EphA-Mediated Regulation of Stomatin Expression in Prostate Cancer Cells.

BACKGROUND AND AIMS: Tumor growth and progression are affected by interactions between tumor cells and stromal cells within the tumor microenvironment. We previously showed that the expression of an integral membrane protein, called stomatin, was increased in cancer cells following their association with stromal cells. Additionally, stomatin impaired the Akt signaling pathway to suppress tumor growth. However, it remains unclear how stomatin expression is regulated. To explore this, we examined the cell surface molecules that can transduce the intercellular communication signals between cancer cells and stromal cells. RESULTS: Among these molecules, EphA3 and EphA7 receptors and their ligand ephrin-A5 were found to be expressed in prostate cancer cells, but not in prostate stromal cells. Cell-to-cell contact of prostate cancer cells through the EphA-ephrin-A interaction suppressed stomatin expression, while knockdown of EphA3/7 or ephrin-A5 increased stomatin expression. This increase contributed to an inhibition of prostate cancer cell proliferation. Intracellularly, the binding of ephrin-A to EphA attenuated extracellular signaling-regulated kinase (ERK) activation that promoted stomatin expression. Furthermore, ELK1 and ELK4, which are Ets family transcription factors phosphorylated by ERK, were involved in the induction of stomatin expression. We also found that higher Gleason score prostate cancer tissue samples had increased activation of EphA, while the stomatin expression and activated ERK and ELK levels were all low. In the mouse xenograft tumor samples generated by implantation of prostate cancer cells, EphA3 phosphorylation was attenuated and the ERK-ELK signaling and stomatin expression were enhanced in the area where stromal cells infiltrated the tumor. CONCLUSION: The EphA-mediated signaling suppresses the ERK-ELK pathway, leading to the reduction of stomatin expression that affects prostate cancer malignancy.

SRF SUMOylation modulates smooth muscle phenotypic switch and vascular remodeling.

Serum response factor (SRF) controls gene transcription in vascular smooth muscle cells (VSMCs) and regulates VSMC phenotypic switch from a contractile to a synthetic state, which plays a key role in the pathogenesis of cardiovascular diseases (CVD). It is not known how post-translational SUMOylation regulates the SRF activity in CVD. Here we show that Senp1 deficiency in VSMCs increased SUMOylated SRF and the SRF-ELK complex, leading to augmented vascular remodeling and neointimal formation in mice. Mechanistically, SENP1 deficiency in VSMCs increases SRF SUMOylation at lysine 143, reducing SRF lysosomal localization concomitant with increased nuclear accumulation and switching a contractile phenotype-responsive SRF-myocardin complex to a synthetic phenotype-responsive SRF-ELK1 complex. SUMOylated SRF and phospho-ELK1 are increased in VSMCs from coronary arteries of CVD patients. Importantly, ELK inhibitor AZD6244 prevents the shift from SRF-myocardin to SRF-ELK complex, attenuating VSMC synthetic phenotypes and neointimal formation in Senp1-deficient mice. Therefore, targeting the SRF complex may have a therapeutic potential for the treatment of CVD.

Baicalein Inhibits Metastasis of Oral Squamous Cell Carcinoma Cells by Regulating ERK/ELK-1/Snail Signaling.

BACKGROUND: Oral squamous cell carcinoma (OSCC) is associated with high recurrence and poor prognosis. Baicalin has multiple pharmacological effects, including anti-inflammatory and anti-proliferative activities. Here, we examine the effect of baicalein on OSCC metastasis and its potential mechanism of action. METHODS: SCC-4 and CAL-27 cells were treated with different concentrations of baicalein. The proliferation of OSCC cells was evaluated by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As for migration and metastasis, baicalein-treated OSCC cells were used for wound healing assay and Transwell assay. The levels of epithelial-mesenchymal transition-related proteins (E-cadherin, N-cadherin, vimentin) and extracellular regulated protein kinases (ERK)/ETS Transcription Factor ELK1 (ELK-1)/Snail signaling pathway-related proteins in baicalein-treated OSCC cells were evaluated by western blotting. RESULTS: The rates of cell proliferation and migration, along with the metastatic potential, of baicalein-treated cells were significantly lower than those of the control (p < 0.05), and the effects were concentration-dependent. Furthermore, compared to the control, baicalein significantly decreased the levels of N-cadherin and vimentin in SCC-4 and CAL-27 cells, and increased the E-cadherin level (p < 0.05). Mechanistically, baicalein downregulated the levels of p-ERK1/2, phospho-ETS Transcription Factor ELK1 (p-ELK-1), and Snail (p < 0.05). Finally, the ERK/ELK-1/Snail pathway inhibitor (U0126) promoted the effect of baicalein in inhibiting the migration and invasion of OSCC cells (p < 0.05). CONCLUSION: Baicalein abates the migration, invasion, and metastasis of OSCC cells through the ERK/ELK-1/Snail signaling pathway. This study provides a basis for the development of baicalein as a compound for the treatment of OSCC.

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

Dysregulation of the Long Noncoding RNA X-Inactive-Specific Transcript Expression in Male Patients with Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a sex-biased disease with female sex as a significant risk factor. Increased expression of the long noncoding RNA X-inactive-specific transcript (Xist), as induced by an intersectin-1s protein fragment with proliferative potential (EHITSN), may explain the sexual dimorphism of female pulmonary artery endothelial cells (ECs) and at least in part, the imbalance sex/ratio of PAH. Xist is essential for X-chromosome inactivation and dosage compensation of X-linked genes. Herein, increased Xist expression was detected in a subset of ECs and lung tissue samples of male patients with PAH. The role of different Xist expression levels in ECs of male patients with PAH (ECPAH) was studied in several lines of male ECPAH in conjunction with molecular, biochemical, morphologic, and functional approaches. Male ECPAH showed on average 10.3-fold increase in high Xist versus low Xist, a significant association between Xist levels and their proliferative potential, and a heterogeneous methylation of the Xist/XIST antisense RNA (Tsix) locus. Interestingly, Xist up-regulation in male ECPAH decreased the expression of Krueppel-like factor 2 (Klf2), via EHITSN interaction with enhancer of zeste polycomb repressive complex 2 subunit (EZH2), the catalytic subunit of the polycomb repressive complex 2. Moreover, the studies demonstrate that EHITSN-triggered p38/ETS domain-containing protein Elk1/AP-1 transcription factor subunit (c-Fos) signaling is a pathologic mechanism central to ECPAH proliferation and the dynamic crosstalk with cell cycle regulatory proteins cyclin A1/cyclin D2 and Xist-EZH2-Klf2 interaction participate directly and differentially in establishing the proliferative profile of male ECPAH.

Elk1 enhances inflammatory cell infiltration and exacerbates acute lung injury/acute respiratory distress syndrome by suppressing Fcgr2b transcription.

OBJECTIVE: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with significant mortality rates. The role of Fcgr2b in the pathogenesis of ALI/ARDS is not fully elucidated. This study aimed to investigate the functions of Fcgr2b in ALI/ARDS and explore its underlying mechanisms. METHODS: Methods: In this study, rat models of ARDS and pulmonary microvascular endothelial cell (PMVEC) injury models were established through the administration of lipopolysaccharide (LPS). The expression levels of Fcgr2b and Elk1 were quantified in both LPS-induced ARDS rats and PMVECs. Subsequent gain- and loss-of-function experiments were conducted, followed by comprehensive assessments of lung tissue for pathomorphological changes, edema, glycogen storage, fibrosis, and infiltration of inflammatory cells. Additionally, bronchoalveolar lavage fluid was analyzed for T-helper 17 (Th17) cell infiltration, inflammatory response, and microvascular permeability to evaluate lung injury severity in ARDS models. Furthermore, the activity, cytotoxicity, apoptosis, and angiogenic potential of PMVECs were assessed to gauge cell injury. The interaction between Elk1 and Fcgr2b was also examined to confirm their regulatory relationship. RESULTS: In the context of LPS-induced ARDS and PMVEC injury, Fcgr2b expression was markedly reduced, whereas Elk1 expression was elevated. Overexpression of Fcgr2b led to a decrease in Th17 cell infiltration and mitigated lung tissue damage in ARDS models, in addition to reducing LPS-induced injury in PMVECs. Elk1 was found to suppress Fcgr2b transcription through the recruitment of histone 3 lysine 9 trimethylation (H3K9me3). Knockdown of Elk1 diminished Th17 cell infiltration and lung tissue damage in ARDS models, and alleviated LPS-induced injury in PMVECs, effects that were reversed upon Fcgr2b upregulation. CONCLUSION: Elk1 negatively regulates Fcgr2b transcription, thereby augmenting the inflammatory response and exacerbating lung injury in LPS-induced ALI/ARDS.

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.

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.