Genotypes of Papillary Thyroid Carcinoma With High Lateral Neck Metastasis in Chinese Population.

Papillary Thyroid Carcinoma (PTC) is one of the most commonly diagnosed cancer types in China, characterized by its early age at diagnosis and high 25-year survival rate. Distinct mutational patterns in PTC have been linked to activation of the mitogen-activated protein kinase (MAPK) signaling pathway. To explore the clinical significance of genomic variation patterns in Chinese patients with thyroid carcinoma, we investigated the genomic variants in 83 PTC cases with complete clinical records. The mutational patterns were evaluated using a 688-gene panel which covered known driver genes in PTC tumorigenesis, and featured genetic markers in various PTC-related pathways. We evaluated the relationship between mutational landscape and various clinical information in PTC patients with lateral lymph node metastasis. BRAF V600E was the most common mutation. Mutations in NF1, CDC27, PMS2 and PPP4R2 were more common in men, and mutations in NF1, PMS2 and TERT were related to lateral lymph node metastasis. According to the clustering of mutational patterns, we show that the underline driving mechanisms in lateral lymph node metastasis can be divided into two major groups (BRAF-TERT pathway, and NF1-PMS2 pathway). When combined with the TERT mutations, the BRAF mutation group was prone to lateral lymph node metastasis, particularly in elderly women. The NF1 mutations usually co-existed with PMS2 mutations, and this group included more men and young patients who had a high tumor mutational burden and lateral lymph node metastasis rate.

GSA: an independent development algorithm for calling copy number and detecting homologous recombination deficiency (HRD) from target capture sequencing.

BACKGROUND: The gain or loss of large chromosomal regions or even whole chromosomes is termed as genomic scarring and can be observed as copy number variations resulting from the failure of DNA damage repair. RESULTS: In this study, a new algorithm called genomic scar analysis (GSA) has developed and validated to calculate homologous recombination deficiency (HRD) score. The two critical submodules were tree recursion (TR) segmentation and filtering, and the estimation and correction of the tumor purity and ploidy. Then, this study evaluated the rationality of segmentation and genotype identification by the GSA algorithm and compared with other two algorithms, PureCN and ASCAT, found that the segmentation result of GSA algorithm was more logical. In addition, the results indicated that the GSA algorithm had an excellent predictive effect on tumor purity and ploidy, if the tumor purity was more than 20%. Furtherly, this study evaluated the HRD scores and BRCA1/2 deficiency status of 195 clinical samples, and the results indicated that the accuracy was 0.98 (comparing with Affymetrix OncoScan™ assay) and the sensitivity was 95.2% (comparing with BRCA1/2 deficiency status), both were well-behaved. Finally, HRD scores and 16 genes mutations (TP53 and 15 HRR pathway genes) were analyzed in 17 cell lines, the results showed that there was higher frequency in HRR pathway genes in high HRD score samples. CONCLUSIONS: This new algorithm, named as GSA, could effectively and accurately calculate the purity and ploidy of tumor samples through NGS data, and then reflect the degree of genomic instability and large-scale copy number variations of tumor samples.

Characterization of genomic clones by targeted deep sequencing of ctDNA to monitor liver cancer.

BACKGROUND: In recent years, the morbidity and mortality of cancer patients have continued to increase in China, and there is an urgent need to develop an effective method to monitor tumor dynamics and measure tumor burden. Derived from the cell-free fraction of blood in cancer patients, circulating tumor DNA (ctDNA) has been regarded as a promising surrogate for tumor tissue biopsies. With the development of sequencing technology, ctDNA has been recognized as a specific and highly sensitive biomarker, and it has become a hot research spot in recent years. METHODS: In this paper, we investigated clonal changes before and after surgery in liver cancer patients using ctDNA. RESULTS: First, we evaluated the accuracy and stability of the method in ctDNA detection using virtual tumor samples with known mutations. The results showed that our method detected variants with an allelic frequency of at least 0.5%. We then applied this method to 34 liver cancer patients. A total of 266 clinically relevant mutations were identified in the pretreatment plasma samples. Through the analysis of plasma DNA samples at different treatment time points, we also investigated the possibility of using ctDNA as a prognostic factor to reflect tumor dynamics and to evaluate clinical responses. CONCLUSIONS: The results demonstrated that targeted high-depth next-generation sequencing can be used in ctDNA detection. Compared to traditional biopsy, the detection of ctDNA provides more information for human liver cancer, which is essential to guide the selection of therapy and predict prognosis.

Variations in Oral Microbiota Composition Are Associated With a Risk of Throat Cancer.

In this study, a next-generation sequencing strategy on 16S ribosomal RNA (16S rRNA) gene was employed to analyze 70 oral samples from 32 patients with throat cancer, nine patients with vocal cord polyp, and 29 healthy individuals (normal controls). Using this strategy, we demonstrated, for the first time, that the salivary microbiota of cancer patients were significantly different from those of patients with a polyp and healthy individuals. We observed that the beta diversity of the cancer group was divergent from both the normal and polyp groups, while alpha-diversity indices such as the Chao1 estimator (P = 8.1e-05), Simpson (P = 0.0045), and Shannon (P = 0.0071) were significantly reduced in cancer patients compared with patients containing a polyp and normal healthy individuals. Linear discriminant analysis (LDA) and Kruskal-Wallis test analyses and real-time quantitative polymerase chain reaction (qPCR) verification test revealed that the genera Aggregatibacter, Pseudomonas, Bacteroides, and Ruminiclostridium were significantly enriched in the throat cancer group compared with the vocal cord polyp and normal control groups (score value >2). Finally, diagnostic models based on putatively important constituent bacteria were constructed with 87.5% accuracy [area under the curve (AUC) = 0.875, 95% confidence interval (CI): 0.695-1]. In summary, in this study we characterized, for the first time, the oral microbiota of throat cancer patients without smoking history. We speculate that these results will help in the pathogenic mechanism and early diagnosis of throat cancer.

Genome-wide methylomic and transcriptomic analyses identify subtype-specific epigenetic signatures commonly dysregulated in glioma stem cells and glioblastoma.

Glioma stem cells (GSCs), a subpopulation of tumor cells, contribute to tumor heterogeneity and therapy resistance. Gene expression profiling classified glioblastoma (GBM) and GSCs into four transcriptomically-defined subtypes. Here, we determined the DNA methylation signatures in transcriptomically pre-classified GSC and GBM bulk tumors subtypes. We hypothesized that these DNA methylation signatures correlate with gene expression and are uniquely associated either with only GSCs or only GBM bulk tumors. Additional methylation signatures may be commonly associated with both GSCs and GBM bulk tumors, i.e., common to non-stem-like and stem-like tumor cell populations and correlating with the clinical prognosis of glioma patients. We analyzed Illumina 450K methylation array and expression data from a panel of 23 patient-derived GSCs. We referenced these results with The Cancer Genome Atlas (TCGA) GBM datasets to generate methylomic and transcriptomic signatures for GSCs and GBM bulk tumors of each transcriptomically pre-defined tumor subtype. Survival analyses were carried out for these signature genes using publicly available datasets, including from TCGA. We report that DNA methylation signatures in proneural and mesenchymal tumor subtypes are either unique to GSCs, unique to GBM bulk tumors, or common to both. Further, dysregulated DNA methylation correlates with gene expression and clinical prognoses. Additionally, many previously identified transcriptionally-regulated markers are also dysregulated due to DNA methylation. The subtype-specific DNA methylation signatures described in this study could be useful for refining GBM sub-classification, improving prognostic accuracy, and making therapeutic decisions.

In vitro study of FUZ as a novel potential therapeutic target in non-small-cell lung cancer.

FUZ is regarded as a planar cell polarity effector that controls multiple cellular processes during vertebrate development. However, the role of FUZ in tumor biology remains poorly studied. Our purpose of this study is to discover the physiological effects and mechanism of FUZ in non-small-cell lung cancer (NSCLC) in vitro. With the help of bioinformatics analysis, we noticed that the expression level of FUZ negatively correlates with prognosis of NSCLC patients. Exogenous FUZ expression markedly promoted cell proliferation of NSCLC cells. The phosphorylation of Erk1/2, STAT3 and related signaling molecules were induced activated after FUZ over-expression. FUZ also plays an important role in cell motility by regulating cell signaling pathways and inducing epithelial to mesenchymal transition (EMT). FUZ promotes EMT along with the up-regulation of N-cadherin, vimentin, Zeb1, Twist1 and decreased level of E-cadherin. Furthermore, we also carried out FUZ directed siRNA treatments to prove the above observations. Knockdown of FUZ resulted in delayed cell growth as well as impaired cell migration and reversed EMT phonotype. Importantly, we reported for the first time that FUZ is a BNIP3-interacting protein. Loss of FUZ resulted in decreased BNIP3 protein level, but no influence on BNIP3 mRNA level, suggesting weakened stability of BNIP3 protein. Overall, our results in vitro show that FUZ is responsible for NSCLC progression and metastasis, suggesting that FUZ can be a potential therapeutic target for NSCLC.

MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma.

ATG4B stimulates autophagy by promoting autophagosome formation through reversible modification of ATG8. We identify ATG4B as a substrate of mammalian sterile20-like kinase (STK) 26/MST4. MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities using genetic approaches or an inhibitor of ATG4B suppresses autophagy and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation, and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM xenograft markedly slows tumor growth and provides a significant survival benefit. Our work describes an MST4-ATG4B signaling axis that influences GBM autophagy and malignancy, and whose therapeutic targeting enhances the anti-tumor effects of radiotherapy.

DCUN1D3 activates SCFSKP2 ubiquitin E3 ligase activity and cell cycle progression under UV damage.

Our previous study showed that knockdown the endogenous expression of DCUN1D3 (also called SCCRO3 or DCNL3) blocked the S phase progression after UV irradiation. Here, we show that the silence of DCUN1D3 can increase the cyclin-dependent kinase inhibitor p27 protein levels after UV irradiation. Through Co-immunoprecipitation experiments, we found that DCUN1D3 bound to CAND1. And DCUN1D3 knockdown synergized with CAND1 over-expression in arresting the S phase. Given the CAND1's established role in Cullin-1 neddylation, we found Cullin-1 was less neddylated in DCUN1D3 deficient cells. So the silence of DCUN1D3 can inhibit the formation of SCFSKP2 complex by reducing Cullin-1 neddylation. Given that p27 is the primary target of SCFSKP2 complex, the cells lost DCUN1D3 showed a remarkable accumulation of p27 to cause S phase block.

The novel secretory protein CGREF1 inhibits the activation of AP-1 transcriptional activity and cell proliferation.

The transcription factor AP-1 plays an important role in inflammation and cell survival. Using a dual-luciferase reporter assay system and a library of 940 candidate human secretory protein cDNA clones, we identified that CGREF1 can inhibit the transcriptional activity of AP-1. We demonstrated that CGREF1 is secreted via the classical secretory pathway through the ER-to-Golgi apparatus. Functional investigations revealed that overexpression of CGREF1 can significantly inhibit the phosphorylation of ERK and p38 MAPK, and suppress the proliferation of HEK293T and HCT116 cells. Conversely, specific siRNAs against CGREF1 can increase the transcriptional activity of AP-1. These results clearly indicated that CGREF1 is a novel secretory protein, and plays an important role in regulation of AP-1 transcriptional activity and cell proliferation.

PC3-secreted microprotein is a novel chemoattractant protein and functions as a high-affinity ligand for CC chemokine receptor 2.

PC3-secreted microprotein (PSMP) or microseminoprotein is a newly discovered secreted protein whose function is currently unknown. In this study, PSMP was found to possess chemotactic ability toward monocytes and lymphocytes, and its functional receptor was identified as CCR2B. PSMP was identified as a chemoattractant protein from a PBMC chemoattractant platform screen that we established. The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, PBLs, and CCR2B-expressing THP-1 cells, but not peripheral blood neutrophils, even though it does not contain the classical structure of chemokines. CCR2B was identified as one receptor for PSMP-mediated chemotaxis by screening HEK293 cells that transiently expressed classical chemokine receptors; results obtained from the chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays all confirmed this finding. To further identify the major function of PSMP, we analyzed its expression profile in tissues. PSMP is highly expressed in benign prostatic hyperplasia and in some prostate cancers, and can also be detected in breast tumor tissue. In response to PSMP stimulation, phosphorylated ERK levels downstream of CCR2B signaling were upregulated in the PC3 cell line. Taken together, our data collectively suggest that PSMP is a chemoattractant protein acting as a novel CCR2 ligand that may influence inflammation and cancer development.

Human TTC5, a novel tetratricopeptide repeat domain containing gene, activates p53 and inhibits AP-1 pathway.

The transcription factor p53 and AP-1 play an important role in cellular proliferation, transformation and death. In this study, we investigated the role of a novel human gene, TTC5 (tetratricopeptide repeat domain 5), in the regulation of cell signaling pathway and cell viability. TTC5 is a member of the TTC family of proteins and has previously been shown to participate in cellular stress response. Here we demonstrate for the first time that TTC5 significantly activates p53 pathway and inhibits AP-1 transcriptional activity. Further investigation revealed that overexpression of TTC5 up-regulated p53 and p21 expression, and significantly inhibited transcriptional activity, expression and phosphorylation of c-Jun. As for the upstream of signaling pathway of AP-1, our study demonstrated that overexpression of TTC5 significantly down-regulated the expression and phosphorylation of JNK/SAPK. Moreover, overexpression of TTC5 repressed cell proliferation and induced S phase cell cycle arrest. These results indicated that TTC5 may regulate cell viability by p53 and AP-1 signaling pathway.

[Comparative analysis on both high risk behaviours, infection of HIV and syphilis between married and unmarried men who have sex with men].

OBJECTIVE: To understand the distribution of marriage status among men who have sex with men (MSM) in the city of Changzhou, and to explore the impact of marriage on AIDS related high risk behaviors and HIV infection in this population. METHODS: Target sampling (snowball sampling) was adopted to carry out a cross-sectional study, and structured questionnaire-based interviews were conducted to collect information on social demography, HIV related high risk behaviors. Blood and urine samples were collected to detect HIV, syphilis, gonorrhea and Chlamydia trachomatis infections. RESULTS: Of the 655 participants, 37.4% were married. Married MSM mostly sought their sexual partners at the public bathing house (61.6%), while unmarried MSM were mainly through bars (33.6%) or internet (31.1%). The proportion of having anal sex with men during the last 6 months was lower in the married group (50.8%) than in the unmarried group (73.3%), (P < 0.001) The percentage of having sex with women in the last 6 months was significantly higher in the married group (68.9%) than that in the unmarried group (33.2%) (P < 0.001), (OR = 4.454, 95%CI: 3.168 - 6.261). The rates of condom use in the last anal sex with men in married and unmarried groups were 71.0% and 77.6%, respectively (P = 0.152). The rate of condom use in the last intercourse with women was significantly lower in the married group (44.0%) than that in the unmarried group (70.4%) (P < 0.001), (OR = 0.331, 95%CI: 0.205 - 0.535). In the sex trade, most of the married MSM would "buy" sex (66.7%), while unmarried MSM would "sell" sex (63.2%) (P < 0.05), (OR = 3.429, 95%CI: 1.255 - 9.366). The percentage of having drugs in the previous year was higher in married group (3.3%) than that in the unmarried group (0.8%) (P < 0.05). In married and unmarried groups, the infection rates of HIV, syphilis, gonorrhea and Chlamydia trachomatis appeared to be (8.6%, 8.6%), (17.1%, 12.3%), (1.6%, 2.4%), and (3.3%, 9.0%), respectively (P > 0.05). CONCLUSION: Marriage seemed to have had limited effects on reducing the high risk behaviors of MSM. Different and multiform interventions should be developed according to the different characteristics of married or unmarried MSM population.

Adenovirus vector-mediated expression of TMEM166 inhibits human cancer cell growth by autophagy and apoptosis in vitro and in vivo.

TMEM166 is a novel programmed cell death-related molecule. In this report, we constructed a recombinant adenovirus 5-TMEM166 vector (Ad5-TMEM166) and evaluated its expression and anti-tumor activities in vitro and in vivo. Cell viability analysis revealed that the adenovirus-mediated increase of TMEM166 inhibited tumor cell growth in a dose- and time-dependent manner. This inhibitory effect was mediated by both autophagy (via inhibition of mTOR and activation of p70S6K) and apoptosis (via caspase-3 activation), both of which contributed to cell death and suppression of tumorigenicity. Our data indicated that Ad5-TMEM166 may be a novel gene therapy candidate for cancer.

Suppression of non-small cell lung cancer proliferation and tumorigenicity by DENND2D.

DENND2D was identified as being down-regulated in lung cancer using a lung cancer low-expression suppression subtractive hybridization (SSH) library. In this study, DENND2D down-regulation has been observed not only in non-small cell lung cancer (NSCLC) cell lines and lung squamous cell carcinoma (SCC) tissues, but also in immortalized human bronchial epithelial (IHBE) cell lines and precancerous lesions, indicating that the down-regulation of DENND2D may be an early event in lung cancer. The relative DNA copy number and mRNA and protein expression levels of DENND2D were determined in vitro, and they revealed a complicated regulatory network at the genomic, transcriptional and translational levels. Over-expression of DENND2D significantly suppressed the proliferation of NSCLC cells in vitro and in vivo by inducing apoptosis. These results indicate that DENND2D might function as a tumor suppressor-like gene to prevent the survival and expansion of cells with genetic damage through apoptosis mechanism, and absence of DENND2D might play a permissive role, as an early event, in tumorigenesis.

[Construction of recombinant NF-κB reporter adenovirus and activity analysis].

OBJECTIVE: To study the feasibility of adenovirus-based nuclear factor-κB (NF-κB) reporter as a model to screen the upstream signal regulators of NF-κB. METHODS: A type 5 (E1/E3 deficient) adenovirus vector pAdxsi was used to construct the NF-κB reporter adenovirus. Multiple adherent and suspending cell lines were infected by the NF-κB reporter adenovirus, and the luciferase activity of the NF-κB reporter gene was measured. RESULTS: An NF-κB reporter adenovirus (Ad-NF-κB-luc) was successfully constructed. The virus was capable of infecting HepG2, MGC803, THP-1 and U937 cell lines and showed high activities of NF-κB-luc reporter gene when stimulated by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). CONCLUSION: The Ad-NF-κB-luc reporter gene transfer system can effectively infect those cells hard-transfected by conventional transfection reagents. It also produces a high activity of NF-κB-luc reporter gene with stability and reliability. Our study expands the application of NF-κB reporter gene.

In vitro biological characterization of DCUN1D5 in DNA damage response.

BACKGROUND: Novel prognostic biomarkers or therapeutic molecular targets for laryngeal squamous cell carcinoma (LSCC) are an urgent priority. We here sought to identify multiple novel LSCC-associated genes. METHODS: Using high-density microarray expression profiling, we identified multiple genes that were significantly altered between human LSCCs and paired normal tissues. Potential oncogenic functions of one such gene, DCUN1D5, were further characterized in vitro. RESULTS: Our results demonstrated that DCUN1D5 was highly expressed in LSCCs. Overexpression of DCUN1D5 in vitro resulted in 2.7-fold increased cellular migration, 67.5% increased invasive capacity, and 2.6-fold increased proliferation. Endogenous DCUN1D5 expression was decreased in a time-dependent manner after genotoxic stress, and silencing of DCUN1D5 by siRNA decreased the number of cells in the S phase by 10.2% and increased apoptosis by 11.7%. CONCLUSION: Our data suggest that DCUN1D5 in vitro might have vital roles in DNA damage response, but further studies are warranted to assess its significance in vivo.

VSTM1-v2, a novel soluble glycoprotein, promotes the differentiation and activation of Th17 cells.

Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.

CCDC134 interacts with hADA2a and functions as a regulator of hADA2a in acetyltransferase activity, DNA damage-induced apoptosis and cell cycle arrest.

Human transcriptional adaptor hADA2a is an important component of the general control nonderepressible 5 (GCN5) histone acetyltransferase complex. Here, we report that coiled-coil domain containing 134 (CCDC134), a novel nuclear protein, binds to hADA2a and enhances the stability of the hADA2a protein in unstressed conditions. Furthermore, CCDC134 was found to participate in the p300/CBP-associated factor (PCAF) complex via hADA2a and affect the histone acetyltransferase activity of the complex. We also found that CCDC134 increased the PCAF-dependent K320 acetylation of p53 and p53 protein stability in the presence of hADA2a overexpression. Moreover, we demonstrated the biological significance of the interaction between CCDC134 and hADA2a. CCDC134 showed obvious nuclear accumulation after ultraviolet (UV) irradiation, and the knockdown of endogenous CCDC134 suppressed hADA2a-induced cell apoptosis activity and G1/S cell cycle arrest. Together, our findings indicate that CCDC134 might act as a novel regulator of hADA2a, and plays roles in the PCAF complex via hADA2a to affect its acetyltransferase activity and UV-induced DNA damage repair.

Human HS1BP3 induces cell apoptosis and activates AP-1.

In the present study, we characterized the function of HS1-binding protein 3 (HS1BP3), which is mutated in essential tremor and may be involved in lymphocyte activation. We found that HS1BP3 localized to the mitochondria and endoplasmic reticulum partially. Overexpression of HS1BP3 induced apoptosis in HEK293T and HeLa cell lines. When these cell lines were transfected with HS1BP3, they exhibited nuclear DNA condensation, externalization of phosphatidylserine (PS), and cleavage of poly ADP ribose polymerase (PARP). Furthermore, suppression of HS1BP3 or HS1 expression attenuates HS1BP3 induced apoptosis. In addition, HS1BP3 enhanced activator protein 1 (AP-1)-mediated transcription in a dose-dependent manner. Therefore, we conclude that HS1BP3 regulates apoptosis via HS1 and stimulates AP-1-mediated transcription.

Identification of C1qTNF-related protein 4 as a potential cytokine that stimulates the STAT3 and NF-κB pathways and promotes cell survival in human cancer cells.

The NF-κB and IL6/STAT3 pathways are major participants in tumor-promoting inflammation. C1qTNF related protein (CTRP) is a family with multiple physiological functions, but their involvement in tumor-promoting inflammation has received little attention. For the first time, we have identified CTRP4 as a novel secretary protein by N-terminal sequencing. Moreover, recombinant CTRP4 can effectively induce the activation of both NF-κB and IL6/STAT3 signaling pathways in the pattern similar to that of classical cytokine. By western blot analysis, we detected the upregulation of CTRP4 in response to IL6. Importantly, functional research revealed that CTRP4 could promote tumor cell survival and tumor resistance against apoptosis induced by chemotherapeutics. These results strongly suggest that CTRP4 is a novel tumor-promoting inflammatory regulator. Our findings might provide a meaningful indication for cancer research.