Activation of the tumor cell-intrinsic STING pathway induced by Fusobacterium nucleatum is associated with poor prognosis in esophageal cancer patients.

Purpose: To investigate the specific role of melanotransferrin antisense RNA (MFI2-AS1) in esophageal cancer (EC) progression. Methods: The differential expression of MFI2-AS1 in EC tissues and cells was determined using quantitative reverse transcription–polymerase chain reaction (qRT-PCR). Silencing MFI2-AS1 was performed by transfection with specific short hairpin RNAs targeting MFI2-AS1. The 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry (FC) were used to assess cell viability and apoptosis of EC cells, respectively. The sponging microRNA (miRNA) of MFI2-AS1 was validated using luciferase activity and RNA immunoprecipitation assays while the downstream target gene of the sponging miRNA was evaluated by luciferase activity assay. Results: MFI2-AS1 was significantly enhanced in EC tissues (p < 0.01) and indicated a poor prognosis in EC patients. Knockdown of MFI2-AS1 in EC cells decreased cell viability and promoted cell apoptosis of EC cells. Functionally, MFI2-AS1 targeted miR-331-3p, and sex-determining region on Ychromosome-related high-mobility-group box4 (SOX4) was identified as a target gene of miR-331-3p. Ectopic expression of SOX4 counteracted the suppressive effect of MFI2-AS1 knockdown on EC cell viability and stimulative effect on EC cell apoptosis. Conclusion: The pro-oncogenic effect of MFI2-AS1 on EC progression occurs via the regulation of the miR-331-3p/SOX4 axis, providing a new potential therapeutic target for EC.

MicroRNAs are endogenous, non-coding, small RNAs that can regulate biological processes. Previous studies have found that microRNA-487a serves as an oncogene. However, the role of microRNA-487a in esophageal cancer (EC) has not been reported. The aim of this investigation was to investigate the biological role of microRNA-487a in EC and its underlying mechanism. The expression of microRNA-487a in 65 pairs of EC tissues and para-cancerous tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Chi-square test was used to analyze the relationship between microRNA-487a expression with age, sex, clinical stage and distant metastasis of OS patients. Kaplan-Meier survival analysis was conducted to evaluate the correlation between microRNA-487a expression and prognosis of EC patients. Subsequently, microRNA-487a expression in EC cell lines was detected as well. After microRNA-487a knockdown, cell counting kit-8 (CCK-8), EdU and transwell assay were conducted to evaluate the role of microRNA-487a in the biological performances of EC cells, respectively. Meanwhile, the apoptosis of EC cells was determined using flow cytometry. Finally, the interaction between microRNA-487a and p62 was explored by Western blot. Transwell assay was carried out in EC cells co-transfected with p62 overexpression plasmid and si-microRNA-487a. Compared with para-cancerous tissues, microRNA-487a expression was significantly higher in EC tissues, and the difference was statistically significant. MicroRNA-487a was highly expressed in EC cells as well. Low expression of microRNA-487a was positively correlated with clinical stage, whereas was not correlated with age, sex, lymph node metastasis and distant metastasis of EC patients. Kaplan-Meier survival curves showed that high expression of microRNA-487a was markedly associated with poor prognosis of EC. The knockdown of microRNA-487a significantly inhibited proliferative, migratory and invasive abilities of EC cells but induced cell apoptosis. Western blot results showed that the protein expression of p62 was remarkably upregulated after microRNA-478a knockdown in EC cells. Transwell assay demonstrated that co-transfection with overexpression plasmid of p62 and si-microRNA-487a in EC cells markedly decreased invasive and migratory abilities. MicroRNA-487a is highly expressed in EC and is closely correlated with clinical stage and poor prognosis of EC. Our findings confirm that microRNA-487a promotes malignant progression of EC by regulating p62 expression.

To explore the molecular mechanisms of FAM129A in regulating the progression of esophageal cancer and its prognosis. FAM129A levels in esophageal cancer tissues and paracancerous ones were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Its influences on clinical features and prognosis in esophageal cancer patients were analyzed. Changes in proliferation and apoptosis in esophageal cancer cells after knockdown of FAM129A were examined by cell counting kit-8 (CCK-8), 5-Ethynyl-2'- deoxyuridine (EdU) assay and flow cytometry, respectively. The feedback loop FAM129A/CXCL14 was finally assessed. FAM129A was upregulated in esophageal cancer tissues. High level of FAM129A predicted advanced tumor staging, large tumor size and poor prognosis in esophageal cancer patients. Knockdown of FAM129A inhibited proliferative ability and induced apoptosis in OE19 and OE33 cells. In addition, knockdown of FAM129A upregulated protein level of CXCL14 in esophageal cancer cells. CXCL14 was downregulated in esophageal cancer tissues and negatively correlated to FAM129A level. The negative feedback loop FAM129A/CXCL14 was responsible for aggravating the malignant phenotypes of esophageal cancer cells. FAM129A is upregulated in esophageal cancer samples, and it is linked to tumor staging, tumor size and poor prognosis. FAM129A aggravates the progression of esophageal cancer by negatively regulating CXCL14 level.

Previous studies have found that IPO5 is a cancer-promoting gene. However, the role of IPO5 in esophageal cancer has not been reported. This study aims to investigate the expression characteristics of IPO5 in esophageal cancer, and to further analyze its relationship with clinical parameters and prognosis of esophageal cancer. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was performed to examine the expression of matrix metalloproteinase 7 (MMP7) in 45 pairs of tumor tissue specimens and adjacent normal ones collected from esophageal cancer patients. The correlation between IPO5 expression and clinical indicators and prognosis of esophageal cancer patients was analyzed. Meanwhile, IPO5 expression in esophageal cancer cell lines was also detected using qRT-PCR. In addition, the influence of IPO5 on esophageal cancer cell functions was analyzed using cell counting kit-8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assays. Finally, Dual-Luciferase reporter assay and cell reverse experiments were conducted to explore its underlying mechanisms. In this experiment, qRT-PCR results indicated that IPO5 expression in tumor tissues of esophageal cancer patients was significantly higher than that in adjacent normal ones, and the difference was statistically significant. Compared with esophageal cancer patients with low expression of IPO5, those with high expression of IPO5 had higher pathological stage and lower overall survival rate. Compared with control group, the proliferation ability of esophageal cancer cells in IPO5 knockdown group was significantly decreased. In addition, Western Blot results indicated that the key protein MMP7 was conspicuously elevated in the esophageal cancer cell line after knockdown of IPO5. Dual-Luciferase reporter assay results suggested that IPO5 can specifically bind MMP7. Additionally, the cell reverse experiment demonstrated that MMP7 was responsible for IPO5-regulated malignant progression of esophageal cancer. IPO5 expression significantly increased in esophageal cancer tissues, which was associated with pathological staging and poor prognosis of esophageal cancer patients. IPO5 may promote malignant progression of esophageal cancer through the regulation of MMP7.

The mechanism by which DNMT3B facilitates esophageal cancer (ESCA) progression is currently unknown, despite its association with adverse prognoses in several cancer types. To investigate the potential therapeutic effects of the Chinese herbal medicine rhubarb on esophageal cancer (ESCA), we adopted an integrated bioinformatics approach. Gene Set Enrichment Analysis (GSEA) was first utilized to screen active anti-ESCA components in rhubarb. We then employed Weighted Gene Co-expression Network Analysis (WGCNA) to identify key molecular modules and targets related to the active components and ESCA pathogenesis. This system-level strategy integrating multi-omics data provides a powerful means to unravel the molecular mechanisms underlying the anticancer activities of natural products, like rhubarb. To investigate module gene functional enrichment, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. In addition, we evaluated the predictive impact of DNMT3B expression on ESCA patients utilizing the Kaplan-Meier method. Finally, we conducted experiments on cell proliferation and the cell cycle to explore the biological roles of DNMT3B. In this study, we identified Rhein as the main active ingredient of rhubarb that exhibited significant anti-ESCA activity. Rhein markedly suppressed ESCA cell proliferation. Utilizing Weighted Gene Co-expression Network Analysis (WGCNA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, we determined that the blue module was associated with Rhein target genes and the cell cycle. Additionally, DNMT3B was identified as a Rhein target gene. Analysis of The Cancer Genome Atlas (TCGA) database revealed that higher DNMT3B levels were associated with poor prognosis in ESCA patients. Furthermore, Rhein partially reversed the overexpression of DNMT3B to inhibit ESCA cell proliferation. In vitro studies demonstrated that Rhein and DNMT3B inhibition disrupted the S phase of the cell cycle and affected the production of cell cycle-related proteins. In this study, we found that Rhein exerts its anti-proliferative effects in ESCA cells by targeting DNMT3B and regulating the cell cycle.

BackgroundEpitranscriptomics studies have contributed greatly to the development of research on human cancers. In recent years, N6-methyladenosine (m6A), an RNA modification on the N-6 position of adenosine, has been found to play a potential role in epigenetic regulation. Therefore, we aimed to evaluate the regulation of cancer progression properties by m6A.ResultsWe found that m6A demethylase fat mass and obesity-associated protein (FTO) was highly expressed in esophageal cancer (EC) stem-like cells, and that its level was also substantially increased in EC tissues, which was closely correlated with a poor prognosis in EC patients. FTO knockdown significantly inhibited the proliferation, invasion, stemness, and tumorigenicity of EC cells, whereas FTO overexpression promoted these characteristics. Furthermore, integrated transcriptome and meRIP-seq analyses revealed that HSD17B11 may be a target gene regulated by FTO. Moreover, FTO promoted the formation of lipid droplets in EC cells by enhancing HSD17B11 expression. Furthermore, depleting YTHDF1 increased the protein level of HSD17B11.ConclusionsThese data indicate that FTO may rely on the reading protein YTHDF1 to affect the translation pathway of the HSD17B11 gene to regulate the formation of lipid droplets in EC cells, thereby promoting the development of EC. The understanding of the role of epitranscriptomics in the development of EC will lay a theoretical foundation for seeking new anticancer therapies.

Background: MicroRNAs (miRNAs) are a class of small non-coding RNAs that inhibit gene expression by binding to 3` untranslated regions of mRNAs and inhibiting their translation. MiRNAs crucially affect cellular processes such as development, proliferation, differentiation and apoptosis, which are deregulated in cancer. MiR-503 is located at Xq26.3 and belongs to an extended miR-16 family of miRNAs. MiR-503 is down-regulated in several types of cancer, including oral, hepatocellular, gastric, and endometrial cancer, suggesting that it plays a tumour-suppressor role in carcinogenesis. In contrast, other recent reports have demonstrated up-regulation of miR-503 in adrenocortical carcinoma, parathyroid and retinoblastoma, compared with adjacent normal tissue. These results suggest that miR-503 shows tissue- or disease-specific expression patterns. However, miR-503 expression patterns in esophageal cancer (EC) and normal esophageal tissue have not been compared and the correlation of miR-503 expression with the clinical features of EC has not been studied. Materials and Methods: A total of 61 formalin-fixed paraffin-embedded (FFPE) tissue samples including 49 EC tissues and 12 adjacent normal esophageal tissues who underwent surgical resection at Mie University Hospital were collected from 2003 to 2013. Tagman qRT-PCR assay was performed to quantify the miR-503 expression in EC and adjacent normal tissues. We analyzed the association between miR-503 expression and clinico-pathological factors in patients with EC. Results: The median age of the patients was 66.2 years (41 - 90). The median follow up time was 24.6 months (3.8 - 80.0). There were 3 patients with stage 0, 16 with stage I, 12 with stage II, 11 with stage III, 3 with stage IVa and 4 with stage IVb by using the criteria of the Japanese Classification of Esophageal Cancer, 10th edition. Forty-four patients had esophageal squamous cell carcinoma (ESCC), five patients had esophageal adenocarcinoma (EAD). MiR-503 was remarkably up-regulated in EC tissues compared to adjacent normal tissue. High miR-503 expression in EC was significantly associated with male sex and large tumor size. Kaplan-Meier analysis showed that patients with high miR-503 expression had significantly poorer disease free and overall survival than those with low expression. Furthermore, high miR-503 expression was an independent predictor for prognosis in EC patients according to multivariate analysis. Conclusions: The results of this study provide novel evidence for associations between miR-503 expression and progression, early recurrence, and poor prognosis in EC, suggesting that it acts as an oncomiR in this type of cancer. MiR-503 may thus be a promising predictive marker for prognosis after surgery in patients with EC. Citation Format: Koichiro Mori, Yuji Toiyama, Shozo Ide, Tomofumi Noguchi, Hiroki Imaoka, Hiromi Yasuda, Susumu Saigusa, Masaki Ohi, Yasuhiro Inoue, Koji Tanaka, Yasuhiko Mohri, Tsutomu Nobori, Masato Kusunoki. MicroRNA-503 promotes tumor progression and acts as a novel biomarker for prognosis in esophageal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3401. doi:10.1158/1538-7445.AM2015-3401

The purpose of this study was to elucidate the biological role of MicroRNA-223 (miRNA-223) in mediating the malignant progression of esophageal cancer and the underlying mechanism. MiRNA-223 levels were measured using qRT-PCR in 50 paired esophageal cancer tissues and adjacent paracancerous tissues. The correlation between miRNA-223 level and pathological indicators in esophageal cancer patients was analyzed. In vitro experiments assessed the impact of miRNA-223 on the proliferative, migratory, and invasive abilities of esophageal cancer cells. Additionally, rescue experiments were conducted to investigate the involvement of miRNA-223 and its downstream target, SMAD4, in the progression of esophageal cancer. Esophageal cancer tissues showed decreased levels of miRNA-223 compared to adjacent tissues. Patients with low miRNA-223 exhibited higher rates of lymphatic and distant metastasis, as well as poorer overall survival than those with high miRNA-223 levels. Increasing miRNA-223 in TE-1 and EC-109 cells reduced their proliferative, migratory, and invasive capabilities. Esophageal cancer tissues and cell lines displayed elevated SMAD4 levels, which were negatively regulated by miRNA-223. Restoring SMAD4 expression partially reversed the inhibitory effects of miRNA-223 overexpression in esophageal cancer cells. MiRNA-223 is closely correlated to lymphatic metastasis, distant metastasis and poor prognosis of esophageal cancer patients. MiRNA-223 suppresses proliferative, migratory and invasive abilities of esophageal cancer cells via negatively regulating SMAD4.

The mutL homolog-1 (MLH1) is a DNA mismatch repair gene and has been reported to be frequently methylated in numerous cancers. However, the association between MLH1 methylation and esophageal cancer (EC), as well as its clinical significance, remains unclear. Hence, we conducted a systematic meta-analysis based on 19 articles (including 1384 ECs, 345 premalignant lesions, and 1244 healthy controls). Our analysis revealed that the frequency of MLH1 methylation was significantly elevated during EC carcinogenesis. In addition, we observed that MLH1 promoter methylation was associated with age (odds ratio [OR]=1.79; 95% CI =1.20–2.66), advanced tumor grade (OR=3.7; 95% CI =2.37–5.77), lymph node metastasis (OR=2.65; 95% CI =1.81–3.88), distant metastasis (OR=7.60; 95% CI =1.23–47.19), advanced clinical stage (OR=4.46; 95% CI =2.88–6.91), and poor prognosis in EC patients (hazard ratio =1.64, 95% CI =1.00–2.69). The pooled sensitivity, specificity, and area under the curve of MLH1 methylation in EC patients versus healthy individuals were 0.15, 0.99, and 0.77, respectively. Our findings indicate that MLH1 methylation is involved in the carcinogenesis, progression, and metastasis of EC. Moreover, methylated MLH1 could be a potential diagnostic and prognostic biomarker for EC.

Survivin over-expression is correlated with a poor prognosis in esophageal cancer patients

Esophageal cancer is a disease showing poor prognosis. Although combination chemotherapy using cisplatin (CDDP) and 5-fluorouracil is standard for unresectable esophageal cancer, the response rate is 35%. Cancer stem cells (CSCs) and inflammation are reportedly responsible for the poor prognosis of esophageal cancer. However, comprehensive analyses have not been conducted and proposals for progress remain lacking. Iron is known to be a key factor in the stemness of CSCs. Our study focused on the therapeutic potential of iron control using iron chelators for CSCs in esophageal cancer. Among 134 immunohistochemically analyzed cases, Nanog expression was high in 98 cases and low in 36 cases. High Nanog expression correlated with low overall and disease-free survivals. The iron chelators deferasirox (DFX) and SP10 suppressed the proliferation and expression of stemness markers in TE8 and OE33 cells. DFX and SP10 did not induce compensatory interleukin (IL)-6 secretion, although CDDP did result in high induction. Moreover, BBI608 and SSZ, as other CSC-targeting drugs, could not suppress the expression of stemness markers. Overall, Nanog expression appears related to poor prognosis in esophageal cancer patients, and inhibition of stemness and compensatory IL-6 secretion by iron chelators may offer a novel therapeutic strategy for esophageal cancer.

The recently identified alphacoronavirus swine acute diarrhea syndrome coronavirus (SADS-CoV) has a high fatality rate in neonatal piglets. Currently, no vaccines or treatment strategies for SADS-CoV infection are available. The stimulator of interferon genes (STING) pathway plays a critical role in initiating innate immune responses against RNA viral infections; however, its role in host defense against SADS-CoV infection remains unexplored. We assessed the pathogenicity of SADS-CoV in 3-day-old, 7-day-old, and 3-week-old mice, revealing striking age-dependent susceptibility-a pattern mirroring clinical observations in piglets. Additionally, SADS-CoV infection activated the STING-dependent pathway, which resulted in significant interferon responses in infected mice. In vitro experimental findings confirmed that STING pathway activation inhibited SADS-CoV replication by modulating the NF-κB and IRF3 signaling pathways and mediating the production of inflammatory cytokines, which underscores the importance of the STING pathway in antiviral defense mechanisms. In vivo studies revealed that the STING inhibitor C176 significantly promoted viral replication, whereas activation of the STING pathway using the STING agonist diABZI increased antiviral immune responses and reduced viral replication. Notably, diABZI protected mice from SADS-CoV infection by reducing viral replication through mechanisms involving both type I interferon-dependent and -independent pathways. These results represent the first demonstration of the in vivo therapeutic efficacy of pharmacological STING activation against SADS-CoV. These findings demonstrate that the STING pathway serves as a critical regulator of host defense against SADS-CoV and suggest that STING-targeted intervention has therapeutic potential.IMPORTANCESwine acute diarrhea syndrome coronavirus (SADS-CoV) is an emerging zoonotic pathogen with significant implications for veterinary and public health; it has a high mortality rate in piglets and the potential for cross-species transmission. Currently, there are no approved vaccines or specific antiviral agents available for this pathogen. In this study, we demonstrated that the stimulator of interferon genes (STING) pathway serves as a critical mediator of host defense against SADS-CoV infection. STING activation inhibits viral replication by coordinating interferon responses and modulating NF-κB/IRF3 signaling, and its inhibition exacerbates infection. Importantly, pharmacological activation of the STING pathway using the agonist diABZI significantly inhibited viral replication in vivo in a STING-dependent manner, with contributions from both type I interferon-dependent and -independent antiviral mechanisms, highlighting its therapeutic potential. These results advance our understanding of antiviral defense strategies against SADS-CoV and identify STING pathway regulation as a viable therapeutic approach for this emerging pathogen.

Methyltransferase like 3 (METTL3) has been proved to be involved in the progression of various cancers. In this study, we explored the role of METTL3 and its underlying mechanism in esophageal cancer progression. The mRNA and protein levels of METTL3 and epiplakin1 (EPPK1) were determined using qRT-PCR and western blot. The proliferative ability was evaluated through 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT), colony formation, and EdU assays. Transwell invasion assay and wound-healing assay were employed for detecting cell invasion and migration, respectively. Cell stemness was evaluated by sphere-formation assay. Xenograft tumor experiments and immunohistochemistry (IHC) were performed to explore the effects of METTL3 knockdown on tumor growth in vivo. The N6-methyladenosine (m6A) modification of EPPK1 was analyzed using MeRIP. RNA-protein immunoprecipitation (RIP) and dual-luciferase reporter assays were used to verify the relationship between EPPK1 and METTL3. METTL3 was upregulated in esophageal cancer tissues and cells, which was related to the poor prognosis of esophageal cancer patients. Knockdown of METTL3 overtly decreased the proliferative, invasive, migrated abilities, and cell stemness of esophageal cancer cells in vitro. Moreover, depletion of METTL3 also observably suppressed the growth of tumor in vivo. EPPK1 was a direct target of METTL3, and METTL3 could mediate the m6A modification of EPPK1. EPPK1 was downregulated in esophageal cancer tissues and cells, and EPPK1 depletion markedly repressed cell proliferation, invasion, migration, and stemness of esophageal cancer cells. The inhibition effects of METTL3 deficiency on these malignant behaviors were harbored by EPPK1 upregulation in esophageal cancer cells. In addition, METTL3 deficiency reduced EPPK1 expression to inactivate the PI3K/AKT pathway. Our results revealed that METTL3 deficiency regulated the m6A modification of EPPK1 to inhibit the PI3K/AKT pathway, thereby restraining the progression of esophageal cancer.

Background: Esophageal cancer represents a significant challenge as most patients present with advanced stage disease. This is due to the development of early nodal or distant metastasis. Metastatic progression is initiated by a process termed epithelial-mesenchymal transition (EMT). Recently, bortezomib, as an anti-tumor reagent, has been used as first-line regimen for patients with metastatic esophageal cancer in a Phase II clinical trial. However, the molecular mechanisms through which bortezomib inhibits tumor progression have not been well described. E-cadherin is a hallmark of EMT in tumor metastatic progression. Its aberrant hypermethylation is significantly associated with a poor prognosis in esophageal cancer. Our prior work indicated that bortezomib increased E-cadherin at transcriptional level in esophageal cancer. We hypothesized that NF-κB inhibition with bortezomib would reduce DNA methylation of E-cadherin and result in increased E-cadherin expression in esophageal cancer. Methods: Esophageal squamous and adenocarcinoma cancer cells were treated with TNF to simulate the pro-inflammatory tumor milieu and TGF-ß, a cytokine critical for the induction of EMT, followed by treatment with bortezomib (50nM) for 24 hrs. Cellular morphological changes were evaluated. E-cadherin promoter methylation status and mRNA was assessed using Methylation Specific PCR (MSP) and real-time PCR, respectively. To evaluate the effect of NF-κB on the methylation of the E-cadherin promoter in esophageal cancer, cells were either stimulated with TNF or transfected with an expression vector encoding the NF-κB subunit RelA/p65 or a super-suppressor IκB (SR- IκB), then treated with or without bortezomib. Results: We examined the effects of bortezomib on cellular morphology and found that bortezomib completely reverses TNF/TGF -induced EMT of esophageal cancer cells, correlating with an increase of E-cadherin transcript (5∼6 fold). Simultaneously, bortezomib dramatically inhibits methylation of the E-cadherin promoter in esophageal cancer cells. Furthermore, treatment with TNF or overexpression of the RelA/p65 induced hypermethylation of E-cadherin promoter. While inactivating NF-κB using SR- IκB expression vector significantly inhibits methylation of E-cadherin promoter in esophageal cancer cell lines. Interestingly, bortezomib failed to induce either hypomethylation or transactivation of E-cadherin in the absence of RelA/p65 when using SR-IκB in esophageal cancer cell lines. Conclusion: These findings indicate that bortezomib inhibits EMT of esophageal cancer cells by demethylation of the E-cadherin promoter, and that this function is NF- B dependent. In addition to functioning as a transcription activator, RelA/p65 appears to function as a transcription repressor by participating in methylation and silencing of E-cadherin. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-279.

MiR-4262 inhibits the development of esophageal cancer by negatively regulating KLF6 level