ILF2 cooperates with E2F1 to maintain mitochondrial homeostasis and promote small cell lung cancer progression

Objective:Mitochondria play multifunctional roles in carcinogenesis.Deciphering uncertainties of molecular interactions within mitochondria will promote further understanding of cancer.Interleukin enhancer binding factor 2(ILF2)is upregulated in several malignancies,however,much remains unknown regarding ILF2 in small cell lung cancer(SCLC).In the current study,we explored ILF2's role in SCLC and demonstrated its importance in mitochondria quality control.Methods:Colony formation,cell proliferation,cell viability and xenograft studies were performed to examine ILF2's role on SCLC progression.Glucose uptake,lactate production,cellular oxygen consumption rate and extracellular acidification rate were measured to examine the effect of ILF2 on glucose metabolism.RNA-sequencing was utilized to explore genes regulated by ILF2.E2 F1 transcriptional activity was determined by dual luciferase reporter assay.Mitochondria quantification and mitochondrial membrane potential assays were performed to examine mitochondrial quality.Gene expression was determined by RT-qPCR,Western blot and IHC assay.Results:ILF2 promotes SCLC tumor growth in vitro and in vivo.ILF2 elevates oxidative phosphorylation expression and declines glucose intake and lactate production.Genome-wide analysis of ILF2 targets identified a cohort of genes regulated by E2 F1.In consistent with this,we found ILF2 interacts with E2 F1 in SCLC cells.Further studies demonstrated that suppression of E2 F1 expression could reverse ILF2-induced tumor growth and enhanced mitochondria function.Significantly,expression of ILF2 is progressively increased during SCLC progression and high ILF2 expression is correlated with higher histologic grades,which indicates ILF2's oncogenic role in SCLC.Conclusions:Our results demonstrate that ILF2 interacts with E2 F1 to maintain mitochondria quality and confers SCLC cells growth advantage in tumorigenesis.

Identification of p100 target promoters by chromatin immunoprecipitation-guided ligation and selection (ChIP-GLAS)

The multifunctional protein p100 is a vital transcriptional regulator that increases gene transcription by forming a physical bridge between promoter-specific transcription factors and the basal transcription machinery.To identify potential signal transduction pathways in which human p100 acts as a coregulator and to find target promoter regions that may interact with p100,we performed a promoter microarray assay called chromatin immunoprecipitation-guided ligation and selection(ChIP-GLAS).From this assay,we determined that a set of promoter fragments,including several factors in the transforming growth factor beta(TGF-β)signaling pathway,exhibited interaction with p100.The ChIP-GLAS data were validated by RT-PCR assessing the mRNA expression of various factors in the TGF-b signaling pathway in cell lines.

Use of copper-cysteamine nanoparticles to simultaneously enable radiotherapy,oxidative therapy and immunotherapy for melanoma treatment

Dear Editor,Melanoma,squamous cell carcinoma(SCC),and basal cell carcinoma(BCC)are three major types of skin cancer.Among them,melanoma is the most severe form and accounts for~4%of all newly diagnosed cancers annually in the United States.It is estimated that approximately 9500 people are diagnosed with skin cancer every day,and more than 1 million Americans are living with melanoma.Melanoma treatment is still a major challenge in the clinic.Photodynamic therapy(PDT)is composed of targeted ablation and immune activation,is less invasive than other therapies and has been widely used in the treatment of various cancers.

Respective IL-17A production by γδ T and Th17 cells and its implication in host defense against chlamydial lung infection

The role of IL-17A is important in protection against lung infection with Chlamydiae,an obligate intracellular bacterial pathogen.In this study,we explored the producers of IL-17A in chlamydial lung infection and specifically tested the role of major IL-17A producers in protective immunity.We found thatγδT cells and Th17 cells are the major producers of IL-17A at the early and later stages of chlamydial infection,respectively.Depletion ofγδT cells in vivo at the early postinfection(p.i.)stage,when mostγδT cells produce IL-17A,failed to alter Th1 responses and bacterial clearance.In contrast,the blockade of IL-17A at the time when IL-17A was mainly produced by Th17(day 7 p.i.)markedly reduced the Th1 response and increased chlamydial growth.The data suggest that theγδT cell is the highest producer of IL-17A in the very early stages of infection,but the protection conferred by IL-17A is mainly mediated by Th17 cells.In addition,we found that depletion ofγδT cells reduced IL-1αproduction by dendritic cells,which was associated with a reduced Th17 response.This finding is helpful to understand the variable role of IL-17A in different infections and to develop preventive and therapeutic approaches against infectious diseases by targeting IL-17A.

Inefficiency of C3H/HeN Mice to Control Chlamydial Lung Infection Correlates with Downregulation of Neutrophil Activation during the Late Stage of Infection

We previously reported that massive infiltration of neutrophils in C3H/HeN （C3H） mice could not efficiently control Chlamydia muridarum （Cm） infection and might contribute to the high susceptibility of these mice to lung infection. To further define the nature of neutrophil responses in C3H mice during chlamydial infection, we examine the expression of adhesion molecules and CDllb related to neutrophils infiltration and activation, respectively, following intranasal Cm infection. The results showed that the expression of selectins （E-selectin, P-selectin and L-selectin）, and intercellular cell adhesion molecule-1 （ICAM-1） in the lung of C3H mice increased more significantly than in C57BL/6 （B6） mice, the more resistant strain. These results correlated well with the massive neutrophils infiltration in C3H mice. In contrast, CDllb expression on peripheral blood and lung neutrophils in C3H mice exhibited a significant reduction compared with B6 mice during the late phage of infection （day 14）. These findings suggest that the high-level expression of adhesion molecules in C3H mice may enhance neutrophils recruitment to the lung, but the decline of CDllb expression on neutrophils may attenuate neutrophil function. Therefore, CDllb down-regulation on neutrophils may contribute to the failure of C3H mice to control chlamydial lung infection.

miR-495 andmiR-5688 are down-regulated in non-small cell lung cancer under hypoxia to maintain interleukin-11 expression

Background:Hypoxia is a hallmark of cancer and is associated with poor prognosis.However,the molecular mechanism by which hypoxia promotes tumor progression remains unclear.MicroRNAs dysregulation has been shown to play a critical role in the tumor and tumor microenvironment.Here,we investigated the roles ofmiR-495 and miR-5688 in human non-small cell lung cancer(NSCLC)and their underlying mechanism.Methods:The expression levels of miR-495 and miR-5688 in human NSCLC tissue specimens were measured by quantitative real-time polymerase chain reaction(qRT-PCR).Deferoxamine(DFO)was used to determine whether the regulation of miR-495 and miR-5688 under hypoxia was dependent on hypoxia-inducible factor 1-alpha(HIF-1α).Furthermore,the functions of miR-495 and miR-5688 in tumor progression were evaluated using colony formation,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium(MTS),wound healing,transwell assays,and xenograft model.Two algorithms,PicTAR and Targetscan,were used to predict the target gene of these two miRNAs,and dual-luciferase reporter assay was conducted to confirm the target.The unpaired two-tailed t test,Pearson correlation analysis,and Fisher’s exact probability test were performed for statistical analyses.Results:Two miRNAs,miR-495 and miR-5688,were found to participate in NSCLC progression under hypoxia.They were down-regulated in NSCLC tissues compared with normal tissues.We determined that hypoxia led to the down-regulation of miR-495 and miR-5688 in NSCLC cells,which was independent of HIF-1αand cellular metabolic energy.In addition,miR-495 and miR-5688 suppressed cell proliferation,migration,and invasion in vitro.The NSCLC xenograft model showed that miR-495 and miR-5688 inhibited tumor formation in vivo.Interestingly,we found that miR-495 and miR-5688 had the same target,interleukin-11(IL-11).Recombinant human IL-11 counteracted the effects of miR-495 and miR-5688 on NSCLC cells,suggesting that miR-495 and miR-5688 executed their tumor suppressive role by repressing IL-11 expression.Conclusion:We found that hypoxia down-regulated the expression levels of miR-495 and miR-5688 in NSCLCto enhance IL-11 expression and tumor progression,indicating that the miR-495/miR-5688/IL-11 axismay serve as a therapeutic target and potential biomarker for NSCLC.

Functions of PARylation in DNA Damage Repair Pathways

Protein poly ADP-ribosylation（PARylation） is a widespread post-translational modification at DNA lesions,which is catalyzed by poly（ADP-ribose） polymerases（PARPs）.This modification regulates a number of biological processes including chromatin reorganization,DNA damage response（DDR）,transcriptional regulation,apoptosis,and mitosis.PARP1,functioning as a DNA damage sensor,can be activated by DNA lesions,forming PAR chains that serve as a docking platform for DNA repair factors with high biochemical complexity.Here,we highlight molecular insights into PARylation recognition,the expanding role of PARylation in DDR pathways,and the functional interaction between PARylation and ubiquitination,which will offer us a better understanding of the biological roles of this unique post-translational modification.

Anti-CD44 mAb remodels biological behaviors of spheroid cells with stemness from human ovarian cancer cell line SKOV-3

There is accumulating evidence that cancer stem cells (CSCs) play an important role in tumor progression. Novel strategies targeting CSCs have been widely researched. In the present study, we explored whether such CSCs existed in human ovarian cancer (OVCA) cell line and whether anti-CD44 antibody had effects on such subpopulation. We isolated and identified spheroid cells from SKOV-3. Then we used A3D8, an anti-CD44 mAb to treat spheroid cells with so-called "stemness". Effects of A3D8 on spheroid cells' biological behaviors were examined. Our findings showed that there was a small subpopulation that had so-called "stemness" in SKOV-3 cell line. Against spheroid cells, A3D8 can (1) inhibit cell proliferation; (2) change cell cycle distribution and expression of p21, CDK2 and cyclinA; (3) enhance cisplatin (DDP)-induced apoptosis; (4) promote cell differentiation; (5) inhibit clone formation efficiency; (6) reduce invasive efficacy; (7) inhibit tumorigenicity. Thus, to sum up points which we have just showed, spheroid cells isolated from SKOV-3 can be used as an appropriate in vitro model for relevant study of human ovarian CSCs. And our results reasoned that anti-CD44 therapy may become a potential promising strategy for OVCA treatment.

Microtubule-associated deacetylase HDAC6 promotes angiogenesis by regulating cell migration in an EB1-dependent manner

Angiogenesis,a process by which the preexisting blood vasculature gives rise to new capillary vessels,is associated with a variety of physiologic and pathologic conditions.However,the molecular mechanism underlying this important process remains poorly understood.Here we show that histone deacetylase 6(HDAC6),a microtubule-associated enzyme critical for cell motility,contributes to angiogenesis by regulating the polarization and migration of vascular endothelial cells.Inhibition of HDAC6 activity impairs the formation of new blood vessels in chick embryos and in angioreactors implanted in mice.The requirement for HDAC6 in angiogenesis is corroborated in vitro by analysis of endothelial tube formation and capillary sprouting.Our data further show that HDAC6 stimulates membrane ruffling at the leading edge to promote cell polarization.In addition,microtubule end binding protein 1(EB1)is important for HDAC6 to exert its activity towards the migration of endothelial cells and generation of capillary-like structures.These results thus identify HDAC6 as a novel player in the angiogenic process and offer novel insights into the molecular mechanism governing endothelial cell migration and angiogenesis.

MicroRNA encoded by the HSV-1 latency-associated transcript anti-apoptotic function in human mesenchymal stem cells

Human mesenchymal stem cells (HMSCs) have emerged as a promising cell type for tissue repopulat-ing and for use as ex vivo gene delivery vehicles. Herpes simplex virus-1 (HSV-1) possesses many vector features, which make it especially suitable for HMSC applications. Here we performed real-time RT-PCR to detect the level of mature microRNA encoded by the HSV-1 latency-associated transcript (microRNA-LAT) in HMSCs cytoplasm with a specific stem loop reverse primer. Three small interfering RNAs (siRNAs) were chemically synthesized towards microRNA-LAT, TGF-β1 and SMAD3. The results demonstrate that HSV-1 and microRNA-LAT prevented HMSCs from undergoing cisplatin-induced apoptosis. In comparison with cells only treated with cisplatin, the apoptosis phenomenon with HSV-1 and microRNA-LAT were markedly reduced. The apoptosis rates of these two groups were both lower (P<0.05) as determined by flow cytometry analysis. The results of RT-PCR and western blotting analysis confirmed that the mRNA and protein levels of TGF-β1 and SMAD3 were significantly decreased with treatment of HSV-1 and microRNA-LAT. Integral TGF-β signalling pathway components were by these means knocked down. Moreover, the levels of the mature microRNA-LAT were decreased in cis-platin-treated HMSCs. In conclusion, HSV-1 and microRNA-LAT exert their anti-apoptotic effect on HMSCs by down-regulation of the TGF-β signalling pathway. Thus HSV-1, having anti-apoptotic effect naturally encoded in its microRNA-LAT, is a good candidate to be developed for HMSC vector.

Type 1 T-cell responses in chlamydial lung infections are associated with local MIP-1a response

Chemokines and their receptors are important mediators of leukocyte trafficking and recruitment and sometimes work as modulators of T-cell responses during infections and inflammation.Modulating the biological activity of chemokines has been found to influence the course of diseases.However,little is known about the role of chemokine responses during chlamydial lung infections.We therefore analyzed the dynamics of multiple chemokines,which are frequently associated with type 1(Th1)T cell immune responses,and their receptors for their expression in the lungs during Chlamydia muridarum(Cm)infections.We also examined the relationship between chemokine responses and the development of Th1 responses as well as the clearance of infection.Our results showed that in parallel with the high levels of gamma interferon(IFN-c)and IL-12 production in the lungs and draining lymph nodes,and the expansion of IFN-c-producing CD4 and CD81 T cells,the production of the cell-related chemokines RANTES,IFN-c-inducible protein-10(IP-10)and macrophage inflammatory protein-1a(MIP-1a)and their receptor CCR1 was elevated in the lung tissues after infection.Interestingly,in a later phase of infection,the expression of RANTES and IP-10 remained elevated but the expression of MIP-1a and CCR1 decreased to a low level,which suggests a closer association with the pattern of Th1 cytokine responses in the process of infection.These results suggest a close association between the MIP-1a response and the Th1-type T-cell responses in chlamydial lung infections .