Esophageal squamous cell carcinoma (ESCC) is a malignancy that severely threatens human health and carries a high incidence rate and a low 5-year survival rate. MicroRNAs (miRNAs) are commonly accepted as a key regulatory function in human cancer, but the potential regulatory mechanisms of miRNA-mRNA related to ESCC remain poorly understood.The GSE55857, GSE43732, and GSE6188 miRNA microarray datasets and the gene expression microarray datasets GSE70409, GSE29001, and GSE20347 were downloaded from Gene Expression Omnibus databases. The differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) were obtained using GEO2R. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for DEGs were performed by Database for Annotation, Visualization and Integrated Discovery (DAVID). A protein-protein interaction (PPI) network and functional modules were established using the STRING database and were visualized by Cytoscape. Kaplan-Meier analysis was constructed based on The Cancer Genome Atlas (TCGA) database.In total, 26 DEMs and 280 DEGs that consisted of 96 upregulated and 184 downregulated genes were screened out. A functional enrichment analysis showed that the DEGs were mainly enriched in the ECM-receptor interaction and cytochrome P450 metabolic pathways. In addition, MMP9, PCNA, TOP2A, MMP1, AURKA, MCM2, IVL, CYP2E1, SPRR3, FOS, FLG, TGM1, and CYP2C9 were considered to be hub genes owing to high degrees in the PPI network. MiR-183-5p was with the highest connectivity target genes in hub genes. FOS was predicted to be a common target gene of the significant DEMs. Hsa-miR-9-3p, hsa-miR-34c-3p and FOS were related to patient prognosis and higher expression of the transcripts were associated with a poor OS in patients with ESCC.Our study revealed the miRNA-mediated hub genes regulatory network as a model for predicting the molecular mechanism of ESCC. This may provide novel insights for unraveling the pathogenesis of ESCC.

BACKGROUND: The FOS gene is located on human chromosome 14q21-31 and encodes the nuclear oncoprotein c-Fos. This study analyzed the correlation between the FOS noncoding region rs7101 and rs1063169 polymorphisms and colorectal cancer susceptibility and prognosis.
METHODS: We analyzed the FOS genotypes in 432 colorectal cancer patients and 315 healthy subjects by PCR/Sanger sequencing. Survival was analyzed by Kaplan-Meier and Cox regression analysis. Western blot was used to detect the expression of c-Fos protein in cancer tissues and adjacent tissues in colorectal cancer patients with different genotypes.
RESULTS: The presence of a T allele at rs7101 and a T allele at rs1063169 in FOS carried a higher risk of colorectal cancer [adjusted odds ratio (OR) = 1.237, 95% confidence interval (95% CI) = 1.131-1.346, P ≤ .001 and adjusted OR = 1.218, 95% CI = 1.111-1.327, P ≤ .001, respectively]. c-Fos protein levels were significantly higher in variant cancer tissues than in normal mucosa tissues (P < .05), and c-Fos proteins levels were also higher in homozygous variant cancer tissues than in heterozygous variant cancer tissues. The 3-year survival rate of patients with wild-type FOS was higher than that of patients with variant FOS (P < .05).
CONCLUSION: The rs7101 and rs1063169 polymorphisms in the noncoding region of FOS are associated with the risk of developing colorectal cancer and the progression of colorectal cancer, which may be because the mutation enhances the expression of c-Fos protein to promote the incidence and development of colorectal cancer.

Gastric cancer (GC) is a common cause of cancer-related death worldwide. As a result of the lack of reliable diagnostic or prognostic biomarkers for GC, patient prognosis is still poor. Therefore, there is an urgent need for studies examining the underlying pathogenesis of GC in order to find effective biomarkers. LRRN1 (leucine-rich repeat neuronal protein-1) is a type I transmembrane protein that plays an important role in the process of nerve development and regeneration. However, its role in cancer, especially in GC, remains unclear. In the present study, we found that LRRN1 expression is upregulated in GC tissues and that high LRRN1 expression is associated with poor prognosis. siRNA and shRNA-mediated knockdowns of LRRN1 expression promoted GC cell apoptosis and activation of the Fas/FasL pathway. LRRN1 knockdown also resulted in upregulation of JUN, a subunit of the transcription factor AP-1 (activator protein-1). This suggests that LRRN1 suppresses GC cell apoptosis by downregulating AP-1, resulting in inhibition of the Fas/FasL pathway. These results confirm that LRRN1 plays a significant role in GC pathogenesis. Moreover, LRRN1 may be a potential prognostic biomarker and therapeutic target for GC.

Ribosomal S6 kinase 2 (RSK2), regulated by Ras/Raf/MEKs/ERKs, transmits upstream activation signals to downstream substrates including kinases and transcription and epigenetic factors. We observed that ELK members, including ELK1, 3, and 4, highly interacted with RSK2. We further observed that the RSK2-ELK3 interaction was mediated by N-terminal kinase and linker domains of RSK2, and the D and C domains of ELK3, resulting in the phosphorylation of ELK3. Importantly, RSK2-mediated ELK3 enhanced

The cancer susceptibility candidate 9 (CASC9) gene has been reported to exert an oncogenic effect in several types of cancer. However, its role in lung squamous cell carcinoma (LUSC) is unknown. Therefore, the present study examined the expression of CASC9 in LUSC and non‑cancer tissues by reverse transcription‑quantitative polymerase chain reaction assays and by data mining of high‑throughput public databases, including The Cancer Genome Atlas, the Gene Expression Omnibus, ArrayExpress and the Cancer Cell Line Encyclopedia. In vitro experiments were conducted to investigate the effects of CASC9 on the viability and the proliferation of LUSC cells. Furthermore, consulting the alteration status of CASC9 in LUSC from cBioPortal, functional enrichment analysis of co‑expressed genes, prediction of potential transcription factors, and inspection of adjacent protein‑coding genes were conducted to explore the potential molecular mechanism of CASC9 in LUSC. The results revealed that CASC9 was overexpressed in LUSC tissue, and significantly associated with the malignant progression of LUSC. In vitro experiments demonstrated that CASC9 knockdown by RNA interference attenuated the viability and proliferation of LUSC cells. Multiple copies of CASC9 gene were detected in 4 of 179 available sequenced LUSC cases. A functional enrichment analysis of 200 co‑expressed genes indicated that these genes were significantly associated with terms, including 'cell‑cell junction organization', 'desmosome organization', 'epidermis development', 'Hippo signaling pathway', 'pathogenic Escherichia coli infection' and 'PID HIF1 TF pathway'. Three genes, Fos‑related antigen 2 (FOSL2), SWI/SNF complex subunit SMARCC2, and transcription factor COE1 (EBF1), were predicted by lncRNAMap to be associated with CASC9. Among these, the expression of FOSL2 and EBF1 was positively and negatively correlated with the expression of CASC9, respectively. Two adjacent protein‑coding genes, cysteine‑rich secretory protein LCCL domain‑containing 1 and hepatocyte nuclear factor 4‑γ, were also positively correlated with CASC9 expression. In conclusion, the present data suggest that CASC9 serves as an oncogene in LUSC and may be a promising target for alternative therapeutic options for patients with this condition.

BACKGROUND: Melanin plays a crucial role in protecting human skin against exposure to ultraviolet (UV) radiation. However, its overproduction induces hyperpigmentation disorders of the skin.
PURPOSE: To investigate effects of phenylethyl resorcinol as one resorcinol derivative on melanogenesis and its mechanisms using B16F10 mouse melanoma cells and human epidermal melanocytes.
METHODS: Effects of phenylethyl resorcinol on melanogenesis and its mechanism of action were examined using several in vitro assays (i.e., cell survival, melanin content, cellular tyrosinase activity, real-time PCR analysis, luciferase-reporter assay, Western blot analysis, and ELISAs for cyclic AMP (cAMP), protein kinase A (PKA), cAMP response element binding (CREB) protein, and mitogen-activated protein kinases (MAPKs)).
RESULTS: Phenylethyl resorcinol reduced both melanin content and tyrosinase activity in these cells. Phenylethyl resorcinol also suppressed tyrosinase activity in cell-free tyrosinase enzyme assay. Although phenylethyl resorcinol decreased mRNA levels of tyrosinase and tyrosinase-related protein (TRP)-2, it did not affect mRNA levels of melanogenic gene microphthalmia-associated transcriptional factor (MITF) or TRP-1. Phenylethyl resorcinol had no effects on cAMP signaling or NF-κB signaling based on results of cyclic AMP response element (CRE)-luciferase reporter assay, cAMP production, protein kinase A (PKA) activity, Western blot assays for phosphorylated CRE-binding protein (CREB), NF-κB-luciferase reporter assay, and Western blot assays for phosphorylated NF-κB. However, phenylethyl resorcinol induced activation of activator protein-1 (AP-1) signaling. Specifically, phenylethyl resorcinol increased AP-1 reporter activity and increased phosphorylation of p44/42 MAPK, but not p38 MAPK or c-Jun N-terminal kinase (JNK). MEK1/2 and Src, upstream molecules of p44/42 MAPK were also phosphorylated by phenylethyl resorcinol. In addition, phenylethyl resorcinol-induced decreases in melanin content, tyrosinase activity, and MITF protein levels were attenuated by PD98059, a p44/42 MAPK inhibitor.
CONCLUSION: These data indicate that the anti-melanogenic activity of phenylethyl resorcinol is mediated by activation of p44/42 MAPK, indicating that phenylethyl resorcinol may be a potential therapeutic agent for treating hyperpigmentation skin disorders.

BACKGROUND/AIM: Triple-negative breast cancers (TNBC) lack expression of three important receptors, and have limited treatment options. High expression of junctional adhesion molecule-A (JAM-A) has been linked with aggressive tumor phenotypes including TNBC. This study aimed to evaluate the bioactivity of a JAM-A-down-regulating compound, Tetrocarcin-A, in TNBC.
MATERIALS AND METHODS: TNBC cell viability, colony formation and xenograft growth were examined in Tetrocarcin-A-treated HCC38 human cells, 4T1 mouse cells or patient-derived primary cells. Protein expression of cell fate signaling effectors was examined by immunoblotting (versus transient JAM-A gene silencing). Apoptotic pathways were investigated in parallel.
RESULTS: Tetrocarcin-A reduced TNBC cell viability in vitro and in an in ovo/semi-in vivo xenograft model. Tetrocarcin-A-induced JAM-A down-regulation and reduced ERK phosphorylation, followed by c-FOS phosphorylation on its transcription-regulating residue, which down-regulated several inhibitor of apoptosis (IAP) proteins and induced caspase-dependent intrinsic pathway of apoptosis.
CONCLUSION: Tetrocarcin-A merits further investigation as a novel anti-tumor agent in TNBC.

BACKGROUND: Mast cells are being increasingly recognized as critical components in the tumor microenvironment. Protein Kinase D (PKD) is essential for the progression of prostate cancer, but its role in prostate cancer microenvironment remains poorly understood.
METHODS: The expression of PKD, mast cells and microvessel density were examined by IHC. The clinical significance was determined by statistical analyses. The biological function of PKD and the underlying mechanisms were investigated using in vitro and in vivo models.
RESULTS: PKD2/3 contributed to MCs recruitment and tumor angiogenesis in the prostate cancer microenvironment. Clinical data showed that increased activation of PKD at Ser744/748 in prostate cancer was correlated with mast cell infiltration and microvascular density. PKD2/3 silencing of prostate cancer cells markedly decreased MCs migration and tube formation of HUVEC cells. Moreover, PKD2/3 depletion not only reduced SCF, CCL5 and CCL11 expression in prostate cancer cells but also inhibited angiogenic factors in MCs. Conversely, exogenous SCF, CCL5 and CCL11 reversed the effect on MCs migration inhibited by PKD2/3 silencing. Mechanistically, PKD2/3 interacted with Erk1/2 and activated Erk1/2 or NF-κB signaling pathway, leading to AP-1 or NF-κB binding to the promoter of scf, ccl5 and ccl11. Finally, PKD-specific inhibitor significantly reduced tumor volume and tumor growth in mice bearing RM-1 prostate cancer cells, which was attributed to attenuation of mast cell recruitment and tumor angiogenesis.
CONCLUSIONS: These results demonstrate a novel PKDs function that contributes to tumor angiogenesis and progression through mast cells recruitment in prostate cancer microenvironment.

BACKGROUND: Focal adhesion kinase (FAK) is a cytoplasmatic protein tyrosine kinase that associates with both integrins and growth factor receptors toward the adhesion, migration and invasion of cancer cells. The G-protein coupled estrogen receptor (GPER) has been involved in the stimulatory action of estrogens in breast tumor. In this study, we have investigated the engagement of FAK by GPER signaling in triple negative breast cancer (TNBC) cells.
METHODS: Publicly available large-scale database and patient data sets derived from "The Cancer Genome Atlas" (TCGA; www.cbioportal.org ) were used to assess FAK expression in TNBC, non-TNBC tumors and normal breast tissues. MDA-MB 231 and SUM159 TNBC cells were used as model system. The levels of phosphorylated FAK, other transduction mediators and target genes were detected by western blotting analysis. Focal adhesion assay was carried out in order to determine the focal adhesion points and the formation of focal adhesions (FAs). Luciferase assays were performed to evaluate the promoters activity of c-FOS, EGR1 and CTGF upon GPER activation. The mRNA expression of the aforementioned genes was measured by real time-PCR. Boyden chamber and wound healing assays were used in order to evaluate cell migration. The statistical analysis was performed by ANOVA.
RESULTS: We first determined by bioinformatic analysis that the mRNA expression levels of the gene encoding FAK, namely PTK2, is higher in TNBC respect to non-TNBC and normal breast tissues. Next, we found that estrogenic GPER signaling triggers Y397 FAK phosphorylation as well as the increase of focal adhesion points (FAs) in TNBC cells. Besides, we ascertained that GPER and FAK activation are involved in the STAT3 nuclear accumulation and gene expression changes. As biological counterpart, we show that FAK inhibition prevents the migration of TNBC cells upon GPER activation.
CONCLUSIONS: The present data provide novel insights regarding the action of FAK in TNBC. Moreover, on the basis of our findings estrogenic GPER signaling may be considered among the transduction mechanisms engaging FAK toward breast cancer progression.

BACKGROUND: Adenocarcinoma of the pancreas is one of the most aggressive tumor diseases affecting the human body. The oncogenic potential of pancreatic cancer is mainly characterized by extremely rapid growth triggered by the activation of oncogenic signaling cascades, which suggests a change in the regulation of important transcription factors. Amongst others, NFAT transcription factors are assumed to play a central role in the carcinogenesis of pancreatic cancer. Recent research has shown the importance of the transcription factor Sp1 in the transcriptional activity of NFATc2 in pancreatic cancer. However, the role of the interaction between these two binding partners remains unclear. The current study investigated the role of Sp1 proteins in the expression of NFATc2 target genes and identified new target genes and their function in cells. A further objective was the domain of the Sp1 protein that mediates interaction with NFATc2. The involvement of Sp1 proteins in NFATc2 target genes was shown by means of a gene expression profile analysis, and the results were confirmed by quantitative RT-PCR. The functional impact of this interaction was shown in a thymidine incorporation assay. A second objective was the physical interaction between NFATc2 and different Sp1 deletion mutants that was investigated by means of immunoprecipitation.
RESULTS: In pancreatic cancer, the proto-oncogene c-Fos, the tumor necrosis factor TNF-alpha, and the adhesion molecule integrin beta-3 are target genes of the interaction between Sp1 and NFATc2. Loss of just one transcription factor inhibits oncogenic complex formation and expression of cell cycle-regulating genes, thus verifiably decreasing the carcinogenic effect. The current study also showed the interaction between the transcription factor NFATc2 and the N-terminal domain of Sp1 in pancreatic cancer cells. Sp1 increases the activity of NFATc2 in the NFAT-responsive promoter.
CONCLUSIONS: The regulation of gene promotors during transcription is a rather complex process because of the involvement of many proteins that - as transcription factors or co-factors - regulate promotor activity as required and control cell function. NFATc2 and Sp1 seem to play a key role in the progression of pancreatic cancer.

BACKGROUND: Lichens, which represent symbiotic associations of fungi and algae, are potential sources of numerous natural products. Physciosporin (PHY) is a potent secondary metabolite found in lichens and was recently reported to inhibit the motility of lung cancer cells via novel mechanisms.
PURPOSE: The present study investigated the anticancer potential of PHY on colorectal cancer (CRC) cells.
METHODS: PHY was isolated from lichen extract by preparative TLC. The effect of PHY on cell viability, motility and tumourigenicity was elucidated by MTT assay, hoechst staining, flow cytometric analysis, transwell invasion and migration assay, soft agar colony formation assay, Western blotting, qRT-PCR and PCR array in vitro as well as tumorigenicity study in vivo.
RESULTS: PHY decreased the viability of various CRC cell lines (Caco2, CT26, DLD1, HCT116 and SW620). Moreover, PHY elicited cytotoxic effects by inducing apoptosis at toxic concentrations. At non-toxic concentrations, PHY dose-dependently suppressed the invasion, migration and colony formation of CRC cells. PHY inhibited the motility of CRC cells by suppressing epithelial-mesenchymal transition and downregulating actin-based motility markers. In addition, PHY downregulated β-catenin and its downstream target genes cyclin-D1 and c-Myc. Moreover, PHY modulated KAI1 C-terminal-interacting tetraspanin and KAI1 expression, and downregulated the downstream transcription factors c-jun and c-fos. Finally, PHY administration showed considerable bioavailability and effectively decreased the growth of CRC xenografts in mice without causing toxicity.
CONCLUSION: PHY suppresses the growth and motility of CRC cells via novel mechanisms.

BACKGROUND/AIM: Certain chromosomal arms are clonally amplified in colorectal cancer (CRC) and may contain novel driver genes. The aim of this study was to identify a novel driver gene for colorectal cancer carcinogenesis on long arm of chromosome 7 and the clarify its biological function.
MATERIALS AND METHODS: We identified ArfGAP with GTPase domain, ankyrin repeat and PH domain 3 (AGAP3) as a putative driver gene using the CRC dataset in The Cancer Genome Atlas (TCGA). Biological functions of AGAP3 and CRMP5-associated GTPase (CRAG), a splicing variant of AGAP3, were explored by overexpression. AGAP3/CRAG expression in our cohort was examined by quantitative reverse transcription polymerase chain reaction. Clinical significance of AGAP3/CRAG expression in TCGA dataset, Gene Expression Omnibus datasets and our clinical cohort was evaluated.
RESULTS: AGAP3 expression was significantly increased in CRC and colorectal adenoma compared to normal tissue. CRAG overexpression up-regulated c-Jun expression, and significantly increased cell proliferation and colony formation capability. AGAP3 expression did not have a concordant association with patient prognosis among datasets.
CONCLUSION: CRAG may contribute to development of CRC via activator protein 1 activation.

The last decade has seen the majority of primary bone tumor subtypes become defined by molecular genetic alteration. Examples include giant cell tumour of bone (H3F3A p.G34W), chondroblastoma (H3F3B p.K36M), mesenchymal chondrosarcoma (HEY1-NCOA2), chondromyxoid fibroma (GRM1 rearrangements), aneurysmal bone cyst (USP6 rearrangements), osteoblastoma/osteoid osteoma (FOS/FOSB rearrangements), and synovial chondromatosis (FN1-ACVR2A and ACVR2A-FN1). All such alterations are mutually exclusive. Many of these have been translated into clinical service using immunohistochemistry or FISH. 60% of central chondrosarcoma is characterised by either isocitrate dehydrogenase (IDH) 1 or IDH2 mutations distinguishing them from other cartilaginous tumours. In contrast, recurrent alterations which are clinically helpful have not been found in high grade osteosarcoma. High throughput next generation sequencing has also proved valuable in identifying germ line alterations in a significant proportion of young patients with primary malignant bone tumors. These findings will play an increasing role in reaching a diagnosis and in patient management.

BACKGROUND: Previously, we have demonstrated that Interleukin 13 receptor alpha 2 (IL-13Rα2) is overexpressed in approximate 78% Glioblastoma multiforme (GBM) samples. We have also demonstrated that IL-13Rα2 can serve as a target for cancer immunotherapy in several pre-clinical and clinical studies. However, the significance of overexpression of IL-13Rα2 in GBM and astrocytoma and signaling through these receptors is not known. IL-13 can signal through IL-13R via JAK/STAT and AP-1 pathways in certain cell lines including some tumor cell lines. Herein, we have investigated a role of IL-13/IL-13Rα2 axis in signaling through AP-1 transcription factors in human glioma samples in situ.
METHODS: We examined the activation of AP-1 family of transcription factors (c-Jun, Fra-1, Jun-D, c-Fos, and Jun-B) after treating U251, A172 (IL-13Rα2 +ve) and T98G (IL-13Rα2 -ve) glioma cell lines with IL-13 by RT-qPCR, and immunocytochemistry (ICC). We also performed colorimetric ELISA based assay to determine AP-1 transcription factor activation in glioma cell lines. Furthermore, we examined the expression of AP-1 transcription factors in situ in GBM and astrocytoma specimens by multiplex-immunohistochemistry (IHC). Student t test and ANOVA were used for statistical analysis of the results.
RESULTS: We have demonstrated up-regulation of two AP-1 transcription factors (c-Jun and Fra-1) at mRNA and protein levels upon treatment with IL-13 in IL-13Rα2 positive but not in IL-13Rα2 negative glioma cell lines. Both transcription factors were also overexpressed in patient derived GBM specimens, however, in contrast to GBM cell lines, c-Fos is also overexpressed in patient derived specimens. Astrocytoma specimens showed lesser extent of immunostaining for IL-13Rα2 and three AP-1 factors compared to GBM specimens. By transcription factor activation assay, we demonstrated that AP-1 transcription factors (C-Jun and Fra-1) were activated upon treatment of IL-13Rα2 + GBM cell lines but not IL-13Rα2 - GBM cell line with IL-13. Our results demonstrate functional activity of AP-1 transcription factor in GBM cell lines in response to IL-13.
CONCLUSIONS: These results indicate that IL-13/IL-13Rα2 axis can mediate signal transduction in situ via AP-1 pathway in GBM and astrocytoma and may serve as a new target for GBM immunotherapy.

Dickkopf‑related protein 3 (DKK3), which is a member of the Dickkopf WNT signaling pathway inhibitor family, is considered to be a tumor suppressor, due to its reduced expression in cancer cells and its ability to induce apoptosis when overexpressed by adenovirus. However, our previous study demonstrated alternative functions for DKK3 in head and neck squamous cell carcinoma (HNSCC). Our study reported that DKK3 expression was predominantly upregulated in HNSCC cell lines and tissue samples, and its expression was significantly correlated with poor prognosis. Furthermore, DKK3 overexpression in HNSCC cells significantly increased cancer cell proliferation, migration, invasion and in vivo tumor growth. These data have led to the hypothesis that DKK3 may exert oncogenic functions and may increase the malignant properties of HNSCC. The present study established a stable DKK3 knockdown cell line (HSC‑3 shDKK3) using lentivirus‑mediated short hairpin RNA, and assessed its effects on cancer cell behavior using MTT, migration and invasion assays. In addition, its effects on in vivo tumor growth were assessed using a xenograft model. Furthermore, the molecular mechanisms underlying the effects of DKK3 knockdown were investigated by microarray analysis, pathway analysis and western blotting. Compared with control cells, HSC‑3 shDKK3 cells exhibited significantly reduced proliferation, migration and invasion, and formed significantly smaller tumor masses when subcutaneously transplanted into nude mice. In addition, in HSC‑3 shDKK3 cells, the expression levels of phosphorylated (p)‑protein kinase B (Akt) (Ser473), p‑phosphoinositide 3‑kinase (PI3K) p85 (Tyr467), p‑PI3K p55 (Try199), p‑3‑phosphoinositide‑dependent protein kinase‑1 (PDK1) (Ser241) and total p38 mitogen‑activated protein kinase (MAPK) were reduced. Furthermore, phosphorylation of mechanistic target of rapamycin (mTOR) (Ser2448) was slightly decreased in HSC‑3 shDKK3 cells, which may be due to the increased expression of DEP domain‑containing mTOR‑interacting protein. Conversely, DKK3 overexpression in HSC‑3 shDKK3 cells rescued cellular proliferation, migration and invasion. With regards to expression levels, p‑PI3K and p‑PDK1 expression was not altered, whereas mTOR and p‑p38 MAPK expression was elevated. These data supported the hypothesis and indicated that DKK3 may contribute to the malignant phenotype of HNSCC cells via the PI3K/Akt/mTOR and MAPK signaling pathways.

One of the still open questions in Ewing sarcoma, a rare bone tumor with weak therapeutic options, is to identify the tumor-driving cell (sub) population and to understand the specifics in the biological network of these cells. This basic scientific insight might foster the development of more specific therapeutic target patterns. The experimental approach is based on a side population (SP) of Ewing cells, based on the model cell line CADO-ES1. The SP is established by flow cytometry and defined by the idea that tumor stem-like cells can be identified by the time-course in clearing a given artificial dye. The SP was characterized by a higher colony forming activity, by a higher differentiation potential, by higher resistance to cytotoxic drugs, and by morphology. Several SP and non-SP cell fractions and bone marrow-derived mesenchymal stem cell reference were analyzed by short read sequencing of the full transcriptome. The double-differential analysis leads to an altered expression structure of SP cells centered around the AP-1 and APC/c complex. The SP cells share only a limited proportion of the full mesenchymal stem cell stemness set of genes. This is in line with the expectation that tumor stem-like cells share only a limited subset of stemness features which are relevant for tumor survival.

Metastasis of hepatocellular carcinoma (HCC) is usually unrecognized before any pathological examination, resulting in time-taking treatment and poor prognosis. As a consequence, HCC patients usually show symptoms of depression. In order to suppress such psychiatric disorders and to facilitate better treatment outcome, antidepressants are prescribed. Up to present, information about the effect of antidepressants on HCC is still lacking. Therefore, we chose fluoxetine (FXT), one of the top five psychiatric prescriptions in the United States, together with the HepG2 cell model to explore its effect on HCC. Our study found that FXT (5 µM) increased the migratory distance of HepG2 cells by a factor of nearly 1.7 compared to control. In addition, our study also investigated the effect of genipin (GNP), which is an active compound from Gardenia jasminoides Ellis fruit (family Rubiaceae), on the FXT-induced HepG2 cells. Our study found that 30 and 60 µM GNP reduced the migratory distance by 42% and 74% respectively, compared to FXT treatment alone. Furthermore, we also found that FXT upregulated matrix metalloproteinases (MMPs) genes, increased the protein expression of MMPs, urokinase-type plasminogen activator (uPA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), activator protein 1 (AP-1), phosphorylated mitogen-activated protein kinase (P-p38), phosphorylated protein kinase B (P-Akt), downregulated tissue inhibitor metalloproteinases (TIMPs) genes and decreased the TIMPs proteins expression whereas, GNP fully counteracted the action of FXT. Conclusively, this study has provided valuable information regarding the possible molecular mechanisms through which FXT affects the metastatic invasiveness of HepG2 cells and evidences to support that GNP counteracts such effect via the same molecular mechanisms.

Tumor suppressor genes (TSGs), including Ten-eleven translocation 1 (TET1), are hypermethylated in hepatocellular carcinoma (HCC). TET1 catalytic domain (TET1-CD) induces genome-wide DNA demethylation to activate TSGs, but so far, anticancer effects of TET1-CD are unclear. Here we showed that after HCC cells were transiently transfected with TET1-CD, the methylation levels of TSGs, namely APC, p16, RASSF1A, SOCS1 and TET1, were distinctly reduced, and their mRNA levels were significantly increased and HCC cells proliferation, migration and invasion were suppressed, but the methylation and mRNA levels of oncogenes, namely C-myc, Bmi1, EMS1, Kpna2 and c-fos, were not significantly change. Strikingly, HCC subcutaneous xenografts in nude mice remained to be significantly repressed even 54 days after transient transfection of TET1-CD. So, transient transfection of TET1-CD may be a great advance in HCC treatment due to its activation of multiple TSGs and persistent anticancer effects.

Prohibitin 1 (PHB1) is a mitochondrial chaperone whose expression is dysregulated in cancer. In liver cancer, PHB1 acts as a tumor suppressor, but the mechanisms of tumor suppression are incompletely understood. Here we aimed to determine PHB1 target genes to better understand how PHB1 influences liver tumorigenesis. Using RNA-Seq analysis, we found interleukin-8 (IL-8) to be one of the most highly up-regulated genes following PHB1 silencing in HepG2 cells. Induction of IL-8 expression also occurred in multiple liver and nonliver cancer cell lines. We examined samples from 178 patients with hepatocellular carcinoma (HCC) and found that

BACKGROUND/AIMS: Circular RNAs (circRNAs) are key regulators in the development and progression of human cancers, however its role in non-small cell lung cancer (NSCLC) tumorigenesis is not well understood. The aim of this study is to identify the expression level of circPVT1 in NSCLC and further investigated its functional relevance with NSCLC progression both in vitro and in vivo.
METHODS: Quantative real-time PCR was used for the measurement of circPVT1 in NSCLC specimens and cell lines. Fluorescence in situ hybridization analysis (FISH) assay was used for the identification of sublocation of circPVT1 in NSCLC cells. Bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation (RIP) were performed to verify the binding of c-Fos at circPVT1 promoter region, and the direct interaction between circPVT1 and miR-125b. Gain- or loss-function assays were performed to evaluate the effects of circPVT1 on cell proliferation and invasion. Western blot and immunohistochemistry assays were performed to detect the protein levels involved in E2F2 pathway.
RESULTS: We found that circPVT1 was upregulated in NSCLC specimens and cells. The transcription factor c-Fos binded to the promoter region of circPVT1, resulting in the overexpression of circPVT1 in NSCLC. Knockdown of circPVT1 suppressed NSCLC cell proliferation, migration and invasion, and increased apoptosis. In addition, circPVT1 mediated NSCLC progression via the regulation of E2F2 signaling pathway. More importantly, circPVT1 was predominantly abundant in the cytoplasm of NSCLC cells, and circPVT1 could serve as a competing endogenous RNA to regulate E2F2 expression and tumorigenesis in a miR-125b-dependent manner, which is further verified by using an in vivo xenograft model.
CONCLUSION: circPVT1 promotes NSCLC cell growth and invasion, and may serve as a promising therapeutic target for NSCLC patients. Therefore, silence of circPVT1 could be a future direction to develop a novel treatment strategy.

Although most cutaneous squamous cell carcinomas (cSCCs) develop from actinic keratoses (AKs), the key events in this evolution remain unclear. We have combined the results of different genomic and expression array platforms on matched concomitant samples of sun-exposed skin (SES), AK, and cSCC from 10 immunocompetent patients. Gene expression analysis and copy number alterations were assessed using GeneChip Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA) and CytoScan HD Cytogenetics Solution (Affymetrix) platforms, respectively. Integration of transcriptome and genome results was evaluated using the DR-Integrator tool. Additional studies (qPCR, immunohistochemistry, and Western blot) were performed for selected genes. FOSL1 and BNC1 encode transcription factors whose expression was increased in cSCC in the expression array and the qPCR. By immunohistochemistry, FOSL1 showed an intense staining at the invasive front of cSCC samples and BNC1 expression varied from a nuclear (SES) to a cytoplasmic location (cSCC). Western blot analyses confirmed the enhancement of FOSL1 and BNC1. In addition, the smallest overlapping regions (SORIs) of genomic imbalance involving at least three of the samples were selected. One of the SORIs was a deletion in the p24.1 band of chromosome 3, shared by seven of the cSCCs. A strong correlation in the integration analysis was found for NEK10, a gene contained in the previously mentioned SORI. Loss of NEK10 expression in cSCC was confirmed by immunohistochemistry and Western blot analyses. In addition, functional studies in NEK10 depleted cells were performed. In conclusion, we identified FOSL1 and BNC1, which could act as tumor drivers, and NEK10, which could function as a tumor suppressor, to be differentially expressed during cSCC development.

INTRODUCTION: Adaptogens are natural compounds or plant extracts that increase adaptability and survival of organisms under stress. Adaptogens stimulate cellular and organismal defense systems by activating intracellular and extracellular signaling pathways and expression of stress-activated proteins and neuropeptides. The effects adaptogens on mediators of adaptive stress response and longevity signaling pathways have been reported, but their stress-protective mechanisms are still not fully understood.
AIM OF THE STUDY: The aim of this study was to identify key molecular mechanisms of adaptogenic plants traditionally used to treat stress and aging-related disorders, i.e., Rhodiola rosea, Eleutherococcus senticosus, Withania somnifera, Rhaponticum carthamoides, and Bryonia alba.
MATERIALS AND METHODS: To investigate the underlying molecular mechanisms of adaptogens, we conducted RNA sequencing to profile gene expression alterations in T98G neuroglia cells upon treatment of adaptogens and analyzed the relevance of deregulated genes to adaptive stress-response signaling pathways using in silico pathway analysis software.
RESULTS AND DISCUSSION: At least 88 of the 3516 genes regulated by adaptogens were closely associated with adaptive stress response and adaptive stress-response signaling pathways (ASRSPs), including neuronal signaling related to corticotropin-releasing hormone, cAMP-mediated, protein kinase A, and CREB; pathways related to signaling involving CXCR4, melatonin, nitric oxide synthase, GP6, Gαs, MAPK, neuroinflammation, neuropathic pain, opioids, renin-angiotensin, AMPK, calcium, and synapses; and pathways associated with dendritic cell maturation and G-coupled protein receptor-mediated nutrient sensing in enteroendocrine cells. All samples tested showed significant effects on the expression of genes encoding neurohormones CRH, GNRH, UCN, G-protein-coupled and other transmembrane receptors TLR9, PRLR, CHRNE, GP1BA, PLXNA4, a ligand-dependent nuclear receptor RORA, transmembrane channels, transcription regulators FOS, FOXO6, SCX, STAT5A, ZFPM2, ZNF396, ZNF467, protein kinases MAPK10, MAPK13, MERTK, FLT1, PRKCH, ROS1, TTN), phosphatases PTPRD, PTPRR, peptidases, metabolic enzymes, a chaperone (HSPA6), and other proteins, all of which modulate numerous life processes, playing key roles in several canonical pathways involved in defense response and regulation of homeostasis in organisms. It is for the first time we report that the molecular mechanism of actions of melatonin and plant adaptogens are alike, all adaptogens tested activated the melatonin signaling pathway by acting through two G-protein-coupled membrane receptors MT1 and MT2 and upregulation of the ligand-specific nuclear receptor RORA, which plays a role in intellectual disability, neurological disorders, retinopathy, hypertension, dyslipidemia, and cancer, which are common in aging. Furthermore, melatonin activated adaptive signaling pathways and upregulated expression of UCN, GNRH1, TLR9, GP1BA, PLXNA4, CHRM4, GPR19, VIPR2, RORA, STAT5A, ZFPM2, ZNF396, FLT1, MAPK10, MERTK, PRKCH, and TTN, which were commonly regulated by all adaptogens tested. We conclude that melatonin is an adaptation hormone playing an important role in regulation of homeostasis. Adaptogens presumably worked as eustressors ("stress-vaccines") to activate the cellular adaptive system by inducing the expression of ASRSPs, which then reciprocally protected cells from damage caused by distress. Functional investigation by interactive pathways analysis demonstrated that adaptogens activated ASRSPs associated with stress-induced and aging-related disorders such as chronic inflammation, cardiovascular health, neurodegenerative cognitive impairment, metabolic disorders, and cancer.
CONCLUSION: This study has elucidated the genome-wide effects of several adaptogenic herbal extracts in brain cells culture. These data highlight the consistent activation of ASRSPs by adaptogens in T98G neuroglia cells. The extracts affected many genes playing key roles in modulation of adaptive homeostasis, indicating their ability to modify gene expression to prevent stress-induced and aging-related disorders. Overall, this study provides a comprehensive look at the molecular mechanisms by which adaptogens exerts stress-protective effects.

Histone deacetylases (HDACs) can regulate cell-cycle, differentiation, and apoptosis of hepatocarcinoma (HCC) cells, while their roles in drug sensitivity remain unclear. Our results showed that the expression of HDAC2 was significantly increased in HCC doxorubicin (Dox) resistant cells as compared with their corresponding control cells. Over expression of HDAC2 can increase the cell viability and decrease the Dox sensitivity. Kaplan-Meier Plotter assay revealed that HCC patients with higher levels of HDAC2 had significantly poor prognosis than that of the lower expression patients. Mechanistically studies revealed that HDAC2 can regulate the transcription of ABCB1 via directly binding with its promoter and increasing its expression in Dox resistant HCC cells. Knockdown of HDAC2 significantly inhibited the expression of ABCB1. Co-immunoprecipitation revealed that HDAC2 can bind with c-fos, an important transcription factor of ABCB1, in HCC/Dox cells. Knockdown of c-Fos decreased the binding between HDAC2 and promoter of ABCB1 in HCC/Dox cells. Collectively, our data revealed that HDAC2 can regulate Dox sensitivity of HCC cells and the transcription of ABCB1.

Casticin is one of the major active components isolated from

The programmed cell death protein 1-programmed death-ligand 1 (PD-L1) axis has been successfully targeted in clinics and the use of immune check-point inhibitors have shown durable antitumor response in untreated or heavily treated advanced stage cancer. PD-L1 upregulation has been found to correlate with poor prognosis in multiple cancer types and expression of PD-L1 in intratumoral compartment has been suggested to influence immune response and act as a key determinant of checkpoint immunotherapy efficacy. Hence it becomes critical to understand the regulation of PD-L1 expression in cancer. Role of oncogenic signaling pathways and transcription factors such as PI3K-AKT, MEK-ERK, JAK-STAT, MYC, HIF-1α, AP-1 and NF-κB is well established in inducing PD-L1 expression. Even the structural variations resulting in the truncation of the 3' untranslated region (UTR) of PD-L1 has been shown to upregulate PD-L1 expression in multiple cancer types. Since microRNAs carry out post-transcriptional gene silencing by binding to the 3' UTR of its target messenger RNA, truncation of PD-L1 3' UTR can result in alleviation of PD-L1 suppression mediated by microRNA, leading to its overexpression. Other epigenetic modifications, such as promoter DNA methylation and histone modifications can also play crucial role in regulating PD-L1 expression. Here, we review recent findings and evidence on epigenetic mechanisms that regulate PD-L1 expression and the biological and clinical implications of such regulation in cancer.

Osteosarcoma (OS) is a rare tumor of the bone occurring mainly in young adults accounting for 5% of all childhood cancers. Because of the limited therapeutic options, there has been no survival improvement for OS patients in the past 40 years. The epidermal growth factor receptor (EGFR) is highly expressed in OS; however, its clinical relevance is unclear. Here, we employed an autochthonous c-Fos-dependent OS mouse model (H2

BACKGROUND: DNA methylation is a key epigenetic mark in mammalian organisms that plays key roles in chromatin organization and gene expression. Although DNA methylation in gene promoters is generally associated with gene repression, recent studies demonstrate that DNA methylation in gene bodies and intergenic regions of the genome may result in distinct modes of gene regulation. Furthermore, the molecular mechanisms underlying the establishment and maintenance of DNA methylation in human health and disease remain to be fully elucidated. We recently demonstrated that a subset of long non-coding RNAs (lncRNAs) associates with the major DNA methyltransferase DNMT1 in human colon cancer cells, and the dysregulation of such lncRNAs contribute to aberrant DNA methylation patterns.
RESULTS: In the current study, we assessed the impact of a key DNMT1-associated lncRNA, DACOR1, on genome-wide DNA methylation using reduced representation bisulfite sequencing (RRBS). Our findings demonstrated that induction of DACOR1 in colon cancer cells restores DNA methylation at thousands of CpG sites throughout the genome including promoters, gene bodies, and intergenic regions. Importantly, these sites overlap with regions of the genome that become hypomethylated in colon tumors. Furthermore, induction of DACOR1 results in repression of FOS and JUN and, consequently, reduced AP-1 transcription factor activity.
CONCLUSION: Collectively, our results demonstrate a key role of lncRNAs in regulating DNA methylation in human cells, and the dysregulation of such lncRNAs could emerge as a key mechanism by which DNA methylation patterns become altered in human tumors.

Preoperative progesterone intervention has been shown to confer a survival benefit to breast cancer patients independently of their progesterone receptor (PR) status. This observation raises the question how progesterone affects the outcome of PR-negative cancer. Here, using microarray and RNA-Seq-based gene expression profiling and ChIP-Seq analyses of breast cancer cells, we observed that the serum- and glucocorticoid-regulated kinase gene (

Undifferentiated pleomorphic sarcoma (UPS) is the second most frequent soft tissue sarcoma. Because of its resistance to chemotherapy, UPS patients are treated with surgical resection and complementary radiotherapy. However, since standard chemotherapy has not been established, unresectable or metastatic cases result in a poor prognosis. Therefore, the identification of a more effective therapy for UPS patients is needed. The development and progression of malignant tumors involve epigenetic alterations, and histone deacetylases (HDAC) have become a promising chemotherapeutic target. In this study, we investigated the potential effects and mechanisms of an HDAC inhibitor, LBH589, in UPS cells. We confirmed that LBH589 exhibits potent antitumor activities in four human UPS cell lines (GBS-1, TNMY-1, Nara-F, and Nara-H) and IC

Estrogen receptor alpha (ERα), which has been detected in over 70% of breast cancer cases, is a driving factor for breast cancer growth. For investigating the underlying genes and networks regulated by ERα in breast cancer, RNA-seq was performed between ERα transgenic MDA-MB-231 cells and wild type MDA-MB-231 cells. A total of 267 differentially expressed genes (DEGs) were identified. Then bioinformatics analyses were performed to illustrate the mechanism of ERα. Besides, by comparison of RNA-seq data obtained from MDA-MB-231 cells and microarray dataset obtained from estrogen (E2) stimulated MCF-7 cells, an overlap of 126 DEGs was screened. The expression level of ERα was negatively associated with metastasis and EMT in breast cancer. We further verified that ERα might inhibit metastasis by regulating of VCL and TNFRSF12A, and suppress EMT by the regulating of JUNB and ID3. And the relationship between ERα and these genes were validated by RT-PCR and correlation analysis based on TCGA database. By PPI network analysis, we identified TOP5 hub genes, FOS, SP1, CDKN1A, CALCR and JUNB, which were involved in cell proliferation and invasion. Taken together, the whole-genome insights carried in this work can help fully understanding biological roles of ERα in breast cancer.