LEF1 | Cancer Genetics Web

Chen Y, Guo Y, Li Y, et al.
miR‑300 regulates tumor proliferation and metastasis by targeting lymphoid enhancer‑binding factor 1 in hepatocellular carcinoma.
Int J Oncol. 2019; 54(4):1282-1294 [PubMed] Free Access to Full Article Related Publications

Accumulating evidence indicates that microRNAs (miRNAs) have a critical role in cell proliferation and metastasis in hepatocellular carcinoma (HCC). However, the effect of miR‑300 on the development and progression of HCC remains unclear. In the present study, it was observed that miRNA (miR)‑300 expression was significantly decreased in HCC cell lines compared with normal liver cells. Furthermore, we detected the effects of miR‑300 on cell proliferation and apoptosis, cell cycle, migration and invasion by using MTT, colony formation assay, wound healing, Transwell assay and flow cytometry methods, respectively. The results demonstrated that miR‑300 overexpression inhibited proliferation, induced apoptosis and G1/S cell cycle arrest, and suppressed migration and invasion in Huh‑7 cells, whereas miR‑300 silencing promoted the proliferation, migration and invasion of Hep3B cells. Mechanistically, the transcription factor lymphoid enhancer‑binding factor 1 (LEF‑1), which was verified as a direct target gene of miR‑300, promoted cell proliferation, migration and invasion and mediates the effects of miR‑300 on HCC cells. In addition, low expression of miR‑300 and high expression of LEF‑1 in HCC tissues were found to be associated with poor prognosis of patients with HCC. These findings indicate that miR‑300 may be a potential prognostic predictor and therapeutic target for patients with HCC.

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Fetisov TI, Lesovaya EA, Yakubovskaya MG, et al.
Alterations in WNT Signaling in Leukemias.
Biochemistry (Mosc). 2018; 83(12):1448-1458 [PubMed] Related Publications

The WNT/β-catenin signaling pathway plays an important role in the differentiation and proliferation of hematopoietic cells. In recent years, special attention has been paid to the role of impairments in the WNT signaling pathway in pathogenesis of malignant neoplasms of the hematopoietic system. Disorders in the WNT/β-catenin signaling in leukemias identified to date include hypersensitivity to the WNT ligands, epigenetic repression of WNT antagonists, overexpression of WNT ligands, impaired β-catenin degradation in the cytoplasm, and changes in the activity of the TCF/Lef transcription factors. At the molecular level, these impairments involve overexpression of the FZD protein, hypermethylation of the SFRP, DKK, WiF, Sox, and CXXC gene promoters, overexpression of Lef1 and plakoglobin, mutations in GSK3β, and β-catenin phosphorylation by the BCR-ABL kinase. This review is devoted to the systematization of these data.

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Leukemia

Legge DN, Shephard AP, Collard TJ, et al.
BCL-3 promotes a cancer stem cell phenotype by enhancing β-catenin signalling in colorectal tumour cells.
Dis Model Mech. 2019; 12(3) [PubMed] Free Access to Full Article Related Publications

To decrease bowel cancer incidence and improve survival, we need to understand the mechanisms that drive tumorigenesis. Recently, B-cell lymphoma 3 (BCL-3; a key regulator of NF-κB signalling) has been recognised as an important oncogenic player in solid tumours. Although reported to be overexpressed in a subset of colorectal cancers (CRCs), the role of BCL-3 expression in colorectal tumorigenesis remains poorly understood. Despite evidence in the literature that BCL-3 may interact with β-catenin, it is perhaps surprising, given the importance of deregulated Wnt/β-catenin/T-cell factor (TCF) signalling in colorectal carcinogenesis, that the functional significance of this interaction is not known. Here, we show for the first time that BCL-3 acts as a co-activator of β-catenin/TCF-mediated transcriptional activity in CRC cell lines and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. We demonstrate that targeting BCL-3 expression (using RNA interference) reduced β-catenin/TCF-dependent transcription and the expression of intestinal stem cell genes

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BCL3

Pineda B, Diaz-Lagares A, Pérez-Fidalgo JA, et al.
A two-gene epigenetic signature for the prediction of response to neoadjuvant chemotherapy in triple-negative breast cancer patients.
Clin Epigenetics. 2019; 11(1):33 [PubMed] Free Access to Full Article Related Publications

BACKGROUND: Pathological complete response (pCR) after neoadjuvant chemotherapy (NAC) in triple-negative breast cancer (TNBC) varies between 30 and 40% approximately. To provide further insight into the prediction of pCR, we evaluated the role of an epigenetic methylation-based signature.
METHODS: Epigenetic assessment of DNA extracted from biopsy archived samples previous to NAC from TNBC patients was performed. Patients included were categorized according to previous response to NAC in responder (pCR or residual cancer burden, RCB = 0) or non-responder (non-pCR or RCB > 0) patients. A methyloma study was performed in a discovery cohort by the Infinium HumanMethylation450 BeadChip (450K array) from Illumina. The epigenetic silencing of those methylated genes in the discovery cohort were validated by bisulfite pyrosequencing (PyroMark Q96 System version 2.0.6, Qiagen) and qRT-PCR in an independent cohort of TN patients and in TN cell lines.
RESULTS: Twenty-four and 30 patients were included in the discovery and validation cohorts, respectively. In the discovery cohort, nine genes were differentially methylated: six presented higher methylation in non-responder patients (LOC641519, LEF1, HOXA5, EVC2, TLX3, CDKL2) and three greater methylation in responder patients (FERD3L, CHL1, and TRIP10). After validation, a two-gene (FER3L and TRIP10) epigenetic score predicted RCB = 0 with an area under the ROC curve (AUC) = 0.905 (95% CI = 0.805-1.000). Patients with a positive epigenetic two-gene score showed 78.6% RCB = 0 versus only 10.7% RCB = 0 if signature were negative.
CONCLUSIONS: These results suggest that pCR in TNBC could be accurately predicted with an epigenetic signature of FERD3L and TRIP10 genes. Further prospective validation of these findings is warranted.

Tanaka T, Kojima K, Yokota K, et al.
Comprehensive Genetic Search to Clarify the Molecular Mechanism of Drug Resistance Identifies ASCL2-LEF1/TSPAN8 Axis in Colorectal Cancer.
Ann Surg Oncol. 2019; 26(5):1401-1411 [PubMed] Related Publications

BACKGROUND: Treatment-resistance genes limiting anticancer therapy have not been well clarified in colorectal cancer (CRC). We explored gene expression profiles to identify biomarkers for predicting treatment resistance to an anticancer drug in CRC.
METHODS: Six CRC cell lines were treated with phenylbutyrate (PB). The gene expression profiles were then compared using microarrays (harboring 54,675 genes), and genes associated with PB resistance were identified. Candidate genes were functionally examined in cell lines and clinically validated for treatment resistance in clinical samples.
RESULTS: Both DLD1 and HCT15 cells were PB resistant, while HCT116 cells were identified as PB sensitive. On microarray analysis, among the PB resistance-related genes, the expression of the genes ASCL2, LEF1, and TSPAN8 was clearly associated with PB resistance. PB-sensitive cells transfected with one of these three genes exhibited significant (P < 0.001) augmentation of PB resistance; ASCL2 induced expression of both LEF1 and TSPAN8, while neither LEF1 nor TSPAN8 induced ASCL2. RNA interference via ASCL2 knockdown made PB-resistant cells sensitive to PB and inhibited both genes. ASCL2 knockdown also played a critical role in sensitivity to treatment by 5-fluorouracil and radiotherapy in addition to PB. Finally, ASCL2 expression was significantly correlated with histological grade of rectal cancer with preoperative chemoradiation therapy.
CONCLUSIONS: ASCL2 was identified as a causative gene involved in therapeutic resistance against anticancer treatments in CRC.

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Cancer Prevention and Risk Reduction

Signal Transduction

Bollaert E, de Rocca Serra A, Demoulin JB
The HMG box transcription factor HBP1: a cell cycle inhibitor at the crossroads of cancer signaling pathways.
Cell Mol Life Sci. 2019; 76(8):1529-1539 [PubMed] Related Publications

HMG box protein 1 (HBP1) is a transcription factor and a potent cell cycle inhibitor in normal and cancer cells. HBP1 activates or represses the expression of different cell cycle genes (such as CDKN2A, CDKN1A, and CCND1) through direct DNA binding, cofactor recruitment, chromatin remodeling, or neutralization of other transcription factors. Among these are LEF1, TCF4, and MYC in the WNT/beta-catenin pathway. HBP1 also contributes to oncogenic RAS-induced senescence and terminal cell differentiation. Collectively, these activities suggest a tumor suppressor function. However, HBP1 is not listed among frequently mutated cancer driver genes. Nevertheless, HBP1 expression is lower in several tumor types relative to matched normal tissues. Several micro-RNAs, such as miR-155, miR-17-92, and miR-29a, dampen HBP1 expression in cancer cells of various origins. The phosphatidylinositol-3 kinase (PI3K)/AKT pathway also inhibits HBP1 transcription by preventing FOXO binding to the HBP1 promoter. In addition, AKT directly phosphorylates HBP1, thereby inhibiting its transcriptional activity. Taken together, these findings place HBP1 at the center of a network of micro-RNAs and oncoproteins that control cell proliferation. In this review, we discuss our current understanding of HBP1 function in human physiology and diseases.

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Khan M, Muzumdar D, Shiras A
Attenuation of Tumor Suppressive Function of FBXO16 Ubiquitin Ligase Activates Wnt Signaling In Glioblastoma.
Neoplasia. 2019; 21(1):106-116 [PubMed] Free Access to Full Article Related Publications

Glioblastoma (GBM) is one of the most aggressive and lethal types of brain tumor. Despite the advancements in conventional or targeted therapies, median survival of GBM patients is less than 12 months. Amongst various signaling pathways aberrantly activated in glioma, active Wnt/β-catenin signaling pathway is one of the crucial oncogenic players. β-catenin, an important mediator of Wnt signaling pathway, gets phosphorylated by GSK3β complex. Phosphorylated β-catenin is specifically recognized by β-Trcp1, a F-box/WD40-repeat protein and with the help of Skp1 it plays a central role in recruiting phosphorylated β-catenin for degradation. In GBM, expression of β-TrCP1 and its affinity for β catenin is reported to be very low. Hence, we investigated whether any other members of the E3 ubiquitin ligase family could be involved in degradation of nuclear β-catenin. We here report that FBXO16, a component of SCF E3 ubiquitin ligase complex, is an interacting protein partner for β-catenin and mediates its degradation. Next, we show that FBXO16 functions as a tumor suppressor in GBM. Under normal growth conditions, FBXO16 proteasomally degrades β-catenin in a GSK-3β independent manner. Specifically, the C-terminal region of FBXO16 targets the nuclear β-catenin for degradation and inhibits TCF4/LEF1 dependent Wnt signaling pathway. The nuclear fraction of β-catenin undergoes K-48 linked poly-ubiquitination in presence of FBXO16. In summary, we show that due to low expression of FBXO16, the β-catenin is not targeted in glioma cells leading to its nuclear accumulation resulting in active Wnt signaling. Activated Wnt signaling potentiates the glioma cells toward a highly proliferative and malignant state.

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Non-Small Cell Lung Cancer

Ye L, Xiang T, Zhu J, et al.
Interferon Consensus Sequence-Binding Protein 8, a Tumor Suppressor, Suppresses Tumor Growth and Invasion of Non-Small Cell Lung Cancer by Interacting with the Wnt/β-Catenin Pathway.
Cell Physiol Biochem. 2018; 51(2):961-978 [PubMed] Related Publications

BACKGROUND/AIMS: Interferon consensus sequence-binding protein 8 (IRF8) belongs to a family of interferon (IFN) regulatory factors that modulates various important physiological processes including carcinogenesis. As reported by others and our group, IRF8 expression is silenced by DNA methylation in both human solid tumors and hematological malignancies. However, the role of IRF8 in lung carcinoma remains elusive. In this study, we determined IRF8 epigenetic regulation, biological functions, and the signaling pathway involved in non-small cell lung cancer (NSCLC).
METHODS: IRF8 expression were determined by Q- PCR. MSP and A+T determined promotor methylation. MTS, clonogenic, Transwell assay, Flow cytometry, three-dimensional culture and AO/EB stain verified cell function. In vivo tumorigenesis examed the in vivo effects. By Chip-QPCR, RT-PCR, Western blot and Immunofluorescence staining, the mechanisms were studied.
RESULTS: IRF8 was significantly downregulated in lung tumor tissues compared with adjacent non-cancerous tissues. Furthermore, methylation-specific PCR analyses revealed that IRF8 methylation in NSCLC was a common event, and demethylation reagent treatment proved that downregulation of IRF8 was due to its promoter CpG hypermethylation. Clinical data showed that the IRF8 methylation was associated with tumor stage, lymph node metastasis status, patient outcome, and tumor histology. Exogenous expression of IRF8 in the silenced or downregulated lung cancer cell lines A549 and H1299 at least partially restored the sensitivity of lung cancer cells to apoptosis, and arrested cells at the G0/G1 phase. Cell viability, clonogenicity, and cell migration and invasive abilities were strongly inhibited by restored expression of IRF8. A three-dimensional culture system demonstrated that IRF8 changed the cells to a more spherical phenotype. Moreover, ectopic expression of IRF8 enhanced NSCLC chemosensitivity to cisplatin. Furthermore, as verified by Chip-qPCR, immunofluorescence staining, and western blotting, IRF8 bound to the T-cell factor/lymphoid enhancer factor (TCF /LEF) promoter, thus repressing β-catenin nuclear translocation and its activation. IRF8 significantly disrupted the effects of Wnt agonist, bml284, further suggesting its involvement in the Wnt/β-catenin pathway.
CONCLUSION: IRF8 acted as a tumor suppressor gene through the transcriptional repression of β-catenin-TCF/LEF in NSCLC. IRF8 methylation may serve as a potential biomarker in NSCLC prognosis.

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Apoptosis

Cisplatin

Lung Cancer

Kafka A, Karin-Kujundžić V, Šerman L, et al.
Hypermethylation of Secreted Frizzled Related Protein 1 gene promoter in different astrocytoma grades.
Croat Med J. 2018; 59(5):213-223 [PubMed] Free Access to Full Article Related Publications

AIM: To identify the involvement of Secreted Frizzled Related Protein 1 (SFRP1) promoter hypermethylation in different malignancy grades of astrocytoma and assess its association with beta-catenin, lymphoid-enhancer factor 1, and T-cell factor 1.
METHODS: Twenty-six astrocytoma samples were collected from 2008-2015. Promoter hypermethylation was evaluated by methylation-specific polymerase-chain-reaction and protein expression by immunohistochemistry and stereological analysis. The staining intensity was scored by comparing immunoreactivity with normal tissue and by using 10% and 50% cut-offs.
RESULTS: SFRP1 promoter methylation was found in 32% of astrocytomas. The number of hypermethylated samples increased in higher astrocytoma grades and was the highest in glioblastoma (P=0.042 compared to other astrocytoma grades). There was 45.8% of samples with the lack of or weak expression of SFRP1 protein and 29.2% with strong expression. Samples with methylated promoter expressed significantly less SFRP1 than samples with unmethylated promoter (P=0.031). Beta-catenin expression levels were elevated. Yet, glioblastomas with unmethylated SFRP1 promoter had significantly less beta-catenin (P=0.033). Strong expression of lymphoid-enhancer factor 1 was associated to higher astrocytoma grades (P=0.006).
CONCLUSION: SFRP1 gene was epigenetically silenced in glioblastomas when compared to low astrocytoma grades, which may suggest that the lack of its protein is involved in astrocytoma progression.

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SFRP1

Wu Y, Fang G, Wang X, et al.
NUP153 overexpression suppresses the proliferation of colorectal cancer by negatively regulating Wnt/β-catenin signaling pathway and predicts good prognosis.
Cancer Biomark. 2019; 24(1):61-70 [PubMed] Related Publications

BACKGROUND: Nucleoporin NUP153 (NUP153) is well known to be involved in the regulating of nuclear transport. Although NUP153 is associated with several cancers, its role in colorectal cancer (CRC) and the underlying mechanism are still unknown.
OBJECTIVE: The aim of this study was to access the effect of NUP153 on the prognosis of patients with CRC, and cancer cell proliferation.
METHODS: The expression levels of NUP153 in CRC tissues and matched normal colon tissues were examined by real-time quantitative PCR and immunohistochemistry. Then the association between NUP153 levels with clinical variables as well as survival time was investigated. Moreover, overexpression of NUP153 in HCT116 cells was established to study its influence on cell proliferation in vitro, and a xenograft model was performed to explore this effect in vivo.
RESULTS: We found that NUP153 was highly expressed in adjacent normal tissues than in cancer tissues, and elevated NUP153 expression was negatively associated with pathological grade (P= 0.015), T stage (P= 0.048) and distant metastasis (P= 0.006). Kaplan-Meier analysis revealed that patients with higher NUP153 expression had a longer overall survival (OS) (P= 0.01) and recurrence free disease (RFS) (P= 0.001). Logistic regression analysis further identified NUP153 as an independent prognostic safe factor for OS and recurrence. Moreover, NUP153 overexpression suppressed CRC cells proliferation and inhibited tumor growth in a xenograft model. Its mechanistic investigations showed that NUP153 overexpression inhibited β-catenin transcriptional activity and down-regulated the mRNA expression levels of Wnt downstream proteins-Axin2, cyclinD1, c-myc and lef-1.
CONCLUSIONS: NUP153 might be a promising prognostic factor, a potential tumor suppressor and therapeutic target in human CRC through an interaction with the Wnt/β-catenin signaling pathway.

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Liu X, Luo X, Wu Y, et al.
MicroRNA-34a Attenuates Paclitaxel Resistance in Prostate Cancer Cells via Direct Suppression of JAG1/Notch1 Axis.
Cell Physiol Biochem. 2018; 50(1):261-276 [PubMed] Related Publications

BACKGROUND/AIMS: Treatment options for metastatic castrate-resistant prostate cancer (mCRPC) are limited and typically centered on paclitaxel-based chemotherapy. In this study, we aimed to evaluate whether miR-34a attenuates chemoresistance to paclitaxel by regulating target genes associated with drug resistance.
METHODS: We used data from The Cancer Genome Atlas to compare miR-34a expression levels in prostate cancer (PC) tissues with normal prostate tissues. The effects of miR-34a inhibition and overexpression on PC proliferation were evaluated in vitro via Cell Counting Kit-8 (CCK-8) proliferation, colony formation, apoptosis, and cell-cycle assays. A luciferase reporter assay was employed to identify the interactions between miR-34a and specific target genes. To determine the effects of up-regulation of miR-34a on tumor growth and chemo-resistance in vivo, we injected PC cells overexpressing miR-34a into nude mice subcutaneously and evaluated the rate of tumor growth during paclitaxel treatment. We examined changes in the expression levels of miR-34a target genes JAG1 and Notch1 and their downstream genes via miR-34a transfection by quantitative reverse transcription PCR (qRT-PCR) and western blot assay.
RESULTS: miR-34a served as an independent predictor of reduced patient survival. MiR-34a was down-regulated in PC-3PR cells compared with PC-3 cells. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to paclitaxel while miR-34a down-regulation resulted in chemoresistance to paclitaxel in vitro. A study of gain and loss in a series of functional assays revealed that PC cells expressing miR-34a were chemosensitive. Furthermore, the overexpression of miR-34a increased the sensitivity of PC-3PR cells to chemotherapy in vivo. The luciferase reporter assay confirmed that JAG1 and Notch1 were directly targeted by miR-34a. Interestingly, western blot analysis and qRT-PCR confirmed that miR-34a inhibited the Notch1 signaling pathway. We found that miR-34a increased the chemosensitivity of PC-3PR cells by directly repressing the TCF1/ LEF1 axis.
CONCLUSION: Our results showed that miR-34a is involved in the development of chemosensitivity to paclitaxel. By regulating the JAG1/Notch1 axis, miR-34a or its target genes JAG1 or Notch1 might serve as potential predictive biomarkers of response to paclitaxel-based chemotherapy and/or therapeutic targets that will help to overcome chemoresistance at the mCRPC stage.

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NOTCH1

Li T, Jian X, He H, et al.
MiR-452 promotes an aggressive colorectal cancer phenotype by regulating a Wnt/β-catenin positive feedback loop.
J Exp Clin Cancer Res. 2018; 37(1):238 [PubMed] Free Access to Full Article Related Publications

BACKGROUND: Aberrant activation of Wnt/β-catenin signaling pathway is considered to be an important issue in progression and metastasis of various human cancers, especially in colorectal cancer (CRC). MiR-452 could activate of Wnt/β-catenin signaling. But the mechanism remains unclear.
METHODS: The expression of miR-452 in CRC and normal tissues was detected by real-time quantitative PCR. The effect of miR-452 on CRC growth and invasion was conducted by functional experiments in vitro and in vivo. Bioinformatics and cell luciferase function studies verified the direct regulation of miR-452 on the 3'-UTR of the GSK3β, which leads to the activation of Wnt/β-catenin signaling.
RESULTS: MiR-452 was upregulated in CRC compared with normal tissues and was correlated with clinical significance. The luciferase reporter system studies affirmed the direct regulation of miR-452 on the 3'-UTR of the GSK3β, which activate the Wnt/β-catenin signaling. The ectopic upregulation of miR-452 significantly inhibited the expression of GSK3β and enhanced CRC proliferation and invasion in vitro and in vivo. Meanwhile, knockdown of miR-452 significantly recovered the expression of GSK3β and attenuated Wnt/β-catenin-mediated cell metastasis and proliferation. More important, T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors, which are crucial downstream molecules of the Wnt/β-catenin signaling pathway was verified as a valid transcription factor of miR-452's promoter.
CONCLUSIONS: Our findings first demonstrate that miR-452-GSK3β-LEF1/TCF4 positive feedback loop induce CRC proliferation and migration.

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TCF4

Wu Y, Chen W, Gong L, et al.
Elevated G-Protein Receptor 125 (GPR125) Expression Predicts Good Outcomes in Colorectal Cancer and Inhibits Wnt/β-Catenin Signaling Pathway.
Med Sci Monit. 2018; 24:6608-6616 [PubMed] Free Access to Full Article Related Publications

BACKGROUND G-protein receptor 125 (GPR125), as a transmembrane signal transducer, is involved in regulating cancer development. Although GPR125 is related with several cancers, its role in colorectal cancer (CRC) and the underlying mechanism are still unknown. Here, we investigated the clinical significance of GPR125 in CRC. MATERIAL AND METHODS We assessed the expression level of GPR125 in CRC tissues by analyzing 3 datasets in the Gene Expression Omnibus (GEO) database and in human samples. The correlation between GPR125 expression and clinicopathological features was further analyzed. Survival analysis was performed to assess the association between GPR125 expression and recurrence-free survival (RFS). Cox logistic regression analysis was used to analyze the role of GPR125 expression in overall survival (OS). Moreover, we activated the Wnt pathway in HCT116 cells to investigate their potential mechanism. RESULTS Analysis of the GEO database showed that the expression of GPR125 was down-regulated in CRC tissues, consistent with our human samples experiments, and patients with higher GPR125 expression had a longer RFS. Also, we found that high GPR125 expression was associated with better tumor outcomes in clinical stage, metastasis, and KRAS status. Cox logistic regression analysis demonstrated that GPR125 was an independent prognostic factor for favorable outcome. Mechanistically, GPR125 overexpression inhibited the β-catenin transcriptional activity, and down-regulated the expression levels of the Wnt downstream proteins-Axin2, c-Myc, cylinD1, and lef-1. CONCLUSIONS GPR125 may be a potential prognosis-related anti-oncogene and its effects on inactivating Wnt/β-catenin signaling pathway might be a key link to inhibiting CRC formation.

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Eskandari E, Mahjoubi F, Motalebzadeh J
An integrated study on TFs and miRNAs in colorectal cancer metastasis and evaluation of three co-regulated candidate genes as prognostic markers.
Gene. 2018; 679:150-159 [PubMed] Related Publications

Molecular alterations that occur in cancer have the potential to be considered as either cancer biomarkers or targeted therapies or even both. In the presented study, we aimed to elucidate the gene regulatory network of metastatic colorectal cancer using data acquired from microarrays to reach the most common DEGs in colorectal cancer metastasis and find their possible regulatory mechanism by DETFs and DEmiRs. In this regards, seven microarray datasets were employed to assess the most important DEGs, DETFs and DEmiRs in colorectal cancer metastasis. Afterward, GRN based on DETFs and DEmiRs were constructed. Also ARACNE algorithm was used to construct an accurate GRN. GRN was analyzed structurally and then, two DETFs (LEF1 and ETV4) and a less-well known DEG (FABP6) by real time qRT-PCR in 50 patients with colorectal cancer were quantified. The constructed GRN highlighted the importance of some DETFs and DEmiRs in colorectal cancer metastasis. Interestingly the gene expression analysis by qRT-PCR on three candidate genes (LEF1, ETV4 and FABP6) indicated that the three genes were co-expressed in tumor samples, and were significantly associated with metastasis in colorectal cancer. Therefore, our experimental results proved a part of our comprehensive data analysis and system biology results. In summary, according to our empirical study we found the importance of three candidate genes as the potent prognostic factors in colorectal cancer metastasis. Also our study in a holistic insight on gene regulatory mechanism revealed the importance of some gene regulatory factors (DETFs and DEmiRs) and their potential as prognostic factors and/or targets in molecular targeted therapies in colorectal cancer.

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ETV4

Wang Q, Yan R, Pinnell N, et al.
Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development.
Blood. 2018; 132(12):1279-1292 [PubMed] Article available free on PMC after 20/09/2019 Related Publications

Notch1 signaling must elevate to high levels in order to drive the proliferation of CD4

Song J, Xie C, Jiang L, et al.
Transcription factor AP-4 promotes tumorigenic capability and activates the Wnt/β-catenin pathway in hepatocellular carcinoma.
Theranostics. 2018; 8(13):3571-3583 [PubMed] Article available free on PMC after 20/09/2019 Related Publications

It has been reported that the transcription factor activating enhancer-binding protein 4 (TFAP4) is upregulated and associated with an aggressive phenotype in several cancers. However, the precise mechanisms underlying the oncogenic role of TFAP4 remain largely unknown.

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Signal Transduction

Bladder Cancer - Molecular Biology

Liu X, Liu X, Wu Y, et al.
MicroRNA-34a Attenuates Metastasis and Chemoresistance of Bladder Cancer Cells by Targeting the TCF1/LEF1 Axis.
Cell Physiol Biochem. 2018; 48(1):87-98 [PubMed] Related Publications

BACKGROUND/AIMS: Chemoresistance is largely responsible for relapses of bladder cancer during clinical therapy. However, the molecular mechanisms involved in the chemoresistance of bladder cancer are unclear. Growing evidence supports the theory that microRNAs (miRNAs) play an important role in chemotherapeutic drug resistance because they are downregulated in many malignancies that have been implicated in the regulation of diverse processes in cancer cells. More specifically, the extent and precise mechanism of the involvement of miR-34as in chemoresistance to epirubicin (EPI) in the treatment of bladder cancer remains unclear.
METHODS: In this study, real-time quantitative polymerase chain reaction (PCR) was used to analyze the expression of miR-34a in bladder cancer cell line BIU87 and its EPI chemoresistant cell line BIU87/ADR. The miR-34a profiles in bladder cancer tissues were obtained from The Cancer Genome Atlas database. The effect of miR-34a on chemosensitivity was evaluated by cell viability assays, colony formation assays, and in vivo experimentation. Apoptosis and the cell cycle were examined by flow cytometry. A luciferase reporter assay was used to assess the target genes of miR-34a. Western blot and qPCR were used to analyze the expression of target proteins and downstream molecules.
RESULTS: The downregulation of miR-34a in bladder cancer serves as an independent predictor of reduced patient survival. The CCK-8 assay showed that miR-34a overexpression resulted in increased sensitivity to EPI, while miR-34a downregulation resulted in chemoresistance to EPI in vitro. Moreover, it was found that miR-34a increased the sensitivity of BIU87/ADR cells to chemotherapy in vivo. The luciferase reporter assay ascertained that TCF1 and LEF1 are direct target genes of miR-34a. It was found that miR-34a increased chemosensitivity in BIU87/ADR cells by inhibiting the TCF1/LEF1 axis.
CONCLUSIONS: The results of this study indicate that miR-34a contributes to the chemosensitivity of BIU87/ADR by inhibiting the TCF1/LEF1 axis. Consequently, miR-34a is a determinant of BIU87 chemosensitivity and may therefore serve as a potential therapeutic target in bladder cancer treatment.

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Gorlov I, Orlow I, Ringelberg C, et al.
Identification of gene expression levels in primary melanoma associated with clinically meaningful characteristics.
Melanoma Res. 2018; 28(5):380-389 [PubMed] Article available free on PMC after 01/10/2019 Related Publications

Factors influencing melanoma survival include sex, age, clinical stage, lymph node involvement, as well as Breslow thickness, presence of tumor-infiltrating lymphocytes based on histological analysis of primary melanoma, mitotic rate, and ulceration. Identification of genes whose expression in primary tumors is associated with these key tumor/patient characteristics can shed light on molecular mechanisms of melanoma survival. Here, we show results from a gene expression analysis of formalin-fixed paraffin-embedded primary melanomas with extensive clinical annotation. The Cancer Genome Atlas data on primary melanomas were used for validation of nominally significant associations. We identified five genes that were significantly associated with the presence of tumor-infiltrating lymphocytes in the joint analysis after adjustment for multiple testing: IL1R2, PPL, PLA2G3, RASAL1, and SGK2. We also identified two genes significantly associated with melanoma metastasis to the regional lymph nodes (PIK3CG and IL2RA), and two genes significantly associated with sex (KDM5C and KDM6A). We found that LEF1 was significantly associated with Breslow thickness and CCNA2 and UBE2T with mitosis. RAD50 was the gene most significantly associated with survival, with a higher level of expression associated with worse survival.

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Melanoma

Skin Cancer

Yang Z, Li K, Liang Q, et al.
Elevated hydrostatic pressure promotes ameloblastoma cell invasion through upregulation of MMP-2 and MMP-9 expression via Wnt/β-catenin signalling.
J Oral Pathol Med. 2018; 47(9):836-846 [PubMed] Related Publications

BACKGROUND: The process of marsupialization involves the release of intracystic pressure and the fluid contained within. Marsupialization of cystic ameloblastoma is controversial; therefore, we investigated how hydrostatic pressure influences biological behaviours of ameloblastoma cells and its underlying mechanisms.
MATERIALS AND METHODS: An ameloblastoma epithelial cell line, hTERT
RESULTS: Elevated hydrostatic pressure promoted migration and invasion of ameloblastoma cells, but inhibited proliferation. Expression of MMP-2, MMP-9, LEF-1, cyclin D1, c-Jun and c-Myc was significantly upregulated under elevated hydrostatic pressure, and these effects could be abolished by DKK1. Expression of RANKL, which is thought to be a downstream target of Wnt signalling, did not significantly change under elevated hydrostatic pressure.
CONCLUSIONS: This study indicates that elevated hydrostatic pressure promotes the migration and invasion of ameloblastoma cells by activating the Wnt/β-catenin pathway, thereby increasing expression of MMP-2, MMP-9 and other Wnt signalling downstream targets. This suggests that marsupialization may reduce invasiveness and reverse the bone resorption process by lowering intracystic hydrostatic pressure in cystic ameloblastoma.

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MMP2

MMP9: matrix metallopeptidase 9

Tan HY, Wang N, Li S, et al.
Repression of WT1-Mediated LEF1 Transcription by Mangiferin Governs β-Catenin-Independent Wnt Signalling Inactivation in Hepatocellular Carcinoma.
Cell Physiol Biochem. 2018; 47(5):1819-1834 [PubMed] Related Publications

BACKGROUND/AIMS: The development of hepatocellular carcinoma (HCC) is a complex process which involves deregulation of multiple signalling pathways. The hyper-activation of Wnt signalling promotes sustained expansion, invasion, and neovascularization of HCC. Mangiferin, a natural small molecule present in Mangifera indica L. has been shown to inactivate β-catenin, which is an indispensable regulator in Wnt pathway. Our study aimed to determine whether mangiferin has any inhibitory effect on HCC and examine how it modulates Wnt signalling.
METHODS: The tumour inhibitory effect of mangiferin was examined by in vitro cellular models and an in vivo orthotopic HCC implantation model. The genes responsible for mangiferin-mediated anti-HCC were delineated by polymerase chain reaction (PCR) microarray. The expression of target genes was further determined by quantitative PCR and immuno-blotting assays. The binding capacity of Wilms' tumour 1 (WT1) to the lymphoid enhancer-binding factor 1 (LEF1) promoter was confirmed by chromatin immunoprecipitation-qPCR.
RESULTS: Oral administration of mangiferin inhibited orthotopic tumour growth. Cellular investigations confirmed the dose-dependent inhibition of mangiferin on HCC expansion and invasion. PCR array combined with Gene Ontology analysis revealed that the Wnt pathway was the predominant target of mangiferin and LEF1 was the most reduced gene in the Wnt pathway. Overexpression of LEF1 diminished repression of Wnt signalling and reduced proliferation activity in mangiferin-treated HCC cells. The mangiferin-mediated down-regulation of LEF1 was independent of β-catenin but associated with WT1 protein. WT1 knock-in in HCC cells further enhanced LEF1 expression. Chromatin immunoprecipitation assays revealed that the mangiferin induced repression of LEF1 was associated with decreased occupancy of WT1 on the LEF1 promoter.
CONCLUSION: Our study identifies a novel mechanism of hepatocellular carcinoma inhibition through β-catenin-independent Wnt signalling, which is regulated by WT1-associated LEF1 repression. The study also highlights mangiferin as a promising Wnt inhibitor for HCC treatment.

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WT1

Cancer Prevention and Risk Reduction

Rosmaninho P, Mükusch S, Piscopo V, et al.
Zeb1 potentiates genome-wide gene transcription with Lef1 to promote glioblastoma cell invasion.
EMBO J. 2018; 37(15) [PubMed] Article available free on PMC after 01/10/2019 Related Publications

Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem-like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial-mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome-wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem-like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells

Zhang S, Li M, Ji H, Fang Z
Landscape of transcriptional deregulation in lung cancer.
BMC Genomics. 2018; 19(1):435 [PubMed] Article available free on PMC after 01/10/2019 Related Publications

BACKGROUND: Lung cancer is a very heterogeneous disease that can be pathologically classified into different subtypes including small-cell lung carcinoma (SCLC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC) and large-cell carcinoma (LCC). Although much progress has been made towards the oncogenic mechanism of each subtype, transcriptional circuits mediating the upstream signaling pathways and downstream functional consequences remain to be systematically studied.
RESULTS: Here we trained a one-class support vector machine (OC-SVM) model to establish a general transcription factor (TF) regulatory network containing 325 TFs and 18724 target genes. We then applied this network to lung cancer subtypes and identified those deregulated TFs and downstream targets. We found that the TP63/SOX2/DMRT3 module was specific to LUSC, corresponding to squamous epithelial differentiation and/or survival. Moreover, the LEF1/MSC module was specifically activated in LUAD and likely to confer epithelial-to-mesenchymal transition, known important for cancer malignant progression and metastasis. The proneural factor, ASCL1, was specifically up-regulated in SCLC which is known to have a neuroendocrine phenotype. Also, ID2 was differentially regulated between SCLC and LUSC, with its up-regulation in SCLC linking to energy supply for fast mitosis and its down-regulation in LUSC linking to the attenuation of immune response. We further described the landscape of TF regulation among the three major subtypes of lung cancer, highlighting their functional commonalities and specificities.
CONCLUSIONS: Our approach uncovered the landscape of transcriptional deregulation in lung cancer, and provided a useful resource of TF regulatory network for future studies.

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Lung Cancer

Katoh M
Multi‑layered prevention and treatment of chronic inflammation, organ fibrosis and cancer associated with canonical WNT/β‑catenin signaling activation (Review).
Int J Mol Med. 2018; 42(2):713-725 [PubMed] Article available free on PMC after 01/10/2019 Related Publications

β‑catenin/CTNNB1 is an intracellular scaffold protein that interacts with adhesion molecules (E‑cadherin/CDH1, N‑cadherin/CDH2, VE‑cadherin/CDH5 and α‑catenins), transmembrane‑type mucins (MUC1/CD227 and MUC16/CA125), signaling regulators (APC, AXIN1, AXIN2 and NHERF1/EBP50) and epigenetic or transcriptional regulators (BCL9, BCL9L, CREBBP/CBP, EP300/p300, FOXM1, MED12, SMARCA4/BRG1 and TCF/LEF). Gain‑of‑function CTTNB1 mutations are detected in bladder cancer, colorectal cancer, gastric cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer and uterine cancer, whereas loss‑of‑function CTNNB1 mutations are also detected in human cancer. ABCB1, ALDH1A1, ASCL2, ATF3, AXIN2, BAMBI, CCND1, CD44, CLDN1, CTLA4, DKK1, EDN1, EOMES, FGF18, FGF20, FZD7, IL10, JAG1, LEF1, LGR5, MITF, MSX1, MYC, NEUROD1, NKD1, NODAL, NOTCH2, NOTUM, NRCAM, OPN, PAX3, PPARD, PTGS2, RNF43, SNAI1, SP5, TCF7, TERT, TNFRSF19, VEGFA and ZNRF3 are representative β‑catenin target genes. β‑catenin signaling is involved in myofibroblast activation and subsequent pulmonary fibrosis, in addition to other types of fibrosis. β‑catenin and NF‑κB signaling activation are involved in field cancerization in the stomach associated with Helicobacter pylori (H. pylori) infection and in the liver associated with hepatitis C virus (HCV) infection and other etiologies. β‑catenin‑targeted therapeutics are functionally classified into β‑catenin inhibitors targeting upstream regulators (AZ1366, ETC‑159, G007‑LK, GNF6231, ipafricept, NVP‑TNKS656, rosmantuzumab, vantictumab, WNT‑C59, WNT974 and XAV939), β‑catenin inhibitors targeting protein‑protein interactions (CGP049090, CWP232228, E7386, ICG‑001, LF3 and PRI‑724), β‑catenin inhibitors targeting epigenetic regulators (PKF118‑310), β‑catenin inhibitors targeting mediator complexes (CCT251545 and cortistatin A) and β‑catenin inhibitors targeting transmembrane‑type transcriptional outputs, including CD44v6, FZD7 and LGR5. Eradicating H. pylori and HCV is the optimal approach for the first‑line prevention of gastric cancer and hepatocellular carcinoma (HCC), respectively. However, β‑catenin inhibitors may be applicable for the prevention of organ fibrosis, second‑line HCC prevention and treating β‑catenin‑driven cancer. The multi‑layered prevention and treatment strategy of β‑catenin‑related human diseases is necessary for the practice of personalized medicine and implementation of precision medicine.

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Kang MH, Choi H, Oshima M, et al.
Estrogen-related receptor gamma functions as a tumor suppressor in gastric cancer.
Nat Commun. 2018; 9(1):1920 [PubMed] Article available free on PMC after 01/10/2019 Related Publications

The principle factors underlying gastric cancer (GC) development and outcomes are not well characterized resulting in a paucity of validated therapeutic targets. To identify potential molecular targets, we analyze gene expression data from GC patients and identify the nuclear receptor ESRRG as a candidate tumor suppressor. ESRRG expression is decreased in GC and is a predictor of a poor clinical outcome. Importantly, ESRRG suppresses GC cell growth and tumorigenesis. Gene expression profiling suggests that ESRRG antagonizes Wnt signaling via the suppression of TCF4/LEF1 binding to the CCND1 promoter. Indeed, ESRRG levels are found to be inversely correlated with Wnt signaling-associated genes in GC patients. Strikingly, the ESRRG agonist DY131 suppresses cancer growth and represses the expression of Wnt signaling genes. Our present findings thus demonstrate that ESRRG functions as a negative regulator of the Wnt signaling pathway in GC and is a potential therapeutic target for this cancer.

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Stomach Cancer

Gastric Cancer

TCF4

Suzuki Y, Ichihara S, Kawasaki T, et al.
β-catenin (CTNNB1) mutation and LEF1 expression in sinonasal glomangiopericytoma (sinonasal-type hemangiopericytoma).
Virchows Arch. 2018; 473(2):235-239 [PubMed] Related Publications

Sinonasal glomangiopericytoma (SN-GPC) is an uncommon mesenchymal tumor with myoid differentiation. Recently, mutations in exon 3 of the gene coding for β-catenin (CTNNB1) and its nuclear expression were discovered in SN-GPC. β-catenin protein is a key regulatory molecule of the canonical Wnt signaling pathway. The expression of β-catenin target proteins is not well characterized in SN-GPC. We examined three SN-GPCs by immunohistochemistry and CTNNB1 mutation analysis. All cases expressed nuclear β-catenin. We identified CTNNB1 exon 3 mutations in two analyzable cases. Lymphoid enhancer-binding factor 1 (LEF1), a protein downstream from β-catenin, was also expressed in all cases. Our results further characterized the activation of the Wnt signaling pathway caused by CTNNB1 exon 3 mutation and suggest the utility of LEF1 immunohistochemistry in the differential diagnosis of SN-GPC.

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Jia P, Wei G, Zhou C, et al.
Upregulation of MiR-212 Inhibits Migration and Tumorigenicity and Inactivates Wnt/β-Catenin Signaling in Human Hepatocellular Carcinoma.
Technol Cancer Res Treat. 2018; 17:1533034618765221 [PubMed] Article available free on PMC after 01/10/2019 Related Publications

BACKGROUND: MicroRNAs are involved in hepatocellular carcinoma metastasis, a principal cause of hepatocellular carcinoma-related death in patients worldwide. MiR-212 is a microRNA that has been identified in several types of cancers and is postulated to influence cell signaling and subsequent malignant pathogenesis. Despite emerging reports suggesting that miR-212 plays a significant role in the onset, progression, and migration of these types of malignant tumors, its involvement in the development of hepatocellular carcinoma has not been fully elucidated.
MATERIALS AND METHODS: Quantitative reverse transcription polymerase chain reaction, wound healing, transwell migration and invasion assays, Western blotting, and xenograft tumor growth models were performed to test the expression levels and functions of miR-212 in hepatocellular carcinoma. Luciferase reporter assay, quantitative reverse transcription polymerase chain reaction, Western blotting, and immunohistochemistry were used to identify and verify the target of miR-212.
RESULTS: In this study, we identify significant repression of miR-212 in hepatocellular carcinoma and demonstrate that overexpression of miR-212 inhibits the migration of hepatocellular carcinoma cells in vitro and in vivo. Furthermore, we identify forkhead box M1, whose expression is inversely related to that of miR-212, as a direct target of miR-212. Additionally, reexpression of forkhead box M1 rescues the miR-212-mediated inhibition of cell migration. We observed that inhibition of miR-212 activates forkhead box M1 but inhibits the Wnt/β-catenin pathway by suppressing Wnt, LEF-1, c-Myc, and nuclear β-catenin. Finally, in vivo studies confirmed the inhibitory effect of miR-212 on hepatocellular carcinoma growth.
CONCLUSION: Our present findings indicate that miR-212 is a potential prognostic biomarker of hepatocellular carcinoma and that the miR-212/forkhead box M1 regulatory axis may represent a new therapeutic objective for hepatocellular carcinoma treatment.

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Gao L, Zhang LJ, Li SH, et al.
Role of miR-452-5p in the tumorigenesis of prostate cancer: A study based on the Cancer Genome Atl(TCGA), Gene Expression Omnibus (GEO), and bioinformatics analysis.
Pathol Res Pract. 2018; 214(5):732-749 [PubMed] Related Publications

BACKGROUND: MiR-452-5p has been reported to be down-regulated in prostate cancer, affecting the development of this type of cancer. However, the molecular mechanism of miR-452-5p in prostate cancer remains unclear. Therefore, we investigated the network of target genes of miR-452-5p in prostate cancer using bioinformatics analyses.
MATERIALS AND METHODS: We first analyzed the expression profiles and prognostic value of miR-452-5p in prostate cancer tissues from a public database. Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), PANTHER pathway analyses, and a disease ontology (DG) analysis were performed to find the molecular functions of the target genes from GSE datasets and miRWalk. Finally, we validated hub genes from the protein-protein interaction (PPI) networks of the target genes in the Human Protein Atlas (HPA) database and Gene Expression Profiling Interactive Analysis (GEPIA). Narrowing down the optimal target genes was conducted by seeking the common parts of up-regulated genes from GEPIA, down-regulated genes from GSE datasets, and predicted genes in miRWalk.
RESULTS: Based on mining of GEO and ArrayExpress microarray chips and miRNA-Seq data in the TCGA database, which includes 1007 prostate cancer samples and 387 non-cancer samples, miR-452-5p is shown to be down-regulated in prostate cancer. GO, KEGG, and PANTHER pathway analyses suggested that the target genes might participate in important biological processes, such as transforming growth factor beta signaling and the positive regulation of brown fat cell differentiation and mesenchymal cell differentiation, as well as the Ras signaling pathway and pathways regulating the pluripotency of stem cells and arrhythmogenic right ventricular cardiomyopathy (ARVC). Nine genes-GABBR, PNISR, NTSR1, DOCK1, EREG, SFRP1, PTGS2, LEF1, and BMP2-were defined as hub genes in the PPI network. Three genes-FAM174B, SLC30A4, and SLIT1-were jointly shared by GEPIA, the GSE datasets, and miRWalk.
CONCLUSIONS: Down-regulated miR-452-5p might play an essential role in the tumorigenesis of prostate cancer.

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