The cytoplasmic expression of FSTL3 correlates with colorectal cancer progression, metastasis status and prognosis.

Follistatin-like (FSTL) family members are associated with cancer progression. However, differences between FSTL members with identical cancer types have not been systematically investigated. Among the most malignant tumours worldwide, colorectal cancer (CRC) has high metastatic potential and chemoresistance, which makes it challenging to treat. A systematic examination of the relationship between the expression of FSTL family members in CRC will provide valuable information for prognosis and therapeutic development. Based on large cohort survival analyses, we determined that FSTL3 was associated with a significantly worse prognosis in CRC at the RNA and protein levels. Immunohistochemistry staining of CRC specimens revealed that FSTL3 expression levels in the cytosol were significantly associated with a poor prognosis in terms of overall and disease-free survival. Molecular simulation analysis showed that FSTL3 participated in multiple cell motility signalling pathways via the TGF-ß1/TWIST1 axis to control CRC metastasis. The findings provide evidence of the significance of FSTL3 in the oncogenesis and metastasis of CRC. FSTL3 may be useful as a diagnostic or prognostic biomarker, and as a potential therapeutic target.

Kinase shRNA screening reveals that TAOK3 enhances microtubule-targeted drug resistance of breast cancer cells via the NF-κB signaling pathway.

BACKGROUND: Chemotherapy is currently one of the most effective treatments for advanced breast cancer. Anti-microtubule agents, including taxanes, eribulin and vinca-alkaloids are one of the primary major anti-breast cancer chemotherapies; however, chemoresistance remains a problem that is difficult to solve. We aimed to discover novel candidate protein targets to combat chemoresistance in breast cancer. METHODS: A lentiviral shRNA-based high-throughput screening platform was designed and developed to screen the global kinome to find new therapeutic targets in paclitaxel-resistant breast cancer cells. The phenotypes were confirmed with alternative expression in vitro and in vivo. Molecular mechanisms were investigated using global phosphoprotein arrays and expression microarrays. Global microarray analysis was performed to determine TAOK3 and genes that induced paclitaxel resistance. RESULTS: A serine/threonine kinase gene, TAOK3, was identified from 724 screened kinase genes. TAOK3 shRNA exhibited the most significant reduction in IC50 values in response to paclitaxel treatment. Ectopic downregulation of TAOK3 resulted in paclitaxel-resistant breast cancer cells sensitize to paclitaxel treatment in vitro and in vivo. The expression of TAOK3 also was correlated to sensitivity to two other anti-microtubule drugs, eribulin and vinorelbine. Our TAOK3-modulated microarray analysis indicated that NF-κB signaling played a major upstream regulation role. TAOK3 inhibitor, CP43, and shRNA of NF-κB both reduced the paclitaxel resistance in TAOK3 overexpressed cells. In clinical microarray databases, high TAOK3 expressed breast cancer patients had poorer prognoses after adjuvant chemotherapy. CONCLUSIONS: Here we identified TAOK3 overexpression increased anti-microtubule drug resistance through upregulation of NF-κB signaling, which reduced cell death in breast cancer. Therefore, inhibition of the interaction between TAOK3 and NF-κB signaling may have therapeutic implications for breast cancer patients treated with anti-microtubule drugs. Video abstract.

The Diverse Roles of TAO Kinases in Health and Diseases.

Thousand and one kinases (TAOKs) are members of the MAP kinase kinase kinase (MAP3K) family. Three members of this subfamily, TAOK1, 2, and 3, have been identified in mammals. It has been shown that TAOK1, 2 and 3 regulate the p38 MAPK and Hippo signaling pathways, while TAOK 1 and 2 modulate the SAPK/JNK cascade. Furthermore, TAOKs are involved in additional interactions with other cellular proteins and all of these pathways modulate vital physiological and pathophysiological responses in cells and tissues. Dysregulation of TAOK-related pathways is implicated in the development of diseases including inflammatory and immune disorders, cancer and drug resistance, and autism and Alzheimer's diseases. This review collates current knowledge concerning the roles of TAOKs in protein-protein interaction, signal transduction, physiological regulation, and pathogenesis and summarizes the recent development of TAOK-specific inhibitors that have the potential to ameliorate TAOKs' effects in pathological situations.

Overexpression of AKR1B10 predicts tumor recurrence and short survival in oral squamous cell carcinoma patients.

BACKGROUND: Aldo-keto reductase family 1 member B10 (AKR1B10) is an enzyme implicated in physiological xenobiotic detoxification and also in pathological carcinogenesis. Overexpression of AKR1B10 has been reported in oral squamous cell carcinoma (OSCC), but its correlation with clinical prognosis is controversial. The aim of this study was to investigate and clarify the role of AKR1B10 in OSCC carcinogenesis. METHODS: Tumor tissue specimens were surgically obtained from 107 patients with OSCC. The expression of AKR1B10 was analyzed by immunohistochemistry to explore the relationship between the level of AKR1B10 and clinicopathological features of OSCC patients. Kaplan-Meier survival and Cox proportional hazard analysis were used to determine the prognostic value of AKR1B10 in OSCC. RESULTS: High expression of AKR1B10 was found to be associated with tumor size (P = 0.043), perineural invasion (P = 0.012), and recurrence (P = 0.001) in OSCC. Cox model analysis revealed that high expression of AKR1B10 is significantly associated with poor overall and disease-free survival in OSCC patients. With the combination of clinicopathological factors in analysis, we found that the expression level of AKR1B10 was a practical indicator that could categorize OSCC patients into different risk groups. High expression of AKR1B10 was associated with a reduced survival in patients with well and moderately differentiated OSCC and even a high incidence of tumor recurrence in the patients with late-stage (III and IV) disease. CONCLUSION: We validated and expanded data on the expression of AKR1B10 in OSCC, suggesting that it is a valuable biomarker for prognostic prediction of recurrence and survival in OSCC.

Inhibition of Epstein-Barr virus reactivation by the flavonoid apigenin.

BACKGROUND: Lytic reactivation of EBV has been reported to play an important role in human diseases, including NPC carcinogenesis. Inhibition of EBV reactivation is considered to be of great benefit in the treatment of virus-associated diseases. For this purpose, we screened for inhibitory compounds and found that apigenin, a flavonoid, seemed to have the ability to inhibit EBV reactivation. METHODS: We performed western blotting, immunofluorescence and luciferase analyses to determine whether apigenin has anti-EBV activity. RESULTS: Apigenin inhibited expression of the EBV lytic proteins, Zta, Rta, EAD and DNase in epithelial and B cells. It also reduced the number of EBV-reactivating cells detectable by immunofluorescence analysis. In addition, apigenin has been found to reduce dramatically the production of EBV virions. Luciferase reporter analysis was performed to determine the mechanism by which apigenin inhibits EBV reactivation: apigenin suppressed the activity of the immediate-early (IE) gene Zta and Rta promoters, suggesting it can block initiation of the EBV lytic cycle. CONCLUSION: Taken together, apigenin inhibits EBV reactivation by suppressing the promoter activities of two viral IE genes, suggesting apigenin is a potential dietary compound for prevention of EBV reactivation.

EGCG inhibits proliferation, invasiveness and tumor growth by up-regulation of adhesion molecules, suppression of gelatinases activity, and induction of apoptosis in nasopharyngeal carcinoma cells.

(-)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, has been shown to inhibit the proliferation of a variety of tumor cells. Epidemiological studies have shown that drinking green tea can reduce the incidence of nasopharyngeal carcinoma (NPC), yet the underlying mechanism is not well understood. In this study, the inhibitory effect of EGCG was tested on a set of Epstein Barr virus-negative and -positive NPC cell lines. Treatment with EGCG inhibited the proliferation of NPC cells but did not affect the growth of a non-malignant nasopharyngeal cell line, NP460hTert. Moreover, EGCG treated cells had reduced migration and invasive properties. The expression of the cell adhesion molecules E-cadherin and ß-catenin was found to be up-regulated by EGCG treatment, while the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 were found to be mediated by suppression of extracellular signal-regulated kinase (ERK) phosphorylation and AP-1 and Sp1 transactivation. Spheroid formation by NPC cells in suspension was significantly inhibited by EGCG. Oral administration of EGCG was capable of suppressing tumor growth in xenografted mice bearing NPC tumors. Treatment with EGCG was found to elevate the expression of p53 and p21, and eventually led to apoptosis of NPC cells via caspase 3 activation. The nuclear translocation of NF-κB and ß-catenin was also suppressed by EGCG treatment. These results indicate that EGCG can inhibit the proliferation and invasiveness, and induce apoptosis, of NPC cells, making it a promising agent for chemoprevention or adjuvant therapy of NPC.

Inhibition of Epstein-Barr virus reactivation in nasopharyngeal carcinoma cells by dietary sulforaphane.

Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcinogenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotoxicity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation.

Tn as a potential predictor for regional lymph node metastasis in T1 colorectal cancer.

BACKGROUND: Approximately 10 percent of T1 colorectal cancer (CRC) has lymph node metastasis. In this study, we aimed to determine possible predictors for nodal involvement in order to aid selection of appropriate patients for organ-preserving strategies. METHODS: We retrospectively reviewed CRC patients underwent radical surgery from January 2009 to December 2016, with final pathology report disclosed as T1 lesion. The paraffin-embedded samples were achieved for glycosylated proteins expression analysis by immunohistochemistry. RESULTS: Totally, 111 CRC patients with T1 lesion were enrolled in this study. Of these patients, seventeen patients had nodal metastases, with the lymph node positive rate of 15.3%. Semiquantitative analysis of immunohistochemical results indicated that mean value of Tn protein expression in T1 CRC specimens was significantly different between patients with and without lymph node metastasis (63.6 vs. 27.4; p = 0.018). CONCLUSIONS: Our data shown that Tn expression may be applied as a molecular predictor for regional lymph node metastasis in T1 CRC. Moreover, the organ-preserving strategy could be improved by proper classification of patients. The mechanism involved in expression of Tn glycosylation protein and CRC metastasis need further investigation.

Overexpression of synaptic vesicle protein Rab GTPase 3C promotes vesicular exocytosis and drug resistance in colorectal cancer cells.

Rab GTPase 3C (RAB3C) is a peripheral membrane protein that is involved in membrane trafficking (vesicle formation) and cell movement. Recently, researchers have noted the exocytosis of RAB proteins, and their dysregulation is correlated with drug resistance and the altered tumor microenvironment in tumorigenesis. However, the molecular mechanisms of exocytotic RABs in the carcinogenicity of colorectal cancer (CRC) remain unknown. Researchers have used various in silico datasets to evaluate the expression profiles of RAB family members. We confirmed that RAB3C plays a key role in CRC progression. Its overexpression promotes exocytosis and is related to the resistance to several chemotherapeutic drugs. We established a proteomic dataset based on RAB3C, and found that dystrophin is one of the proteins that is upregulated with the overexpression of RAB3C. According to our results, RAB3C-induced dystrophin expression promotes vesicle formation and packaging. A connectivity map predicted that the cannabinoid receptor 2 (CB2) agonists reverse RAB3C-associated drug resistance, and that these agonists have synergistic effects when combined with standard chemotherapy regimens. Moreover, we found high dystrophin expression levels in CRC patients with poor survival outcomes. A combination of the dystrophin and RAB3C expression profiles can serve as an independent prognostic factor in CRC and is associated with several clinicopathological parameters. In addition, the RAB3C-dystrophin axis is positively correlated with the phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (PIK3CA) genetic alterations in CRC patients. These findings can be used to provide novel combined therapeutic options for the treatment of CRC.

The activation of EP300 by F11R leads to EMT and acts as a prognostic factor in triple-negative breast cancers.

Cancer progression is influenced by junctional adhesion molecule (JAM) family members. The relationship between JAM family members and different types of cancer was examined using The Cancer Genome Atlas dataset. mRNA levels of the F11R (F11 receptor) in tumours were inversely correlated to the expression of JAM-2 and JAM-3. This relationship was unique to breast cancer (BCa) and was associated with poor prognosis (p = 0.024, hazard ratio = 1.44 [1.05-1.99]). A 50-gene molecular signature (prediction analysis of microarray 50) was used to subtype BCa. F11R mRNA expression significantly increased in human epidermal growth factor receptor 2 (HER2)-enriched (p = 0.0035) and basal-like BCa tumours (p = 0.0005). We evaluated F11R protein levels in two different compositions of BCa subtype patient tissue array cohorts to determine the relationship between BCa subtype and prognosis. Immunohistochemistry staining revealed that a high F11R protein level was associated with poor overall survival (p < 0.001; Taipei Medical University [TMU] cohort, p < 0.001; Kaohsiung Veterans General Hospital [KVGH] cohort) or disease-free survival (p < 0.001 [TMU cohort], p = 0.034 [KVGH cohort]) in patients with BCa. Comparison of F11R levels in different subtypes revealed the association of poor prognosis with high levels of F11R among luminal (p < 0.001 [TMU cohort], p = 0.027 [KVGH cohort]), HER2 positive (p = 0.018 [TMU cohort], p = 0.037 [KVGH cohort]), and triple-negative (p = 0.013 [TMU cohort], p = 0.037 [KVGH cohort]) BCa. F11R-based RNA microarray analysis and Ingenuity Pathway Analysis were successful in profiling the detailed gene ontology of triple-negative BCa cells regulated by F11R. The EP300 transcription factor was highly correlated with F11R in BCa (R = 0.51, p < 0.001). By analysing these F11R-affected molecules with the L1000CDs datasets, we were able to predict some repurposing drugs for potential application in F11R-positive BCa treatment.

The stability and immunogenicity of formalin-inactivated Enterovirus A71 whole virion vaccine after ten years of low temperature storage.

BACKGROUND: Vaccine stability is an important issue for vaccine development, which affects whether the vaccine product is effective within a certain period of time in each progress. Hand, foot, and mouth diseases (HFMD) is an epidemic disease in young children usually caused by Enterovirus A group viruses, and the Enterovirus A71 (EV-A71) had caused several pandemics and public health issues around the world. After two decades of research and development, formalin-inactivated EV-A71 (FI-EV-A71) vaccines are the first to complete the phase III clinical trials for protection against EV-A71 infection. Currently, the shelf life of FI-EV-A71 vaccine product is set to be within 18 months, but the stability and the effectiveness of the FI-EV-A71 whole virion when stored long-term at low temperature remains undetermined. METHODS: Assessing the long-term storage properties of viral particles facilitates flexibility in manufacturing of vaccine products. In this study, the stability profiles of FI-EV-A71 vaccine lots and bulks after long-term of low temperature storage were analyzed by protein tests, particle measurement and animal immunization study. RESULTS: After over ten years of storage, the reduction of protein concentration in the FI-EV-A71 bulk samples is less than 30 % and the antigenic content remained in a suspended, particulate state. Both the packed FI-EV-A71 final vaccine products and the FI-EV-A71 antigens adjuvant premix bulk could elicit strong neutralizing responses in mice. CONCLUSION: After ten years of low temperature storage, the FI-EV-A71 vaccine still presents decent stability and good immunogenicity.

Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system.

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

Epstein-Barr virus DNase (BGLF5) induces genomic instability in human epithelial cells.

Epstein-Barr Virus (EBV) DNase (BGLF5) is an alkaline nuclease and has been suggested to be important in the viral life cycle. However, its effect on host cells remains unknown. Serological and histopathological studies implied that EBV DNase seems to be correlated with carcinogenesis. Therefore, we investigate the effect of EBV DNase on epithelial cells. Here, we report that expression of EBV DNase induces increased formation of micronucleus, an indicator of genomic instability, in human epithelial cells. We also demonstrate, using gammaH2AX formation and comet assay, that EBV DNase induces DNA damage. Furthermore, using host cell reactivation assay, we find that EBV DNase expression repressed damaged DNA repair in various epithelial cells. Western blot and quantitative PCR analyses reveal that expression of repair-related genes is reduced significantly in cells expressing EBV DNase. Host shut-off mutants eliminate shut-off expression of repair genes and repress damaged DNA repair, suggesting that shut-off function of BGLF5 contributes to repression of DNA repair. In addition, EBV DNase caused chromosomal aberrations and increased the microsatellite instability (MSI) and frequency of genetic mutation in human epithelial cells. Together, we propose that EBV DNase induces genomic instability in epithelial cells, which may be through induction of DNA damage and also repression of DNA repair, subsequently increases MSI and genetic mutations, and may contribute consequently to the carcinogenesis of human epithelial cells.

Novel strategies for the development of hand, foot, and mouth disease vaccines and antiviral therapies.

INTRODUCTION: Hand, foot, and mouth disease (HFMD) poses a great threat to young children in the Asia-Pacific region. HFMD is usually caused by enterovirus A, and infection with enterovirus A71 (EV-A71) is particularly associated with severe complications. However, coxsackievirus CV-A16, CV-A6, and CV-A10 pandemics have been observed in recent HFMD outbreaks. Inactivated monovalent EV-A71 vaccines are available to prevent EV-A71 infection; however, they cannot prevent infections by non-EV-A71 enteroviruses. Anti-enteroviral drugs are still in the developmental stage. Application of novel strategies will facilitate the development of new therapies against these emerging HFMD-associated enteroviruses. AREAS COVERED: The authors highlight the current approaches for anti-enterovirus therapeutic development and discuss the application of these novel strategies for the discovery of vaccines and antiviral drugs for enteroviruses. EXPERT OPINION: The maturation of DNA/RNA vaccine technology could be applied for rapid and robust development of multivalent enterovirus vaccines. Structure biology and neutralization antibody studies decipher the immunodominant sites of enteroviruses for vaccine design. Nucleotide aptamer library screening is a novel, fast, and cost-effective strategy for the development of antiviral agents. Animal models carrying viral receptors and attachment factors are required for enterovirus study and vaccine/antiviral development. Currently developed antivirals require effectiveness evaluation in clinical trials.

Separation and purification of highly infectious enterovirus A71 particles using a strong anion-exchange column.

Virions produced from cell culture is the primary source for production of formalin-inactivated whole virus vaccines for enteroviruses. EV-A71 particles produced from culture system comprise two major types, the immature/empty (E)-particle and the mature/full (F)-particle, which both exhibit low isoelectric point (pI) values but have distinct differences in infectivity and immunogenicity. Although EV-A71 particles can conventionally be separated into E-particle and F-particle using sucrose gradient ultracentrifugation, this procedure is cumbersome and difficult to put into practice for vaccine production. Methods based on ion-exchange chromatography have been exploited to improve the purification efficacy; however, none of them are capable of separating the E- and F-particles efficiently. In this study, we aimed to develop an approach to isolate and purify the highly immunogenic mature EV-A71 particles. By applying a step gradient elution procedure, we successfully isolated the viral structure protein VP0-cleaved particles of EV-A71 from a mixture of cultured viral solution using the Q-membrane anion-exchange chromatography. The elution started with 0.1x phosphate buffered saline (PBS) solution while increasing the percentage of 1x PBS containing 1M NaCl in sequential steps. By this procedure, the VP0-cleaved mature particles and VP0-uncleaved immature particles of EV-A71 could be separated into different fractions in Q-membrane with gradually increased NaCl concentration in elution buffer. The purified VP0-cleaved particles were shown to have characteristics equivalent to those of the highly infectious F-particles of EV-A71. The overall recovery rate for the mature EV-A71 particles by Q-membrane is 56% and its purity was shown to be equivalent to those isolated by the sucrose gradient ultracentrifugation. Our approach provides a simple and efficient purification method for recovering mature, highly infectious virus particles from the EV-A71 culture bulk.

An Advanced In Situ Magnetic Resonance Imaging and Ultrasonic Theranostics Nanocomposite Platform: Crossing the Blood-Brain Barrier and Improving the Suppression of Glioblastoma Using Iron-Platinum Nanoparticles in Nanobubbles.

Glioblastoma (GBM) is one of the deadliest and most invasive brain cancers/gliomas, and there is currently no established way to treat this disease. The treatment of GBM typically involves intracranial surgery followed by chemotherapy. However, the blood-brain barrier (BBB) impedes the delivery of the chemotherapeutic drug, making the treatment challenging. In this study, we embedded a chemotherapeutic drug and other nanomaterials into a nanobubble (NB), utilized active tracking and other guidance mechanisms to guide the nanocomposite to the tumor site, and then used high-intensity focused ultrasound oscillation to burst the nanobubbles, generating a transient cavitation impact on the BBB and allowing the drug to bypass it and reach the brain. FePt enhances the resolution of T2-weighted magnetic resonance imaging images and has magnetic properties that help guide the nanocomposite to the tumor location. FePt nanoparticles were loaded into the hydrophobic core of the NBs along with doxorubicin to form a bubble-based drug delivery system (Dox-FePt@NB). The surface of the NBs is modified with a targeting ligand, transferrin (Dox-FePt@NB-Tf), giving the nanocomposite active tracking abilities. The Dox-FePt@NB-Tf developed in the present study represents a potential breakthrough in GBM treatment through improved drug delivery and biological imaging.

Exosomal Components and Modulators in Colorectal Cancer: Novel Diagnosis and Prognosis Biomarkers.

The relatively high incidence and mortality rates for colorectal carcinoma (CRC) make it a formidable malignant tumor. Comprehensive strategies have been applied to predict patient survival and diagnosis. Various clinical regimens have also been developed to improve the therapeutic outcome. Extracellular vesicles (EVs) are recently proposed cellular structures that can be produced by natural or artificial methods and have been extensively studied. In addition to their innate functions, EVs can be manipulated to be drug carriers and exert many biological functions. The composition of EVs, their intravesicular components, and the surrounding tumor microenvironment are closely related to the development of colorectal cancer. Determining the expression profiles of exocytosis samples and using them as indicators for selecting effective combination therapy is an indispensable direction for EV study and should be regarded as a novel prediction platform in addition to cancer stage, prognosis, and other clinical assessments. In this review, we summarize the function, regulation, and application of EVs in the colon cancer research field. We provide an update on and discuss potential values for clinical applications of EVs. Moreover, we illustrate the specific markers, mediators, and genetic alterations of EVs in colorectal carcinogenesis. Furthermore, we outline the vital markers present in the EVs and discuss their plausible uses in colon cancer patient therapy in combination with the currently used clinical strategies. The development and application of these EVs will significantly improve the accuracy of diagnosis, lead to more precise prognoses, and may lead to the improved treatment of colorectal cancer.

Nonenzymatic function of Aldolase A downregulates miR-145 to promote the Oct4/DUSP4/TRAF4 axis and the acquisition of lung cancer stemness.

Drug resistance remains a serious issue of clinical importance and is a consequence of cancer stemness. In this study, we showed that the level of Aldolase A (ALDOA) expression is significantly associated with the IC50 value of chemotherapy drugs in lung cancer. Our data revealed that ALDOA overexpression resulted in a significant increase of lung tumor spheres. The use of ingenuity pathway analysis (IPA) resulted in the identification of POU5F1 (Oct4) as the leading transcription factor of ALDOA. We observed high expression of ALDOA, Oct4 and stemness markers in collected spheroid cells. DUSP4 and TRAF4 were confirmed as major downstream targets of the ALDOA-Oct4 axis. Knockdown of these molecules significantly decreased the stemness ability of cells. In addition, we investigated whether miR-145 targets the 3'-UTR of Oct4 and is regulated by ALDOA due to the involvement of ALDOA in glycolysis and metabolic reprogramming. Furthermore, we constructed several mutant forms of ALDOA that disrupted its enzymatic activity and showed that they still induced significant in vitro sphere formation and in vivo tumorigenicity. These results demonstrated that ALDOA-mediated spheroid formation is independent of its enzymatic activity. In the clinical component, we also showed that the combination of ALDOA and TRAF4 or DUSP4 is positively correlated with poor overall survival in a xenograft model and cancer patients through immunohistochemical analyses. The results of our study revealed novel functional roles of ALDOA in inducing cancer stemness via the inhibition of miR-145 expression and the activation of Oct4 transcription. These findings offer new therapeutic strategies for modulation of lung cancer stemness to enhance chemotherapeutic responses in lung cancer patients.

Recurrent chemical reactivations of EBV promotes genome instability and enhances tumor progression of nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is an endemic malignancy prevalent in South East Asia. Epidemiological studies have associated this disease closely with Epstein-Barr virus (EBV) infection. Previous studies also showed that EBV reactivation is implicated in the progression of NPC. Thus, we proposed that recurrent reactivations of EBV may be important for its pathogenic role. In this study, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 (TW01) and HONE-1, were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium n-butyrate (SB) for lytic cycle induction. A single treatment with TPA/SB revealed that DNA double-strand breaks and formation of micronuclei (a marker for genome instability) were associated with EBV reactivation in NA and HA cells. Examination of EBV early genes had identified several lytic proteins, particularly EBV DNase, as potent activators that induced DNA double-strand breaks and contribute to genome instability. Recurrent reactivations of EBV in NA and HA cells resulted in a marked increase of genome instability. In addition, the degree of chromosomal aberrations, as shown by chromosome structural variants and DNA copy-number alterations, is proportional to the frequency of TPA/SB-induced EBV reactivation. Whereas these DNA abnormalities were limited in EBV-negative TW01 cells with mock or TPA/SB treatment, and were few in mock-treated NA cells. The invasiveness and tumorigenesis assays also revealed a profound increase in both characteristics of the repeatedly reactivated NA cells. These results suggest that recurrent EBV reactivations may result in accumulation of genome instability and promote the tumor progression of NPC.

Increased Expression of Programmed Death-Ligand 1 in Infiltrating Immune Cells in Hepatocellular Carcinoma Tissues after Sorafenib Treatment.

OBJECTIVE: Programmed death-ligand 1 (PD-L1) expression in the tumor microenvironment (TME) has been reported to be related to prognosis in patients with hepatocellular carcinoma (HCC) after hepatectomy. The impact of sorafenib on PD-L1 expression in the TME of advanced HCC is unclear. PATIENTS AND METHODS: Patients with HCC who received sorafenib for advanced disease at National Taiwan University Hospital, Taipei, Taiwan, and who had paired HCC tissues obtained before and after sorafenib treatment were included in the study group. HCC patients not treated with sorafenib who had paired primary and recurrent or metastatic tissues were identified as the reference group. The membrane PD-L1 staining, detected by immunohistochemistry (IHC) using SP142 antibody, was semiquantitatively scored in tumor cells (TCs) or tumor-infiltrating immune cells (ICs). Additional IHC assays were employed to characterize the PD-L1-expressing ICs. RESULTS: Twenty-three advanced HCC patients with pre- and post-sorafenib paired HCC tissues were included in the study group. The median duration of sorafenib treatment was 4.3 months (range: 1.3-18.7). PD-L1 expression in ICs was significantly higher in post-sorafenib HCC tissues than in pre-sorafenib HCC tissues (pre-sorafenib vs. post-sorafenib IHC 0/1/2/3: 11/5/5/2 vs. 5/5/2/11, p = 0.016). However, PD-L1 expression in TCs was not significantly different between pre- and post-sorafenib tissues (IHC 0/1/2/3: 19/2/0/2 vs. 14/5/0/4, p = 0.094). In the reference group of 44 patients not treated with sorafenib, PD-L1 expression in ICs and TCs was not significantly different between the paired primary and metastatic HCC tissues. By performing IHC double staining with PD-L1 and CD68, we found the PD-L1-expressing ICs were mainly CD68-positive macrophages. PD-L1 expression levels of pre- and post-sorafenib tissues were not associated with patients' overall survival or duration of sorafenib treatment. CONCLUSIONS: PD-L1 expression in ICs was significantly increased in post-sorafenib HCC tissues. The mechanisms and clinical significance of this observation warrants further investigation.