Acquired immune resistance is associated with interferon signature and modulation of KLF6/c-MYB transcription factors in myeloid leukemia.

Resistance to immunity is associated with the selection of cancer cells with superior capacities to survive inflammatory reactions. Here, we tailored an ex vivo immune selection model for acute myeloid leukemia (AML) and isolated the residual subpopulations as "immune-experienced" AML (ieAML) cells. We confirmed that upon surviving the immune reactions, the malignant blasts frequently decelerated proliferation, displayed features of myeloid differentiation and activation, and lost immunogenicity. Transcriptomic analyses revealed a limited number of commonly altered pathways and differentially expressed genes in all ieAML cells derived from distinct parental cell lines. Molecular signatures predominantly associated with interferon and inflammatory cytokine signaling were enriched in the AML cells resisting the T-cell-mediated immune reactions. Moreover, the expression and nuclear localization of the transcription factors c-MYB and KLF6 were noted as the putative markers for immune resistance and identified in subpopulations of AML blasts in the patients' bone marrow aspirates. The immune modulatory capacities of ieAML cells lasted for a restricted period when the immune selection pressure was omitted. In conclusion, myeloid leukemia cells harbor subpopulations that can adapt to the harsh conditions established by immune reactions, and a previous "immune experience" is marked with IFN signature and may pave the way for susceptibility to immune intervention therapies.

Temporal regulation of gene expression and pathways in chemotherapy-induced senescence in HeLa cervical cancer cell line.

Cellular senescence is the state of permanent growth arrest. Chemotherapeutic drugs induce senescence, known as therapy-induced senescence. Although there are studies deciphering processes in senescence, more studies providing detailed information on therapy-induced senescence at the transcriptome level are needed. In order to understand temporal molecular changes of doxorubicin treatment in the course of senescence formation, two data sets from HeLa cells at 16 h and 72 h doxorubicin treatment were analyzed. GO BP enrichment, KEGG pathways and hub genes specific to or shared between 16 h and 72 h doxorubicin treated HeLa cells were identified. Genes functioning in p53 signaling were upregulated only in 16 h, while genes functioning in extracellular matrix organization were upregulated only in 72 h doxorubicin treated HeLa cells. Wound healing genes were gradually upregulated from 16 h to 72 h doxorubicin treatment and metabolic pathways were downregulated at both. ncRNA processing and ribosome biogenesis GO BP terms were enriched in upregulated genes at 16 h, while these terms were enriched in downregulated genes at 72 h senescent HeLa cells. According to our results, genes functioning in p53 signaling may be involved in the induction of senescence, but may not be required to maintain senescence in HeLa cells.

Genetic Analysis of RASD1 as a Candidate Gene for Schizophrenia

Background: RASD1 encodes Dexamethasone-induced Ras-related protein 1 (Dexras1), a protein with a critical role in signal transduction in neurons. There is a strong suspicion that dysfunction of Dexras1 might contribute to the pathogenesis of neuropsychiatric diseases. Related to its functions in intracellular signaling pathways, Dexras1 has a potential role in the etiology of schizophrenia because of its close interaction with NOS1, NOS1AP, and NMDAR, which have previously been associated with schizophrenia. Aims: To investigate the association of RASD1 variants with schizophrenia in a selected cohort from Turkey. Study Design: A case-control study. Methods: We performed targeted sequencing for the two exons, single intron, and untranslated regions of RASD1 gene in 200 individuals with schizophrenia and 100 healthy controls of Turkish origin. Results: Two rare variants, RASD1 (NM_016084.5): c.722A>T and c*31G>A were identified in individuals with schizophrenia but not in any controls. The c.722A>T was found in a single individual with schizophrenia and is a missense heterozygous variant in the second exon of RASD1, which is extremely rare in GnomAD. The other variant, c*31G>A, which was found in another individual from this schizophrenia cohort, has not been reported previously. Seven previously identified common single nucleotide polymorphisms were also detected; however, they were not significantly associated with schizophrenia in this study cohort. Conclusion: Our findings suggest that rare variants of RASD1 might be contributing to the etiopathogenesis of schizophrenia. Further studies are needed to elucidate the underlying mechanism of this association.

Identification of BRIP1, NSMCE2, ANAPC7, RAD18 and TTL from chromosome segregation gene set associated with hepatocellular carcinoma.

INTRODUCTION: Hepatocellular carcinoma is one of the most frequent cancers with high mortality rate worldwide. METHODS: TCGA LIHC HTseq counts were analyzed. GSEA was performed with GO BP gene sets. GO analysis was performed with differentially expressed genes. The subset of genes contributing most of the enrichment result of GO_BP_CHROMOSOME_SEGREGATION of GSEA were identified. Five genes have been selected in this subset of genes for further analysis. A microarray data set, GSE112790, was analyzed as a validation data set. Survival analysis was performed. RESULTS: According to GSEA and GO analysis several gene sets and processes related to chromosome segregation were enriched in LIHC. GO_BP_CHROMOSOME_SEGREGATION gene set from GSEA had the highest size of the genes contributing most of the enrichment. Five genes in this gene set; BRIP1, NSMCE2, ANAPC7, RAD18 and TTL, whose expressions and prognostic values have not been studied in hepatocellular carcinoma in detail, have been selected for further analyses. Expression of these five genes were identified as significantly upregulated in LIHC RNA-seq and HCC microarray data set. Survival analysis showed that high expression of the five genes was associated with poor overall survival in HCC patients. CONCLUSION: Selected genes were upregulated and had prognostic value in HCC.

The Expression Profile of Protease Inhibitors in the Airway Epithelial Cells after Allergen (Der p 1) Stimulation.

BACKGROUND: Airway epithelial cells are constantly exposed to intracellular and extracellular proteases that play a pivotal role in several airway diseases. Dermatophagoides pteronyssinus (Der p) 1 derived from house dust mite has protease activity that causes epithelial barrier defect and inflammatory response. Protease inhibitors released against proteases are involved in the maintenance of homeostasis. A disruption of the balance between proteases and protease inhibitors can lead to distortion of the cellular structures and cellular activities and thus culminate in disease processes. Although the effects of Der p 1 allergen on epithelial barrier integrity and inflammatory response are well-established, its contribution to protease inhibitor production is highly limited. OBJECTIVE: This study aimed to determine the profile of the protease inhibitor response to Der p 1 allergen in human airway epithelial cells, A549 and BEAS-2B. METHODS: Differentiated cells by the air-liquid interface were exposed to Der p 1 with or without Th2 type cytokines (IL-4 and IL-13). Gene expression of protease inhibitors was determined by qPCR at 2 different time points. RESULTS: We found that the effect of allergen exposure on the protease inhibitor profile can vary depending on the antigen concentration, treatment duration, and the presence or absence of type 2 cytokines. Gene expressions of serine protease inhibitor (SERPIN)B3 and SERPINB4 were increased following Th2 cytokine stimulation in both cell types at both time points, whereas SERPINB2 and TFPI-2 expressions were induced by 24-h Der p 1 stimulation in both cells. CONCLUSIONS: Our study suggests that Der p 1 exposure of the airway epithelium may have consequences related to its protease activity in the presence as well as in the absence of Th2 cytokines in the microenvironment.

The implication of ROCK 2 as a potential senotherapeutic target via the suppression of the harmful effects of the SASP: Do senescent cancer cells really engulf the other cells?

Chemotherapy-induced senescent cancer cells secrete several factors in their microenvironment called SASP. Accumulated evidence states that SASP is responsible for some of the harmful effects of chemotherapy such as drug resistance and the induction of cancer cell proliferation, migration, and invasion. Therefore, to develop senolytic and/or senomorphic drugs, targeting the senescent cells gains importance as a new strategy for preventing the damage that senescent cancer cells cause. In the current work, we evaluated whether Rho/Rho kinase pathway has the potential to be used as a target pathway for the development of senolytic and/or senomorphic drugs in doxorubicin-induced senescent cancer cell lines. We have determined that inhibition of Rho/Rho kinase pathway with CT04 and Y27632 reduced the secretory activity of senescent cancer cells and changed the composition of SASP. Our results indicate that ROCK 2 isoform was responsible for these observed effects on the SASP. In addition, non-senescent cancer cell proliferation and migration accelerated by senescent cells were set back to the pre-induction levels after ROCK inhibition. Moreover, contrary to the previous observations, another important finding of the current work is that senescent HeLa and A549 cells did not engulf the non-senescent HeLa, A549 cells, and non-cancer HUVEC. These results indicate that ROCK inhibitors, in particular ROCK 2 specific inhibitors, have the potential to be developed as novel senomorphic drugs. In addition, we found that all senescent cancer cells do not share the same engulfment ability, and this process should not be generalized.

CHRNA5 belongs to the secondary estrogen signaling network exhibiting prognostic significance in breast cancer.

PURPOSE: Cholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored. METHODS: Meta-analyses of large breast cancer microarray cohorts were used to evaluate the association of CHRNA5 expression with estrogen (E2) treatment, estrogen receptor (ER) status and patient prognosis. The results were validated through RT-qPCR analyses of multiple E2 treated cell lines, CHRNA5 depleted MCF7 cells and across a breast cancer patient cDNA panel. We also calculated a predicted secondary (PS) score representing direct/indirect induction of gene expression by E2 based on a public dataset (GSE8597). Co-expression analysis was performed using a weighted gene co-expression network analysis (WGCNA) pipeline. Multiple other publicly available datasets such as CCLE, COSMIC and TCGA were also analyzed. RESULTS: Herein we found that CHRNA5 expression was induced by E2 in a dose- and time-dependent manner in breast cancer cell lines. ER- breast tumors exhibited higher CHRNA5 expression levels than ER+ tumors. Independent meta-analysis for survival outcome revealed that higher CHRNA5 expression was associated with a worse prognosis in untreated breast cancer patients. Furthermore, CHRNA5 and its co-expressed gene network emerged as secondarily induced targets of E2 stimulation. These targets were largely downregulated by exposure to CHRNA5 siRNA in MCF7 cells while the response of primary ESR1 targets was dependent on the direction of the PS-score. Moreover, primary and secondary target genes were uncoupled and clustered distinctly based on multiple public datasets. CONCLUSION: Our findings strongly associate increased expression of CHRNA5 and its co-expression network with secondary E2 signaling and a worse prognosis in breast cancer.

RAB25 confers resistance to chemotherapy by altering mitochondrial apoptosis signaling in ovarian cancer cells.

Ovarian cancer remains one of the most frequent causes of cancer-related death in women. Many patients with ovarian cancer suffer from de novo or acquired resistance to chemotherapy. Here, we report that RAB25 suppresses chemotherapy-induced mitochondrial apoptosis signaling in ovarian cancer cell lines and primary ovarian cancer cells. RAB25 blocks chemotherapy-induced apoptosis upstream of mitochondrial outer membrane permeabilization by either increasing antiapoptotic BCL-2 proteins or decreasing proapoptotic BCL-2 proteins. In particular, BAX expression negatively correlates with RAB25 expression in ovarian cancer cells. BH3 profiling assays corroborated that RAB25 decreases mitochondrial cell death priming. Suppressing RAB25 by means of RNAi or RFP14 inhibitory hydrocarbon-stapled peptide sensitizes ovarian cancer cells to chemotherapy as well as RAB25-mediated proliferation, invasion and migration. Our data suggest that RAB25 is a potential therapeutic target for ovarian cancer.

Preparation and characterization of novel albumin-sericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment.

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.

High-throughput gene expression analysis identifies p53-dependent and -independent pathways contributing to the adrenocortical dysplasia (acd) phenotype.

In mammalian cells TPP1, encoded by the Acd gene, is a key component of the shelterin complex, which is required for telomere length maintenance and telomere protection. In mice, a hypomorphic mutation in Acd causes the adrenocortical dysplasia (acd) phenotype, which includes limb and body axis anomalies, and perinatal lethality. p53 deficiency partially rescues limb and body axis anomalies in acd mutant embryos, but not perinatal lethality, implicating p53-independent mechanisms in the acd phenotype. Loss of function of most shelterin proteins results in early embryonic lethality. Thus, study of the hypomorphic acd allele provides a unique opportunity to understand telomere dysfunction at an organismal level. The aim of this study was to identify transcriptome alterations in acd mutant and acd, p53 double mutant embryos to understand the p53-dependent and -independent factors that contribute to the mutant phenotypes in the context of the whole organism. Genes involved in developmental processes, cell cycle, metabolic pathways, tight junctions, axon guidance and signaling pathways were regulated by p53-driven mechanisms in acd mutant embryos, while genes functioning in immune response, and RNA processing were altered independently of p53 in acd, p53 double mutant embryos. To our best of knowledge, this is the first study revealing detailed transcriptomic alterations, reflecting novel p53-dependent and -independent pathways contributing to the acd phenotype. Our data confirm the importance of cell cycle and DNA repair pathways, and suggest novel links between telomere dysfunction and immune system regulation and the splicing machinery. Given the broad applicability of telomere maintenance in growth, development, and genome stability, our data will also provide a rich resource for others studying telomere maintenance and DNA damage responses in mammalian model systems.

Computational prediction and analysis of deleterious cancer associated missense mutations in DYNC1H1.

Dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) gene encodes a subunit of human cytoplasmic dynein complex, which has several crucial functions in the cell, such as intracellular transport of DNA damage proteins and mitotic spindle positioning. Recent studies reported the altered expression of DYNC1H1 in different cancers and DYNC1H1 was suggested to be potential biomarker in colorectal cancers. Previously, DYNC1H1 mutations have been associated with neurodegenerative diseases, however mutations of DYNC1H1 have not been fully investigated in cancers except for different types of pancreatic cancers. In this study we aimed to identify the cancer related mutations in DYNC1H1, which are deleterious for the DYNC1H1 structure and/or function. We investigated 523 cancer related missense mutations in DYNC1H1, which were collected from COSMIC database, to predict the effect of mutations on DYNC1H1 function. Of the 523 mutations, we identified 28 amino acid substitutions, which were predicted to be deleterious by PredictSNP1.0. When we searched for the effect of 28 deleterious mutations on protein stability by MUpro and I-Mutant2.0, we observed that most of the mutations decrease the protein stability. We analyzed the localization of deleterious mutations on primary protein structure and identified that predicted deleterious mutations were mainly located in the motor domain, which is crucial for the DYNC1H1 function. In addition, we detected close positioning of mutated residues in AAA + regions on 3D structure by STRUM and UCSF Chimera. When we searched the mutations in COSMIC database, we observed the occurrence of the mutations in different cancers, which might show the importance of these regions in corresponding cancers. Therefore, our findings provide potential structural and functional mutations and hotspots for DYNC1H1.

Functionally conserved effects of rapamycin exposure on zebrafish.

Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well­known anti­cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta­analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose­dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin­modulated functional pathways between zebrafish and mice, in addition to the dose­dependent growth curves of zebrafish embryos upon rapamycin exposure.

Dose- and time-dependent expression patterns of zebrafish orthologs of selected E2F target genes in response to serum starvation/replenishment.

Targets of E2F transcription factors effectively regulate the cell cycle from worms to humans. Furthermore, the dysregulation of E2F transcription modules plays a highly conserved role in cancers of human and zebrafish. Studying E2F target expression under a given cellular state, such as quiescence, might lead to a better understanding of the conserved patterns of expression in different taxa. In the present study, we used literature searches and phylogeny to identify several targets of E2F transcription factors that are known to be serum-responsive; namely, PCNA, MYBL2, MCM7, TYMS, and CTGF. The transcriptional serum response of zebrafish orthologs of these genes were quantified under different doses (i.e., 0, 0.1, 1, 3, and 10% FBS) and time points (i.e., 6, 24 and 48 hours, h) using quantitative RT-PCR (qRT-PCR) in the zebrafish fibroblast cells (ZF4). Our results indicated that mRNA expression of zebrafish pcna, mybl2, mcm7 and tyms drastically decreased while that of ctgf increased with decreasing serum levels as observed in mammals. These genes responded to serum starvation at 24 and 48 h and to the mitogenic stimuli as early as 6 h except for ctgf whose expression was significantly altered at 24 h. The zebrafish Mcm7 protein levels also were modulated by serum starvation/replenishment. The present study provides a foundation for the comparative analysis of quantitative expression patterns for genes involved in regulation of cell cycle using a zebrafish serum response model.

Alternatively spliced Robo2 isoforms in zebrafish and rat.

Robo2, a member of the robo gene family, functions as a repulsive axon guidance receptor as well as a regulator of cell migration and tissue morphogenesis in different taxa. In this study, a novel isoform of the zebrafish robo2 (robo2_tv2), which included an otherwise alternatively spliced exon (CAE), has been characterized. Robo2_tv2 is expressed differentially in most non-neuronal tissues of adult zebrafish whereas robo2_tv1 expression to a great extent is restricted to the brain and eye. In zebrafish, robo2_tv2 exhibits a very-low-level basal expression starting from 1 day post fertilization until the mid-larval stages, at which time its expression increases dramatically and could be detected throughout adulthood. Our findings demonstrate that the amino acid sequence coded by CAE of the robo2 gene is highly conserved between zebrafish and mammals, and also contains conserved motifs shared with robo1 and robo4 but not with robo3. Furthermore, we provide an account of differential transcription of the CAE homolog in various tissues of the adult rat. These results suggest that the alternatively spliced robo2 isoforms may exhibit tissue specificity.