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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.

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.

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) 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.

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.

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) 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.

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.

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.

The immune spectrum of severe acute respiratory syndrome (SARS) is poorly understood. To define the dynamics of the immune spectrum in SARS, serum levels of cytokines, chemokines, immunoglobulins, complement and specific antibodies against SARS-associated coronavirus (SARS-CoV) were assayed by enzyme-linked immunosorbent assay (ELISA), and phenotypes of peripheral lymphocytes were analyzed by flow cytometry in 95 SARS-infected patients. Results showed that interleukin (IL)-10 and transforming growth factor β (TGF-β) were continuously up-regulated during the entirety of SARS. Regulated on activation normally T cell-expressed and secreted (RANTES) levels were decreased, while monocyte chemoattractant protein-1 (MCP-1) was elevated in acute patients. Immunoglobulins and complement were elevated during the first month of SARS. Both serum-positive rates and titers of specific IgM and IgG antibodies responding to SARS-CoV peaked at days 41–60 from the onset of SARS. CD4+ and CD8+ T lymphocytes decreased significantly in acute-phase. CD3+CD8+CD45RO+ T lymphocytes were decreased by 36.78% in the convalescent patients. Conclusion: SARS-CoV seemed to elicit effective humoral immunity but inhibited cellular immunity, especially CD8+ memory T lymphocytes over time. Prolonged overproduction of IL-10 and TGF-β may play an important role in the disease.

HER2 (c-erbB-2) has been suggested to be a prognostic factor in a variety of human cancers including breast, gastric and ovarian cancers. This study is therefore designed to identify changes of HER2 in nasopharyngeal carcinoma (NPC), an epithelia-derived malignancy with strong racial and geographic distribution. Interphase FISH and immunohistochemical (IHC) staining were used to analyze the gene copy number and protein expression of HER2 in 45 cases of NPC from Guangzhou, Southern China, an area with the highest incidence of NPC in the world. Our results, however, found no significant alterations in gene copy number for HER2, although IHC staining detected expression of HER2 oncoprotein in 33% of the 45 NPC tumors. No correlation was observed between HER2 expression and sex, age and clinical outcome of the patients, T stage, lymph node status, site and histopathological grading of the tumors. These results cast doubt on the value of HER2 as a prognostic factor for NPC.

Nasopharyngeal carcinoma (NPC), is one of the most common human malignancies in south China, it has the highest recurrence rate and treatment resistance. The underlying molecular mechanisms of NPC relapse and treatment tolerance are not fully understood. In this study, the effects of NEDD8 and NEDD8-activating enzyme inhibitor (MLN4924) on NPC were studied both in vitro and in vivo. Immunohistochemical staining of 197 NPC tissues revealed an elevated NEDD8 expression as an unfavorable independent factor in overall survival and disease-free survival rates. NEDD8 expression was positively correlated with a high risk of death and positivity of lymph node metastasis. Depleted NEDD8 expression by shRNA and inhibited by specific inhibitor MLN4924 dramatically suppressed cell proliferation, cell apoptosis, cell cycle arrest, while ectopic NEDD8 exhibited opposing effects. NEDD8 affected cancer stem cell phenotypes of NPC as assessed in vitro using the cell number of side population (SP) by flow cytometry analysis, colony formation assay, sphere formation assay, and tumor initiation ability in vivo. Downregulation of NEDD8 enhanced the susceptibility of NPC cells to cisplatin and radiation. Moreover, we found that MLN4924 suppressed c-Jun degradation in human NPC cells. Taken together, this report revealed that NEDD8 may act as a novel prognostic marker and MLN4924 may serve as a promising therapeutic target for patients with NPC.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies in southern China and Southeast Asia, with the highest metastasis rate among head and neck cancers. The mechanisms underlying NPC progression remain poorly understood. Genome-wide expression profiling on 18 NPC vs. 18 noncancerous nasopharyngeal tissues together with GeneGo pathway analysis and expression verification in NPC cells and tissues revealed a potential role of urokinase-type plasminogen activator receptor (uPAR) in NPC progression, which has not been investigated in NPC. We then observed that uPAR expression is increased in poorly differentiated, highly metastatic NPC cells compared with lowly metastatic cells or differentiated NPC cells. In vitro studies demonstrated that uPAR regulates NPC cell growth, colony formation, migration, and invasion and promotes the epithelial–mesenchymal transition (EMT). Additional tumor xenograft and spontaneous metastasis experiments revealed that uPAR promotes NPC cell growth and metastasis in vivo. The JAK–STAT pathway is involved in uPAR-regulated signaling in NPC cells as determined by immunoblotting. Moreover, uPAR-mediated growth and motility is partially abolished upon treatment with the Jak1/Jak2 inhibitor INCB018424. We suppressed uPA expression in uPAR-overexpressing NPC cells and found that uPAR-mediated cellular growth and motility is not exclusively dependent on uPA. In summary, uPAR is a significant regulator of NPC progression and could serve as a promising therapeutic target.

Dicer11 plays an important role in generation of microRNA and is dysregulated in a variety of human cancers. The purpose of this study was to evaluate Dicer11 expression and its prognostic value in nasopharyngeal carcinoma (NPC). The protein expression of Dicer1 was examined by immunohistochemistry in 276 NPC specimens, and the mRNA levels of Dicer1 were analyzed by qRT-PCR in 56 NPC and 11 nasopharyngitis tissues. Cox regression analysis was used to identify independent prognostic factors, and a prognostic score model was constructed for survival prediction. Expression of Dicer1 was downregulated in NPC tissues at both the mRNA and the protein levels, and there was a notable positive correlation between the expression levels of Dicer1 mRNA and protein. Low Dicer1 expression was positively correlated with distant metastasis (P

Objectives: The role of heparanase (HPSE) gene in cancers including hepatocellular carcinoma (HCC) is currently controversial. This study was aimed at investigating the impact of genetic alteration and expression change of HPSE on the progression and prognosis of HCC. Methods: The HPSE gene was studied in three different aspects: (1) loss of heterozygosity (LOH) by a custom SNP microarray and DNA copy number by real-time PCR; (2) mRNA level by qRT-PCR; and (3) protein expression by immunohistochemistry. The clinical significances of allele loss and expression change of HPSE were analyzed. Results: Microarray analysis showed that the average LOH frequency for 10 SNPs located within HPSE gene was 31.6%, three of which were significantly correlated with tumor grade, serum HBV-DNA level, and AFP concentration. In agreement with SNP LOH data, DNA copy number loss of HPSE was observed in 38.74% (43/111) of HCC cases. HPSE mRNA level was notably reduced in 74.1% (83/112) of tumor tissues compared with non-tumor liver tissues, which was significantly associated with DNA copy number loss, increased tumor size, and post-operative metastasis. HPSE protein level was also remarkably reduced in 66.3% (53/80) of tumor tissues, which was correlated with tumor grade. Patients with lower expression level of HPSE mRNA or protein had a significantly lower survival rate than those with higher expression. Cox regression analysis suggested that HPSE protein was an independent predictor of overall survival in HCC patients. Conclusions: The results in this study demonstrate that genetic alteration and reduction of HPSE expression are associated with tumor progression and poor prognosis of HCCs, suggesting that HPSE behaves like a tumor suppressor gene and is a potential prognostic marker for HCC patients. Citation: Huang G-L, Li B-K, Zhang M-Y, Wei R-R, Yuan Y-F, et al. (2012) Allele Loss and Down-Regulation of Heparanase Gene Are Associated with the Progression

Background Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene. This study aimed to investigate the impact of BRMS1 on metastasis in nasopharyngeal carcinoma (NPC) and to evaluate the prognostic significance of BRMS1 in NPC patients. Methods BRMS1 expression was examined in NPC cell lines using quantitative reverse transcription-polymerase chain reaction and Western blotting. NPC cells stably expressing BRMS1 were used to perform wound healing and invasion assays in vitro and a murine xenograft assay in vivo. Immunohistochemical staining was performed in 274 paraffin-embedded NPC specimens divided into a training set (n = 120) and a testing set (n = 154). Results BRMS1 expression was down-regulated in NPC cell lines. Overexpression of BRMS1 significantly reversed the metastatic phenotype of NPC cells in vitro and in vivo. Importantly, low BRMS1 expression was associated with poor distant metastasis-free survival (DMFS, P P Conclusions Low expression of the metastasis suppressor BRMS1 may be an independent prognostic factor for poor prognosis in NPC patients.

To investigate the anti-inflammatory effect of bone marrow stromal cells (MSCs) transfected with recombinant adenovirus-mediated ciliary neurotrophic factor (CNTF) gene in C57BL/6 mice with experimental allergic encephalomyelitis (EAE).An adenovirus vector containing CNTF gene Ad-CNTF-IRES-GFP was constructed and transfected in the MSCs (MSC-CNTF). After examination of CNTF expression, the transfected cells were transplanted in C57BL/6 mice with MOG 35-55-induced EAE, which were monitored for the changes in the symptoms scores. The levels of tumor necrosis factor-alpha (TNF-alpha), inteferon-gamma (IFN-gamma), interleukin-12P35 (IL-12P35), and IL-10 in the peripheral blood of the mice were detected, and the number of MSC-CNTF cells in the spleen and spinal cord was counted. CD3+ T cell infiltration and TNF-alpha and IFN-gamma expressions in the lesions were also observed after the cell transplantation.CNTF gene transfection resulted in significantly increased CNTF expression in the MSCs. The mice receiving MSC-CNTF transplantation exhibited significantly improved symptoms with shortened disease course and lessened disease severity. The cell transplantation also resulted in significantly decreased peripheral blood TNF-alpha levels, ameliorated CD3+T cell infiltrations and lowered TNF-alpha expression in the lesions, while the levels of IFN-gamma underwent no significant changes.Transplantation of CNTF gene-transfected MSCs results in decreased peripheral blood TNF-alpha and IFN-gamma levels and reduced inflammatory cells, CD3-positive cells and TNF-alpha expression in the lesion of EAE, therefore providing better effect than MSCs in relieving the symptoms of EAE in mice.

E10A, a recombinant adenovirus type 5 vector carrying the human endostatin gene, may be a promising gene therapy drug in the treatment of solid tumors by antiangiogenesis, but a preclinical safety evaluation of E10A has not yet been performed. With high and low doses equivalent to 30 and 7.5 times the human curative dose, respectively, intramuscular injections of E10A were given once daily, 6 days/week, for 3 months, followed by a 1-month recovery period. As of 4 months, all experimental animals appeared generally healthy: normal behavior and eating habits, no nausea, vomiting, or salivation, no abnormal changes in urination or defecation, and increased body weight with the time of experiment. Urinalysis, hemogram, blood biochemistry, electrocardiogram, macroscopic and microscopic studies of organs and tissues were done before treatment, at month 3 of treatment, and 1 month posttreatment. At all time points, no significant abnormal toxic effects were noted. Preliminary investigation of E10A immunotoxicity in dogs indicated that anti-adenoviral antibodies were generated, in a dose- and time-independent manner, after E10A injection. Our data demonstrated that, long term, high-dose intramuscular administration of recombinant human endostatin-carrying adenovirus (E10A) was not notably toxic and might be safe for clinical therapeutic use, although additional long-term toxicity studies by other administration routes are still necessary.

The p53 codon 72 polymorphism affects human papillomavirus (HPV) E6-mediated degradation of p53. This study was to investigate distribution of p53 polymorphism in Guangdong women, and relationship between p53 polymorphism and tumorigenesis of cervical cancer.Cervical smears of 46 patients with cervical cancer (case group), and 84 patients with benign gynecologic tumor (control group) treated in our hospital from Sept. 2002 to May 2003 were collected. DNA, extracted from cervical smears,were examined by polymerase chain reaction (PCR) for detection of HPV DNA and p53 codon 72 polymorphism.Positive rate of HPV DNA in case group was 47.8%, in control group was 20.2%. Proportions of genotypes Arg/Arg, Pro/Pro, and Arg/Pro in case group were 56.5%, 21.7%,and 21.7%, respectively; in control group were 71.4%,20.2%, and 8.3%,respectively. There were no significant differences in proportions of Arg/Arg (OR,0.520; 95% CI,0.245-1.102), and Arg/Pro (OR, 1.095; 95% CI,0.454-2.639) between 2 groups; proportion of Pro/Pro in case group was significantly higher than control group (OR, 3.056; 95% CI,1.076-8.678), but no significant difference was found in women with HPV infection.Arg/Arg genotype is not a high-risk factor for cervical cancer in Chinese population, and individuals with Pro/Pro genotype are likely to develop cervical cancer.

To determine the accuracy of p53 gene mutations predicted by overexpression of p53 protein immunohistochemically, and to investigate the co-mutation of p53 and K-ras genes in rectal cancer and its effect on promoting malignant biologic behaviors of tumors.Ninety-seven specimens of rectal cancer were surgically resected in our hospital from August 1996 to October 1997. The hot mutation areas of p53 gene (in exons 5-8) and K-ras gene (in codon 5/12 and 13) were detected with polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), and overexpression of p53 protein was detected with immunohistochemistry (IHC) in the 97 specimens of rectal cancer. Correlation between gene mutations and tumor clinicopathologic factors was studied, and survival analysis was penfomed as well.There were 36 cases of p53 gene mutations in 61 p53 protein positive cases, and 21 cases of p53 gene non-mutation in 36 p53 protein negative cases respectively. The coincidence rate of p53 gene mutation by IHC method with PCR-SSCP method was 58.8% (57/97). The mutation rate of p53 gene was 52.6% (51/97), while K-ras gene mutation was observed in codons 12 and 13 in 61 cases with a mutation rate of 62.9% (61/97). Single gene mutation of p53 or K-ras was found in 32 cases. Both p53 and K-ras gene mutation were found in 48 cases. Statistical analysis showed that p53 and K-ras gene mutations were not related to the clinicopathologic factors, including tumor size, gross tumor type, histological classification, differentiation, invasion to intestinal veins, lymphatics and nerves, invasive depth to wall, lymph node metastasis, and Dukes' stages (P0.05). The survival in patients with no gene mutation, single gene mutation and both gene mutations were similar (P0.05).IHC has a certain false positive and false negative rate in detecting p53 gene mutations. Malignant biological behaviours of rectal cancer are not enhanced by p53 and K-ras gene mutations. Co-mutation of p53 and K-ras gene has neither synergic carcinogenesis-promoting effect, nor prognostic effect on rectal cancer.

Molecular cytogenetics of oncogene HER-2 amplification in primary hepatocellular carcinoma (HCC) is still unknown. The aim of this study was to investigate the frequency of HER-2 oncogene amplification in primary HCC and its relations to clinicopathological parameters and prognosis.Forty-two surgical samples from patients with primary HCC were detected for their HER-2 oncogene amplification. The number of chromosome 17 and their ratio were tested by dual fluorescence in situ hybridization (FISH) technique, and then the correlations between HER-2 amplification, clinicopathological characteristics and prognosis were analyzed statistically.HER-2 oncogene amplification was detected in 9 (21.4%) of the 42 primary HCCs, including 4 patients with high copy (HC) (9.5%) and 5 patients with low copy (LC) (11.9%). HER-2 amplification was associated significantly with tumor size and postoperative survival time of HCC patients (P0.05), and the presence of HER-2 gene amplification was correlated with postoperative relapse (P=0.257), but not related to sex, age, AFP level, HBV infection, histopathological grading and clinical staging of HCC patients (P0.05). The HER-2 oncogene copy was examined in 31 (73.8%) of the 42 primary HCCs, consisting of 9 patients with HER-2 amplification (21.4%) and 22 patients with aneuploidy (52.4%). No significant relations were observed between the HER-2 oncogene copy, patient sex, tumor size, histopathological grading, clinical staging, postoperative relapse and survival time (P0.05); but the HER-2 oncogene copy was correlated significantly to age, AFP level and HBV infection (P0.05).There are a lower frequency of HER-2 oncogene amplification and a higher frequency of chromosome 17 aneuploidy in primary HCC. HER-2 oncogene amplification may be involved in the development and progression of large HCC in some patients, and seems to be a valuably independent prognostic factor predicting the recurrence and poor survival in patients with large HCC.

To investigate the deletion of p53 gene and amplification of HER-2 oncogene at chromosome 17 in primary hepatocellular carcinoma (HCC) and the clinical significance.Interphase dual fluorescence in situ hybridization (FISH) was applied to detect the ratio of the number of p53 gene copy or HER-2 oncogene copy to that of chromosome 17 copy, to determine the p53 gene deletion and HER-2 oncogene amplification in nuclei prepared from 42 surgical specimens of HCC. Statistical analysis for their clinical significance was performed.Loss of p53 gene and amplification of HER-2 oncogene were detected in 27 (64.3%) and 9 (21.4%) of the 42 HCC respectively including 4 cases with low and 5 with high copy amplification. Six (14.3%) of 42 HCC showed simultaneously p53 gene deletion and HER-2 oncogene amplification. 61.9% (26/42) of HCC were polysomy 17, which correlated positively with p53 gene deletion (chi(2) = 12.286, P0.001). No close correlation between p53 gene loss and HER-2 oncogene amplification was found (chi(2) = 0.00, P = 1.00). Loss of p53 gene was related to the serum alpha-fetoprotein (AFP) level and the tumor size (P0.05). The postoperative 2-year survival rate (18.5%) of HCC patients with p53 gene deletion was significantly lower than postoperative 2-year survival rate (60.0%) of those without p53 gene loss (chi(2) = 7.467, P = 0.006). Meanwhile, HER-2 oncogene amplification showed a tendency of correlation with the tumor size (chi(2) = 2.973, P = 0.085), and the postoperative 2-year survival rate (0/9) of HCC patients with HER-2 oncogene amplification was significantly lower than those (42.4%) without HER-2 oncogene amplification (chi(2) = 3.977, P = 0.046).There were a high frequency of p53 gene deletion and a low frequency of HER-2 oncogene amplification in primary HCC, which might be involved in initiation and development of a subset of primary HCC.

To explore the therapeutic effect of Hejie Decoction (HJD) in treating chronic hepatitis B and its relationship with T-cell receptor V beta 7 (TCRV beta 7) gene expression.Forty-five patients of chronic hepatitis B were randomly divided into two groups. The 30 patients in the treated group were treated by HJD, and the 15 patients in the control group were treated by conventional western medicine. The therapeutic effect and changes of TCRV beta 7 gene expression after treatment were observed.After 6 months treatment, the ALT level in the two groups were obviously decreased (P0.01). No significant difference was shown in comparison of the total effective rate between the two groups but it did show in comparison of markedly effective rate between them. TCRV beta 7 expression was detected in 5 patients of the treated group, and HBV-DNA and HBeAg in the 5 patients were all negatively converted. While in the control group, no one had TCRV beta 7 expression detected, either no one with negative conversion of HBV-DNA and HBeAg. TCRV beta 7 could not be detected in all the patients whose HBV-DNA and HBeAg hasn't negatively converted, though their liver function could be normalized.HJD might have the effect of regulation on TCRV beta 7 expression, it possibly is the important way for HBV replication inhibition and virucidal action of HJD.

AIM: To investigate genes around the locus D4S2964 affected by loss of heterozygosity (LOH) and their clinical implications. METHODS: Four hundred and forty single nucleotide polymorphisms (SNPs) located at 49 genes around D4S2964 were selected from the National Center for Biotechnology Information website for the SNPs microarray fabrication. LOH of SNPs markers in 112 cases of hepatocellular carcinoma (HCC) tissues and paired adjacent liver tissues were investigated by the SNPs microarray. The correlation between allelic losses with clinicopathological features and overall survival was analyzed. RESULTS: A fine map of LOH of SNPs in genes around D4S2964 was plotted. The average frequency of LOH in genes was 0.39. A correlation between cirrhosis and the FAL index (fractional allelic loss) was found (P = 0.0202). Larger tumor size was found to be significantly associated with LOH in genes ADP-ribosyltransferase 3 (ART3), nucleoporin 54 kDa (NUP54), scavenger receptor class B, member 2 (SCARB2) and coiled-coil domain containing 158 (CCDC158) (P = 0.043, P = 0.019, P = 0.001, P = 0.037, respectively). Kaplan-Meier analysis showed that patients with LOH in ARD1 homolog B (ARD1B) and septin 11 (SEPT11) had a significantly lower survival rate than those with retention (P = 0.021 and P = 0.004, respectively). A Cox regression model suggested that LOH in ARD1B and SEPT11, respectively, were predictors of the overall survival in HCC (P = 0.006 and P = 0.026, respectively). CONCLUSION: LOH in genes around D4S2964 may play an important role in HCC development and progression. LOH in ARD1B and SEPT11 could serve as novel prognostic predictors in HCC patients.

AIM: To explore the effect of Hejie decoction (HJD) (mediation decoction) on T cellular immune state of chronic hepatitis B patients. METHODS: Sixty-five patients with chronic hepatitis B were randomly divided into 2 groups. Forty patients in the treatment group were treated by HJD, and 25 patients in the control group were treated by routine Western medicine. The TCRVβ7 gene expression, T lymphocyte subsets (CD3+, CD4+, CD8+, CD4+/CD8+) levels were observed before and after treatment. RESULTS: The level of CD4+ cells was lower whereas the level of CD8+ cells was higher in patients than in the normal group. There was no significant difference between the levels of CD3+ cells in patients and normal persons. After 6 months of treatment, ALT, AST, TB levels of the 2 groups were obviously decreased, and the level of CD4+ cells was increased whereas the level of CD8+ cells was decreased in the treatment group. However, the level of CD4+ cells and CD8+ cells had no significant difference in the control group. TCRVβ7 expressions were detected in 6 patients of the treatment group, whose HBV-DNA and HBeAg turned negative and ALT became normal. HBeAg in another 3 patients turned negative while HBV-DNA did not, and TCRVβ7 expressions were not detectable. TCRVβ7 expression could not be detected in the control group, HBV-DNA of the control group did not turn negative. HBeAg in 1 patient turned negative while HBV-DNA did not, and TCRVβ7 expressions were not detectable. The total effective rate was not significantly different between the 2 groups and the markedly effective rate was significantly different (P < 0.01). CONCLUSION: HJD is effective for treating chronic hepatitis B, and its effect seems to relate with the improvement of the TCRVβ7 expression of chronic hepatitis B patients, thus activating T cells and eliminating HBV. T cellular immune function plays an important role in HBV infection and virus elimination.

Nasopharyngeal carcinoma (NPC) is a common malignancy in Southern China and Southeast Asia. In this study, we investigated the functional and molecular mechanisms by which CDC42-interacting protein 4 (CIP4) influences NPC. The expression levels of CIP4 were examined by Western blot, qRT-PCR or IHC. MTT assay was used to detect the proliferative rate of NPC cells. The invasive abilities were examined by matrigel and transwell assay. The metastatic abilities of NPC cells were revealed in BALB/c nude mice. We report that CIP4 is required for NPC cell motility and invasion. CIP4 promotes the activation of N-WASP that controls invadopodia formation and activates EGFR signaling, which induces downstream MMP2 (matrix metalloproteinase 2) upregulation. In addition, CIP4 could promote NPC metastasis by activating the EGFR pathway. In nude mouse models, distant metastasis was significantly inhibited in CIP4-silenced groups. High CIP4 expression is an independent adverse prognostic factor of overall survival (OS) and distant metastasis-free survival (DMFS). We identify the critical role of CIP4 in metastasis of NPC which suggest that CIP4 may be a potential therapeutic target of NPC patients.