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Ultra-high dose rate FLASH irradiation (FLASH-IR) has been shown to cause less normal tissue damage compared with conventional irradiation (CONV-IR), this is known as the "FLASH effect." It has attracted immense research interest because its underlying mechanism is scarcely known. The purpose of this study was to determine whether FLASH-IR and CONV-IR induce differential inflammatory cytokine expression using a modified clinical linac.An Elekta Synergy linac was used to deliver 6 MeV CONV-IR and modified to deliver FLASH-IR. Female FvB mice were randomly assigned to three different groups: a non-irradiated control, CONV-IR, or FLASH-IR. The FLASH-IR beam was produced by single pulses repeated manually with a 20-s interval (Strategy 1), or single-trigger multiple pulses with a 10 ms interval (Strategy 2). Mice were immobilized in the prone position in a custom-designed applicator with Gafchromic films positioned under the body. The prescribed doses for the mice were 6 to 18 Gy and verified using Gafchromic films. Cytokine expression of three pro-inflammatory cytokines (tumor necrosis factor-α [TNF-α], interferon-γ [IFN-γ], interleukin-6 [IL-6]) and one anti-inflammatory cytokine (IL-10) in serum samples and skin tissue were examined within 1 month post-IR.The modified linac delivered radiation at an intra-pulse dose rate of around 1 × 10Ultra-high dose rate electron FLASH caused lower pro-inflammatory cytokine levels in serum and skin tissue which might mediate differential tissue damage between FLASH-IR and CONV-IR.

Suppression of PTPN3 increased the cell colony numbers. Representative micrographs and quantification of crystal violet stained cells from 3 independent experiments. Columns, average of three independent experiments; bars, SD. *P < 0.05, **P < 0.01, Student t test.

PTPN3 mRNA was significantly lower in all tumor samples than in paired normal tissues of the TCGA database. a-c. http://gepia.cancer-pku.cn/detail.php?gene=PTPN3

Supplementary Table S1. Clinical characteristics of 534 NPC patients. Supplementary Table S2. Sequences used for shRNA construction and siRNAs. Supplementary Table S3. Primers used for real-time PCR. Supplementary Table S4. Association of SPINK6 expression with clinicopathological characteristics of NPC patients.

Supplementary Materials and Methods

Supplementary Fig. S1. Expression of SPINK6 in condition medium of NPC cells. Supplementary Fig. S2. Cellular growth of NPC cells. Supplementary Fig. S3. Wound-healing ability of NPC cells upon SPINK6 knockdown. Supplementary Fig. S4. Migration and invasion of NPC cells upon silencing of KLKs. Supplementary Fig. S5. Promoted motility of NPC cells by recombinant SPINK6. Supplementary Fig. S6. Interaction between SPINK6 and EGFR. Supplementary Fig. S7. Phosphorylation of EGFR and downstream signaling molecules in NPC cells. Supplementary Fig. S8. Wound-healing ability of NPC cells. Supplementary Fig. S9. The expression of E-cadherin and vimentin in NPC cells. Supplementary Fig. S10. The expression of EGF, EGFR and KLKs in NPC and NP tissues.

Nasopharyngeal carcinoma has the highest rate of metastasis among head and neck cancers, and distant metastasis is the major reason for treatment failure. The underlying molecular mechanisms of nasopharyngeal carcinoma metastasis are not fully understood. Here, we report the identification of serine protease inhibitor Kazal-type 6 (SPINK6) as a functional regulator of nasopharyngeal carcinoma metastasis via EGFR signaling. SPINK6 mRNA was upregulated in tumor and highly metastatic nasopharyngeal carcinoma cells. Immunohistochemical staining of 534 nasopharyngeal carcinomas revealed elevated SPINK6 expression as an independent unfavorable prognostic factor for overall, disease-free, and distant metastasis–free survival. Ectopic SPINK6 expression promoted in vitro migration and invasion as well as in vivo lymph node metastasis and liver metastasis of nasopharyngeal carcinoma cells, whereas silencing SPINK6 exhibited opposing effects. SPINK6 enhanced epithelial–mesenchymal transition by activating EGFR and the downstream AKT pathway. Inhibition of EGFR with a neutralizing antibody or erlotinib reversed SPINK6-induced nasopharyngeal carcinoma cell migration and invasion. Erlotinib also inhibited SPINK6-induced metastasis in vivo. Notably, SPINK6 bound to the EGFR extracellular domain independent of serine protease–inhibitory activity. Overall, our results identified a novel EGFR-activating mechanism in which SPINK6 has a critical role in promoting nasopharyngeal carcinoma metastasis, with possible implications as a prognostic indicator in nasopharyngeal carcinoma patients. Cancer Res; 77(2); 579–89. ©2016 AACR.

The immune regulator galectin-9 (Gal-9) is commonly involved in the regulation of cell proliferation, but with various impacts depending on the cell type. Here, we revealed that Gal-9 expression was persistently increased in Epstein-Barr virus (EBV)-infected primary B cells from the stage of early infection to the stage of mature lymphoblastoid cell lines (LCLs). This sustained upregulation paralleled that of gene sets related to cell proliferation, such as oxidative phosphorylation, cell cycle activation, and DNA replication. Knocking down or blocking Gal-9 expression obstructed the establishment of latent infection and outgrowth of EBV-infected B cells, while exogenous Gal-9 protein promoted EBV acute and latent infection and outgrowth of EBV-infected B cells at the early infection stage. Mechanically, stimulator of interferon gene (STING) activation or signal transducer and activator of transcription 3 (STAT3) inhibition impeded the outgrowth of EBV-infected B cells and promotion of Gal-9-induced lymphoblastoid cell line (LCL) transformation. Accordingly, Gal-9 expression was upregulated by forced EBV nuclear antigen 1 (EBNA1) expression in 293T cells

Background Hepatocellular carcinoma (HCC), a common type of cancer, has a strong metastatic ability and poor prognosis. The tumor microenvironment is the “soil” for the occurrence and development of tumors, with exosomes playing an important role in these processes. In traditional Chinese medicine(TCM), the tumor microenvironment corresponds to the internal environment of the syndrome known as spleen deficiency (SD). Numerous studies have shown that exosomes contain high levels of miRNAs, which have been shown to contribute to tumor immune regulation and metastasis. The aim of this study was to explore the mechanisms underlying the changes in the tumor microenvironment under the condition of spleen deficiency in order to find better treatments for cancer. Methods The effects of exosomal miR-29a-3p on lung metastasis from hepatocellular carcinoma (HCC) were evaluated using the scratch test, migration test, mouse SD model, HCC model, and tail-vein injection model of lung metastasis. The western blot assay, ELISA, flow cytometry, luciferase reporter gene analysis, qRT-PCR and immunofluorescence staining were among the methods used to study the molecular mechanism of lung metastasis promotion under the SD internal environment. Results Compared with the mice with HCC only, the mice with HCC and SD symptoms secreted more miR-29a- 3p-enriched exosomes, and their tumor tissue expressed significantly higher levels of α1-integrin and lower levels of FAM167A. These changed the immune microenvironment of mice (Decreased infiltration of T cells (CD3+CD4+ and CD3+CD8+), activated α1-integrin-NF-κB signaling pathway, and secreted more interleukin inflammatory factors(IL-1β, IL-6, and IL-8), which promoted the invasion and infiltration of HCC and its lung metastasis both in vivo and in vitro. In a series of patients with liver cancer, SD was found to have affected their overall survival and relapse-free survival. Conclusion Our study showed that under conditions of SD, the body releases more miRNA-containing exosomes, changes the immune microenvironment of the body, and ultimately promotes tumor metastasis and growth. These results highlight potential therapeutic targets and methods for the prevention of cancer metastasis, which may help to screen possible anticachexia TCMs and elucidate its mechanism in the future.

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor that commonly occurs worldwide. Usually, Asia, especially China, has a high incidence of esophageal cancer. ESCC often has a poor outcome because of a late diagnosis and lack of effective treatments. To build foundations for the early diagnosis and treatment of ESCC, we used the gene expression datasets GSE20347 and GSE17351 from the GEO database and a private dataset to uncover differentially expressed genes (DEGs) and key genes in ESCC. Notably, we found that replication factor C subunit 4 (RFC4) and guanine monophosphate synthase (GMPS) were upregulated but have been rarely studied in ESCC. In particular, to the best of our knowledge, our study is the first to explore GMPS and ESCC. Furthermore, we found that high levels of RFC4 and GMPS expression may result from an increase in DNA copy number alterations. Furthermore, RFC4 and GMPS were both upregulated in the early stage and early nodal metastases of esophageal carcinoma. The expression of RFC4 was strongly correlated with GMPS. In addition, we explored the relationship between RFC4 and GMPS expression and tumor-infiltrating immune cells (TILs) in esophageal carcinoma. The results showed that the levels of RFC4 and GMPS increased with a decrease in some tumor-infiltrating cells. Upregulated RFC4 and GMPS with high TILs indicate a worse prognosis. In summary, our study shows that RFC4 and GMPS have potential as biomarkers for the early diagnosis of ESCC and may played a crucial role in the process of tumor immunity in ESCC.

Background It has been known for years that the same genetic defects drive breast cancer formation, yet, the onset of breast cancer in different individuals among the same population differs greatly in their life spans with unknown mechanisms. Methods We used a MMTV-PyMT mouse model with different genetic backgrounds (FVB/NJ vs. C57BL/6J) to generate different cancer onset phenotypes, then profiled and analyzed the gene expression of three tumor stages in both Fvb.B6 and Fvb mice to explore the underlying mechanisms. Results We found that in contrast with the FVB/N-Tg (MMTV-PyMT) 634Mul mice (Fvb mice), mammary tumor initiation was significantly delayed and tumor progression was significantly suppressed in the Fvb.B6 mice (generated by crossing FVB/NJ with C57BL/6J mice). Transcriptome sequencing and analysis revealed that the differentially expressed genes were enriched in immune-related pathways. Flow cytometry analysis showed a higher proportion of matured dendritic cells in the Fvb.B6 mice. The plasma levels of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) were significantly reduced in the Fvb.B6 mice. IL-6 also impaired the maturation of bone marrow dendritic cells (BMDCs) of the Fvb mice in vitro. Conclusion All these findings suggest that immunity levels (characterized by a reduced IL-6 level and intact DC maturation in Fvb.B6 mice) are the key factors affecting tumor onset in a murine mammary cancer model.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignant tumors in China. However, there are no targets to treat ESCC because the molecular mechanism behind the cancer is still unclear. Here, we found a novel long noncoding RNA LINC02820 was upregulated in ESCC and associated with the ESCC clinicopathological stage. Through a series of functional experiments, we observed that LINC02820 only promoted the migration and invasion capabilities of ESCC cell lines. Mechanically, we found that LINC02820 may affect the cytoskeletal remodeling, interact with splice factor 3B subunit 3 (SF3B3), and cooperate with TNFα to amplify the NF-κB signaling pathway, which can lead to ESCC metastasis. Overall, our findings revealed that LINC02820 is a potential biomarker and therapeutic target for the diagnosis and treatment of ESCC.

Distant metastasis is the major cause of short survival in ccRCC patients. However, the development of effective therapies for metastatic ccRCC is limited. Herein, we reported that ETV4 was selected from among 150 relevant genes with in vivo evidence of promoting metastasis. In this study, we identified that ETV4 promoted ccRCC cell migration and metastasis in vitro and in vivo, and a positive correlation between ETV4 and FOSL1 expression was found in ccRCC tissues and cell lines. Further investigation suggested that ETV4 increase FOSL1 expression through direct binding with the FOSL1 promoter. Furthermore, ETV4/FOSL1 was proved as a novel upstream and downstream causal relationship in ccRCC in an AKT dependent manner. In addition, both ETV4 and FOSL1 serve as an independent, unfavorable ccRCC prognostic indicator, and the accumulation of the ETV4 and FOSL1 in ccRCC patients result in a worse survival outcome in ccRCC patients. Taken together, our results suggest that the ETV4/FOSL1 axis acts as a prognostic biomarker and ETV4 directly up-regulates FOSL1 by binding with its promoter in a PI3K-AKT dependent manner, leading to metastasis and disease progression of ccRCC.

The aldo‐keto reductases family 1 member C2 (AKR1C2) has critical roles in the tumorigenesis and progression of malignant tumours. However, it was also discovered to have ambiguous functions in multiple cancers and till present, its clinical significance and molecular mechanism in oesophageal squamous cell carcinoma (ESCC) has been unclear. The aim of this study was to explore the role of AKR1C2 in the tumorigenesis of ESCC. Here, we showed that AKR1C2 expression was found to be up‐regulated in ESCC tissues and was significantly associated with pathological stage, lymph node metastasis and worse outcomes. Functional assays demonstrated that an ectopic expression of AKR1C2 in ESCC cells resulted in increased proliferation, migration and cisplatin resistance, while knockdown led to inversing effects. Bioinformation analyses and mechanistic studies demonstrated that AKR1C2 activated the PI3K/AKT signalling pathway, furthermore, the inhibitor of PI3K or the selective inhibitor of AKR1C2 enzyme activity could reverse the aggressiveness and showed synergistic antitumour effect when combined with cisplatin, both in vitro and in vivo. In conclusion, Our findings revealed that AKR1C2 could function as an oncogene by activating the PI3K/AKT pathway, as a novel prognostic biomarker and/or as a potential therapeutic target to ESCC.

Nasopharyngeal carcinoma (NPC) is a unique head and neck cancer with highly aggressive and metastatic potential in which distant metastasis is the main reason for treatment failure. Till present, the underlying molecular mechanisms of NPC metastasis remains poorly understood. Here, we identified S100 calcium-binding protein A14 (S100A14) as a functional regulator suppressing NPC metastasis by inhibiting the NF-kB signaling pathway and reversing the epithelial-mesenchymal transition (EMT). S100A14 was found to be downregulated in highly metastatic NPC cells and tissues. Immunohistochemical staining of 202 NPC samples revealed that lower S100A14 expression was significantly correlated with shorter patient overall survival (OS) and distant metastasis-free survival (DMFS). S100A14 was also found as an independent prognostic factor for favorable survival. Gain- and loss-of-function studies confirmed that S100A14 suppressed the in vitro and in vivo motility of NPC cells. Mechanistically, S100A14 promoted the ubiquitin-proteasome-mediated degradation of interleukin-1 receptor-associated kinase 1 (IRAK1) to suppress NPC cellular migration. Moreover, S100A14 and IRAK1 established a feedback loop that could be disrupted by the IRAK1 inhibitor T2457. Overall, our findings showed that the S100A14-IRAK1 feedback loop could be a promising therapeutic target for NPC metastasis.

Background: Dietary fat absorption involves the re-esterification of digested triacylglycerol in the enterocytes, it is a biological process catalyzed by monoacylglycerol O-acyltransferase 2 (MOGAT2, aka MGAT2), which is highly expressed in the small intestine. A previous study showed that the loss of the Mogat2 gene can prevent high-fat diet-induced obesity in mice. Obesity is associated with an increased risk of several types of cancer including postmenopausal breast cancer.Methods: We collected 147 patients with triple negative breast adenocarcinoma to explore the relationship between the expression of MOGAT2 and patient overall survival. And we generated a Mogat2-deficient mouse mammary tumor model by crossing Mogat2-deficient mice with MMTV-PyMT mice to examine the effect of losing MOGAT2 in vivo.Results: Our founding suggest that obesity was induced by a relatively high-fat diet (37% of calories from fat) in the mice with or without Mogat2 knockout. Mammary tumor development was deteriorated by a relatively high-fat diet regardless of Mogat2 deficiency. As a compensation mechanism, upregulation of diacylglycerol O-acyltransferases 1 and 2 (Dgat1 and Dgat2) in the Mogat2 deficient mice was found. Conclusions: Elevated expression of MOGAT2 in triple negative breast adenocarcinoma predicts poorer patient overall survival. With the compensation of Dgat1 and Dgat2, Mogat2 deficiency alone cannot prevent fat diet-induced obesity, nor prevent mammary tumor development in a mouse model.

Purpose: Ultra-high dose rate FLASH irradiation has been shown to cause less normal tissue damage compared with conventional irradiation, also termed “FLASH effect”. However, the underlying mechanism was scarcely known. The purpose of the present study was to determine whether FLASH and conventional irradiation would induce differential inflammatory cytokines expression. Materials and methods: Female FvB mice were randomly assigned to three different groups: non-irradiated control, conventional (CONV) and FLASH groups. Mice were irradiated at 6 to 19 Gy of CONV (0.1 Gy/s) or FLASH (38.5-600 Gy/s) irradiation using an Elekta Synergy linac (6 MeV). Mice were immobilized in prone position in a custom-designed applicator with dosimetry films positioned under the body. Dose were verified by Gafchromic films. Enzyme linked immunosorbent assay (ELISA) were performed in serum samples of the mice at 6, 18 and 31 days after irradiation for four inflammatory cytokines: tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-6 (IL-6) and IL-10. Flow cytometry using antibodies for CD3, CD8, CD4 and CD45 in blood were performed pre- and 1-week post irradiation. Results: At D6 (18-19 Gy), both IL-6 and TNF-α were elevated, and IL-10 was reduced in FLASH and CONV group, while IFN-γ was only significantly increased in conventional group, compared with control group. At D18 (10 Gy) and D31 (13-19 Gy), conventional RT significantly elevated levels of IL-6, IFN-γ and TNF-α and reduced IL-10 level compared with FLASH group and control group. Additionally, even low dose conventional irradiation (13 Gy) could induce higher level of pro-inflammatory cytokines and lower level of anti-inflammatory cytokine than high dose (17-19 Gy) FLASH irradiation at D31. Flow cytometry showed that the CD8+/CD45+ ratio in the blood were higher in the conventional than in FLASH. These data indicate that minor inflammatory cytokine levels of serum in FLASH could be result of the absent of immune overactivation induced by conventional irradiation. Conclusions: Ultra-high dose rate electron FLASH caused less inflammatory cytokine levels of serum which might be a result from less CD8+/CD45+ ratio in the blood. Thus, differential cytokines and CD8+ T cell expression between FLASH and conventional irradiation would be a potential mechanism for “FLASH effect”.

Hong-Fa Xu,1,2,* Tie-Jun Huang,3,* Qin Yang,1 Liang Xu,1 Fen Lin,1 Yan-Hong Lang,1 Hao Hu,4 Li-Xia Peng,1 Dong-Fang Meng,1 Yu-Jie Xie,1 Li Tan,5 Chao-Nan Qian,1 Bi-Jun Huang11Department of Experimental Research, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-Sen University Cancer Center, Guangzhou 510060, People’s Republic of China; 2Zhuhai Precision Medical Center, Zhuhai People’s Hospital Affiliated to Jinan University, Zhuhai 519000, People’s Republic of China; 3Department of Nuclear Medicine, The Second People’s Hospital of Shenzhen, Shenzhen 518035, People’s Republic of China; 4Department of Traditional Chinese Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou 510060, People’s Republic of China; 5Center of Hematology, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510230, People’s Republic of China*These authors contributed equally to this work Background/Aims: The tumor-suppressive functions of interferon regulatory factor 6 (IRF6) in some tumors have been preliminarily established, but its pathogenesis and underlying molecular mechanisms in breast cancer, the most common malignancy in women, remains poorly understood.Methods: Pairs of typical breast cancer cell lines (high- and low-aggressive) in addition to 27 breast cancer tissue samples and 31 non-cancerous breast tissues were used to investigate the expression level of IRF6 and Lentivirus-mediated gain-of-function studies, short hairpin RNA-mediated loss-of-function studies in vivo and in vitro were used to validate the role of IRF6 in breast cancer. Next, we performed RNA-Seq analysis to identify the molecular mechanisms of IRF6 involved in breast cancer progression.Results: Our findings showed that IRF6 was downregulated in highly invasive breast cancer cell lines but upregulated in poorly aggressive ones. Functional assays revealed that elevated IRF6 expression could suppress cell proliferation and tumorigenicity, and enhanced cellular chemotherapeutic sensitivity. To identify the molecular mechanisms involved, we performed a genome-wide and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis in breast cancer cells using RNA sequencing of gene expression profiles following the overexpression of IRF6. Genome-wide and KEGG analyses showed that IRF6 might mediate the PI3K-regulatory subunit PIK3R2, which in turn modulated the PI3K/AKT pathway to control breast cancer pathogenesis.Conclusion: We provide the first evidence of the involvement of IRF6 in breast cancer pathogenesis, which was found to modulate the PI3K/AKT pathway via mediating PIK3R2; indicating that IRF6 can be targeted as a potential therapeutic treatment of breast cancer.Keywords: breast cancer, IRF6, proliferation, RNA sequencing, PIK3R2, PI3K/AKT

BackgroundAccumulating evidence has shown that dysregulated expression of microRNAs plays a key role in tumorigenesis. To explore the mechanisms of this we conducted this study.MethodsFive Gene Expression Omnibus datasets (GEO) datasets , GSE32960, GSE36682, GSE43039, GSE70970 and GSE118613 and head and neck squamous cell carcinoma data of The Cancer Genome Atlas (TCGA) were analysis in this study.ResultsBy analyzing the microRNA expression profile of nasopharyngeal carcinoma (NPC) in the five GEO datasets, we identified miR-150-5p as potential biomarker for patient survival. To explore the mechanisms of this, We examined the head and neck squamous cell carcinoma data of TCGA and found that miR-150-5p was correlated with high enrichment of tumor-infiltrating B cells, low enrichment of cancer-associated fibroblasts and down-regulated oncogenic pathways. miR-150-5p may also improve the immune response in the tumor microenvironment. These findings may explain how miR-150-5p improves outcome of head and neck squamous cell carcinoma patients including NPC. Additionally, the exosomal long non-coding RNA AC073130.1 was identified as a potential regulator of miR-150-5p. As miR-150-5p can also be released via exosomes, this study provides insight into the cross-talk of tumor cells and B cells in the tumor microenvironment via exosomal AC073130.1 and miR-150-5p. ConclusionMiR-150-5p improves outcome of head and neck squamous cell carcinoma patients by improving the immune response. There might be a cross-talk of tumor cells and B cells in the tumor microenvironment via exosomal AC073130.1 and miR-150-5p.

Background: Dietary fat absorption involves the re esterification of digested triacylglycerol in the enterocytes, it is a biological process catalyzed by monoacylglycerol O acyltransferase 2 (MOGAT2, aka MGAT2), which is highly expressed in the small intestine. A previous study showed that the loss of the Mogat2 gene can prevent high fat diet induced obesity in mice. Obesity is associated with an increased risk of several types of cancer including postmenopausal breast cancer.Methods: We collected 147 patients with triple negative breast adenocarcinoma to explore the relationship between the expression of MOGAT2 and patient overall survival. And we generated a Mogat2 deficient mouse mammary tumor model by crossing Mogat2 deficient mice with MMTV PyMT mice to examine the effect of losing MOGAT2 in vivo. Results: Our founding suggest that obesity was induced by a relatively high fat diet (37% of calories from fat) in the mice with or without Mogat2 knockout. Mammary tumor development was deteriorated by a relatively high fat diet regardless of Mogat2 deficiency. As a compensation mechanism, upregulation of diacylglycerol O acyltransferases 1 and 2 (Dgat1 and Dgat2) in the Mogat2 deficient mice was found. Conclusions: Elevated expression of MOGAT2 in triple negative breast adenocarcinoma predicts poorer patient overall survival. With the compensation of Dgat1 and Dgat2, Mogat2 deficiency alone cannot prevent fat diet induced obesity, nor prevent mammary tumor development in a mouse model.

Purpose/Objective(s) To clarify the efficacy of FLASH radiotherapy (FLASH-RT) in inhibiting mammary tumorigenesis and normal tissue sparing. Materials/Methods We used a spontaneous mammary tumor model of MMTV-PyMT mice for the study. Prior to the formation of mammary tumor, MMTV-PyMT mice (3–6-week-old) were treated with a single fraction dose of 9-16 Gy FLASH-RT or conventional radiotherapy (CONV-RT) using a mice-applicator designed targeting five pair of mammary glands. Tumor growth was detected by thorough palpation of all 10 mammary glands twice a week. Liver function, HE however, IFN-g and IL-6 also significantly increased in FLASH compared with control, while IL-10 significantly decreased in CONV compared with control. Serum IL-6, IFN-g and TNF-a levels in FLASH did not markedly change on D6, D18 and D31 post-IR compared with control. Serum IL-6, IFN-g and TNF-a levels were elevated on D6 and continuously increased on D18 and D31 in CONV, but began to increase on D6, and then recovered to the control level on D18 or D31 in FLASH. Serum IL-10 levels decreased at D6, D18 and D31 in CONV, but began to decrease at D6, and then recovered to the control level at D18 or D31 in FLASH. FLASH induced reactive oxygen species (ROS) accumulation in the tumor tissue but not in normal tissue; while absence of ROS accumulation in either tumor or normal tissue under CONV. Notably, the ROS level of normal liver tissue did not differ under CONV or FLASH. Conclusion We found that mammary tumorigenesis was inhibited by FLASH without significant liver injury compared with CONV-RT. The alteration of serum and liver inflammatory cytokines induced by CONV-RT was mildly altered by FLASH. This unique benefit can by partially explained by the accumulatio of ROS in tumor tissue after FLASH.

Background. The occurrence and development of hepatocellular carcinoma (HCC) are closely related to immune function, as is the capacity of hepatoma cells to escape. Immunosurveillance is a key mechanism. Catgut implantation at acupoint (CIAA) is a promising acupuncture improvement method that can regulate immunity and has been widely used in the clinical treatment of a variety of diseases. The aim of this study is to observe the therapeutic effect of CIAA on HCC and to investigate the potential mechanism of immune escape. Materials and Methods. A total of 40 mice were randomly divided into three groups: the HCC model group (n = 15), the CIAA treatment group (n = 15), and the control group (n = 10). HCC was chemically induced in 30 mice by the combination of DEN, carbon tetrachloride, and ethanol for 150 days. Among them, 15 were selected for CIAA treatment to ascertain the therapeutic effect. The mRNA expression levels of AFP, IL-10, PD-1, and CTLA-4 in three groups were examined by using RT-PCR. AFP and AKT expressions were measured by using western blotting. PD1, CTLA-4, IL-10, CD4+, and CD8+ protein expression levels were evaluated by using IHC. The mortality rate, body weight, and psychological conditions of three groups were also compared. Results. The mRNA and protein expression levels of AFP, PD-1, CTLA-4, and IL-10 were significantly downregulated in the CIAA-treated mice in comparison with HCC mice. IHC assay shows that CD4+ and CD8+ expression levels were notably upregulated after CIAA treatment. Western blotting assay shows that AKT pathway was deactivated in CIAA-treated mice. CIAA notably reduced the mortality rate and inhibited weight loss caused by HCC and improved the overall psychological condition of the mice. Conclusions. Taken together, our data corroborate the effective potency of CIAA in the treatment of HCC by and inhibiting immune escape and deactivating the AKT pathway.

Background: The extracellular matrix (ECM) component serglycin promotes the epithelial-to-mesenchymal transition (EMT) and metastasis in an autocrine manner. However, the detailed mechanism underlying the roles of serglycin in Hepatocellular carcinoma (HCC) metastasis progression remains to be clarified.Methods: Analyzing 123 patients’ serum by ELISA to investigate the association between serglycin expression and HCC metastasis and prognosis. Serglycin, CRISPLD2, and YAP were overexpressed or knocked down with vector or RNAi. The RNA - sequence was used to screen serglycin downstream effectors, followed by bioinformatics, ChIP-PCR, luciferase, and promoter site mutation to identify novel target genes. The metastatic abilities of serglycin were demonstrated by a series of in vitro and in vivo experiments. Immunofluorescence, flow cytometry, and western blotting were carried out to demonstrate the potential mechanisms of serglycin.Results: We observed that serglycin was overexpressed in HCC tissues and serum, and its level was associated with metastasis and poor prognosis. By gain- and loss-of-function approaches, we found serglycin affects migratory motility and metastatic capacity in vitro and in vivo, and it's significantly associated with the stemness-like properties. Interestingly, serglycin activated the effector YAP of Hippo pathway, and further study verified the crucial role of serglycin/YAP in tumorigenesis by the DEN-induced HCC mouse model. Furthermore, the CRISPLD2 was selectively upregulated by serglycin, the axis of serglycin / YAP / CRISPLD2 regulated metastatic capacity in HCC cells, it revealed CRISPLD2 was the direct target gene of serglycin-mediated YAP/TEAD1 complex. Besides, serglycin-mediated YAP pathway enhanced cell sorafenib resistance, sorafenib combined with verteporfin reversed serglycin-mediated cellular insensitivity to sorafenib therapy.Conclusions: Autocrine ECM serglycin plays a crucial role in the process of stemness maintenance, tumorigenesis, and metastasis via selective reactivation of the novel YAP/CRISPLD2 developmental axis promotes metastasis and sorafenib resistance in Hepatocellular carcinoma, YAP-TEAD1 inhibitor verteporfin reversed serglycin-mediated cellular sorafenib resistance.

Nasopharyngeal carcinoma (NPC) originates in the nasopharyngeal epithelium and has the highest metastatic rate among head and neck cancers. Distant metastasis is the main reason for treatment failure with the underlying mechanisms remaining unclear. By comparing the expression profiling of NPCs versus non-cancerous nasopharyngeal tissues, we found LACTB was highly expressed in the tumor tissues. We found that elevated expression of the LACTB protein in primary NPCs correlated with poorer patient survival. LACTB is known to be a serine protease and a ubiquitous mitochondrial protein localized in the intermembrane space. Its role in tumor biology remains controversial. We found that the different methylation pattern of LACTB promoter led to its differential expression in NPC cells. Overexpressing LACTB in NPC cells promoted their motility in vitro and metastasis in vivo. While knocking down LACTB reduced the metastasis capability of NPC cells. However, LACTB did not influence cellular proliferation. We further found the role of LACTB in promoting NPC metastasis depended on the activation of ERBB3/EGFR-ERK signaling, which in turn, affected the stability and the following acetylation of histone H3. These findings may shed light on unveiling the mechanisms of NPC metastasis.

Nasopharyngeal carcinoma (NPC) is relatively sensitive to ionizing radiation, and radiotherapy is the main treatment modality for non-metastatic NPC. Radiation therapy generates overproduction of reactive oxygen species (ROS), which can cause DNA damage and induce apoptosis in tumors, thereby killing the malignant cells. Although dietary antioxidant supplementation reduces oxidative stress and promotes tumor progression, the effects of antioxidants on the NPC cells upon radiation have not been reported. In the present study, we showed that antioxidants (β-Carotene, NAC, GSH) played an anti-apoptotic role in response to radiation via decreasing ROS production and inhibiting MAPK pathway in NPC cells. Based on that, we conclude that the use of supplemental antioxidants during radiotherapy should be avoided because of the possibility of tumor protection and reduced treatment efficacy.

It is believed that the alteration of tissue microenvironment would affect cancer initiation and progression. However, little is known in terms of the underlying molecular mechanisms that would affect the initiation and progression of breast cancer. In the present study, we use two murine mammary tumor models with different speeds of tumor initiation and progression for whole genome expression profiling to reveal the involved genes and signaling pathways. The pathways regulating PI3K-Akt signaling and Ras signaling were activated in Fvb mice and promoted tumor progression. Contrastingly, the pathways regulating apoptosis and cellular senescence were activated in Fvb.B6 mice and suppressed tumor progression. We identified distinct patterns of oncogenic pathways activation at different stages of breast cancer, and uncovered five oncogenic pathways that were activated in both human and mouse breast cancers. The genes and pathways discovered in our study would be useful information for other researchers and drug development.

Background CLCA2 was reported as a tumor suppressor and disregulated in breast cancer. However, its function in tumor growth and metastasis in NPC has rarely been reported. In this study, we investigated the functional and molecular mechanisms by which CLCA2 influences NPC. Methods CLCA2 expression in human NPC cell lines and tissues was examined via real-time PCR (RT-PCR), Western blot and IHC. The biological roles of CLCA2 in proliferative, migration and invasion of NPC cell lines was evaluated in 5-8F, S18, S26 and SUNE-1 cells. Cell viability, migration and invasion were assessed in vitro by MTS, colony formation and transwell assay, respectively. CLCA2 in growth and metastasis of NPC were evaluated in vivo through NPC xenograft tumor growth, lung metastatic mice model and popliteal lymph node (LN) metastasis model. Results Overexpression of CLCA2 significantly decreased proliferation, migration and invasion of NPC cells. In contrast, knockdown of CLCA2 elicited the opposite effects. CLCA2 overexpression suppressed xenograft tumor growth and lung, popliteal lymph node (LN) metastasis in vivo. CLCA2 inhibited tumor metastasis through suppressing epithelial-Mesenchymal transition (EMT) and in-activating FAK/ERK1/2 signaling pathway in NPC cells. Immunohistochemical staining of 143 NPC samples revealed that CLCA2 expression was an independent, favorable prognostic factor for overall survival and distant metastasis-free survival of patients. In addition, inhibition of FAK and ERK1/2 reversed CLCA2 silencing-induced tumor cell migration. Furthermore, inhibitors against chloride channels suppressed NPC cellular migration which could have been enhanced by the presence of CLCA2. Conclusion CLCA2 suppress NPC proliferation, migration, invasion and epithelial-mesenchymal transition through inhibiting FAK/ERK signaling. Electronic supplementary material The online version of this article (10.1186/s13046-018-0692-8) contains supplementary material, which is available to authorized users.

Purpose: To date, no guidelines exist for elderly nasopharyngeal carcinoma (NPC) patients (60 years of age or older) due to a lack of prospective clinical trials. This study evaluated the efficacy of concurrent chemotherapy (CCRT) for NPC in elderly patients treated with intensity-modulated radiotherapy (IMRT). Methods: Patients were identified from a prospectively maintained database. A total of 198 consecutive cases of elderly patients with NPC receiving IMRT, including 103 patients treated with IMRT plus CCRT and 95 patients treated with IMRT alone, were analysed from January 2002 to December 2013. Multivariate analysis (MVA) using the Cox proportional hazards model and propensity score analysis (PSA) were performed for overall survival (OS) and disease-free survival (DFS). Finally, sensitivity analysis was performed. Results: The median follow-up time was 55.3 months (range, 3-135.6 months). In the entire cohort, both MVA and PSA models showed that compared with IMRT alone, IMRT plus CCRT significantly improved survival (hazard ratio [HR] 2.143, 95% confidence interval [95% CI] 1.180-3.890; HR 1.961, 95% CI, 1.117-3.443, for OS and DFS, respectively). Similar results were found in the subgroups with high levels of Epstein-Barr virus (EBV) DNA, except in the low-EBV-DNA cohort. The total rates of severe acute toxicity, including leukopenia, neutropenia, stomatitis, and emesis, were significantly higher in the IMRT+CCRT group than in the IMRT-alone group (P < 0.001) but were similar to the rates of severe late toxicity (P = 0.818). Sensitivity analysis confirmed the robustness of our analysis. Conclusions: In the era of IMRT, CCRT retained survival benefits at high EBV DNA levels but not at low EBV DNA levels for elderly NPC patients. Randomized clinical trials are needed to confirm our findings.

The underlying molecular mechanism driving clear cell renal cell carcinoma (ccRCC) progression is still to be explored. The significant downregulation of protein tyrosine phosphatase nonreceptor type 3 (PTPN3) expression in the tumor tissues suggested its protective role in ccRCC progression. IHC analysis of PTPN3 protein in 172 ccRCC tissue revealed that PTPN3 was an independently favorable prognostic factor for progression-free survival (P = 0.0166) and overall survival (P = 0.0343) of patients. The ccRCC cell lines SN12C, 1932, ACHN, and Caki-1 were used to evaluate, both in vitro and in vivo, the biological roles of PTPN3. We observed that overexpression of PTPN3 significantly inhibited the proliferation, migration, and invasion of ccRCC cells. In contrast, the knocking down of PTPN3 elicited opposite effects. Overexpressing PTPN3 inhibited xenograft tumor growth and lung metastasis displayed by the in vivo mice models. PTPN3 inhibited tumor cell motility by suppressing the phosphorylation of AKT, and subsequently inactivating the PI3K/AKT signaling pathway of renal cell carcinoma cells. Furthermore, the inhibition of phospho-AKTThr308 and phospho-AKTSer473 reversed PTPN3-induced silencing in tumor cell migration. Our work revealed that the overexpression of PTPN3 could suppress kidney cancer progression by negatively regulating the AKT signaling pathway, and served as a favorable prognostic factor in patients with ccRCC. Our findings provided insight that PTPN3 could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC. Implications: PTPN3 is an independent favorable prognostic factor for patients with ccRCC and could be a potential target for therapy aiming to inhibit the malignant behaviors of ccRCC.

Background With the rapid development of the high throughput detection techniques, tumor-related Omics data has become an important source for studying the mechanism of tumor progression including breast cancer, one of the major malignancies worldwide. A previous study has shown that the G2 and S phase-expressed-1 (GTSE1) can act as an oncogene in several human cancers. However, its functional roles in breast cancer remain elusive. Method In this study, we analyzed breast cancer data downloaded from The Cancer Genome Atlas (TCGA) databases and other online database including the Oncomine, bc-GenExMiner and PROGgeneV2 database to identify the molecules contributing to the progression of breast cancer. The GTSE1 expression levels were investigated using qRT-PCR, immunoblotting and IHC. The biological function of GTSE1 in the growth, migration and invasion of breast cancer was examined in MDA-MB-231, MDA-MB-468 and MCF7 cell lines. The in vitro cell proliferative, migratory and invasive abilities were evaluated by MTS, colony formation and transwell assay, respectively. The role of GTSE1 in the growth and metastasis of breast cancer were revealed by in vivo investigation using BALB/c nude mice. Results We showed that the expression level of GTSE1 was upregulated in breast cancer specimens and cell lines, especially in triple negative breast cancer (TNBC) and p53 mutated breast cancer cell lines. Importantly, high GTSE1 expression was positively correlated with histological grade and poor survival. We demonstrated that GTSE1 could promote breast cancer cell growth by activating the AKT pathway and enhance metastasis by regulating the Epithelial-Mesenchymal transition (EMT) pathway. Furthermore, it could cause multidrug resistance in breast cancer cells. Interestingly, we found that GTSE1 could regulate the p53 function to alter the cell cycle distribution dependent on the mutation state of p53. Conclusion Our results reveal that GTSE1 played a key role in the progression of breast cancer, indicating that GTSE1 could serve as a novel biomarker to aid in the assessment of the prognosis of breast cancer. Electronic supplementary material The online version of this article (10.1186/s13046-019-1157-4) contains supplementary material, which is available to authorized users.

Nasopharyngeal carcinoma (NPC) has the highest metastatic rate among head and neck cancers, with its underlying mechanism not yet fully unveiled. High- versus low-metastasis, NPC cell lines have been established. The footpad-popliteal lymph node metastasis model and other in vivo models have been stably used to study NPC metastasis. The histological appearance and the expression of epithelial-to-mesenchymal transition (EMT) markers might be helpful in selecting high-risk NPC patients for developing post-treatment metastasis. Tested EMT markers and their protein expression levels that correlate with patient disease-free survival in large patient cohorts include E-cadherin, N-cadherin, CD44, Twist, Snail, and Cyclin D1. Epstein-Barr virus (EBV) infection can trigger NPC metastasis from multiple angles via multiple signaling pathways. High endothelial venules are commonly seen in NPC tissues, with their role in NPC metastasis requiring clarification. The molecules that promote and inhibit NPC metastasis are introduced, with a focus on cytokines SPINK6, serglycin, interleukin 8 (IL8), Wnt family member 5A (WNT5A), and chemokine C-C motif ligand 2 (CCL2). Two videos showing NPC cells with and without SPINK6 knocked down are presented. Future directions for studying NPC metastasis are also discussed.

Cancer patients are more susceptible to severe coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Angiotensin-converting enzyme 2 (ACE2) is the functional host receptor for SARS-CoV-2 entering into human cells. Bioinformatics’ analyses have revealed that ACE2 is upregulated in some cancer cells. In the present study, we evaluated ACE2 protein expression levels in several common malignancies compared to non-cancerous normal tissues. ACE2 expression was elevated in colorectal adenocarcinoma, pancreatic adenocarcinoma, gastric adenocarcinoma, and papillary renal cell carcinoma cancer. Yet, it was suppressed in chromophobe renal cell carcinoma, testicular germ cell tumors, and papillary thyroid carcinoma. Two tumor tissue microarrays were used to evaluate the prognostic value of ACE2 expression in patients with gastric adenocarcinoma, and colorectal adenocarcinoma without COVID-19. No significant survival benefit was found for patients with overexpression of ACE2 in the tumor. In the tumor vasculature, ACE2 expression was observed in only 54% of the tumor micro-vessels. Using an in vitro co-culture of endothelial cells and tumor cells overexpressing fusion protein ACE2-red fluorescent protein, we did not observe any clear and convincing intercellular transfer of ACE2 from cancer cells into endothelial cells. In summary, alteration of ACE2 expression was found in common malignancies, but there is no evidence of intercellular transfer of ACE2 from cancer cells to endothelial cells.

Nasopharyngeal carcinoma (NPC) is well known as one of the most common malignancies in southern China and Southeast Asia. However, the mechanisms underlying NPC progression remain poorly understood. Herein, through overlapping the differentially expressed genes from 3 microarray data sets with the human kinome, we identified PBK, a serine-threonine kinase, is highly upregulated and has not been intensively investigated in NPC. PBK was required for malignant phenotypes of NPC, as PBK depletion by RNAi and inhibition by specific inhibitor HI-TOPK-032 obviously reduced cell proliferation and xenograft tumor growth in mice. Moreover, we determined that targeting PBK could accelerate apoptosis by inducing ROS that activates JNK/p38 signaling pathway. In NPC patients, elevated PBK expression in primary tumor positively correlated to clinical severity such as advanced T stage, high death risk and disease progression, and it could serve as an unfavorable independent indicator of overall survival and disease-free survival. Altogether, our results indicate that PBK is a novel significant regulator of NPC progression and a potential therapeutic target for NPC patients.

ObjectivesGeographical disparities have been identified as a specific barrier to cancer screening and a cause of worse outcomes for patients with cancer. In the present study, our aim was to assess the influence of geographical disparities on the survival outcomes of patients with nasopharyngeal carcinoma (NPC) treated with intensity-modulated radiation therapy (IMRT).DesignCohort study.SettingGuangzhou, China.ParticipantsA total of 1002 adult patients with NPC (724 males and 278 females) who were classified by area of residence (rural or urban) received IMRT from 1 January 2010 to 31 December 2014, at Sun Yat-sen University Cancer Center. Following propensity score matching (PSM), 812 patients remained in the analysis.Main outcome measuresWe used PSM to reduce the bias of variables associated with treatment effects and outcome prediction. Survival outcomes were estimated using the Kaplan-Meier method and compared by the log-rank test. Multivariate Cox regression was used to identify independent prognostic factors.ResultsIn the matched cohort, 812 patients remained in the analysis. Kaplan-Meier survival analysis revealed that the rural group was significantly associated with worse overall survival (OS, pConclusionsThe survival outcomes of patients with NPC who received the same standardised treatment were significantly better in urban regions than in rural regions. By analysing the geographic disparities in outcomes for NPC, we can guide the formulation of healthcare policies.

The controversy of (p53) in nasopharyngeal carcinoma persists, despite the fact that many studies have been conducted on its correlation with latent membrane protein 1 (LMP1), bcl-2, and prognosis. To better understand this postulated relationship, a meta-analysis was performed based on existing relevant studies. A total of 19 individual studies with a total of 1189 patients were included in the meta-analysis. Overall, the results revealed a significant association of p53-positive status with a poor 5-year survival of nasopharyngeal carcinoma (NPC) patients as the risk difference (RD) was -0.17 (95% CI, -0.31, -0.03; P=0.02, Pheterogeneity =0.01).The overall odds ratio (OR) for LMP1 in the p53 positive group vs. negative group revealed that a significantly elevated risk of positive LMP1 in the former was achieved (OR 5.52 95% CI, 2.66-11.46; P0.00001, Pheterogeneity =0.78). Similarly, a strong correlation between bcl-2 and p53 was found with an OR 6.85 (95% CI, 2.37-19.74; P=0.0004, Pheterogeneity =0.48). However, there did not appear to be any correlations with clinical parameters such as gender, tumor site, lymph node metastasis,pathological type and TNM stage. In conclusion, p53 expression is related to the survival of nasopharyngeal carcinoma. It can be considered as the auxiliary detection index in treatment and prognosis of nasopharyngeal carcinoma.

Purpose: Trophinin-associated protein (TROAP) is a cytoplasmic protein that plays a significant role in the processes of embryo transplantation and microtubule regulation. However, the relevant survival analysis and cancer progression analysis have not yet been reported. Methods: Eighteen matched pairs of tumor and adjacent non-tumor samples were evaluated to detect the TROAP mRNA level. Immunohistochemistry (IHC) was used to evaluate the TROAP expression in 108 hepatocellular carcinoma patients who underwent surgical resection. Meanwhile, data from the TCGA database was statistically evaluated. Results: In the present study, we detected a significant increase in the TROAP mRNA level in tumor tissues when compared with adjacent non-tumor tissues. Moreover, the upregulation of TROAP was associated with increased serum AFP and GGT; the greater the tumor number was, the larger the tumor size, differentiation grade, and cancer embolus in clinical analysis. In HCC patients, elevated TROAP expression in the primary tumor was positively related to clinical severity, such as poor overall survival and disease-free survival. In addition, both univariate and multivariate survival analysis validated that TROAP expression was a promising independent risk factor for overall survival and disease-free survival in HCC patients. Furthermore, the results derived from the analysis of data from the TCGA database were consistent with previous results. Altogether, our results show that TROAP is a novel crucial regulator of HCC progression and is a potential therapeutic biomarker for HCC patients. Conclusions: Elevated TROAP expression predicted a poor prognosis, and TROAP may serve as a potential biomarker for application in oncotherapy.

Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44(+/High) stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which subsequently stimulated expression of CSC markers, development of side population and tumor sphere formation. This suggests that PI3K/AKT pathway has an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44(+/High) cells. In addition, LMP1 greatly increased expression of miR-21 and downregulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by an miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and upregulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to the development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and have a key role in LMP1-induced CSCs in NPC.

Nasopharyngeal carcinoma (NPC) has the highest metastasis rate among head and neck cancers with unclear mechanism. WNT5A belongs to the WNT family of cysteine-rich secreted glycoproteins. Our previous high-throughput gene expression profiling revealed that WNT5A was up-regulated in highly metastatic cells. In the present study, we first confirmed the elevated expression of WNT5A in metastatic NPC tissues at both the mRNA and protein levels. We then found that WNT5A promoted epithelial-mesenchymal transition (EMT) in NPC cells, induced the accumulation of CD24-CD44+ cells and side population, which are believed to be cancer stem cell characteristics. Moreover, WNT5A promoted the migration and invasion of NPC cells in vitro, while in vivo treatment with recombinant WNT5A promoted lung metastasis. Knocking down WNT5A diminished NPC tumorigenesis in vivo. When elevated expression of WNT5A coincided with the elevated expression of vimentin in the primary NPC, the patients had a poorer prognosis. Among major signaling pathways, protein kinase C (PKC) signaling was activated by WNT5A in NPC cells. A positive feedback loop between WNT5A and phospho-PKC to promote EMT was also revealed. Taken together, these data suggest that WNT5A is an important molecule in promoting stem cell characteristics in NPC, leading to tumorigenesis and metastasis.

Nasopharyngeal carcinoma (NPC), which originates from the nasopharynx, is highly prevalent in Southern China and Southeast Asia, and more than 90% of all NPCs are non-keratinizing undifferentiated cells or poorly differentiated squamous cells. Cancer stem cells (CSCs) are capable of self-renewal and have differentiation potential. These properties form the basis of cancer initiation, development, and radiochemoresistance. However, the molecular mechanisms underlying NPC CSC maintenance remain poorly understood. Here, genomic expression profiling using our previously established monoclonal cellular and animal models revealed that interferon regulatory factor 6 (IRF6) was downregulated in highly metastatic NPC cells, cancer stem-like NPC cells and animal models. Functional assays revealed that elevated IRF6 expression suppressed cell proliferation, growth, CSCs properties and enhanced cell chemotherapeutic sensitivity. However, silencing IRF6 resulted in opposing effects. Moreover, we determined that as a tumor suppressor gene and transcription factor, IRF6 directly bound the upstream region of the ATP-binding cassette sub-family G member 2 (ABCG2) DNA element and suppressed target ABCG2 expression in NPC cells. Consistently, an inverse correlation was observed between the mRNA levels of IRF6 and ABCG2 in clinical NPC samples. With these results, we provide the first evidence that IRF6 directly targets the ABCG2 gene and selectively kills CSCs in NPC and that IRF6 may be a valuable tool for developing new CSC-targeted treatment strategies for undifferentiated NPC patients.

Purpose: To compare the long-term survival outcomes and acute toxicity between locoregionally advanced nasopharyngeal carcinoma (NPC) patients who received either weekly or 3-weekly cisplatin during concurrent chemoradiotherapy (CCRT). Methods: Between November 2008 and August 2011, 241 biopsy-proved NPC patients receiving concurrent cisplatin with intensity modulated radiotherapy (IMRT) were included. 90 patients treated with 4-7 weeks of 30-40 mg/m2 cisplatin weekly were matched with 90 patients who received two or three cycles of 80 mg/m2 cisplatin three-weekly by sex, age, T stage, N stage, Karnosky performance score (KPS). IMRT was presented to the nasopharyngeal gross target volume at 66-72 Gy/30-32 fractions and those involved neck area at 60-66 Gy/30-32 fractions. Results: The median follow-up time was 69 months (range, 2-91 months), and the 5-year overall survival (OS), disease-free survival (DFS), locoregional relapse-free survival (LRFS), and distant metastasis-free survival (DMFS) rates were 85.6% vs. 90.0% (P = 0.207), 85.6% vs. 92.6% (P = 0.152), 94.4% vs. 96.7% (P = 0.411), and 88.9% vs. 95.6% (P = 0.107) for the group treated weekly and 3-weekly cisplatin, respectively. No statistically significant survival differences were found between the two treatment groups in both univariate and multivariate analyses. The similar incidence of acute toxicities was observed between two groups. Conclusions: Concurrent cisplatin-based chemotherapy administered weekly or three-weekly in combination with IMRT leads to similar acute toxicities and long-term survival outcomes in locoregionally advanced NPC patients.

Emerging evidence confirms that cancer stem cells (CSCs) are responsible for the chemoradioresistance of malignancies. EBV-encoded latent membrane protein 1 (LMP1) is associated with tumor relapse and poor prognosis of nasopharyngeal carcinoma (NPC). However, whether LMP1 induces the development of CSCs and the mechanism by which this rare cell subpopulation leads to radioresistance in NPC remain unclear. In the present study, LMP1-transformed NPC cells showed significant radioresistance compared to the empty vector control. We found that LMP1 up-regulated the expression of several stemness-related genes, increased the cell number of side population (SP) by flow cytometry analysis, enhanced the self-renewal properties of the cells in a spherical culture and enhanced the in vivo tumor initiation ability. We also found that LMP1 positively regulated the expression of the CSC marker CD44. The CD44(+/High) subpopulation of the LMP1-transformed NPC cells displayed more significant CSC characteristics than the CD44(-/Low) subpopulation of the LMP1-transformed NPC cells; these characteristics included the upregulation of stemness-related genes, in vitro self-renewal and in vivo tumor initiation ability. Importantly, the CD44(+/High) subpopulation displayed more radioresistance than the CD44(-/Low) subpopulation. Our results also demonstrated that phosphorylation of the DNA damage response (DDR) proteins, ATM, Chk1, Chk2 and p53, was inactivated in the LMP1-induced CD44(+/High) cells in response to DNA damage, and this was accompanied by a downregulation of the p53-targeted proapoptotic genes, which suggested that the inactivation of the p53-mediated apoptosis pathway was responsible for the radioresistance in the CD44(+/High) cells. Taken together, we found that LMP1 induced an increase in CSC-like CD44(+/High) cells, and we determined the molecular mechanism underlying the radioresistance of the LMP1-activated CSCs, highlighting the need of CSC-targeted radiotherapy in EBV-positive NPC.

An E1B55K-attenuated adenovirus, dl1520, has been shown to replicate selectively in and lyse tumor cells. In this study, the antitumor activities of dl1520, alone or in combination with the chemotherapeutic agent cisplatin, were investigated in nasopharyngeal carcinoma (NPC) cells. The results demonstrated that dl1520 replicated in and destroyed NPC cells, and induced apoptosis in vitro. In a nude mouse xenograft model, dl1520 significantly inhibited the growth of NPC cell xenografts, and the viral replication was associated with tumor regression. Importantly, the antitumor activity of dl1520 was augmented by the addition of cisplatin both in vitro and in vivo, showing that dl1520 and cisplatin have a synergistic anti-NPC effect. These data suggest that dl1520 exerts an efficient anti-NPC activity through oncolysis and the induction of apoptosis, which is enhanced synergistically by cisplatin. These findings indicate that oncolytic viral therapeutics using the E1B55K-attenuated adenovirus dl1520 could be promising in the comprehensive treatment of NPC, especially in combination with platinum-based chemotherapy.

Based on microarray analysis, we previously reported that miR-29c is significantly downregulated in nasopharyngeal carcinoma (NPC). However, little is known about the effect and molecular mechanisms of action of miR-29c deregulation during the development and progression of NPC. Quantitative RT-PCR demonstrated that miR-29c was significantly downregulated in NPC cell lines and clinical specimens. Wound healing, Transwell migration and lung metastasis assays demonstrated that ectopic expression of miR-29c inhibited NPC cell migration and invasion in vitro and suppressed the formation of lung metastases in vivo. T cell lymphoma invasion and metastasis 1 (TIAM1) was confirmed as a miR-29c target gene using luciferase reporter assays, quantitative RT-PCR and Western blotting. Ectopic expression of TIAM1 significantly promoted the migration and invasion of SUNE-1 cell line stably overexpressing miR-29c. The prognostic value of TIAM1 was analyzed in 217 NPC patients using immunohistochemistry. Strikingly, patients with high TIAM1 expression had poorer overall, disease-free and distant metastasis-free survival than patients with low TIAM1 expression. Furthermore, multivariate Cox regression analysis revealed that TIAM1 could serve as an independent prognostic factor in NPC. The newly identified miR-29c/TIAM1 pathway further elucidates the molecular mechanisms regulating invasion and metastasis in NPC, and may provide novel prognostic and treatment strategies for NPC patients.

Nasopharyngeal carcinoma has the highest rate of metastasis among head and neck cancers, and distant metastasis is the major reason for treatment failure. The underlying molecular mechanisms of nasopharyngeal carcinoma metastasis are not fully understood. Here, we report the identification of serine protease inhibitor Kazal-type 6 (SPINK6) as a functional regulator of nasopharyngeal carcinoma metastasis via EGFR signaling. SPINK6 mRNA was upregulated in tumor and highly metastatic nasopharyngeal carcinoma cells. Immunohistochemical staining of 534 nasopharyngeal carcinomas revealed elevated SPINK6 expression as an independent unfavorable prognostic factor for overall, disease-free, and distant metastasis–free survival. Ectopic SPINK6 expression promoted in vitro migration and invasion as well as in vivo lymph node metastasis and liver metastasis of nasopharyngeal carcinoma cells, whereas silencing SPINK6 exhibited opposing effects. SPINK6 enhanced epithelial–mesenchymal transition by activating EGFR and the downstream AKT pathway. Inhibition of EGFR with a neutralizing antibody or erlotinib reversed SPINK6-induced nasopharyngeal carcinoma cell migration and invasion. Erlotinib also inhibited SPINK6-induced metastasis in vivo. Notably, SPINK6 bound to the EGFR extracellular domain independent of serine protease–inhibitory activity. Overall, our results identified a novel EGFR-activating mechanism in which SPINK6 has a critical role in promoting nasopharyngeal carcinoma metastasis, with possible implications as a prognostic indicator in nasopharyngeal carcinoma patients. Cancer Res; 77(2); 579–89. ©2016 AACR.

Summary Background MicroRNAs (miRNAs) can be used as prognostic biomarkers in many types of cancer. We aimed to identify miRNAs that were prognostic in patients with nasopharyngeal carcinoma. Methods We retrospectively analysed miRNA expression profiles in 312 paraffin-embedded specimens of nasopharyngeal carcinoma from Sun Yat-sen University Cancer Center (Guangzhou, China) and 18 specimens of non-cancer nasopharyngitis. Using an 873 probe microarray, we assessed associations between miRNA signatures and clinical outcome in a randomly selected 156 samples (training set) and validated findings in the remaining 156 samples (internal validation set). We confirmed the miRNAs signature using quantitative RT-PCR analysis in 156 samples from a second randomisation of the 312 samples, and validated the miRNA signature in 153 samples from the West China Hospital of Sichuan University in Chengdu, China (independent set). We used the Kaplan-Meier method and log-rank tests to estimate correlations of the miRNA signature with disease-free survival (DFS), distant metastasis-free survival (DMFS), and overall survival. Findings 41 miRNAs were differentially expressed between nasopharyngeal carcinoma and non-cancer nasopharyngitis tissues. A signature of five miRNAs, each significantly associated with DFS, was identified in the training set. We calculated a risk score from the signature and classified patients as high risk or low risk. Compared with patients with low-risk scores, patients with high risk scores in the training set had shorter DFS (hazard ratio [HR] 2·73, 95% CI 1·46–5·11; p=0·0019), DMFS (3·48, 1·57–7·75; p=0·0020), and overall survival (2·48, 1·24–4·96; p=0·010). We noted equivalent findings in the internal validation set for DFS (2·47, 1·32–4·61; p=0·0052), DMFS (2·28, 1·09–4·80; p=0·030), and overall survival (2·87, 1·38–5·96; p=0·0051) and in the independent set for DFS (3·16, 1·65–6·04; p=0·0011), DMFS (2·39, 1·05–5·42; p=0·037), and overall survival (3·07, 1·34–7·01; p=0·0082). The five-miRNA signature was an independent prognostic factor. A combination of this signature and TNM stage had better prognostic value than did TNM stage alone in the training set (area under receiver operating characteristics 0·68 [95% CI 0·60–0·76] vs 0·60 [0·52–0·67]; p=0·013), the internal validation set (0·70 [0·61–0·78] vs 0·61 [0·54–0·68]; p=0·012), and the independent set (0·70 [0·62–0·78] vs 0·63 [0·56–0·69]; p=0·032). Interpretation Identification of patients with the five-miRNA signature might add prognostic value to the TNM staging system and inform treatment decisions for patients at high risk of progression. Funding Science Foundation of Chinese Ministry of Health, National Natural Science Foundation of China, Pearl River Scholar Funded Scheme, Guangdong Key Scientific and Technological Innovation Program, Guangdong Natural Science Foundation, Fundamental Research Funds for the Central Universities.

DNA replication is initiated by assembly of the kinase cell division cycle 7 (CDC7) with its regulatory activation subunit, activator of S-phase kinase (ASK), to activate DNA helicase. However, the mechanism underlying regulation of CDC7-ASK complex is unclear. Here, we show that ADP generated from CDC7-mediated MCM phosphorylation binds to an allosteric region of CDC7, disrupts CDC7-ASK interaction, and inhibits CDC7-ASK activity in a feedback way. EGFR- and ERK-activated casein kinase 2α (CK2α) phosphorylates nuclear phosphoglycerate kinase (PGK) 1 at S256, resulting in interaction of PGK1 with CDC7. CDC7-bound PGK1 converts ADP to ATP, thereby abrogating the inhibitory effect of ADP on CDC7-ASK activity, promoting the recruitment of DNA helicase to replication origins, DNA replication, cell proliferation, and brain tumorigenesis. These findings reveal an instrumental self-regulatory mechanism of CDC7-ASK activity by its kinase reaction product ADP and a nonglycolytic role for PGK1 in abrogating this negative feedback in promoting tumor development.

Endostatin is a potent angiogenic inhibitor that has been approved for the treatment of cancer. However, endostatin is unstable in vitro and difficult to produce in large quantities. Endostatin gene therapy is an alternative to overcome these difficulties by expressing sustained bioactive endostatin in vivo. We previously developed a recombinant replication-defective adenovirus, E10A, which carries the human endostatin gene. Phase I trial of E10A given as a weekly intratumoral injection in adult patients with solid tumors has been finished. The clinical application of endostatin gene therapy was limited by the high cost of large-scale production. In the current study, we found that there was a high level (100 mg/L) of endostatin in the fermentation supernatant of 293 cells transfected with Ad/rhEndo. A protocol was developed to purify recombinant endostatin in the fermentation supernatant to a yield of 24 mg/L and 98% purity by the use of SP Sepharose FF cation exchange chromatography, Sepharose-heparin Hi Trap affinity chromatography and gel filtration chromatography. The anti-proliferative activity of 293 cell-expressed endostatin (ArhEndo) is comparable to that of yeast-expressed endostatin (YrhEndo). Cell migration assay indicated that ArhEndo is more effective than YrhEndo. Moreover, ArhEndo is of higher stability than YrhEndo. These results suggested that purification of recombinant endostatin from fermentation supernatant provided an economic and available strategy for Ad/rhEndo production.