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Natural killer (NK) cells are unique from other immune cells in that they can rapidly kill multiple neighboring cells without the need for antigenic pre-sensitization once the cells display surface markers associated with oncogenic transformation. Given the dynamic role of NK cells in tumor surveillance, NK cell-based immunotherapy is rapidly becoming a "new force" in tumor immunotherapy. However, challenges remain in the use of NK cell immunotherapy in the treatment of solid tumors. Many metabolic features of the tumor microenvironment (TME) of solid tumors, including oxygen and nutrient (e.g., glucose, amino acids) deprivation, accumulation of specific metabolites (e.g., lactate, adenosine), and limited availability of signaling molecules that allow for metabolic reorganization, multifactorial shaping of the immune-suppressing TME impairs tumor-infiltrating NK cell function. This becomes a key barrier limiting the success of NK cell immunotherapy in solid tumors. Restoration of endogenous NK cells in the TME or overt transfer of functionally improved NK cells holds great promise in cancer therapy. In this paper, we summarize the metabolic biology of NK cells, discuss the effects of TME on NK cell metabolism and effector functions, and review emerging strategies for targeting metabolism-improved NK cell immunotherapy in the TME to circumvent these barriers to achieve superior efficacy of NK cell immunotherapy. [ABSTRACT FROM AUTHOR]

Currently, women around the world are still suffering from various female common diseases with the high incidence, such as ovarian cancer, uterine fibroids and preeclampsia (PE), and some diseases are even with the high mortality rate. As a negative feedback regulator in G Protein-Coupled Receptor signaling (GPCR), the Regulator of G-protein Signaling (RGS) protein family participates in regulating kinds of cell biological functions by destabilizing the enzyme–substrate complex through the transformation of hydrolysis of G Guanosine Triphosphate (GTP). Recent work has indicated that, the Regulator of G-protein Signaling 2 (RGS2), a member belonging to the RGS protein family, is closely associated with the occurrence and development of certain female diseases, providing with the evidence that RGS2 functions in sustaining women's health. In this review paper, we summarize the current knowledge of RGS2 in female common diseases, and also tap and discuss its therapeutic potential by targeting multiple mechanisms. [ABSTRACT FROM AUTHOR]

Background: Intratumoral injection of oncolytic viruses (OVs) shows promise in immunotherapy: ONCOS-102, a genetically engineered OV that encodes Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) demonstrated efficacy in early clinical trials, enhancing T cell infiltration in tumors. This suggests OVs may boost various forms of immunotherapy, including tumor-specific bi-specific antibodies (BsAbs). Methods: Our study investigated in vitro, how ONCOS-204, a variant of ONCOS-virus expressing the ligand of inducible T-cell co-stimulator (ICOSL), modulates the process of T cell activation induced by a BsAb. ONCOS-102 was used for comparison. Phenotypic and functional changes induced by combination of different OVs, and BsAb in T cell subsets were assessed by flow cytometry, viability, and proliferation assays. Results: Degranulation and IFNγ and TNF production of T cells, especially CD4 + T cells was the most increased upon target cell exposure to ONCOS-204. Unexpectedly, ONCOS-204 profoundly affected CD8 + T cell proliferation and function through ICOS-L/ICOS interaction. The effect solely depended on cell surface expression of ICOS-L as soluble ICOSL did not induce notable T cell activity. Conclusions: Together, our data suggests that oncolytic adenoviruses encoding ICOSL may enhance functional activity of tumor-specific BsAbs thereby opening a novel avenue for clinical development in immunotherapeutics. [ABSTRACT FROM AUTHOR]

Background: Radioresistance is a primary factor contributing to the failure of rectal cancer treatment. Immune suppression plays a significant role in the development of radioresistance. We have investigated the potential role of phosphatidylinositol transfer protein cytoplasmic 1 (PITPNC1) in regulating immune suppression associated with radioresistance. Methods: To elucidate the mechanisms by which PITPNC1 influences radioresistance, we established HT29, SW480, and MC38 radioresistant cell lines. The relationship between radioresistance and changes in the proportion of immune cells was verified through subcutaneous tumor models and flow cytometry. Changes in the expression levels of PITPNC1, FASN, and CD155 were determined using immunohistochemistry and western blotting techniques. The interplay between these proteins was investigated using immunofluorescence co-localization and immunoprecipitation assays. Additionally, siRNA and lentivirus-mediated gene knockdown or overexpression, as well as co-culture of tumor cells with PBMCs or CD8+ T cells and establishment of stable transgenic cell lines in vivo, were employed to validate the impact of the PITPNC1/FASN/CD155 pathway on CD8+ T cell immune function. Results: Under irradiation, the apoptosis rate and expression of apoptosis-related proteins in radioresistant colorectal cancer cell lines were significantly decreased, while the cell proliferation rate increased. In radioresistant tumor-bearing mice, the proportion of CD8+ T cells and IFN-γ production within immune cells decreased. Immunohistochemical analysis of human and animal tissue specimens resistant to radiotherapy showed a significant increase in the expression levels of PITPNC1, FASN, and CD155. Gene knockdown and rescue experiments demonstrated that PITPNC1 can regulate the expression of CD155 on the surface of tumor cells through FASN. In addition, co-culture experiments and in vivo tumor-bearing experiments have shown that silencing PITPNC1 can inhibit FASN/CD155, enhance CD8+ T cell immune function, promote colorectal cancer cell death, and ultimately reduce radioresistance in tumor-bearing models. Conclusions: PITPNC1 regulates the expression of CD155 through FASN, inhibits CD8+ T cell immune function, and promotes radioresistance in rectal cancer. [ABSTRACT FROM AUTHOR]

Background: Non-alcoholic Fatty Liver Disease (NAFLD), now better known as Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and its progression to Nonalcoholic Steatohepatitis (NASH), more recently referred to as Metabolic (Dysfunction)-Associated Steatohepatitis (MASH) are the most common causes of liver failure and chronic liver damage. The new names emphasize the metabolic involvement both in relation to liver function and pathological features with extrahepatic manifestations. This study aims to explore the role of the immunometabolic enzyme ATP citrate lyase (ACLY), with a critical function in lipogenesis, carbohydrate metabolism, gene expression and inflammation. Methods: ACLY function was investigated in TNFα-triggered human hepatocytes and in PBMC-derived macrophages from MASH patients. Evaluation of expression levels was carried out by western blotting and/or RT-qPCR. In the presence or absence of ACLY inhibitors, ROS, lipid peroxidation and GSSG oxidative stress biomarkers were quantified. Chromatin immunoprecipitation (ChIP), transient transfections, immunocytochemistry, histone acetylation quantitation were used to investigate ACLY function in gene expression reprogramming. IL-6 and IL-1β were quantified by Lumit immunoassays. Results: Mechanistically, ACLY inhibition reverted lipid accumulation and oxidative damage while reduced secretion of inflammatory cytokines in TNFα-triggered human hepatocytes. These effects impacted not only on lipid metabolism but also on other crucial features of liver function such as redox status and production of inflammatory mediators. Moreover, ACLY mRNA levels together with those of malic enzyme 1 (ME1) increased in human PBMC-derived macrophages from MASH patients when compared to age-matched healthy controls. Remarkably, a combination of hydroxycitrate (HCA), the natural ACLY inhibitor, with red wine powder (RWP) significantly lowered ACLY and ME1 mRNA amount as well as IL-6 and IL-1β production in macrophages from subjects with MASH. Conclusion: Collectively, our findings for the first time highlight a broad spectrum of ACLY functions in liver as well as in the pathogenesis of MASH and its diagnostic and therapeutic potential value. [ABSTRACT FROM AUTHOR]

Background: Disruption of N6 methyl adenosine (m6A) modulation hampers gene expression and cellular functions, leading to various illnesses. However, the role of m6A modification in osteoarthritis (OA) synovitis remains unclear. This study aimed to explore the expression patterns of m6A regulators in OA synovial cell clusters and identify key m6A regulators that mediate synovial macrophage phenotypes. Methods: The expression patterns of m6A regulators in the OA synovium were illustrated by analyzing bulk RNA-seq data. Next, we built an OA LASSO-Cox regression prediction model to identify the core m6A regulators. Potential target genes of these m6A regulators were identified by analyzing data from the RM2target database. A molecular functional network based on core m6A regulators and their target genes was constructed using the STRING database. Single-cell RNA-seq data were collected to verify the effects of m6A regulators on synovial cell clusters. Conjoint analyses of bulk and single-cell RNA-seq data were performed to validate the correlation between m6A regulators, synovial clusters, and disease conditions. After IGF2BP3 was screened as a potential modulator in OA macrophages, the IGF2BP3 expression level was tested in OA synovium and macrophages, and its functions were further tested by overexpression and knockdown in vitro. Results: OA synovium showed aberrant expression patterns of m6A regulators. Based on these regulators, we constructed a well-fitting OA prediction model comprising six factors (FTO, YTHDC1, METTL5, IGF2BP3, ZC3H13, and HNRNPC). The functional network indicated that these factors were closely associated with OA synovial phenotypic alterations. Among these regulators, the m6A reader IGF2BP3 was identified as a potential macrophage mediator. Finally, IGF2BP3 upregulation was verified in the OA synovium, which promoted macrophage M1 polarization and inflammation. Conclusions: Our findings revealed the functions of m6A regulators in OA synovium and highlighted the association between IGF2BP3 and enhanced M1 polarization and inflammation in OA macrophages, providing novel molecular targets for OA diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Background: Diabetic cardiomyopathy (DCM) has been considered as a major threat to health in individuals with diabetes. GrpE-like 2 (Grpel2), a nucleotide exchange factor, has been shown to regulate mitochondrial import process to maintain mitochondrial homeostasis. However, the effect and mechanism of Grpel2 in DCM remain unknown. Methods: The streptozotocin (STZ)-induced DCM mice model and high glucose (HG)-treated cardiomyocytes were established. Overexpression of cardiac-specific Grpel2 was performed by intramyocardial injection of adeno-associated virus serotype 9 (AAV9). Bioinformatics analysis, co-immunoprecipitation (co-IP), transcriptomics profiling and functional experiments were used to explore molecular mechanism of Grpel2 in DCM. Results: Here, we found that Grpel2 was decreased in DCM induced by STZ. Overexpression of cardiac-specific Grpel2 alleviated cardiac dysfunction and structural remodeling in DCM. In both diabetic hearts and HG-treated cardiomyocytes, Grpel2 overexpression attenuated apoptosis and mitochondrial dysfunction, including decreased mitochondrial ROS production, increased mitochondrial respiratory capacities and increased mitochondrial membrane potential. Mechanistically, Grpel2 interacted with dihydrolipoyl succinyltransferase (DLST), which positively mediated the import process of DLST into mitochondria under HG conditions. Furthermore, the protective effects of Grpel2 overexpression on mitochondrial function and cell survival were blocked by siRNA knockdown of DLST. Moreover, Nr2f6 bond to the Grpel2 promoter region and positively regulated its transcription. Conclusion: Our study provides for the first time evidence that Grpel2 overexpression exerts a protective effect against mitochondrial dysfunction and apoptosis in DCM by maintaining the import of DLST into mitochondria. These findings suggest that targeting Grpel2 might be a promising therapeutic strategy for the treatment of patients with DCM. [ABSTRACT FROM AUTHOR]

Background: Cellular compositions of solid tumor microenvironments are heterogeneous, varying across patients and tumor types. High-resolution profiling of the tumor microenvironment cell composition is crucial to understanding its biological and clinical implications. Previously, tumor microenvironment gene expression and DNA methylation-based deconvolution approaches have been shown to deconvolve major cell types. However, existing methods lack accuracy and specificity to tumor type and include limited identification of individual cell types.Results: We employed a novel tumor-type-specific hierarchical model using DNA methylation data to deconvolve the tumor microenvironment with high resolution, accuracy, and specificity. The deconvolution algorithm is named HiTIMED. Seventeen cell types from three major tumor microenvironment components can be profiled (tumor, immune, angiogenic) by HiTIMED, and it provides tumor-type-specific models for twenty carcinoma types. We demonstrate the prognostic significance of cell types that other tumor microenvironment deconvolution methods do not capture.Conclusion: We developed HiTIMED, a DNA methylation-based algorithm, to estimate cell proportions in the tumor microenvironment with high resolution and accuracy. HiTIMED deconvolution is amenable to archival biospecimens providing high-resolution profiles enabling to study of clinical and biological implications of variation and composition of the tumor microenvironment. [ABSTRACT FROM AUTHOR]

Background: The tumour microenvironment and cirrhotic liver are excellent sources of cancer-associated fibroblasts (CAFs), which participate in carcinogenesis. Thus, it is important to clarify the crosstalk between CAFs and HCC cells and the related mechanism in regulating carcinogenesis.Methods: Human hepatocellular carcinoma (HCC) tissues and matched adjacent normal tissues were obtained from HCC patients. Immunohistochemistry, Western blotting (WB) and RT-qPCR were performed to detect the expression of SCUBE1. The roles of SCUBE1 in inducing stemness features in HCC cells were explored and investigated in vitro and in vivo. Student's t tests or Mann-Whitney U tests were used to compare continuous variables, while chi-square tests or Fisher's exact tests were used to compare categorical variables between two groups.Results: SCUBE1 was confirmed to be highly expressed in CAFs in HCC and had a strong connection with stemness and a poor prognosis. In addition, CAFs were found to secrete SCUBE1 to enhance the malignancy of HCC cells and increase the proportion of CD133-positive cells. Silencing SCUBE1 expression had the opposite effect. The Shh pathway was activated by SCUBE1 stimulation. Inhibition of cyclopamine partially reversed the stimulating effect of SCUBE1 both in vivo and in vitro. Moreover, based on the RT-qPCR, ELISA and WB results, a high SCUBE1 expression level was found in HCC tissue and serum.Conclusion: This study revealed that CAFs-derived SCUBE1 can enhance the malignancy and stemness of HCC cells through the Shh pathway. This study aims to provide new perspectives for future HCC studies and provide new strategies for HCC treatment. [ABSTRACT FROM AUTHOR]

Background: Radiotherapy is the primary therapeutic option for glioblastoma. Some studies proved that radiotherapy increased the release of exosomes from cells. The mechanism by which these exosomes modify the phenotype of microglia in the tumor microenvironment to further determine the fate of irradiated glioblastoma cells remains to be elucidated.Methods: We erected the co-culture system of glioblastoma cells and microglia. After radiation, we analyzing the immunophenotype of microglia and the proliferation of radiated glioblastoma cells. By whole transcriptome sequencing, we analyzed of circRNAs in exosomes from glioblastoma cells and microglia. We used some methods, which included RT-PCR, dual-luciferase reporter, et al., to identify how circ_0012381 from radiated glioblastoma cell-derived exosomes regulated the immunophenotype of microglia to further affect the proliferation of radiated glioblastoma cells.Results: Radiated glioblastoma cell-derived exosomes markedly induced M2 microglia polarization. These M2-polarized microglia promoted the proliferation of irradiated glioblastoma cells. Circ_0012381 expression was increased in the irradiated glioblastoma cells, and circ_0012381 entered the microglia via exosomes. Circ_0012381 induced M2 microglia polarization by sponging with miR-340-5p to increase ARG1 expression. M2-polarized microglia suppressed phagocytosis and promoted the growth of the irradiated glioblastoma cells by CCL2/CCR2 axis. Compared with the effects of radiotherapy alone, the inhibition of exosomes significantly inhibited the growth of irradiated glioblastoma cells in a zebrafish model.Conclusions: Our data suggested that the inhibition of exosome secretion might represent a potential therapeutic strategy to increase the efficacy of radiotherapy in patients with glioblastoma. [ABSTRACT FROM AUTHOR]

Cancer is one of the leading causes of death in both men and women worldwide. One of the main changes associated with cancer progression, metastasis, recurrence, and chemoresistance is the change in the tumor immune microenvironment, especially immunosuppression. Cancer immunosuppression appears in multiple forms, such as inhibition of immuno-stimulant cells with downregulation of immuno-stimulant mediators or through stimulation of immuno-suppressive cells with upregulation of immunosuppressive mediators. One of the most immunosuppressive mediators that approved potency in lung cancer progression is indoleamine 2,3-dioxygenase (IDO) and its metabolite kynurenine (Kyn). The current review tries to elucidate the role of IDO/Kyn on cancer proliferation, apoptosis, angiogenesis, oxidative stress, and cancer stemness. Besides, our review investigates the new therapeutic modalities that target IDO/Kyn pathway and thus as drug candidates for targeting lung cancer and drugs that potentiate IDO/Kyn pathway and thus can be cancer-promoting agents. [ABSTRACT FROM AUTHOR]

Background: The resistance to radiotherapy remains a major obstacle that limits the efficacy of radiotherapy in non-small cell lung cancer (NSCLC). This study aims to illustrate the molecular mechanism underlying the role of LINC00665 in the radiosensitivity of NSCLC, which involves ubiquitin C-terminal hydrolase L3 (UCHL3).Methods and Results: The expression of UCHL3 was determined in clinical tissue samples collected from NSCLC patients and NSCLC cell lines. We found that UCHL3 overexpression occurred in both NSCLC tissues and cells, associated with poor prognosis in NSCLC patients. Mechanistically, UCHL3 stabilized aryl hydrocarbon receptor (AhR) protein through deubiquitination, thereby promoting PD-L1 expression. UCHL3 reduced the radiosensitivity of NSCLC cells by stabilizing AhR protein. Upstream microRNAs (miRNAs) and lncRNAs of UCHL3 were predicted by microarray profiling and validated by functional experiments. LINC00665 functioned as a sponge of miR-582-5p and thus up-regulated the expression of the miR-582-5p target UCHL3. Gain- and loss- of function assays were performed to assess the effects of LINC00665, UCHL3 and miR-582-5p on the in vitro cell malignant behaviors and immune escape as well as on the in vivo tumor growth. Silencing LINC00665 or overexpressing miR-582-5p enhanced the sensitivity of NSCLC cells to radiotherapy. LINC00665 augmented the immune escape of NSCLC cells in vitro and in vivo through stabilizing AhR protein via the miR-582-5p/UCHL3 axis.Conclusions: Overall, LINC00665 reduced the radiosensitivity of NSCLC cells via stabilization of AhR through the miR-582-5p/UCHL3 axis. [ABSTRACT FROM AUTHOR]

Background: We hypothesized that a gender difference in clinical response may exist to adjuvant CTLA4 blockade with ipilimumab versus high-dose IFNα (HDI). We investigated differences in candidate immune biomarkers in the circulation and tumor microenvironment (TME).Patients and Methods: This gender-based analysis was nested within the E1609 trial that tested adjuvant therapy with ipilimumab 3 mg/kg (ipi3) and 10 mg/kg (ipi10) versus HDI in high risk resected melanoma. We investigated gender differences in treatment efficacy with ipi3 and ipi10 versus HDI while adjusting for age, stage, ECOG performance (PS), ulceration, primary tumor status and lymph node number. Forest plots were created to compare overall survival (OS) and relapse free survival (RFS) between ipi and HDI. Gene expression profiling (GEP) was performed on tumors of 718 (454 male, 264 female) patients. Similarly, serum and peripheral blood mononuclear cells (PBMC) samples were tested for soluble and cellular biomarkers (N = 321 patients; 109 female and 212 male).Results: The subgroups of female, stage IIIC, PS = 1, ulcerated primary, in-transit metastasis demonstrated significant improvement in RFS and/or OS with ipi3 versus HDI. Female gender was significant for both OS and RFS and was further explored. In the RFS comparison, a multivariate Cox regression model including significant variables indicated a significant interaction between gender and treatment (P = 0.024). In peripheral blood, percentages of CD3+ T cells (P = 0.024) and CD3+ CD4+ helper T cells (P = 0.0001) were higher in females compared to males. Trends toward higher circulating levels of IL1β (P = 0.07) and IL6 (P = 0.06) were also found in females. Males had higher percentages of monocytes (P = 0.03) with trends toward higher percentages of regulatory T cells (T-reg). Tumor GEP analysis supported enhanced infiltration with immune cells including gammadelta T cells (P = 0.005), NK cells (P = 0.01), dendritic cells (P = 0.01), CD4+ T cells (P = 0.03), CD8+ T cells (P = 0.03) and T-reg (P = 0.008) in the tumors of females compared to males and a higher T-effector and IFNγ gene signature score (P = 0.0244).Conclusion: Female gender was associated with adjuvant CTLA4 blockade clinical benefits and female patients were more likely to have evidence of type1 immune activation within the TME and the circulation. Trial registration ClinicalTrials.gov NCT01274338. Registered 11 January 2011, https://www.Clinicaltrials: gov/ct2/show/NCT01274338. [ABSTRACT FROM AUTHOR]

Gastric cancer is a common type of gastrointestinal malignant tumor in China. The mechanism of the development and progression of gastric cancer remains the continuing research focus. The tumor microenvironment plays an important role in the development and progression of tumors. The present study used single-cell sequencing data to characterize the microenvironment of gastric cancer, investigate the effects of oxidative stress on gastric cancer microenvironmental cells through the comparison between cancer tissue and normal tissue, and identify the key genes associated with gastric cancer patients' survival. The results showed that compared with normal gastric tissue, gastric cancer tissue had a decreased oxidative stress response, weaker oxidative detoxification ability, and increased oxidative stress-induced cell death. In the different types of single cells of gastric cancer microenvironment, the oxidative stress response of T cell was increased, the ability of oxidative detoxification was enhanced, and the oxidative stress-induced cell death was exacerbate. Mucous cell showed the same trend as gastric cancer cells: decreased oxidative stress response, weak oxidative detoxification ability, and weakened oxidative stress-induced cell death. Moreover, TRIM62, MET, and HBA1, which were significantly associated with oxidative stress, may be biomarkers for the prognosis of gastric cancer. High expression of TRIM62 indicated a good prognosis, while MET and HBA1 indicated a poor prognosis, which will be confirmed by further clinical studies. [ABSTRACT FROM AUTHOR]

Harnessing the effector mechanisms of the immune system to combat brain tumors with antigen specificity and memory has been in research and clinical testing for many years. Government grant mechanisms and non-profit organizations have supported many innovative projects and trials while biotech companies have invested in the development of needed tools, assays and novel clinical approaches. The National Brain Tumor Society and the Parker Institute for Cancer Immunotherapy partnered to host a workshop to share recent data, ideas and identify both hurdles and new opportunities for harnessing immunotherapy against pediatric and adult brain tumors. Adoptively transferred cell therapies have recently shown promising early clinical results. Local cell delivery to the brain, new antigen targets and innovative engineering approaches are poised for testing in a new generation of clinical trials. Although several such advances have been made, several obstacles remain for the successful application of immunotherapies for brain tumors, including the need for more representative animal models that can better foreshadow human trial outcomes. Tumor and tumor microenvironment biopsies with multiomic analysis are critical to understand mechanisms of response and patient stratification, yet brain tumors are especially challenging for such biopsy collection. These workshop proceedings and commentary shed light on the status of immunotherapy in pediatric and adult brain tumor patients, including current research as well as opportunities for improving future efforts to bring immunotherapy to the forefront in the management of brain tumors. [ABSTRACT FROM AUTHOR]

Background: Triosephosphate isomerase 1 (TPI1), as a key glycolytic enzyme, is upregulated in multiple cancers. However, expression profile and regulatory mechanism of TPI1 in breast cancer (BRCA) remain mysterious.Methods: Western blotting and immunohistochemistry (IHC) assays were used to investigate the expression of TPI1 in BRCA specimens and cell lines. TPI1 correlation with the clinicopathological characteristics and prognosis of 362 BRCA patients was analyzed using a tissue microarray. Overexpression and knockdown function experiments in cells and mice models were performed to elucidate the function and mechanisms of TPI1-induced BRCA progression. Related molecular mechanisms were clarified using co-IP, IF, mass spectrometric analysis, and ubiquitination assay.Results: We have found TPI1 is highly expressed in BRCA tissue and cell lines, acting as an independent indicator for prognosis in BRCA patients. TPI1 promotes BRCA cell glycolysis, proliferation and metastasis in vitro and in vivo. Mechanistically, TPI1 activates phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway to regulate epithelial-mesenchymal transformation (EMT) and aerobic glycolysis, which is positively mediated by cell division cycle associated 5 (CDCA5). Moreover, TPI1 interacts with sequestosome-1 (SQSTM1)/P62, and P62 decreases the protein expression of TPI1 by promoting its ubiquitination in MDA-MB-231 cells.Conclusions: TPI1 promotes BRCA progression by stabilizing CDCA5, which then activates the PI3K/AKT/mTOR pathway. P62 promotes ubiquitin-dependent proteasome degradation of TPI1. Collectively, TPI1 promotes tumor development and progression, which may serve as a therapeutic target for BRCA. [ABSTRACT FROM AUTHOR]

Background: Targeting glutamine metabolism is previously indicated as a potential and attractive strategy for gastric cancer (GC) therapy. However, the underlying mechanisms responsible for the modification of glutamine metabolism in GC cells have not been fully elucidated. Accordingly, the current study sought to investigate the physiological mechanisms of RUNX3-mediated circDYRK1A in glutamine metabolism of GC.Methods: Firstly, GC tissues and adjacent normal tissues were obtained from 50 GC patients to determine circDYRK1A expression in GC tissues. Next, the binding affinity among RUNX3, circDYRK1A, miR-889-3p, and FBXO4 was detected to clarify the mechanistic basis. Moreover, GC cells were subjected to ectopic expression and knockdown manipulations of circDYRK1A, miR-889-3p, and/or FBXO4 to assay GC cell malignant phenotypes, levels of glutamine, glutamic acid, and α-KG in cell supernatant and glutamine metabolism-related proteins (GLS and GDH). Finally, nude mice were xenografted with GC cells to explore the in vivo effects of circDYRK1A on the tumorigenicity and apoptosis.Results: circDYRK1A was found to be poorly expressed in GC tissues. RUNX3 was validated to bind to the circDYRK1A promoter, and circDYRK1A functioned as a miR-889-3p sponge to up-regulate FBXO4 expression. Moreover, RUNX3-upregulated circDYRK1A reduced levels of glutamine, glutamic acid, and α-KG, and protein levels of GLS and GDH, and further diminished malignant phenotypes in vitro. Furthermore, in vivo experimentation substantiated that circDYRK1A inhibited the tumorigenicity and augmented the apoptosis in GC.Conclusion: In conclusion, these findings highlighted the significance and mechanism of RUNX3-mediated circDYRK1A in suppressing glutamine metabolism in GC via the miR-889-3p/FBXO4 axis. [ABSTRACT FROM AUTHOR]

Background: Long noncoding RNA NEAT1 has been implicated in glioma progression. However, the effect of NEAT1 on glycolysis of glioma cell and the potential mechanism remain unclear.Methods: In vitro experiments, including CCK-8, colony formation, ECAR, and lactate detection assays were performed to evaluate the effect of NEAT1 on proliferation and glycolysis of glioma cell. RNA pulldown and RIP assays were performed to identify the interaction between NEAT1 and PGK1. Truncated mutation of NEAT1 and PGK1 was used to confirm the specific interactive domains between NEAT1 and PGK1. Animal studies were performed to analyze the effect of NEAT1/PGK1 on glioma progression.Results: NEAT1 knockdown significantly suppressed the proliferation and glycolysis of glioma cells. NEAT1 could specifically interact with PGK1, which promotes PGK1 stability. Hairpin A of NEAT1 is essential for interaction with M1 domain of PGK1. Depletion of NEAT1 markedly inhibited tumor growth in mice, while PGK1 could reverse this effect. Higher expression of NEAT1 was associated with poor overall survival of GBM patients.Conclusions: NEAT1 over expression promotes glioma progression through stabilizing PGK1. NEAT1/PGK1 axis is a candidate therapeutic target for glioma treatment. [ABSTRACT FROM AUTHOR]

Background: LINC00491 was involved in some tumors development, but its function in liver cancer has not been reported. This study aimed to investigate LINC00491 expression and function in liver cancer progression.Methods: Sixty liver cancer cases were enrolled. LINC00491, miR-324-5p and rho-associated kinase 1 (ROCK1) expression in liver cancer patients and cells were detected by quantitative reverse transcription-polymerase chain reaction and Western blot. HUH-7 and SK-Hep-1 cells were transfected to modulate LINC00491, miR-324-5p and ROCK1 expression. Cell counting kit-8 assay, colony formation assay, wound healing assay, Transwell experiment, Tunel assay and flow cytometry were performed to detected HUH-7 and SK-Hep-1 cells proliferation, migration, invasion, apoptosis and cell cycle. Biotin-RNA pull-down assay and Dual-Luciferase Reporter Assay was performed to detect the binding among LINC00491, miR-324-5p and ROCK1. Xenograft tumor and lung metastasis was performed using nude mice. Xenograft tumor and lung tissues of mice were experienced immunohistochemistry and hematoxylin-eosin staining.Results: LINC00491 was highly expressed in liver cancer cases, associating with poor prognosis. si-LINC00491 inhibited proliferation, colony formation, invasion, migration, and induced cell cycle G1 arrest and apoptosis in HUH-7 and SK-Hep-1 cells. LINC00491 overexpression showed opposite effects. LINC00491 promoted ROCK1 expression by reducing miR-324-5p. miR-324-5p up-regulation or ROCK1 knockdown reversed LINC00491 promotion on liver SK-Hep-1 cells malignant phenotype. LINC00491 facilitated xenograft tumor growth and lung metastasis in mice.Conclusion: LINC00491 was highly expressed in liver cancer patients, associating with poor prognosis. LINC00491 facilitated liver cancer progression by sponging miR-324-5p/ROCK1. LINC00491 might be a potential treatment target of liver cancer. [ABSTRACT FROM AUTHOR]

Background: The characterization of the immune component of the tumor microenvironment (TME) of human epidermal growth factor receptor 2 positive (HER2+) breast cancer has been limited. Molecular and spatial characterization of HER2+ TME of primary, recurrent, and metastatic breast tumors has the potential to identify immune mediated mechanisms and biomarker targets that could be used to guide selection of therapies.Methods: We examined 15 specimens from eight patients with HER2+ breast cancer: 10 primary breast tumors (PBT), two soft tissue, one lung, and two brain metastases (BM). Using molecular profiling by bulk gene expression TME signatures, including the Tumor Inflammation Signature (TIS) and PAM50 subtyping, as well as spatial characterization of immune hot, warm, and cold regions in the stroma and tumor epithelium using 64 protein targets on the GeoMx Digital Spatial Profiler.Results: PBT had higher infiltration of immune cells relative to metastatic sites and higher protein and gene expression of immune activation markers when compared to metastatic sites. TIS scores were lower in metastases, particularly in BM. BM also had less immune infiltration overall, but in the stromal compartment with the highest density of immune infiltration had similar levels of T cells that were less activated than PBT stromal regions suggesting immune exclusion in the tumor epithelium.Conclusions: Our findings show stromal and tumor localized immune cells in the TME are more active in primary versus metastatic disease. This suggests patients with early HER2+ breast cancer could have more benefit from immune-targeting therapies than patients with advanced disease. [ABSTRACT FROM AUTHOR]

Background: The development of Bruton's tyrosine kinase inhibitors (BTKi) for the treatment of chronic lymphocytic leukaemia (CLL) has provided a highly effective and relatively non-toxic alternative to conventional chemotherapy. Some studies have shown that BTKi can also lead to improvements in T cell immunity in patients despite in vitro analyses suggesting an immunosuppressive effect of BTKi on T cell function.Methods: In this study, we examined both the in vitro effect and long-term in vivo effect of two clinically available BTKi, ibrutinib and zanubrutinib. Additional in vitro assessments were undertaken for a third BTKi, acalabrutinib. Immune subset phenotyping, cytokine secretion, T cell degranulation and proliferation assays were performed on peripheral blood mononuclear cells isolated from untreated CLL patients, and CLL patients on long-term (> 12 months) BTKi treatment.Results: Similar to prior studies we observed that long-term BTKi treatment normalises lymphocyte subset frequency and reduces PD-1 expression on T cells. We also observed that T cells from patients taken prior to BTKi therapy showed an abnormal hyper-proliferation pattern typical of senescent T cells, which was normalised by long-term BTKi treatment. Furthermore, BTKi therapy resulted in reduced expression of the T cell exhaustion markers PD-1, TIM3 and LAG3 in late generations of T cells undergoing proliferation.Conclusions: Collectively, these findings indicate that there are critical differences between the in vitro effects of BTKi on T cell function and the effects derived from long-term BTKi exposure in vivo. Overall long-term exposure to BTKi, and particularly ibrutinib, resulted in improved T cell fitness in part due to suppressing the abnormal hyper-proliferation of CLL T cells and the associated development of T cell senescence. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma is a common type of bone tumors and frequently occurs in children and adolescents. Cancer stem cells (CSCs) are a unique sub-type of self-renewal cancer cells and the stemness of cancer cells are involved in the spread, recurrence, metastasis, and even therapeutic resistance. However, the regulation mechanisms of CSCs in osteosarcoma are poorly understood. Circular RNA (circRNA) is a unique sort of non-coding RNAs and widely participate in the modulation of cancer progression.Methods: In this study, we identified the critical function of circular RNA circPIP5K1A in stemness of osteosarcoma cells.Results: CircPIP5K1A expression was significantly enhanced in clinical osteosarcoma tissues compared with the adjacent normal tissues. The depletion of circPIP5K1A by siRNA repressed osteosarcoma cell viabilities and induced osteosarcoma cell apoptosis. The suppression of circPIP5K1A attenuated the capabilities of invasion and migration of osteosarcoma cells. The circPIP5K1A knockdown increased E-Cadherin expression and decreased Vimentin expression in osteosarcoma cells. The sphere formation abilities of osteosarcoma cells were repressed by the depletion of circPIP5K1A. The CD133+CD44+ cell population of osteosarcoma cells was reduced by circPIP5K1A knockdown. The expression of ALDH1 and Nanog was decreased by the inhibition of circPIP5K1A in osteosarcoma cells. Mechanically, circPIP5K1A enhanced YAP expression by targeting miR-515-5p. MiR-515-5p inhibited stemness of osteosarcoma cells. The CSCs properties of osteosarcoma cells were repressed by circPIP5K1A knockdown or miR-515-5p mimic, while miR-515-5p inhibitor or YAP overexpression reversed circPIP5K1A knockdown-induced repression. Tumor xenograft analysis in nude mice demonstrated that the depletion of circPIP5K1A represses osteosarcoma cell growth in vivo.Conclusion: In conclusion, we identified that circular RNA circPIP5K1A contributed to cancer stemness of osteosarcoma by miR-515-5p/YAP axis. Targeting circPIP5K1A may be considered as a potential therapeutic strategy for osteosarcoma treatment. [ABSTRACT FROM AUTHOR]

Background: The function and regulatory mechanism of FBXO43 in breast cancer (BC) are still unclear. Here, we intended to determine the role and mechanism of FBXO43 in BC.Methods: FBXO43 expression in BC was evaluated by analysis of The Cancer Genome Atlas (TCGA). RT-qPCR and western blotting were utilized to detect FBXO43 expression in BC cell lines. Lentivirus was applied to downregulate FBXO43 in human BC cells. Proliferation assays were performed to evaluate the proliferative ability of BC cells. The apoptosis and cell cycle analysis of BC cells were analyzed by flow cytometry. Cell migration and invasion were investigated via Transwell assays. The function of FBXO43 in vivo was evaluated by constructing a xenograft mouse model. The proteins that might interact with FBXO43 in BC were identified by mass spectrometry, bioinformatics analysis, and co-immunoprecipitation (Co-IP) assays. Finally, rescue experiments were conducted to validate the recovery effects of the proteins interacting with FBXO43.Results: FBXO43 was highly expressed in BC and was significantly downregulated after FBXO43 knockdown. The proliferation, migration, and invasion of BC cells were inhibited, and cell apoptosis was induced by FBXO43 knockdown. In addition, an in vivo experiment indicated that FBXO43 knockdown could inhibit the cell growth of BC. The results of the Co-IP assay showed that FBXO43 interacted with PCNA. Further rescue experiments confirmed that overexpression of PCNA significantly reversed the effects of FBXO43 knockdown on BC cells.Conclusion: Downregulation of FBXO43 inhibits the tumor growth of BC by limiting its interaction with PCNA. FBXO43 might be a new potential oncogene and a therapeutic target for BC. [ABSTRACT FROM AUTHOR]

Background: Antiangiogenic therapy has increasingly become an important strategy for the treatment of colorectal cancer. Recent studies have shown that the tumour microenvironment (TME) promotes tumour angiogenesis. Bufalin is an active antitumour compound whose efficacy has been indicated by previous studies. However, there are very few studies on the antiangiogenic effects of bufalin.Methods: Herein, human umbilical vein endothelial cell (HUVEC) tube formation, migration and adhesion tests were used to assess angiogenesis in vitro. Western blotting and quantitative PCR were used to detect relevant protein levels and mRNA expression levels. A subcutaneous xenograft tumour model and a hepatic metastasis model were established in mice to investigate the influence of bufalin on angiogenesis mediated by the TME in vivo.Results: We found that angiogenesis mediated by cells in the TME was significantly inhibited in the presence of bufalin. The results demonstrated that the proangiogenic genes in HUVECs, such as VEGF, PDGFA, E-selectin and P-selectin, were downregulated by bufalin and that this downregulation was mediated by inhibition of the STAT3 pathway. Overexpression of STAT3 reversed the inhibitory effects of bufalin on angiogenesis. Furthermore, there was little reduction in angiogenesis when bufalin directly acted on the cells in the tumour microenvironment.Conclusion: Our findings demonstrate that bufalin suppresses tumour microenvironment-mediated angiogenesis by inhibiting the STAT3 signalling pathway in vascular endothelial cells, revealing that bufalin may be used as a new antiangiogenic adjuvant therapy medicine to treat colorectal cancer. [ABSTRACT FROM AUTHOR]

Advances in immune checkpoint therapy and targeted therapy have led to improvement in overall survival for patients with advanced melanoma. Single agent checkpoint PD-1 blockade and combination with BRAF/MEK targeted therapy demonstrated benefit in overall survival (OS). Superior response rates have been demonstrated with combined PD-1/CTLA-4 blockade, with a significant OS benefit compared with single-agent PD-1 blockade. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic therapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers but they have yet to be fully characterized and implemented clinically. Overall, the progress in melanoma therapeutics and translational research will help to optimize treatment regimens to overcome resistance and develop robust biomarkers to guide clinical decision-making. During the Melanoma Bridge meeting (December 3rd-5th, 2020, Italy) we reviewed the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine. [ABSTRACT FROM AUTHOR]

Background: Studies of molecular mechanisms underlying tumor cell signaling highlighted a critical role for kinases in carcinogenesis and cancer progression. To this regard, protein kinases regulates a number of critical cellular pathways by adding phosphate groups to specific substrates. For this reason, their involvement in the complex interactions between the human microbiota and cancer cells to determine therapy and tumor progression outcome is becoming increasingly relevant. Mycoplasmas are components of the normal human microbiota, and several species have also been associated to human diseases, including certain cancers. It is also important to note that Mycoplasmas and their proteins are a component of the common tumor microenvironment. In addition, several epidemiological, in vivo and in vitro studies indicate a close involvement of Mycoplasmas in cellular transformation and cancer progression.Methods: In this study, we investigate the effect of exogenous Mycoplasma DnaK on kinases activity by treating in vitro four different eukaryotic cancer cell lines, namely lung and prostate cancer, colon adenocarcinoma, and neuroblastoma. Phosphorylation of kinases and specific substrates was measured at 20 and 60 min.Results: Kinome analysis of our data indicates that Mycoplasma DnaK promotes the dysregulation of the activity of specific kinases and their substrates, with a known involvement in carcinogenesis and cancer progression.Conclusions: Given the similarity in structure and amino acid composition of this protein with other bacterial DnaKs we provide a novel mechanism whereby components of the human microbiota and present in the tumor microenvironment are able to deregulate phosphorylation events occurring during carcinogenesis and cancer progression. [ABSTRACT FROM AUTHOR]

Background: HIV-1 Vpr encodes a 14 kDa protein that has been implicated in viral pathogenesis through modulation of several host cell functions. In addition to pro-apoptotic and cytostatic properties, Vpr can redirect cellular E3 ubiquitin ligases (such as DCAF1-Cul4A E3 ligase complex) to target many host proteins and interfere with their functions. Among them, Vpr binds the uracil DNA glycosylase UNG2, which controls genome uracilation, and induces its specific degradation leading to loss of uracil removal activity in infected cells. Considering the essential role of UNG2 in antibody diversification in B-cells, we evaluated the impact of Vpr on UNG2 fate in B lymphocytes and examined the functional consequences of UNG2 modulations on class switch recombination (CSR).Methods: The impact of Vpr-induced UNG2 deregulation on CSR proficiency was evaluated by using virus-like particles able to deliver Vpr protein to target cells including the murine model CSR B cell line CH12F3 and mouse primary B-cells. Co-culture experiments were used to re-examine the ability of Vpr to be released by HIV-1 infected cells and to effectively accumulate in bystander B-cells. Vpr-mediated UNG2 modulations were monitored by following UNG2 protein abundance and uracil removal enzymatic activity.Results: In this study we report the ability of Vpr to reduce immunoglobulin class switch recombination (CSR) in immortalized and primary mouse B-cells through the degradation of UNG2. We also emphasize that Vpr is released by producing cells and penetrates bystander B lymphocytes.Conclusions: This work therefore opens up new perspectives to study alterations of the B-cell response by using Vpr as a specific CSR blocking tool. Moreover, our results raise the question of whether extracellular HIV-1 Vpr detected in some patients may manipulate the antibody diversification process that engineers an adapted response against pathogenic intruders and thereby contribute to the intrinsic B-cell humoral defect reported in infected patients. [ABSTRACT FROM AUTHOR]

Background: The increased bone marrow angiogenesis is involved in the progression of multiple myeloma (MM) with the underlying mechanism poorly understood. Cancer-released exosomes could play an important role in the pathological angiogenesis through exosomal microRNAs (miRs) delivery. It is reported that miR-29b played an important role in regulating the tumor angiogenesis.Methods: In this study, we explored the role of C6-ceramide (C6-cer, a Ceramide pathway activator) in the angiogenic effect of MM exosomes and its potential mechanism. MM cells (OPM2 and RPMI-8226) treated with C6-cer were studied for its effects on the endothelial cell (EC) functions.Results: Our results showed that exosomes released from MM cells treated by C6-cer (ExoC6-cer) significantly inhibited the proliferation, migration and tube formation of ECs. For mechanism studies, we found that the level of miR-29b was increased in ECs treated by ExoC6-cer, while mRNA and protein expressions of Akt3, PI3K and VEGFA were decreased in ECs, indicating the involvement of Akt pathway. Furthermore, downregulation of miR-29b by inhibitor administration could prevent the ExoC6-cer-induced cell proliferation, migration and angiogenesis of ECs, accompanied with the increased expressions of Akt3, PI3K and VEGFA.Conclusions: Collectively, our data suggest that ExoC6-cer-mediated miR-29b expression participates in the progression of MM through suppressing the proliferation, migration and angiogenesis of ECs by targeting Akt signal pathway. [ABSTRACT FROM AUTHOR]

Background: Immunotherapeutic regulation of the tumor microenvironment in prostate cancer patients is not understood. Most antibody immunotherapies have not succeeded in prostate cancer. We showed previously that high-risk PCa patients have a higher density of tumor infiltrating B-cells in prostatectomy specimens. In mouse models, anti-CD20 antibody ablation of B-cells delayed PCa regrowth post-treatment. We sought to determine whether neoadjuvant anti-CD20 immunotherapy with rituximab could reduce CD20+ B cell infiltration of prostate tumors in patients.Methods: An open label, single arm clinical trial enrolled eight high-risk PCa patients to receive one cycle of neoadjuvant rituximab prior to prostatectomy. Eleven clinical specimens with similar characteristics were selected as controls. Treated and control samples were concurrently stained for CD20 and digitally scanned in a blinded fashion. A new method of digital image quantification of lymphocytes was applied to prostatectomy sections of treated and control cases. CD20 density was quantified by a deconvolution algorithm in pathologist-marked tumor and adjacent regions. Statistical significance was assessed by one sided Welch's t-test, at 0.05 level using a gatekeeper strategy. Secondary outcomes included CD3+ T-cell and PD-L1 densities.Results: Mean CD20 density in the tumor regions of the treated group was significantly lower than the control group (p = 0.02). Mean CD3 density in the tumors was significantly decreased in the treated group (p = 0.01). CD20, CD3 and PD-L1 staining primarily occurred in tertiary lymphoid structures (TLS). Neoadjuvant rituximab was well-tolerated and decreased B-cell and T-cell density within high-risk PCa tumors compared to controls.Conclusions: This is the first study to treat patients prior to surgical prostate removal with an immunotherapy that targets B-cells. Rituximab treatment reduced tumor infiltrating B and T-cell density especially in TLSs, thus, demonstrating inter-dependence between B- and T-cells in prostate cancer and that Rituximab can modify the immune environment in prostate tumors. Future studies will determine who may benefit from using rituximab to improve their immune response against prostate cancer. Trial registration NCT01804712, March 5th, 2013 https://clinicaltrials.gov/ct2/show/NCT01804712?cond=NCT01804712&draw=2&rank=1. [ABSTRACT FROM AUTHOR]

Background: As a nucleolar protein associated with ribosome biogenesis, pescadillo homolog 1 (PES1) has been reported to participate in the development of many cancers. However, its role in prostate cancer is not clearly defined. Therefore, the aim of this study is to explore the effects and the specific mechanism of PES1 in prostate cancer.Methods: A microarray-based analysis was performed to analyze differentially expressed genes (DEGs) between prostate cancer and normal samples. Next, the interaction between PES1 and microRNA-1271 (miR-1271) was investigated using bioinformatics analysis in combination with dual-luciferase reporter gene assay. The expression of miR-1271 in prostate cancer cells and tissues was determined using RT-qPCR. Its effects on downstream estrogen receptor β (ERβ) signaling pathway were further examined. Moreover, we analyzed whether miR-1271 affects proliferation, apoptosis, migration and invasion of prostate cancer cells by EdU assay, flow cytometry, and Transwell assay. Lastly, a prostate cancer mouse model was conducted to measure their roles in the tumor growth.Results: PES1 was identified as a prostate cancer-related DEG and found to be upregulated in prostate cancer. miR-1271, which was poorly expressed in both cells and tissues of prostate cancer, can specifically bind to PES1. Additionally, overexpression of miR-1271 activated the ERβ signaling pathway. Overexpression of miR-1271 or depletion of PES1 inhibited prostate cancer cell proliferation, migration and invasion, promoted apoptosis in vitro and suppressed tumor growth in vivo.Conclusions: Taken together, overexpression of miR-1271 downregulates PES1 to activate the ERβ signaling pathway, leading to the delayed prostate cancer development. Our data highlights the potential of miR-1271 as a novel biomarker for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers worldwide. Due to its high morbidity and mortality rates, it is urgent to find a molecular target that contributes to esophageal carcinogenesis and progression. In this research, we aimed to investigate the functions of Latent transforming growth factor β binding protein 1(LTBP1) in ESCC progression and elucidate the underlying mechanisms.Methods: The tandem mass tag-based quantitative proteomic approach was applied to screen the differentially expressed proteins (DEPs) between 3 cases of ESCC tumor samples and paired normal tissues. Then the DEPs were validated in human ESCC tissues using western blot assays and GEPIA database respectively. The expression level of LTBP1 was detected in 152 cases of ESCC tissues and paired normal tissues. Loss-of-function assays were performed to detect the function of LTBP1 in vivo and in vitro. Immunofluorescence and Western blot assays were used to detect the expression of apoptosis, epithelial-mesenchymal transition (EMT) and cancer-associated fibroblasts (CAFs) markers.Results: A total of 39 proteins were screened to be up-regulated (ratio > 2.0) in all three ESCC tissues. The results of immunohistochemistry assays indicated that the expression level of LTBP1 was higher in ESCC tissues than that in paired normal tissues (p < 0.001). Overexpression of LTBP1 was positively associated with lymphatic metastasis in ESCC (p = 0.002). Down-regulation of LTBP1 inhibited the invasion and migration as well as metastatic abilities in vitro and in vivo. It was also observed the down-regulation of LTBP1 not only decreased the mesenchymal phenotypes but also inhibited TGFβ-induced EMT in ESCC cells. We further found that down-regulation of LTBP1 enhanced ESCC cells' sensitivity to 5-FU treatment. Inhibition of LTBP1 expression could also attenuate induction of CAFs transformation and restrain fibroblast express fibronectin (FN1) in ESCC cells.Conclusion: Overexpression of LTBP1 was associated with lymph node metastasis in ESCC. Our results indicated that LTBP1 not only increased the malignant behaviors of ESCC cells but also induced EMT and CAFs transformation. Our studies suggested an oncogenic role of LTBP1 in ESCC progression and it may serve as a potential therapeutic target for ESCC patients. [ABSTRACT FROM AUTHOR]

Background: Gastric cancer is associated with chronic inflammation, but there is still much to understand about the tumor microenvironment and the underlying tumor-promoting mechanisms. The Map kinase-activated protein kinase 2 (MK2) pathway is a regulator of inflammatory cytokine production that we have been studying in gastrointestinal cancers. Here, we set out to determine the significance of this gene in gastric cancer along with its downstream mediators and if there were differences in the primary tumors with and without metastasis.Methods: Human gastric cancer tissues with and without metastasis were examined for MK2 expression and cytokine profile in organ culture supernatants. Advanced statistical methods including a lower triangular correlation matrix, novel rooted correlation network, linear and logistic regression modeling along with Kruskal-Wallis testing with Sidak correction for multiple testing were applied to gain understanding of cytokines/chemokines linked to metastasis.Results: The MK2 pathway is strongly linked with metastasis and a panel of cytokines. Gene expression was able to classify gastric cancer metastasis 85.7% of the time. A significant association with a panel of cytokines was found, including G-CSF, GM-CSF, Mip-1β, IFN-α, MCP-1, IL-1β, IL-6, and TNF-α. Mip-1β was found to have the strongest association with MK2 and metastasis after Sidak correction for multiple testing.Conclusions: MK2 gene expression and a novel associated cytokine panel are linked to gastric cancer metastasis. G-CSF is the strongest cytokine to differentiate between metastasis and non-metastasis patients and had the lowest P value, while Mip-1β showed the strongest association with MK2 and metastasis after Sidak correction. MK2 and associated cytokines are potential biomarkers for gastric cancer metastasis. The novel intercorrelation analysis approach is a promising method for understanding the complex nature of cytokine/chemokine regulation and links to disease outcome. [ABSTRACT FROM AUTHOR]

Background: Clear cell renal cell carcinoma (ccRCC) is a malignancy characterized by metabolic reprogramming. ABAT and ALDH6A1 are metabolic enzymes. In this study, we aim to investigate the associations of ABAT and ALDH6A1 with the malignancy of ccRCC cells.Methods: The gene expression levels of ABAT and ALDH6A1 in ccRCC were analyzed from gene expression microarray datasets and RNA sequencing data. Clinical information was analyzed from The Cancer Genome Atlas (TCGA) data. The distributions of ABAT and ALDH6A1 in ccRCC clinical tissues were screened by reverse transcription-quantitative polymerase chain reaction (RT-QPCR) and immunohistochemical assays. The effect of overexpression of ABAT or ALDH6A1 was measured by detecting the cell viability, migration ability, and the ratio of lactate and nicotinamide adenine dinucleotide phosphate (NADPH). Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were carried out to investigate the transcript regulation of HNF4A in ABAT and ALDH6A1.Results: Remarkable downregulated ABAT and ALDH6A1 expression levels were observed in ccRCC patients and low expression of ABAT and ALDH6A1 was correlated with poor survival. Overexpression of ABAT or ALDH6A1 significantly attenuated cell proliferation and migration, and impaired lactate production. In ABAT increased ccRCC cells, the ratio of NADPH/NADP+ was reduced. Finally, we demonstrated that ABAT and ALDH6A1 were directly regulated by a tumor suppressor, HNF4A.Conclusions: These observations identified HNF4A-regulated low-expressed ABAT and ALDH6A1 as promising diagnostic and prognostic biomarkers for ccRCC. [ABSTRACT FROM AUTHOR]

The concept of cancer as a cell-autonomous disease has been challenged by the wealth of knowledge gathered in the past decades on the importance of tumor microenvironment (TM) in cancer progression and metastasis. The significance of endothelial cells (ECs) in this scenario was initially attributed to their role in vasculogenesis and angiogenesis that is critical for tumor initiation and growth. Nevertheless, the identification of endothelial-derived angiocrine factors illustrated an alternative non-angiogenic function of ECs contributing to both physiological and pathological tissue development. Gene expression profiling studies have demonstrated distinctive expression patterns in tumor-associated endothelial cells that imply a bilateral crosstalk between tumor and its endothelium. Recently, some of the molecular determinants of this reciprocal interaction have been identified which are considered as potential targets for developing novel anti-angiocrine therapeutic strategies. [ABSTRACT FROM AUTHOR]

The use of patient-derived primary cell cultures in cancer preclinical assays has increased in recent years. The management of resected tumor tissue remains complex and a number of parameters must be respected to obtain complete sample digestion and optimal vitality yield. We provide an overview of the benefits of correct primary cell culture management using different preclinical methodologies, and describe the pros and cons of this model with respect to other kinds of samples. One important advantage is that the heterogeneity of the cell populations composing a primary culture partially reproduces the tumor microenvironment and crosstalk between malignant and healthy cells, neither of which is possible with cell lines. Moreover, the use of patient-derived specimens in innovative preclinical technologies, such as 3D systems or bioreactors, represents an important opportunity to improve the translational value of the results obtained. In vivo models could further our understanding of the crosstalk between tumor and other tissues as they enable us to observe the systemic and biological interactions of a complete organism. Although engineered mice are the most common model used in this setting, the zebrafish (Danio rerio) species has recently been recognized as an innovative experimental system. In fact, the transparent body and incomplete immune system of zebrafish embryos are especially useful for evaluating patient-derived tumor tissue interactions in healthy hosts. In conclusion, ex vivo systems represent an important tool for cancer research, but samples require correct manipulation to maximize their translational value. [ABSTRACT FROM AUTHOR]

Background: The coiled-coil domain is a structural motif found in proteins that participate in a variety of biological processes. Aberrant expression of such proteins has been shown to be associated with the malignant behavior of human cancers. In this study, we investigated the role of a specific family member, coiled-coil domain containing 109B (CCDC109B), in human gliomas.Methods and Results: We confirmed that CCDC109B was highly expressed in high grade gliomas (HGG; WHO III-IV) using immunofluorescence, western blot analysis, immunohistochemistry (IHC) and open databases. Through Cox regression analysis of The Cancer Genome Atlas (TCGA) database, we found that the expression levels of CCDC109B were inversely correlated with patient overall survival and it could serve as a prognostic marker. Then, a serious of cell functional assays were performed in human glioma cell lines, U87MG and U251, which indicated that silencing of CCDC109B attenuated glioma proliferation and migration/invasion both in vitro and in vivo. Notably, IHC staining in primary glioma samples interestingly revealed localization of elevated CCDC109B expression in necrotic areas which are typically hypoxic. Moreover, small interfering RNA (siRNA) and specific inhibiters of HIF1α led to decreased expression of CCDC109B in vitro and in vivo. Transwell assay further showed that CCDC109B is a critical factor in mediating HIF1α-induced glioma cell migration and invasion.Conclusion: Our study elucidated a role for CCDC109B as an oncogene and a prognostic marker in human gliomas. CCDC109B may provide a novel therapeutic target for the treatment of human glioma. [ABSTRACT FROM AUTHOR]

Background: Chronic fatigue syndrome (CFS) is a prevalent and disabling condition affecting adolescents. The pathophysiology is poorly understood, but immune alterations might be an important component. This study compared whole blood gene expression in adolescent CFS patients and healthy controls, and explored associations between gene expression and neuroendocrine markers, immune markers and clinical markers within the CFS group.Methods: CFS patients (12-18 years old) were recruited nation-wide to a single referral center as part of the NorCAPITAL project. A broad case definition of CFS was applied, requiring 3 months of unexplained, disabling chronic/relapsing fatigue of new onset, whereas no accompanying symptoms were necessary. Healthy controls having comparable distribution of gender and age were recruited from local schools. Whole blood samples were subjected to RNA sequencing. Immune markers were blood leukocyte counts, plasma cytokines, serum C-reactive protein and immunoglobulins. Neuroendocrine markers encompassed plasma and urine levels of catecholamines and cortisol, as well as heart rate variability indices. Clinical markers consisted of questionnaire scores for symptoms of post-exertional malaise, inflammation, fatigue, depression and trait anxiety, as well as activity recordings.Results: A total of 29 CFS patients and 18 healthy controls were included. We identified 176 genes as differentially expressed in patients compared to controls, adjusting for age and gender factors. Gene set enrichment analyses suggested impairment of B cell differentiation and survival, as well as enhancement of innate antiviral responses and inflammation in the CFS group. A pattern of co-expression could be identified, and this pattern, as well as single gene transcripts, was significantly associated with indices of autonomic nervous activity, plasma cortisol, and blood monocyte and eosinophil counts. Also, an association with symptoms of post-exertional malaise was demonstrated.Conclusion: Adolescent CFS is characterized by differential gene expression pattern in whole blood suggestive of impaired B cell differentiation and survival, and enhanced innate antiviral responses and inflammation. This expression pattern is associated with neuroendocrine markers of altered HPA axis and autonomic nervous activity, and with symptoms of post-exertional malaise. Trial registration Clinical Trials NCT01040429. [ABSTRACT FROM AUTHOR]

Background: Rare nucleated CD45 negative cells in peripheral blood may be malignant such as circulating tumor cells. Untouched isolation thereof by depletion of normal is favored yet still technological challenging. We optimized and evaluated a novel magnetic bead-based negative selection approach for enhanced enrichment of rare peripheral blood nucleated CD45 negative cells and investigated the problem of rare cell contamination during phlebotomy.Methods: Firstly, the performance of the magnetic cell separation system was assessed using leukocytes and cultivated fibroblast cells in regard to depletion efficiency and the loss of cells of interest. Secondly, a negative selection assay was optimized for high performance, simplicity and cost efficiency. The negative selection assay consisted of; a RBC lysis step, two depletion cycles comprising direct magnetically labelling of leukocytes using anti-CD45 magnetic beads followed by magnetic capture of leukocytes using a duopole permanent magnet. Thirdly, assay evaluation was aligned to conditions of rare cell frequencies and comprised cell spike recovery, cell viability and proliferation, and CD45 negative cell detection. Additionally, the problem of CD45 negative cell contamination during phlebotomy was investigated.Results: The depletion factor and recovery of the negative selection assay measured at most 1600-fold and 96%, respectively, leaving at best 1.5 × 104 leukocytes unseparated and took 35 min. The cell viability was negatively affected by chemical RBC lysis. Proliferation of 100 spiked ovarian cancer cells in culture measured 37% against a positive control. Healthy donor testing revealed findings of nucleated CD45 negative cells ranging from 1 to 22 cells /2.5 × 107 leukocytes or 3.5 mL whole blood in 89% (23/26) of the samples.Conclusion: Our assay facilitates high performance at shortest assay time. The enrichment assay itself causes minor harm to cells and allows proliferation. Our findings suggest that rare cell contamination is unavoidable. An unexpected high variety of CD45 negative cells have been detected. It is hypothesized that a rare cell profile may translate into tumor marker independent screening. [ABSTRACT FROM AUTHOR]

Background: Cytokine-induced-killer (CIK) cells are a promising immunotherapeutic approach for impending relapse following hematopoietic stem cell transplantation (HSCT). However, there is a high risk for treatment failure associated with severe graft versus host disease (GvHD) necessitating pharmaceutical intervention post-transplant. Whether immunosuppression with mycophenolate mofetil (MMF) or Ciclosporin A (CsA) influences the cytotoxic effect of CIK cell immunotherapy is still an open issue.Methods: CIK cells were generated from PBMC as previously described followed by co-incubation with mycophenolic acid (MPA) or CsA. Proliferation, cytotoxicity and receptor expression were investigated following short- (24 h), intermediate- (3 days) and long-term (7 days) MPA incubation with the intention to simulate the in vivo situation when CIK cells were given to a patient with relevant MPA/CsA plasma levels.Results: Short-term MPA treatment led to unchanged proliferation capacity and barely had any effect on viability and cytotoxic capability in vitro. The composition of CIK cells with respect to T-, NK-like T- and NK cells remained stable. Intermediate MPA treatment lacked effects on NKG2D, FasL and TRAIL receptor expression, while an influence on proliferation and viability was detectable. Furthermore, long-term treatment significantly impaired proliferation, restricted viability and drastically reduced migration-relevant receptors accompanied by an alteration in the CD4/CD8 ratio. CD3(+)CD56(+) cells upregulated receptors relevant for CIK cell killing and migration, whereas T cells showed the most interference through significant reductions in receptor expression. Interestingly, CsA treatment had no significant influence on CIK cell viability and the cytotoxic potential against K562.Conclusions: Our data indicate that if immunosuppressant therapy is indispensable, efficacy of CIK cells is maintained at least short-term, although more frequent dosing might be necessary. [ABSTRACT FROM AUTHOR]

Background: Aspirin (ASA) is the most widely used medicine to prevent cardiovascular diseases; however, the mechanisms by which ASA exerts its anti-proliferative effect remain not fully understood. This study was designed to investigate whether miR-145 is involved in the regulation of vascular smooth muscle cells' (VSMCs) proliferation and to determine the anti-inflammatory effects of ASA via its regulation of CD40 to provide a new theoretical basis for the pharmacological effect of aspirin.Methods: The TNF-α induced proliferation model of VSMCs was divided into different groups with or without aspirin. Cell proliferation was detected by EdU; Real-time PCR was used to detect the mRNA expression of miR-145, CD40, and Calponin, a VSMCs differentiation marker gene. Western blot was used to detect the protein expression of CD40; ELISA was used to determine the concentrations of the inflammatory cytokine IL-6 in cell supernatants.Results: The proliferation of VSMCs was stimulated by TNF-α and accompanied by decreased levels of Calponin. TNF-α also decreased the levels of miR-145 and increased the levels of CD40 and IL-6. Pretreatment with 20 μg/mL of aspirin in VSMCs could partially block the above-mentioned effects induced by TNF-α. The protective effects of ASA in VSMCs were reversed by a pretreatment with a miR-145 inhibitor. We also found that the expression of miR-145 in peripheral blood mononuclear cells in ischemic stroke patients was significantly increased after a 10-day treatment with aspirin.Conclusion: miR-145 is involved in the anti-proliferation and anti-inflammation effects of aspirin on VSMCs by inhibiting the expression of CD40. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is a leading cause of cancer mortality in the Western world and commonly treated with genotoxic chemotherapy. Stress in the endoplasmic reticulum (ER) was implicated to contribute to chemotherapeutic resistance. Hence, ER stress related protein may be of prognostic or therapeutic significance.Methods: The expression levels of ER stress proteins calnexin, calreticulin, GRP78 and GRP94 were determined in n = 23 Stage II and III colon cancer fresh frozen tumour and matched normal tissue samples. Data were validated in a cohort of n = 11 rectal cancer patients treated with radiochemotherapy in the neoadjuvant setting. The calnexin gene was silenced using siRNA in HCT116 cells.Results: There were no increased levels of ER stress proteins in tumour compared to matched normal tissue samples in Stage II or III CRC. However, increased calnexin protein levels were predictive of poor clinical outcome in the patient cohort. Data were validated in the rectal cancer cohort treated in the neoadjuvant setting. Calnexin gene-silencing significantly reduced cell survival and increased cancer cell susceptibility to 5FU chemotherapy.Conclusion: Increased tumour protein levels of calnexin may be of prognostic significance in CRC, and calnexin may represent a potential target for future therapies. [ABSTRACT FROM AUTHOR]

Background: Cytokine fusion protein that modulates the immune response holds great potential for cancer immunotherapy. IL-2 is an effective treatment against advanced cancers. However, the therapeutic efficacy of IL-2 is limited by severe systemic toxicity. Several mutants recombinant IL-2 can increase antitumor activity and minimize systemic toxicity. Melittin is an attractive anticancer candidate because of its wide-spectrum lytic properties. We previously generated a bifunctional fusion protein melittin-MIL-2, composed of melittin and a mutant IL-2. The melittin-MIL-2 inhibited the growth of human ovarian cancer SKOV3 cells in vitro and in vivo tumor growth. However, whether this antitumor effect could also be used in cancer immunotherapy was unknown. To assess its cancer immunotherapy potential, we further investigated its more effective antitumor immune response and antitumor effect against cancers of different tissue origins in vitro and in vivo.Methods: The specific IL-2 activity of the melittin-MIL-2 fusion protein was tested on the cytokine growth dependent cell line CTLL-2. The cytolytic activity was detected by standard 4-h (51)Cr-release assays. PBMC stimulation in response to the melittin-MIL-2 was determined by IFN-γ release assay. We observed the cancer cell proliferation of different tissue origins by MTT assay. The ability of melittin-MIL-2 to inhibit tumor growth in vivo was evaluated by using human liver (SMMC-7721 cancer cells), lung (A549 cancer cells) and ovarian (SKOV3 cancer cells) cancer xenograft models. To assess the immunity within the tumor microenvironment, the level of some cytokines including IFN-γ, TNF-α, IL-12 and IL-4 was analyzed by ELISA. We injected the MDA-MB-231 cells and the melittin-MIL-2 into mice, and the anti-metastatic effect was examined by counting nodules in the lung.Results: The melittin-MIL-2 was more effective in inducing T cell and NK-cell cytotoxicity. The fusion protein significantly increased IFN-γ production in PBMCs. In vitro, the melittin-MIL-2 mediated immune cells killing or directly killed the cancer cell lines of different tissue origins. In vivo, the fusion protein exhibited stronger inhibition on the growth of transplanted human tumors compared to rIL-2. The melittin-MIL-2 treatment promoted the IFN-γ secretion in tumor tissues and decreased the immunosuppressive cells in vivo. Furthermore, the fusion protein reduced lung metastasis of breast cancer.Conclusions: This study provides the evidence that the melittin-MIL-2 can produce stronger immune stimulation and antitumor effects, and the fusion protein is a potent candidate for cancer immunotherapy. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stromal cells (MSC) are largely investigated in clinical trials aiming to control inappropriate immune reactions (GVHD, Crohn's disease, solid organ transplantation). As the percentage of MSC precursors in bone marrow is very low, these must be expanded in vitro to obtain therapeutic cell doses. We describe here the constitution of an allogeneic human third-party MSC bank from screened healthy volunteer donors in compliance with quality specifications and ISCT-release criteria and report follow-up of different aspects of this activity since 2007.Methods: 68 clinical-grade large-scale MSC cultures were completed and analyzed. The whole process was described, including volunteer donor screening, bone marrow collection, mononuclear cell isolation and expansion over 4 weeks, harvesting, cryopreservation, release, administration and quality controls of the cells (including microbiology, phenotype, and potency assays).Results: From 59 validated donors, 68 cultures were completed (mean of final yields: 886 × 10(6) cells/culture) and a total of 464 MSC aliquots have been produced and stored in liquid nitrogen (mean of 132.8 × 10(6) cells/bag). Each MSC batch underwent extensive testing to verify its conformity with EBMT and ISCT release criteria and was individually validated. As of June 1 2015, 314 bags have been released and infused to patients included in 6 different clinical protocols. All thawed MSC units satisfied to release criteria and no infusion-related toxicity was reported.Conclusion: In conclusion, despite low passage cultures, we have been able to create an allogeneic "off-the-shelf" MSC bank with a large number of frozen aliquots and report here an efficient clinical-grade MSC banking activity in place for more than 7 years. Our challenge now is to produce MSC in compliance with good manufacturing practices (GMP) as, in the meantime, MSC have become considered as advanced therapy medicinal products (ATMP). Another significant challenge remains the development of relevant potency assay. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRs, miRNAs) are small molecules of 18-22 nucleotides that serve as important regulators of gene expression at the post-transcriptional level. One of the mechanisms through which miRNAs regulate gene expression involves the interaction of their "seed" sequences primarily with 3'-end and more rarely with 5'-end, of mRNA transcribed from target genes. Numerous studies over the past decade have been devoted to quantitative and qualitative assessment of miRNAs expression and have shown remarkable changes in miRNA expression profiles in various diseases. Thus, profiling of miRNA expression can be an important tool for diagnostics and treatment of disease. However, less attention has been paid towards understanding the underlying reasons for changes in miRNA expression, especially in cancer cells. The purpose of this review is to analyze and systematize current data that explains reasons for changes in the expression of miRNAs. The review will cover both transcriptional (changes in gene expression and promoter hypermethylation) and post-transcriptional (changes in miRNA processing) mechanisms of regulation of miRNA expression, as well as effects of endogenous (hormones, cytokines) and exogenous (xenobiotics) compounds on the miRNA expression. The review will summarize the complex multilevel regulation of miRNA expression, in relation to cell type, physiological state of the body and various external factors. [ABSTRACT FROM AUTHOR]

Background: TLR4/MD-2 complex-mediated MyD88-dependent activation of NF-κB and Akt promotes tumor-associated immunosuppression in epithelial ovarian cancer (EOC) via induction of immunesuppressive cytokines and indoleamine 2,3-dioxygenase (IDO). Atractylenolide I (AO-1) is a naturally occurring sesquiterpene lactone known to change the conformational ensemble of human MD-2 on EOC cells. This study examined the modulation by AO-1 of TLR4/MD-2 complex-mediated MyD88/NF-κB signaling.Methods: The expression and activation of NF-κB, Akt and IDO1 by MyD88(+) EOC SKOV3 cells was determined using western blot; the TLR4/MD-2 complex on SKOV3 cells and the phenotype of T lymphocytes were determined using flow cytometry; IDO activity was evaluated by measuring L-kynurenine; Immunesuppressive cytokines were detected using ELISA; T-cell proliferation to mitogen stimulation was assessed by MTT assay; the cytotoxicity of lymphocytes and NK cells was measured using LDH-cytotoxicity assay.Results: AO-1 could down-regulate expression of TLR4/MD-2 complex, resulting in downregulation of MyD88/NF-κB signaling and activation of NF-κB, Akt and IDO1 and secretion of IL-6, TGF-β1, VEGF and IL-17A by EOC SKOV3 cells, and further reduce increased levels of regulatory T cells (Treg cells) and improve decreased proliferative response and antitumor cytotoxicity of T lymphocytes exposed to EOC SKOV3 cell supernatant.Conclusion: AO-1 may reverse EOC cell-mediated immunosuppression through blocking TLR4/MD-2 complex-mediated MyD88/NF-κB signaling. [ABSTRACT FROM AUTHOR]

Background: PRL-3 is a phosphatase implicated in oncogenesis in multiple cancers. In some cancers, notably carcinomas, PRL-3 is also associated with inferior prognosis and increased metastatic potential. In this study we investigated the expression of PRL-3 mRNA in fresh-frozen samples from patients undergoing radical prostatectomy because of prostate cancer (PC) and the biological function of PRL-3 in prostate cancer cells.Methods: Samples from 41 radical prostatectomy specimens (168 samples in total) divided into low (Gleason score ≤ 6), intermediate (Gleason score = 7) and high (Gleason score ≥ 8) risk were analyzed with gene expression profiling and compared to normal prostate tissue. PRL-3 was identified as a gene with differential expression between healthy and cancerous tissue in these analyses. We used the prostate cancer cell lines PC3 and DU145 and a small molecular inhibitor of PRL-3 to investigate whether PRL-3 had a functional role in cancer. Relative ATP-measurement and thymidine incorporation were used to assess the effect of PRL-3 on growth of the cancer cells. We performed an in vitro scratch assay to investigate the involvement of PRL-3 in migration. Immunohistochemistry was used to identify PRL-3 protein in prostate cancer primary tumor and corresponding lymph node metastases.Results: Compared to normal prostate tissue, the prostate cancer tissue expressed a significantly higher level of PRL-3. We found PRL-3 to be present in both PC3 and DU145, and that inhibition of PRL-3 led to growth arrest and apoptosis in these two cell lines. Inhibition of PRL-3 led to reduced migration of the PC3 cells. Immunohistochemistry showed PRL-3 expression in both primary tumor and corresponding lymph node metastases.Conclusions: PRL-3 mRNA was expressed to a greater extent in prostate cancer tissue compared to normal prostate tissue. PRL-3 protein was expressed in both prostate cancer primary tumor and corresponding lymph node metastases. The results from our in vitro assays suggest that PRL-3 promotes growth and migration in prostate cancer. In conclusion, these results imply that PRL-3 has a role in the pathogenesis of prostate cancer. [ABSTRACT FROM AUTHOR]

Background: Autoimmune Addison's disease (AAD) is caused by multiple genetic and environmental factors. Variants of genes encoding immunologically important proteins such as the HLA molecules are strongly associated with AAD, but any environmental risk factors have yet to be defined. We hypothesized that primary or reactivating infections with cytomegalovirus (CMV) could represent an environmental risk factor in AAD, and that CMV specific CD8(+) T cell responses may be dysregulated, possibly leading to a suboptimal control of CMV. In particular, the objective was to assess the HLA-B8 restricted CD8(+) T cell response to CMV since this HLA class I variant is a genetic risk factor for AAD.Methods: To examine the CD8(+) T cell response in detail, we analyzed the HLA-A2 and HLA-B8 restricted responses in AAD patients and healthy controls seropositive for CMV antibodies using HLA multimer technology, IFN-γ ELISpot and a CD107a based degranulation assay.Results: No differences between patients and controls were found in functions or frequencies of CMV-specific T cells, regardless if the analyses were performed ex vivo or after in vitro stimulation and expansion. However, individual patients showed signs of reactivating CMV infection correlating with poor CD8(+) T cell responses to the virus, and a concomitant upregulation of interferon regulated genes in peripheral blood cells. Several recently diagnosed AAD patients also showed serological signs of ongoing primary CMV infection.Conclusions: CMV infection does not appear to be a major environmental risk factor in AAD, but may represent a precipitating factor in individual patients. [ABSTRACT FROM AUTHOR]

Background: Proteasome inhibitors are attractive cancer therapeutic agents because they can regulate apoptosis-related proteins. Bortezomib also known as Velcade(®), a proteasome inhibitor that has been approved by the food and drug administration for treatment of patients with multiple myeloma, and many clinical trials are ongoing to examine to the efficacy of bortezomib for the treatment of other malignancies. Bortezomib has been shown to induce apoptosis and inhibit cell growth of many cancer cells. In current study, we determine whether bortezomib induces cell death/apoptosis in CML.Methods: Cell viability was measured using MTT assays. Apoptosis was measured by annexin V/PI dual staining and DNA fragmentation assays. Immunoblotting was performed to examine the expression of proteins. Colony assays were performed using methylcellulose.Results: Treatment of CML cells with bortezomib results in downregulation of S-phase kinase protein 2 (SKP2) and concomitant stabilization of the expression of p27Kip1. Furthermore, knockdown of SKP2 with small interference RNA specific for SKP2 caused accumulation of p27Kip1. CML cells exposed to bortezomib leads to conformational changes in Bax protein, resulting in loss of mitochondrial membrane potential and leakage of cytochrome c to the cytosol. In the cytosol, cytochrome c causes sequential activation of caspase-9, caspase-3, PARP cleavage and apoptosis. Pretreatment of CML cells with a universal inhibitor of caspases, z-VAD-fmk, prevents bortezomib-mediated apoptosis. Our data also demonstrated that bortezomib treatment of CML downregulates the expression of inhibitor of apoptosis proteins. Finally, inhibition of proteasome pathways by bortezomib suppresses colony formation ability of CML cells.Conclusions: Altogether, these findings suggest that bortezomib suppresses the cell proliferation via induction of apoptosis in CML cells by downregulation of SKP2 with concomitant accumulation of p27Kip1, suggesting that proteasomal pathway may form novel therapeutic targets for better management of CML. [ABSTRACT FROM AUTHOR]

Background: Nitrobenzoxadiazole derivatives (NBDs), including NBDHEX and the recently developed MC3181, are promising anticancer agents able to target glutathione transferase and inhibit both its catalytic activity and ability to sequester TNF-receptor associated factor 2 (TRAF2) and c-Jun N-terminal kinase (JNK). NBDs have been shown to impair the growth and survival of a broad-spectrum of tumor types, in vitro and in vivo. Herein, we evaluated the effects of the new compound MC3181 on U-2OS osteosarcoma cells and investigated the impact of both NBDHEX and MC3181 on autophagy.Methods: Cell viability was evaluated by sulforhodamine B assay. The dissociation of the TRAF2-GSTP1-1 complex was detected by proximity ligation assay, while the phospho-activation of JNK was assessed by western blotting. The effects of NBDs on autophagy were evaluated by GFP-LC3 puncta formation, western blotting for LC3-II and p62, and LC3 turnover assay in the presence of bafilomycin A1. The role of JNK in the reduction of autophagic flux caused by NBDs was investigated using JNK1 shRNA-transfected cells. Fluorogenic caspase activity assay and flow cytometric analysis of DNA content were used to determine the cytotoxic effects of NBDs on JNK1-silenced cells.Results: Similar to NBDHEX, MC3181 reduced viability and activated TRAF2/JNK signaling in U-2OS cells. Moreover, NBDs induced the accumulation of autophagic vesicles and LC3-II while reducing both basal and nutritional stress-induced autophagic flux. Furthermore, increased levels of both LC3-II and the autophagy selective substrate p62 were observed in different tumor cell lines treated with NBDs, the concurrent increase of these markers being consistent with an impairment of autophagosome clearance. Autophagy inhibition by NBDs required JNK activity: NBDs caused autophagy inhibition and caspase-3 activation in JNK-positive U-2OS, but no autophagic flux inhibition or caspase-3 activation in JNK-silenced cells.Conclusions: Our demonstration that NBDs can act as late-phase autophagy inhibitors opens new opportunities to fully exploit their therapeutic potential. This may not rely solely on their effectiveness in inducing cell cycle arrest and apoptosis, but also on their ability to weaken the capacity of tumor cells to endure stress conditions via autophagy. In addition, this study provides evidence that JNK can participate in impairing autophagy. [ABSTRACT FROM AUTHOR]

Background: Fei-Liu-Ping (FLP) ointment is an oral prescription medication that has been widely applied to treat lung cancer patients in China. Regulation of the metastatic microenvironment is an important therapeutic approach for prevention and treatment of tumor recurrence and metastasis. The advantage of Traditional Chinese Medicine management of lung cancer lies in the prevention of recurrence and metastasis. Our previous study has demonstrated that FLP ointment could regulate lung inflammatory microenvironment in vitro. However, the effects of FLP on the tumor microenvironment in vivo are still poorly understood. The objective of this study is to investigate the effect of FLP alone or in combination with celecoxib in the prevention of lung cancer progression by Cyclooxygenase (Cox)-2 mediated tumor inflammatory microenvironment in vivo.Methods: 120 Lewis lung carcinoma xenograft mice were divided equally into four groups: vehicle, FLP, celecoxib, and FLP plus celecoxib. The dynamic growth of the xenografted tumors was observed using an in vivo fluorescence imaging system. Mice were sacrificed on day 14, day 21, and day 28, and tumor specimens and lung tissues were harvested to detect the metastasis-associated protein expression.Results: Tumor inhibition rate was 15.4, 44.2, 47.4 % at day 14, 37.3, 34.7, 61.5 % at day 21, and 15.5, 10.3, 32.5 % at day 28 after treatment of FLP, celecoxib, and FLP plus celecoxib, respectively. Upon treatment of FLP and celecoxib together, lung metastasis rate was 30 % (8 metastatic nodules) lower than other groups. FLP inhibited Cox-2 expression in a time-dependent manner. Moreover, FLP inhibited N-cadherin, matrix metalloproteinases (MMP)-9, and Vimentin expression. Treatment of FLP in combination with celecoxib was more effective than FLP or celecoxib alone in inhibiting vascular endothelial growth factor, platelet-derived growth factor receptors β, microsomal Prostaglandin E synthase-1, MMP-2, MMP-9, N-cadherin, and Vimentin expression, but increased E-cadherin expression.Conclusions: FLP inhibited tumor growth and metastasis in a Lewis lung xenograft mice model through the Cox-2 pathway. FLP in combination with celecoxib enhanced the antitumor growth and anti-metastasis effects. Traditional Chinese herbs combined with anti-inflammatory drugs might offer a promising strategy to prevent tumor metastasis. [ABSTRACT FROM AUTHOR]