Small-molecule α-lipoic acid targets ELK1 to balance human neutrophil and erythrocyte differentiation.

BACKGROUND: Erythroid and myeloid differentiation disorders are commonly occurred in leukemia. Given that the relationship between erythroid and myeloid lineages is still unclear. To find the co-regulators in erythroid and myeloid differentiation might help to find new target for therapy of myeloid leukemia. In hematopoiesis, ALA (alpha lipoic acid) is reported to inhibit neutrophil lineage determination by targeting transcription factor ELK1 in granulocyte-monocyte progenitors via splicing factor SF3B1. However, further exploration is needed to determine whether ELK1 is a common regulatory factor for erythroid and myeloid differentiation. METHODS: In vitro culture of isolated CD34+, CMPs (common myeloid progenitors) and CD34+ CD371- HSPCs (hematopoietic stem progenitor cells) were performed to assay the differentiation potential of monocytes, neutrophils, and erythrocytes. Overexpression lentivirus of long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 transduced CD34+ HSPCs were transplanted into NSG mice to assay the human lymphocyte and myeloid differentiation differences 3 months after transplantation. Knocking down of SRSF11, which was high expressed in CD371+GMPs (granulocyte-monocyte progenitors), upregulated by ALA and binding to ELK1-RNA splicing site, was performed to analyze the function in erythroid differentiation derived from CD34+ CD123mid CD38+ CD371- HPCs (hematopoietic progenitor cells). RNA sequencing of L-ELK1 and S-ELK1 overexpressed CD34+ CD123mid CD38+ CD371- HPCs were performed to assay the signals changed by ELK1. RESULTS: Here, we presented new evidence that ALA promoted erythroid differentiation by targeting the transcription factor ELK1 in CD34+ CD371- hematopoietic stem progenitor cells (HSPCs). Overexpression of either the long isoform (L-ELK1) or the short isoform (S-ELK1) of ELK1 inhibited erythroid-cell differentiation, but knockdown of ELK1 did not affect erythroid-cell differentiation. RNAseq analysis of CD34+ CD123mid CD38+ CD371- HPCs showed that L-ELK1 upregulated the expression of genes related to neutrophil activity, phosphorylation, and hypoxia signals, while S-ELK1 mainly regulated hypoxia-related signals. However, most of the genes that were upregulated by L-ELK1 were only moderately upregulated by S-ELK1, which might be due to a lack of serum response factor interaction and regulation domains in S-ELK1 compared to L-ELK1. In summary, the differentiation of neutrophils and erythrocytes might need to rely on the dose of L-ELK1 and S-ELK1 to achieve precise regulation via RNA splicing signals at early lineage commitment. CONCLUSIONS: ALA and ELK1 are found to regulate both human granulopoiesis and erythropoiesis via RNA spliceosome, and ALA-ELK1 signal might be the target of human leukemia therapy.

High expression of RTEL1 predicates worse progression in gliomas and promotes tumorigenesis through JNK/ELK1 cascade.

Gliomas are the most common primary intracranial tumor worldwide. The maintenance of telomeres serves as an important biomarker of some subtypes of glioma. In order to investigate the biological role of RTEL1 in glioma. Relative telomere length (RTL) and RTEL1 mRNA was explored and regression analysis was performed to further examine the relationship of the RTL and the expression of RTEL1 with clinicopathological characteristics of glioma patients. We observed that high expression of RTEL1 is positively correlated with telomere length in glioma tissue, and serve as a poor prognostic factor in TERT wild-type patients. Further in vitro studies demonstrate that RTEL1 promoted proliferation, formation, migration and invasion ability of glioma cells. In addition, in vivo studies also revealed the oncogene role of RTEL1 in glioma. Further study using RNA sequence and phospho-specific antibody microarray assays identified JNK/ELK1 signaling was up-regulated by RTEL1 in glioma cells through ROS. In conclusion, our results suggested that RTEL1 promotes glioma tumorigenesis through JNK/ELK1 cascade and indicate that RTEL1 may be a prognostic biomarker in gliomas.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

BRCA1 and ELK-1 regulate neural progenitor cell fate in the optic tectum in response to visual experience in Xenopus laevis tadpoles.

In developing Xenopus tadpoles, the optic tectum begins to receive patterned visual input while visuomotor circuits are still undergoing neurogenesis and circuit assembly. This visual input regulates neural progenitor cell fate decisions such that maintaining tadpoles in the dark increases proliferation, expanding the progenitor pool, while visual stimulation promotes neuronal differentiation. To identify regulators of activity-dependent neural progenitor cell fate, we profiled the transcriptomes of proliferating neural progenitor cells and newly differentiated neurons using RNA-Seq. We used advanced bioinformatic analysis of 1,130 differentially expressed transcripts to identify six differentially regulated transcriptional regulators, including Breast Cancer 1 (BRCA1) and the ETS-family transcription factor, ELK-1, which are predicted to regulate the majority of the other differentially expressed transcripts. BRCA1 is known for its role in cancers, but relatively little is known about its potential role in regulating neural progenitor cell fate. ELK-1 is a multifunctional transcription factor which regulates immediate early gene expression. We investigated the potential functions of BRCA1 and ELK-1 in activity-regulated neurogenesis in the tadpole visual system using in vivo time-lapse imaging to monitor the fate of GFP-expressing SOX2+ neural progenitor cells in the optic tectum. Our longitudinal in vivo imaging analysis showed that knockdown of either BRCA1 or ELK-1 altered the fates of neural progenitor cells and furthermore that the effects of visual experience on neurogenesis depend on BRCA1 and ELK-1 expression. These studies provide insight into the potential mechanisms by which neural activity affects neural progenitor cell fate.

Mutant p53 and ELK1 co-drive FRA-1 expression to induce metastasis in breast cancer.

Tumor-associated p53 mutations induce activities different from wild-type p53, thus causing loss of the protein's tumor inhibition function. The cells carrying p53 mutations have more aggressive characteristics related to invasion, metastasis, proliferation, and cell survival. By comparing the gene expression profiles of mutant p53 (mutp53) and mutp53 silenced cohorts, we found that FOS-related antigen-1 (FRA-1), which is encoded by FOSL1, is a potential effector of mutp53-mediated metastasis. We demonstrate that the expression of FRA-1, a gatekeeper of mesenchymal-epithelial transition, is elevated in the presence of p53 mutations. Mechanistically, mutant p53 cooperates with the transcription factor ELK1 in binding and activating the promoter of FOSL1, thus fostering lung metastasis. This study reveals new insights into how mutant p53 contributes to metastasis in breast cancer.

Aberrant expression of CKS2 induced by ELK1 contributes to malignant progression of pancreatic cancer.

Cyclin-dependent kinase subunit 2 (CKS2) has been reported to promote various malignancies. This study investigated the functional role of CKS2 in pancreatic cancer (PC). An analysis of abnormally expressed genes and their prognostic value for PC was performed by using the Gene Expression Profiling Interactive Analysis (GEPIA) database and performing immunohistochemical staining on 64 samples of tumor tissue. CCK-8 assays, EdU staining, colony formation assays, flow cytometry, and a xenograft tumor model were used to analyze the biological function of CKS2 in PC. Our results revealed that CKS2 was expressed at significantly higher levels in PC tissues than in adjacent normal tissues, and a high level of CKS2 expression was associated with a poor prognosis for patients with PC. Moreover, functional assays revealed that CKS2 knockdown suppressed cell proliferation, induced cell cycle S phase, G2/M phase arrest, and apoptosis in vitro, and also reduced tumor growth in vivo. In addition, CKS2 knockdown increased the levels of Bax, caspase-3, P53, P21, and GADD45α expression, but decreased Bcl-2, Cyclin B1, CDK1, Cyclin A, and Cdc25C expression. CKS2 overexpression produced the opposite effects of CKS2 knockdown. Furthermore, we found that ELK1 protein regulated transcription of the CKS2 gene. In conclusion, our findings suggest that CKS2 expression is regulated by ELK1, which could possibly serve as prognostic indicator and therapeutic target for PC.

Role of ELK1 in regulating colorectal cancer progression: miR-31-5p/CDIP1 axis in CRC pathogenesis.

Background and Objective: Colorectal cancer (CRC) is a malignant tumor that affects the digestive system. With the increased of modernization of society, the incidence of colorectal cancer has increased throughout the world. As a transcription factor, ELK1 has been widely studied in colorectal cancer. However, there are still many unknown factors regarding its specific mechanism of action.This study explored the role of ELK1 and its downstream pathway in CRC pathogenesis. Methods: Based on clinical samples, this study examined miR-31-5p expression in CRC cells and its impact on malignant behaviors (migration, invasion, apoptosis) and autophagy. The promoter sequence of miR-31-5p was obtained from the UCSC database, and ELK1 was identified as its transcription factor. In ELK1-knockdown CRC cells, miR-31-5p was overexpressed, and its response in malignant behaviors and autophagy was analyzed. The target gene CDIP1 was predicted and verified using a dual-luciferase assay. The influence of CDIP1 on malignant behavior in CRC cells was assessed, and CDIP1 siRNA was used as a rescue treatment for miR-31-5p inhibition. The role of ELK1/miR-31-5p in tumor growth was validated in vivo. Results: miR-31-5p expression was upregulated in the colorectal cancer tissues and cells. The knockdown of miR-31-5p markedly inhibited cancer cells' malignant behaviors and mediated autophagy. ELK1 was confirmed to bind with the miR-31-5p promoter and enhance miR-31-5p transcription. miR-31-5p was found to bind with the CDIP1 3'UTR and inhibit CDIP1 expression. CDIP1 siRNA partially rescued the effects of miR-31-5p knockdown on cell metastatic ability, autophagy, and apoptosis. Based on the in vivo experiments, results showed that the ELK1/miR-31-5p axis positively regulated tumor growth in nude mice. Conclusion: Our findings indicate that ELK1 regulates the progression of colorectal cancer via an miR-31-5p/CDIP1 axis, and the ELK1/miR-31-5p/CDIP1 axis could be a therapeutic target for colorectal cancer.

Androgen receptor knockdown enhances prostate cancer chemosensitivity by down-regulating FEN1 through the ERK/ELK1 signalling pathway.

PURPOSE: Flap endonuclease 1 (FEN1) is highly upregulated in prostate cancer and promotes the growth of prostate cancer cells. Androgen receptor (AR) is the most critical determinant of the occurrence, progression, metastasis, and treatment of prostate cancer. However, the effect of FEN1 on docetaxel (DTX) sensitivity and the regulatory mechanisms of AR on FEN1 expression in prostate cancer need to be further studied. METHODS: Bioinformatics analyses were performed using data from the Cancer Genome Atlas and the Gene Expression Omnibus. Prostate cancer cell lines 22Rv1 and LNCaP were used. FEN1 siRNA, FEN1 overexpression plasmid, and AR siRNA were transfected into cells. Biomarker expression was evaluated by immunohistochemistry and Western blotting. Apoptosis and the cell cycle were explored using flow cytometry analysis. Luciferase reporter assay was performed to verify the target relationship. Xenograft assays were conducted using 22Rv1 cells to evaluate the in vivo conclusions. RESULTS: Overexpression of FEN1 inhibited cell apoptosis and cell cycle arrest in the S phase induced by DTX. AR knockdown enhanced DTX-induced cell apoptosis and cell cycle arrest at the S phase in prostate cancer cells, which was attenuated by FEN1 overexpression. In vivo experiments showed that overexpression of FEN1 significantly increased tumour growth and weakened the inhibitory effect of DTX on prostate tumour growth, while AR knockdown enhance the sensitivity of DTX to prostate tumour. AR knockdown resulted in FEN1, pho-ERK1/2, and pho-ELK1 downregulation, and the luciferase reporter assay confirmed that ELK1 can regulate the transcription of FEN1. CONCLUSION: Collectively, our studies demonstrate that AR knockdown improves the DTX sensitivity of prostate cancer cells by downregulating FEN1 through the ERK/ELK1 signalling pathway.

ELK1/KIFC1 axis promotes breast cancer cell proliferation by regulating glutathione metabolism.

BACKGROUND: KIFC1 exerts an important function in centrosome aggregation in breast cancer (BC) cells and a variety of other cancer cells, but its potential mechanisms in BC pathogenesis are yet fully elucidated. The aim of this study was to investigate the effects of KIFC1 on BC progression and its underlying mechanisms. METHODS: Expression of ELK1 and KIFC1 in BC was analyzed by The Cancer Genome Atlas database and quantitative real-time polymerase chain reaction. Cell proliferative capacity was examined by CCK-8 and colony formation assays, respectively. Glutathione (GSH)/glutathione disulfide (GSSG) ratio and GSH level were measured using the kit. Expression of GSH metabolism-related enzymes (G6PD, GCLM, and GCLC) was detected by western blot. Intracellular reactive oxygen species (ROS) levels were measured by the ROS Assay Kit. The transcription factor ELK1 upstream of KIFC1 was identified by hTFtarget, KnockTFv2 database and Pearson correlation. Their interaction was validated by dual-luciferase reporter assay and chromatin immunoprecipitation. RESULTS: This study demonstrated the upregulation of ELK1 and KIFC1 in BC and found that ELK1 could bind to the KIFC1 promoter to promote KIFC1 transcription. KIFC1 overexpression increased cell proliferation and intracellular GSH levels, while decreasing intracellular ROS levels. The addition of the GSH metabolism inhibitor BSO attenuated the promotion of BC cell proliferation induced by KIFC1 overexpression. In addition, KIFC1 overexpression reversed the inhibitory effect of knockdown of ELK1 on BC cell proliferation. CONCLUSION: ELK1 was a transcriptional factor of KIFC1. ELK1/KIFC1 axis reduced ROS level by increasing GSH synthesis, thus facilitating BC cell proliferation. Current observations suggest that ELK1/ KIFC1 may be a potential therapeutic target for BC treatment.

Transcription factor ELK1 regulates the expression of histone 3 lysine 9 to affect developmental potential of porcine preimplantation embryos.

A series of changes occur in the early embryo that are critical for subsequent development, and the pig is an excellent animal model of human disease, so understanding the regulatory mechanisms of early embryonic development in the pig is of very importance. To find key transcription factors regulating pig early embryonic development, we first profiled the transcriptome of pig early embryos, and confirmed that zygotic gene activation (ZGA) in porcine embryos starts from 4 cell stage. Subsequent enrichment analysis of up-regulated gene motifs during ZGA revealed that the transcription factor ELK1 ranked first. The expression pattern of ELK1 in porcine early embryos was analyzed by immunofluorescence staining and qPCR, and the results showed that the transcript level of ELK1 reached the highest at the 8 cell stage, while the protein level reached the highest at 4 cell stage. To further investigate the effect of ELK1 on early embryo development in pigs, we silenced ELK1 in zygotes and showed that ELK1 silencing significantly reduced cleavage rate, blastocyst rate as well as blastocyst quality. A significant decrease in the expression of the pluripotency gene Oct4 was also observed in blastocysts from the ELK1 silenced group by immunofluorescence staining. Silencing of ELK1 also resulted in decreased H3K9Ac modification and increased H3K9me3 modification at 4 cell stage. To investigate the effect of ELK1 on ZGA, we analyzed transcriptome changes in 4 cell embryos after ELK1 silencing by RNA seq, which revealed that ELK1 silencing resulted in significant differences in the expression of a total of 1953 genes at the 4 cell stage compared with their normal counterparts, including 1106 genes that were significantly upregulated and 847 genes that were significantly downregulated. Through GO and KEGG enrichment, we found that the functions and pathways of down-regulated genes were concentrated in protein synthesis, processing, cell cycle regulation, etc., while the functions of up-regulated genes were focused on aerobic respiration process. In conclusion, this study demonstrates that the transcription factor ELK1 plays an important role in regulation of preimplantation embryo development of pigs and deficiency of ELK1 leads to abnormal epigenetic reprogramming as well as zygotic genome activation, thus adversely affecting embryonic development. This study will provide important reference for the regulation of transcription factors in porcine embryo development.

Hsa_circ_0008500 inhibits apoptosis of adipose-derived stem cells under high glucose through hsa-miR-1273h-5p/ELK1 axis.

Preliminary researches have confirmed that the number of apoptosis of adipose tissue-derived stem cells (ADSCs) in patients with diabetes is significantly increased, leading to a difficult healing wound. Increasing researches revealed that circular RNAs (circRNAs) can control apoptosis. However, it is still unclear whether and how circRNAs are critical for regulating ADSCs apoptosis. In this study, we utilized in vitro model in which ADSCs were cultivated with normal glucose (NG) (5.5 mM) or high glucose (HG) (25 mM) medium, respectively, and found that more apoptotic ADSCs were observed in HG medium comparing to ADSCs in NG medium. Furthermore, we found that hsa_circ_0008500 attenuated HG-mediated ADSCs apoptosis. In addition, Hsa_circ_0008500 could directly interact with hsa-miR-1273h-5p, acting as a miRNA sponge, which subsequently suppressed Ets-like protein-1(ELK1) expression, the downstream target of hsa-miR-1273h-5p. Thus, these results indicated that targeting the hsa_circ_0008500/hsa-miR-1273h-5p/ELK1 signaling pathway in ADSCs may be a potential target for repairing diabetic wounds.

The Small Molecule Antagonist KCI807 Disrupts Association of the Amino-Terminal Domain of the Androgen Receptor with ELK1 by Modulating the Adjacent DNA Binding Domain.

The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a glutathione S-transferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.

Comparative analysis of protein expression systems and PTM landscape in the study of transcription factor ELK-1.

Post-translational modifications (PTMs) are important for protein folding and activity, and the ability to recreate physiologically relevant PTM profiles on recombinantly-expressed proteins is vital for meaningful functional analysis. The ETS transcription factor ELK-1 serves as a paradigm for cellular responses to mitogens and can synergise with androgen receptor to promote prostate cancer progression, although in vitro protein function analyses to date have largely overlooked its complex PTM landscapes. We expressed and purified human ELK-1 using mammalian (HEK293T), insect (Sf9) and bacterial (E. coli) systems in parallel and compared PTMs imparted upon purified proteins, along with their performance in DNA and protein interaction assays. Phosphorylation of ELK-1 within its transactivation domain, known to promote DNA binding, was most apparent in protein isolated from human cells and accordingly conferred the strongest DNA binding in vitro, while protein expressed in insect cells bound most efficiently to the androgen receptor. We observed lysine acetylation, a hitherto unreported PTM of ELK-1, which appeared highest in insect cell-derived ELK-1 but was also present in HEK293T-derived ELK-1. Acetylation of ELK-1 was enhanced in HEK293T cells following starvation and mitogen stimulation, and modified lysines showed overlap with previously identified regulatory SUMOylation and ubiquitination sites. Our data demonstrate that the choice of recombinant expression system can be tailored to suit biochemical application rather than to maximise soluble protein production and suggest the potential for crosstalk and antagonism between different PTMs of ELK-1.

Circ_0022340 promotes colorectal cancer progression via HNRNPC/EBF1/SYT7 or miR-382-5p/ELK1 axis.

Circular RNAs (circRNAs) are characterized as a class of new noncoding RNAs and function in tumorigenesis of colorectal cancer (CRC). In our study, the molecule mechanism of circ_0022340 in CRC was investigated. For this aim, quantitative real-time polymerase chain reaction (RT-qPCR) was used to test gene expression in CRC cells. Cell function assays including 5-ethynyl-20-deoxyuridine (EdU), colony formation and transwell investigated the proliferation and migration capacity in CRC cells. Luciferase reporter and RNA immunoprecipitation (RIP)assays determined the interaction between circRNA, miRNA and mRNA. Western blot was used to test protein expression. An immunohistochemistry assay was used to assess the tumor growth in vivo. Results showed that Circ_0022340 was highly expressed in CRC cells. Circ_0022340 was formed from exon 5 to 6 of the synaptotagmin 7 (SYT7). Silencing of circ_0022340 suppressed CRC cell proliferation and migration. Functionally, circ_0022340 recruited heterogeneous nuclear ribonucleoprotein C (HNRNPC) to stabilize EBF1 mRNA and thereby activated SYT7. Moreover, circ_0022340 targeted miR-382-5p to up-regulate ETS transcription factor ELK1 (ELK1). It is concluded that Circ_0022340 promoted colorectal cancer progression via recruiting HNRNPC to stabilize EBF1 mRNA and thereby activated SYT7 or miR-382-5p/ELK1 axis, which might provide a novel target for CRC treatment.

The autism risk factor CHD8 is a chromatin activator in human neurons and functionally dependent on the ERK-MAPK pathway effector ELK1.

The chromodomain helicase DNA-binding protein CHD8 is the most frequently mutated gene in autism spectrum disorder. Despite its prominent disease involvement, little is known about its molecular function in the human brain. CHD8 is a chromatin regulator which binds to the promoters of actively transcribed genes through genomic targeting mechanisms which have yet to be fully defined. By generating a conditional loss-of-function and an endogenously tagged allele in human pluripotent stem cells, we investigated the molecular function and the interaction of CHD8 with chromatin in human neurons. Chromatin accessibility analysis and transcriptional profiling revealed that CHD8 functions as a transcriptional activator at its target genes in human neurons. Furthermore, we found that CHD8 chromatin targeting is cell context-dependent. In human neurons, CHD8 preferentially binds at ETS motif-enriched promoters. This enrichment is particularly prominent on the promoters of genes whose expression significantly changes upon the loss of CHD8. Indeed, among the ETS transcription factors, we identified ELK1 as being most highly correlated with CHD8 expression in primary human fetal and adult cortical neurons and most highly expressed in our stem cell-derived neurons. Remarkably, ELK1 was necessary to recruit CHD8 specifically to ETS motif-containing sites. These findings imply that ELK1 and CHD8 functionally cooperate to regulate gene expression and chromatin states at MAPK/ERK target genes in human neurons. Our results suggest that the MAPK/ERK/ELK1 axis potentially contributes to the pathogenesis caused by CHD8 mutations in human neurodevelopmental disorders.

ELK1-mediated YTHDF1 drives prostate cancer progression by facilitating the translation of Polo-like kinase 1 in an m6A dependent manner.

Background: N6-methyladenosine (m6A) is one of the most prevalent mRNA modifications in mammals, and it regulates the fate of modified RNA transcripts. In the current study, we aimed to elucidate the role of YTH m6A RNA-binding protein 1 (YTHDF1), a "reader" of m6A modification, in prostate cancer tumorigenesis. Methods: We employed a multi-omics approach to detect the direct target of YTHDF1 upon manipulation of YTHDF1 expression in prostate cancer cells. Expression of YTHDF1 was also evaluated in human prostate tumors and either adjacent or paired normal tissues. Additionally, in vivo tumor growth and metastasis experimental assays were performed to evaluate the role of YTHDF1 in tumorigenesis. Finally, luciferase reporter assays and Chromatin immunoprecipitation (ChIP) were conducted to elucidate the transcriptional regulators of YTHDF1. Results: We demonstrated that polo-like kinase 1 (PLK1) is a direct target of YTHDF1. YTHDF1 facilitated the translation efficiency of PLK1 in an m6A-dependent manner by identifying the m6A-modified PLK1 mRNA and subsequently promoted the hyperactivation of the PI3K/AKT signaling pathway. Moreover, our results indicated that YTHDF1 was upregulated in prostate cancer tissue and that high YTHDF1 expression was associated with adverse prognosis in patients with prostate cancer. Furthermore, upregulation of YTHDF1 promoted prostate cancer tumorigenesis and metastasis in vitro and in vivo. Additionally, dysregulation of ETS transcription factor ELK1 activated the transcription of YTHDF1 by directly binding to its promoter region. Conclusions: Collectively, our findings suggest that the ELK1/YTHDF1/PLK1/PI3K/AKT axis is critical for prostate cancer progression and may serve as a potential therapeutic target for prostate cancer treatment.

BCL6 is regulated by the MAPK/ELK1 axis and promotes KRAS-driven lung cancer.

Mutational activation of KRAS is a common oncogenic event in lung cancer, yet effective therapies are still lacking. Here, we identify B cell lymphoma 6 (BCL6) as a lynchpin in KRAS-driven lung cancer. BCL6 expression was increased upon KRAS activation in lung tumor tissue in mice and was positively correlated with the expression of KRAS-GTP, the active form of KRAS, in various human cancer cell lines. Moreover, BCL6 was highly expressed in human KRAS-mutant lung adenocarcinomas and was associated with poor patient survival. Mechanistically, the MAPK/ERK/ELK1 signaling axis downstream of mutant KRAS directly regulated BCL6 expression. BCL6 maintained the global expression of prereplication complex components; therefore, BCL6 inhibition induced stalling of the replication fork, leading to DNA damage and growth arrest in KRAS-mutant lung cancer cells. Importantly, BCL6-specific knockout in lungs significantly reduced the tumor burden and mortality in the LSL-KrasG12D/+ lung cancer mouse model. Likewise, pharmacological inhibition of BCL6 significantly impeded the growth of KRAS-mutant lung cancer cells both in vitro and in vivo. In summary, our findings reveal a crucial role of BCL6 in promoting KRAS-addicted lung cancer and suggest BCL6 as a therapeutic target for the treatment of this intractable disease.

ELK1 suppresses SYTL1 expression by recruiting HDAC2 in bladder cancer progression.

ETS transcription factor (ELK1) stimulates the expression of genes at the onset of the cell cycle and participates in early developmental programming. Here, we investigated whether alterations of ELK1 lead to progression of bladder cancer (BCa), a main neoplasm of urinary tract, and clarified the function of ELK1 in BCa. Using the GEO database, we identified ELK1 as the most significantly overexpressed gene in BCa, which was substantiated in the acquired clinical samples and cells. Silencing of ELK1 inhibited the malignant phenotype of BCa cells. Further analysis revealed that ELK1 synergized with histone deacetylase 2 (HDAC2) to specifically bind to the synaptotagmin like 1 (SYTL1) promoter, thereby repressing SYTL1 transcription and protein expression. Depletion of SYTL1 reversed the repressive effects of ELK1 depletion on the malignant phenotype of BCa cells. Our in vitro findings were reproduced in vivo on a nude mouse tumorigenic model. Together, our results reveal that ELK1, through suppression of SYTL1 via HDAC2, supports the malignant phenotype of BCa cells.

GPX4 suppresses ferroptosis to promote malignant progression of endometrial carcinoma via transcriptional activation by ELK1.

BACKGROUND: Glutathione Peroxidase 4 (GPX4) is a key protein that inhibits ferroptosis. However, its biological regulation and mechanism in endometrial cancer (EC) have not been reported in detail. METHODS: The expression of GPX4 in EC tissues was determined by TCGA databases, qRT-PCR, Western blot, and immunohistochemistry (IHC). The effects of GPX4 on EC cell proliferation, migration, apoptosis, and tumorigenesis were studied in vivo and in vitro. In addition, ETS Transcription Factor ELK1 (ELK1) was identified by bioinformatics methods, dual-luciferase reporter assay, and chromatin immunoprecipitation (ChIP). Pearson correlation analysis was used to evaluate the association between ELK1 and GPX4 expression. RESULTS: The expression of GPX4 was significantly up-regulated in EC tissues and cell lines. Silencing GPX4 significantly inhibited the proliferation, migration ability, induced apoptosis, and arrested the cell cycle of Ishikawa and KLE cells. Knockdown of GPX4 accumulated intracellular ferrous iron and ROS, disrupted MMP, and increased MDA levels. The xenograft tumor model also showed that GPX4 knockdown markedly reduced tumor growth in mice. Mechanically, ELK1 could bind to the promoter of GPX4 to promote its transcription. In addition, the expression of ELK1 in EC was positively correlated with GPX4. Rescue experiments confirmed that GPX4 knockdown could reverse the strengthens of cell proliferation and migration ability and the lower level of Fe2+ and MDA caused by upregulating ELK1. CONCLUSION: The results of the present study suggest that ELK1 / GPX4 axis plays an important role in the progress of EC by promoting the malignant biological behavior and inducing ferroptosis of EC cells, which provides evidence for investigating the potential therapeutic strategies of endometrial cancer.

Silencing circ_0074371 inhibits the progression of sepsis-induced acute kidney injury by regulating miR-330-5p/ELK1 axis.

BACKGROUND: Sepsis-induced acute kidney injury (AKI) is a common in clinic. Circular RNAs (circRNAs) play significant roles in ameliorating AKI. The purpose of this study was aimed to identify the role of circ_0074371 and the potential action mechanism in sepsis-induced AKI. METHODS: AKI patients and healthy individual serum samples were collected and the relative expression of circ_0074371 was measured by real-time polymerase chain reaction (RT-PCR). HK2 cells were treated with different dose (0, 2.5, 5 and 10 µg/ml) lipopolysaccharide (LPS) to establish the AKI cell model. The cell viability and apoptosis of HK2 cells were detected using cell counting kit-8 (CCK-8) and flow cytometry, respectively. The contents of malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using the relative commercial kits. The IL-1ß and TNF-α levels in cell culture supernatants were measured by ELISA. The interaction relationship between miR-330-5p and circ_0074371 or ELK1 was predicted by Targetscan database and further confirmed by the dual-luciferase reporter assay system. RESULTS: The circ_0074371 expression was up-regulated in sepsis patients and LPS-induced HK2 cells. Silencing circ_0074371 promoted HK2 cells viability and inhibited the HK2 cells apoptosis. miR-330-5p inhibitor weakened circ_0074371 inhibitor-induced cell viability, apoptosis and oxidative stress. Further mechanism analysis showed that circ_0074371 acted as a sponge for miR-330-5p to increase ELK1 expression level. Importantly, miR-330-5p downregulation or ELK1 upregulation reversed the action of circ_0074371 knockdown on LPS-induced HK2 cells. CONCLUSION: Knockdown of circ_0074371 ameliorated LPS-induced HK2 cells apoptosis, inflammation and oxidative stress via regulating miR-330-5p/ELK1, opening a new window into the pathogenesis AKI.