delineates a crosstalk between EGFR and TLR4 pathways and identified TLR4 as a potential biomarker as well as a therapeutic target in overcoming the resistance to anti-EGFR therapy of HNSCC.

Cisplatin based induction chemotherapy modified by ERCC1 improved the outcome of young adults with locally advanced oral squamous cell carcinoma.

Objective: To evaluate the efficacy of induction chemotherapy in young adults with locally advanced oral squamous cell carcinoma (OSCC) and the usefulness of ERCC1 as a prognostic indicator. Methods: A total of 156 young adults with locally advanced OSCC were retrospectively analyzed from May 2007 to May 2017. Cisplatin based induction chemotherapy followed by surgery and upfront surgery were the primary treatment options for locally advanced OSCC. ERCC1 was evaluated by immunohistochemistry. Multivariate analysis was performed to identify significant prognostic factors for the overall survival (OS) in young adults with locally advanced OSCC. Results: Extracapsular spread (ECS) (p<0.0001) and UICC staging (p<0.0001) were critical prognostic factors for OS in young adults with locally advanced OSCC. The 5-year OS was 83.2% in N0 patients received induction chemotherapy and 61.7% in N0 patients received upfront surgery (p<0.05). Patients with a low ERCC1 expression were more likely to benefit from induction chemotherapy, as the 5-year OS was 22.4% in patients with a high ERCC1 expression and 84.7% in patients with a low ERCC1 expression, respectively (p<0.0001). However, induction chemotherapy resulted in a higher 5-year OS (84.7%) than upfront surgery (59.1%) in patients with a low ERCC1 expression (p=0.03). Conclusions: Induction chemotherapy can improve the outcome of N0 patients. However, the ERCC1 expression should be determined in young patients with locally advanced OSCC prior to induction chemotherapy, as it is a useful biomarker for predicting the outcome after induction chemotherapy.

Blocking autophagy flux promotes interferon-alpha-mediated apoptosis in head and neck squamous cell carcinoma.

Despite multiple antitumor activities, interferon-alpha (IFNα) therapy alone is less effective in solid tumors. Autophagy has been reported to play a key role in tumor chemoresistance. Therefore, it is meaningful to explore whether autophagy can be activated by IFNα in head and neck squamous cell carcinoma (HNSCC) and serve as a potential target to improve efficacy of IFNα therapy. In this study, we report that IFNα not only exhibits anti-proliferation activity and induces apoptosis, but also activates autophagy in HNSCC cells. Moreover, silencing autophagy-related protein 5 (ATG5) and signal transducer and activator of transcription 1 (STAT1) suppresses autophagy flux. Furthermore, IFNα and autophagy inhibitors (hydroxychloroquine and wortmannin) show clear synergistic effects on inhibiting growth and promoting apoptosis in HNSCC cells and xenograft models. Our findings indicate that IFNα-induced autophagy plays a cytoprotective role and blocking autophagy flux promotes IFNα-mediated apoptosis in HNSCC. These results suggest that the combination of IFNα and autophagy inhibitors represents a novel strategy for HNSCC treatment.

Cell division cycle 7 is a potential therapeutic target in oral squamous cell carcinoma and is regulated by E2F1.

Cell division cycle 7 (Cdc7) plays important roles in the regulation of the initiation of DNA replication throughout S phase. Whether inhibition of Cdc7 has a direct antitumour effect in oral squamous cell carcinoma (OSCC) remains unclear. In this study, XL413, a novel Cdc7 inhibitor, markedly inhibited the viability of OSCC cells but not that of non-tumour primary cells. There was a synergistic effect between XL413 and DNA-damaging agents (e.g. cisplatin and 5-fluorouracil) on OSCC in vitro and in vivo. Moreover, XL413 exhibited a notable antitumour effect on OSCC patients with high Cdc7 expression in mini patient-derived xenografts model. The proliferation was significantly inhibited in OSCC cells after Cdc7 silencing. Cdc7 knockdown significantly induced apoptosis in OSCC cell lines. Furthermore, we demonstrated that Cdc7 was overexpressed and transcriptionally regulated by E2F1 in OSCC by using chromatin immunoprecipitation and luciferase assays. Our results reveal that XL413 has an excellent antitumour effect in OSCC. Importantly, it does not inhibit the proliferation of non-tumour cells. These findings suggest that the overexpression of Cdc7 promotes progression in OSCC and that inhibition of Cdc7 is a very promising therapy for OSCC patients.

A comprehensive genome-wide analysis of the long noncoding RNA expression profile in metastatic lymph nodes of oral mucosal melanoma.

BACKGROUND & AIM: Oral mucosal melanoma (OMM) is a kind of malignancy with extremely rare morbidity. It exhibits a poorer biological behavior and clinical outcome compared with cutaneous melanoma. lncRNAs are endogenous cellular RNA transcripts with no protein-coding potential and are associated with oncogenesis through cis- or trans-acting mechanisms. Despite increased evidence that proved lncRNAs have vital roles in tumorigenesis of mucosal melanoma, little is known about their functions in the progress of lymph node dissemination of OMM. METHOD: Here, we constructed a lncRNA and mRNA microarray using six metastatic lymph nodes and paired-matched non-metastatic lymph nodes. Then, we performed RT-PCR to validate the microarray data both in primary and metastases. We further constructed lncRNA and mRNA co-expressing networks and analyzed the biological functions by Gene Ontology (GO) and pathway analyses for dysregulated lncRNAs and mRNAs. Cis- and trans-regulation analysis were also performed to explore the specific mechanism of lncRNAs in OMM. RESULT: Our results showed that 570 lncRNAs were upregulated with 292 lncRNAs downregulated in the metastatic OMM tissues. The results of RT-PCR were consistent with our microarray dataset both in primary and metastases. Gene Ontology (GO) and pathway analyses indicated that they play an important role in the melanin biosynthetic process, new growing cell tip and lysosomes in metastatic OMM. In the cis-regulation analysis, we observed metastasis-associated gene, PLEKHA5, the cis gene of lnc-AEBP2-1_1 and lnc-AEBP2-2_1, and microphthalmia-associated transcription factor (MITF), the cis gene of SAMMSON_3, SAMMSON_5 and lnc-MITF-5_1. In the trans-regulation analysis, CTBP2 and SUZ12 regulated lncRNA expression in the core TF-lncRNA-gene network. CONCLUSION: Our results suggest that lncRNAs may be involved in the metastasis of OMM, and further investigation is needed to focus on the biological functions and the underlining molecular mechanisms exerted by these dysregulated lncRNAs in OMM.

Altered expression pattern of circular RNAs in metastatic oral mucosal melanoma.

Circular RNAs (circRNAs) are known to be associated with carcinogenesis, and can serve as potential biomarkers for cancer diagnosis and therapeutic implications. However, little is known about their expression patterns in oral mucosal melanoma (OMM), an extremely rare cancer that is distinct from cutaneous melanoma for its clinical course and prognosis. To investigate circRNAs expression profile in OMM, we performed a circRNAs microarray to analyze 6 primary OMM samples with lymph nodes dissemination, and constructed a genome-wide circRNA profile. Our results revealed that 90 circRNAs were significantly dysregulated in the metastatic OMM tissues when compared to the paired adjacent tissues. Among them, hsa_circ_0005320, hsa_circ_0067531, hsa_circ_0008042 were significantly upregulated in primary tumor and metastatic lymph nodes compared to paired adjacent normal tissues and non-metastatic lymph nodes, whereas the expression of hsa_circ_0000869 and hsa_circ_0000853 were downregulated relatively. Gene Ontology (GO) and pathway analyses of differentially expressed circRNAs indicated that these identified circRNAs might play important roles in protein modification, protein binding and cellular metabolism in metastatic OMM. Functions of several selected circRNA were also identified. In addition, by using bioinformatics predictions, we further demonstrated that hsa_circ_0005320, hsa_circ_0067531 and hsa_circ_0000869 could serve as competing endogenous RNA (ceRNA), which might regulate tumorigenesis and metastatic of OMM by binding to specific microRNAs. Our results not only suggested that circRNAs might play critical roles in metastasis of OMM, but also provided critical information of circRNAs in regulating OMM progression. The findings would help us to develop potential biomarkers for clinical diagnosis and design therapeutic strategies for OMM.