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

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

Comprehensive impacts of different integrated rice-animal co-culture systems on rice yield, nitrogen fertilizer partial factor productivity and nitrogen losses: A global meta-analysis.

Integrated rice-animal co-culture (IRAC) is an ecological agricultural system combining rice cultivation with animal farming, which holds significant implications for food security and agriculture sustainable development. However, the comprehensive impacts of the co-culture on rice yield, nitrogen (N) losses, and N fertilizer partial factor productivity (NPFP) remain elusive and may vary under different environmental conditions and N management. Here, we conducted a meta-analysis of data from various IRAC systems on a global scale, including 371, 298, and 115 sets of data for rice yield, NPFP, and N losses, respectively. The results showed that IRAC could significantly increase rice yield (by 3.47 %) and NPFP (by 4.26 %), and reduce N2O emissions (by 16.69 %), NH3 volatilization (by 11.03 %), N runoff (by 17.72 %), and N leaching (by 19.10 %). Furthermore, there were significant differences in rice yield, NPFP, and N loss among different IRAC systems, which may be ascribed to variations in regional climate, soil variables, and N fertilizer management practices. The effect sizes of rice yield and NPFP were notably correlated with the rate and frequency of N application and the soil clay content. Moreover, a higher amount of precipitation corresponded to a larger effect size on rice NPFP. N2O emissions were closely associated with mean annual air temperature, annual precipitation, N application frequency, soil pH level, soil organic matter content, soil clay content, and soil bulk density. However, NH3 volatilization, N runoff, and N leaching exhibited no correlation with either the environmental conditions or the N management. Multivariate regression analysis further demonstrated that the soil clay content and N application rate are pivotal in predicting the effect sizes of rice yield, NPFP, and N2O emissions under IRAC. Specifically, IRAC with a low N application rate in soils with a high clay content could augment the effect size to increase rice NPFP and yield and reduce N2O emissions. In conclusion, IRAC offers a potent strategy to optimize rice yield and NPFP as well as mitigate N losses.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Gene sequencing and expression of Raf-1 in lymphatic metastasis of hypopharyngeal carcinoma.

OBJECTIVE: We performed differential gene screening for lymphatic metastasis of hypopharyngeal carcinoma by gene sequencing. We also aimed to investigate the expression and clinicopathological significance of the screened gene in hypopharyngeal carcinoma lymphatic metastasis. METHODS: The clinicopathological characteristics of 98 patients with hypopharyngeal carcinoma were collected to make survival analysis by Kaplan-Meier & log-rank test. Six cases of tumor tissues from patients with or without lymphatic metastasis were used for gene sequencing of differentially expressed genes. The most frequently differently expressed genes were validated by RT-PCR and Western blot in another 20 patients diagnosed for hypopharyngeal carcinoma. A total of 70 cases of hypopharyngeal carcinoma tumor tissues and normal tissues were investigated to examine the immunohistochemical expression and to explore the prognostic value by Kaplan-Meier & log-rank test and Cox's test. RESULTS: Lymphatic metastasis has been proved to cause a reduction in postoperative survival of patients with hypopharyngeal carcinoma. The results of gene sequencing analysis showed that Raf-1 was a differentially expressed gene in lymphatic metastasis of hypopharyngeal carcinoma. Moreover, the expression of Raf-1 was significantly up-regulated in tumor tissues of lymphatic metastasis patients compared to non-lymphatic metastasis tumor tissues and normal tissues. Meanwhile, Raf-1 had been verified to be an independent risk factor affecting the prognosis of hypopharyngeal carcinoma. CONCLUSIONS: For the first time, we investigated Raf-1 as an independent prognostic risk factor of lymphatic metastasis in hypopharyngeal carcinoma. It suggests that Raf-1 may serve as an important potential biomarker in preventing and diagnosing lymphatic metastasis in patients with hypopharyngeal carcinoma and improving the prognosis of patients.

Silencing Snail Reverses Epithelial-Mesenchymal Transition and Increases Radiosensitivity in Hypopharyngeal Carcinoma.

PURPOSE: Radioresistance in response to radiotherapy leads to cancer recurrence and poor survival in hypopharyngeal carcinoma patients. Previous studies indicate that ionizing radiation (IR) can induce epithelial-mesenchymal transition (EMT) that promotes the radioresistance, migration and invasiveness of tumors. The aim of this study was to explore the role of Snail in EMT and acquired radioresistance in hypopharyngeal carcinoma. METHODS: Radioresistance human hypopharyngeal carcinoma cells (FaduRR) were previously established from the Fadu cell line. Radiosensitivity was measured by colony forming assay. Western blot and Quantitative real-time PCR were used to detect the expression of EMT phenotypes and AKT/GSK-3ß/Snail signaling pathway related proteins in Fadu+4Gy and FaduRR cells. Transwell and wound-healing assays were used to measure cell migration and invasiveness. EMT-related proteins and Snail expression were assessed in 80 hypopharyngeal carcinoma patient samples from radiosensitive and radioresistance groups using immunohistochemistry. Snail was silenced to evaluate its effects on EMT, radioresistance, migration, and invasiveness of FaduRR cells. RESULTS: The molecular characteristics of EMT were observed following radiation treatment, with migration, invasiveness and radioresistance enhanced in Fadu+4Gy and FaduRR cells. Moreover, we demonstrated that IR-induced EMT by activating the AKT/GSK-3ß/Snail signaling pathway and that Snail silencing reversed EMT and attenuated radioresistance in FaduRR cells. Significant differences in EMT-related proteins and Snail expression were observed between radiosensitive and resistant group. CONCLUSION: We demonstrate that IR can trigger EMT and enhance the migration, invasiveness, and radioresistance of FaduRR cells through the AKT/GSK-3ß/Snail axis. Snail silencing could attenuate these effects and represents a novel therapeutic target for EMT-induced radioresistance in hypopharyngeal carcinoma.

The Homologous Recombination Repair Pathway is Associated with Resistance to Radiotherapy in Nasopharyngeal Carcinoma.

Radiotherapy plays a major role in the management of nasopharyngeal carcinoma (NPC). However, the radioresistant cells limit its efficiency and drive recurrence inside the irradiated tumor volume leading to poor outcome for patients. To illuminate the signal pathway involved in the radioresistance and evaluate the potential for predicting NPC response to radiotherapy, we established the radioresistant NPC cell line (CNE2-RR) derived from NPC cell line CNE2 by gradually increased the radiation dose (total 60 Gy), and the radioresistance of CNE2-RR cells was evaluated by the colony formation, FCM and comet assays. Furthermore, comparison of established CNE2-RR cell line to parental cell line found the homologous recombination repair (HRR) proteins differences involved in NPC radioresistance. In addition, the differentially expressed proteins were further validated by western blotting, immunofluorescence and IHC in tumor xenografs and radioresistant NPC tissues. Furthermore, the correlation of HRR proteins expression levels with NPC radioresistance were evaluated. The results showed that the upregulation of HRR proteins were significantly correlated with NPC radioresistance. In addition, using the Youden Index cutoff value, a panel of the HRR proteins analyses revealed a sensitivity of 70%, specificity of 72%. Furthermore, silencing NFBD1 enhanced the radiosensitivity of CNE2-RR cells by impairing IR-inducing γ-H2AX and HR proteins foci formation. These results suggest that controlling the HRR signaling pathway may hold promise to overcome NPC radioresistance.

PARP inhibitor Olaparib increases the sensitization to radiotherapy in FaDu cells.

Radioresistance causes a major problem for improvement of outcomes of patients treated with radiation. Targeting for DNA repair deficient mechanisms is a hallmark of sensitization to resistance. We tested whether Olaparib, a (poly) ADP-ribose polymerase (PARP) inhibitor, can sensitize the radioresistant FaDu cells to radiotherapy. Radioresistant FaDu cells, called FaDu-RR cells, were used as the radioresistant hypopharyngeal cancer models. The expression of PARP1 was detected in both FaDu and FaDu-RR cells. The role of Olaparib in radiosensitization was analysed with several assays including clonogenic cell survival, cell proliferation and cell cycle, and radioresistant xenograft. High expression of PARP1 had a significant effect on enhancing radioresistance in FaDu-RR cells compared with FaDu cells. After treatment of Olaparib, FaDu-RR cells showed significantly less and smaller surviving colonies, lower proliferation ability and G2/M arrest than those in the group without treatment. Moreover, Olaparib significantly reduced growth of tumours in FaDu-RR cell xenografts treated with ionizing radiation. Olaparib can significantly inhibit PARP1 expression and consequently has significant effects on radiosensitization in FaDu-RR cells. These results indicate that Olaparib may help individualize treatment and improve their outcomes of hypopharyngeal cancer patients treated with radiation.

Protocadherin 17 is a tumor suppressor and is frequently methylated in nasopharyngeal carcinoma.

PURPOSE: Several PCDH genes were shown to be downregulated or silenced in carcinomas and act as candidate tumor suppressor genes. However, the functions of PCDH17 in nasopharyngeal carcinoma (NPC) remain unclear. Here, we investigated the PCDH17 promoter methylation status and its impact on the expression and functions of PCDH17 in NPC. PATIENTS AND METHODS: To determine the mRNA levels and promoter methylation status of PCDH17 in NPC cell lines as well as 42 NPC patient specimens, we performed reverse transcription PCR, methylation-specific PCR, and bisulfite genome sequencing. The effects of ectopic PCDH17 expression in NPC cell lines were determined by colony formation, cell proliferation, wound healing, in vitro human umbilical vein endothelial cells tube formation, migration, invasion, cell cycle, and apoptosis assays and an in vivo subcutaneous tumor model. RESULTS: PCDH17 expression was almost absent or significantly reduced in 100% of the NPC cell lines (5/5). However, 5-aza-2'-deoxycytidine and trichostatin A treatment restored PCDH17 expression. Promoter methylation was involved in PCDH17 silencing. Ectopic expression of PCDH17 in silenced NPC cells reduced colony formation, cell migration, angiogenesis, VEGF secretion, and tumorigenicity. CONCLUSION: PCDH17 plays a tumor suppressor role in NPC. PCDH17 methylation may be a tumor-specific event and can be used as an epigenetic biomarker for NPC.

Loss of NFBD1/MDC1 disrupts homologous recombination repair and sensitizes nasopharyngeal carcinoma cells to PARP inhibitors.

BACKGROUND: Nasopharyngeal carcinoma (NPC), a highly invasive tumor, exhibits a distinctive racial and geographic distribution. As options of agents for effective combination chemoradiotherapy for advanced NPC are limited, novel therapeutic approaches are desperately needed. Here the potential of silencing NFBD1 in combination with PARP inhibition as a novel therapeutic strategy for NPC was investigated. METHODS: To investigate the function of NFBD1, we created NFBD1-depleted NPC cell lines via lentivirus mediated shRNA, and the colony formation, MTS assay, comet assay and apoptosis analysis were used to evaluate the sensitivity of NFBD1 knockdown on PARP inhibition. The signaling change was assessed by western blot, Immunofluorescence and flow cytometry. Furthermore, Xenografts model was used to evaluate the role of silencing NFBD1 in combination with PARP inhibition. RESULTS: We find that silencing NFBD1 in combination with PARP inhibition significantly inhibits the cell proliferation and cell cycle checkpoint activity, and increases the apoptosis and DNA damage. Mechanistic studies reveal that NFBD1 loss blocks olaparib-induced homologous recombination repair by decreasing the formation of BRCA1, BRCA2 and RAD51 foci. Furthermore, the xenograft tumor model demonstrated significantly increases sensitivity towards PARP inhibition under NFBD1 deficiency. CONCLUSIONS: We show that NFBD1 depletion may possess sensitizing effects of PARP inhibitor, and consequently offers novel therapeutic options for a significant subset of patients.

IL-37 attenuates allergic process via STAT6/STAT3 pathways in murine allergic rhinitis.

Allergic rhinitis (AR) is a common upper airway allergic disease caused by allergens triggering a type 2 immune response. The imbalance of CD4+ T cell subsets is the essential immunological feature of AR, which is mainly characterized by the predominance of T helper (Th) 2 cells. Recent studies indicated that the anti-inflammatory factor interleukin (IL)-37 is involved in the immune regulation of AR. However, the mechanism of IL-37 acts on AR has not been fully elucidated. Thus, we sought to assess the protective role of IL-37 in AR and further explore the possible mechanism. An ovalbumin (OVA)-induced AR murine model was established. After IL-37 treatment, the allergic symptoms (sneezes and nasal rubbings), nasal mucosal infiltration with eosinophils, and serum IgE production were found significantly attenuated. For CD4+ T cell subsets, the proliferation and differentiation of Th2 and Th17 cells were restrained. The relevant effector cytokines of IL-4, IL-5, IL-6, and IL-17a protein expression and transcription factors GATA3 and RORγt mRNA levels were obviously decreased. However, IL-37 had no significant effect on Th1 and Treg response including in IFN-γ, IL-10, T-bet, and Foxp3 expression. Furthermore, IL-37 was found down-regulated the STAT6, STAT3, phospho-STAT6, and phospho-STAT3 expression. In conclusion, IL-37 alleviates allergic inflammation in AR possibly through repressing STAT6 and STAT3 signaling pathways.

RPA1 downregulation enhances nasopharyngeal cancer radiosensitivity via blocking RAD51 to the DNA damage site.

BACKGROUND/AIM: Nasopharyngeal cancer (NPC) has a high local recurrence rate due to its resistance to ionizing radiation (IR). Replication protein A1 (RPA1) is one of the main elements in the homologous repair (HR) pathway, which is closely associated with the repair of DNA double strand breaks (DDBs). Studies on the relationship between RPA1 and the radiosensitivity of NPC are substantially limited. It was hypothesized that RPA1 plays a crucial role in predicting the radiosensitivity of NPC. METHODS: The protein expression of RPA1 in 182 patients with NPC in the complete response (CR) and non-complete response (nCR) groups was evaluated using immunohistochemistry. Then, univariate and multivariate analysis were performed using SPSS software vision 22 to determine the relationship between the expression of RPA1 and the clinicopathological features. In addition, the mRNA expression of RPA1 was tested in 24 fresh samples using qRT-PCR. RPA1 was silenced in CNE-2R cell lines combined with IR to measure the radiosensitivity, proliferation, DNA damage repair and cell cycle of CNE-2R cells. Xenograft models in nude mice were used to determine the effect of RPA1 on tumor growth after IR. Immunoblotting and immunofluorescence staining were performed to identify proteins that interacted with RPA1. All statistical tests were two-sided. RESULTS: RPA1 protein was overexpressed in NPC patients with nCR (65.31%), and was an independent predictor of radiosensitivity (HR: 3.755, 95% CI: 1.990-7.085), in addition to Epstein-Barr virus (EBV; HR: 3.984; 95% CI: 1.524-10.410). The silencing of RPA1 increased the radiosensitivity of CNE-2R cells, blocked the repair of DNA, impaired cell proliferation, and contributed to G2/M cell cycle arrest. Furthermore, the xenograft models in nude mice revealed that silencing RPA1 combined with irradiation significantly retarded the growth of tumors. Moreover, the knockdown of RPA1 decreased Rad51 collection to the damage site and prolonged the time of DNA repair. CONCLUSION: RPA1 protein is frequently overexpressed in NPC patients with nCR. The silencing of RPA1 enhanced the radiosensitivity of CNE-2R cells. These present findings reveal that RPA1 is a potential biomarker for predicting the radiosensitivity in NPC.