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The RAS family of proto-oncogenes are among the most commonly mutated genes in human cancers and predict poor clinical outcome. Several mechanisms underlying oncogenic RAS transformation are well documented, including constitutive signaling through the RAF-MEK-ERK proproliferative pathway as well as the PI3K-AKT prosurvival pathway. Notably, control of redox balance has also been proposed to contribute to RAS transformation. However, how homeostasis between reactive oxygen species (ROS) and antioxidants, which have opposing effects in the cell, ultimately influence RAS-mediated transformation and tumor progression is still a matter of debate and the mechanisms involved have not been fully elucidated. Here, we show that oncogenic KRAS protects fibroblasts from oxidative stress by enhancing intracellular GSH levels. Using a whole transcriptome approach,we discovered that this is attributable to transcriptional up-regulation of xCT, the gene encoding the cystine/glutamate antiporter. This is in line with the function of xCT, which mediates the uptake of cystine, a precursor for GSH biosynthesis. Moreover, our results reveal that the ETS-1 transcription factor downstream of the RAS-RAF-MEK-ERK signaling cascade directly transactivates the xCT promoter in synergy with the ATF4 endoplasmic reticulum stress-associated transcription factor. Strikingly, xCT was found to be essential for oncogenic KRAS-mediated transformation in vitro and in vivo bymitigating oxidative stress, as knockdown of xCT strongly impaired growth of tumor xenografts established from KRAS-transformed cells. Overall, this study uncovers a mechanism by which oncogenic RAS preserves intracellular redox balance and identifies an unexpected role for xCT in supporting RAS-induced transformation and tumorigenicity.

The dynamic cross correlation (DCC) analysis is a popular method for analyzing the trajectories of molecular dynamics (MD) simulations. However, it is difficult to detect correlative motions that appear transiently in only a part of the trajectory, such as atomic contacts between the side-chains of amino acids, which may rapidly flip. In order to capture these multi-modal behaviors of atoms, which often play essential roles, particularly at the interfaces of macromolecules, we have developed the \"multi-modal DCC (mDCC)\" analysis. The mDCC is an extension of the DCC and it takes advantage of a Bayesian-based pattern recognition technique. We performed MD simulations for molecular systems modeled from the (Ets1)2-DNA complex and analyzed their results with the mDCC method. Ets1 is an essential transcription factor for a variety of physiological processes, such as immunity and cancer development. Although many structural and biochemical studies have so far been performed, its DNA binding properties are still not well characterized. In particular, it is not straightforward to understand the molecular mechanisms how the cooperative binding of two Ets1 molecules facilitates their recognition of Stromelysin-1 gene regulatory elements. A correlation network was constructed among the essential atomic contacts, and the two major pathways by which the two Ets1 molecules communicate were identified. One is a pathway via direct protein-protein interactions and the other is that via the bound DNA intervening two recognition helices. These two pathways intersected at the particular cytosine bases (C110/C11), interacting with the H1, H2, and H3 helices. Furthermore, the mDCC analysis showed that both pathways included the transient interactions at their intermolecular interfaces of Tyr396-C11 and Ala327-Asn380 in multi-modal motions of the amino acid side chains and the nucleotide backbone. Thus, the current mDCC approach is a powerful tool to reveal these complicated behaviors and scrutinize intermolecular communications in a molecular system.

Transcriptional reactivation of TERT, the catalytic subunit of telomerase, is necessary for cancer progression in about 90% of human cancers. The recent discovery of two prevalent somatic mutations—C250T and C228T—in the TERT promoter in various cancers has provided insight into a plausible mechanism of TERT reactivation. Although the two hotspot mutations create a similar binding motif for E-twenty-six (ETS) transcription factors, we show that they are functionally distinct, in that the C250T unlike the C228T TERT promoter is driven by non-canonical NF-κB signalling. We demonstrate that binding of ETS to the mutant TERT promoter is insufficient in driving its transcription but this process requires non-canonical NF-κB signalling for stimulus responsiveness, sustained telomerase activity and hence cancer progression. Our findings highlight a previously unrecognized role of non-canonical NF-κB signalling in tumorigenesis and elucidate a fundamental mechanism for TERT reactivation in cancers, which if targeted could have immense therapeutic implications. The TERT promoter is mutated in many cancers. Li et al.  show that non-canonical NF-κB signalling and ETS1/2 transcription factors are jointly needed to activate the C250T mutant TERT promoter, leading to telomerase activity and glioblastoma growth.

Aberrant vascularization is a hallmark of cancer progression and treatment resistance. Here, we have shown that endothelial cell (EC) plasticity drives aberrant vascularization and chemoresistance in glioblastoma multiforme (GBM). By utilizing human patient specimens, as well as allograft and genetic murine GBM models, we revealed that a robust endothelial plasticity in GBM allows acquisition of fibroblast transformation (also known as endothelial mesenchymal transition [Endo-MT]), which is characterized by EC expression of fibroblast markers, and determined that a prominent population of GBM-associated fibroblast-like cells have EC origin. Tumor ECs acquired the mesenchymal gene signature without the loss of EC functions, leading to enhanced cell proliferation and migration, as well as vessel permeability. Furthermore, we identified a c-Met/ETS-1/matrix metalloproteinase-14 (MMP-14) axis that controls VE-cadherin degradation, Endo-MT, and vascular abnormality. Pharmacological c-Met inhibition induced vessel normalization in patient tumor-derived ECs. Finally, EC-specific KO of Met inhibited vascular transformation, normalized blood vessels, and reduced intratumoral hypoxia, culminating in suppressed tumor growth and prolonged survival in GBM-bearing mice after temozolomide treatment. Together, these findings illustrate a mechanism that controls aberrant tumor vascularization and suggest that targeting Endo-MT may offer selective and efficient strategies for antivascular and vessel normalization therapies in GBM, and possibly other malignant tumors.

Background N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer. Methods We determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay. Results We demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1. Conclusion We have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment.

Interleukin 17 (IL-17)-producing helper T cells (T H -17 cells) are associated with the pathogenesis of multiple sclerosis. Pei and colleagues have now identified a T H -17 cell–associated microRNA, miR-326, whose expression correlates with disease severity in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis. Interleukin 17 (IL-17)-producing T helper cells (T H -17 cells) are increasingly recognized as key participants in various autoimmune diseases, including multiple sclerosis. Although sets of transcription factors and cytokines are known to regulate T H -17 differentiation, the role of noncoding RNA is poorly understood. Here we identify a T H -17 cell–associated microRNA, miR-326, whose expression was highly correlated with disease severity in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis (EAE). In vivo silencing of miR-326 resulted in fewer T H -17 cells and mild EAE, and its overexpression led to more T H -17 cells and severe EAE. We also found that miR-326 promoted T H -17 differentiation by targeting Ets-1, a negative regulator of T H -17 differentiation. Our data show a critical role for microRNA in T H -17 differentiation and the pathogenesis of multiple sclerosis.

Cancer cells have metabolic features that allow them to preferentially metabolize glucose through aerobic glycolysis, providing them with a progression advantage. However, microRNA (miRNA) regulation of aerobic glycolysis in cancer cells has not been extensively investigated. We addressed this in the present study by examining the regulation of miR-139-5p on aerobic glycolysis of hepatocellular carcinoma (HCC) using clinical specimens, HCC cells, and a mouse xenograft model. We found that overexpressing miR-139-5p restrained aerobic glycolysis, suppressing proliferation, migration, and invasion in HCC cells. miR-139-5p regulated hexokinase 1 (HK1) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) expression by directly targeting the transcription factor ETS1, which bound to the promoters of the HK1 and PFKFB3 genes. miR-139-5p-induced aerobic glycolysis, proliferation, migration, and invasion were reversed by ETS1 overexpression, while ETS1 silencing induced the expression of miR-139-5p via a post-transcriptional regulation mode involving Drosha. miR-139-5p expression was reduced in HCC compared to para-carcinoma tissue, which was confirmed in The Cancer Genome Atlas and GSE54751 HCC cohorts. Notably, the lower expression of mir-139 was correlated with worse prognosis. These outcomes indicate that reciprocal regulatory interactions between miR-139-5p and ETS1 modulate aerobic glycolysis, proliferation, and metastasis in HCC cells, suggesting new targets for HCC treatment.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic predisposition, characterized by an upregulated type I interferon pathway. MicroRNAs are important regulators of immune homeostasis, and aberrant microRNA expression has been demonstrated in patients with autoimmune diseases. We recently identified miR-146a as a negative regulator of the interferon pathway and linked the abnormal activation of this pathway to the underexpression of miR-146a in SLE patients. To explore why the expression of miR-146a is reduced in SLE patients, we conducted short parallel sequencing of potentially regulatory regions of miR-146a and identified a novel genetic variant (rs57095329) in the promoter region exhibiting evidence for association with SLE that was replicated independently in 7,182 Asians (P(meta) = 2.74×10(-8), odds ratio = 1.29 [1.18-1.40]). The risk-associated G allele was linked to reduced expression of miR-146a in the peripheral blood leukocytes of the controls. Combined functional assays showed that the risk-associated G allele reduced the protein-binding affinity and activity of the promoter compared with those of the promoter containing the protective A allele. Transcription factor Ets-1, encoded by the lupus-susceptibility gene ETS1, identified in recent genome-wide association studies, binds near this variant. The manipulation of Ets-1 levels strongly affected miR-146a promoter activity in vitro; and the knockdown of Ets-1, mimicking its reduced expression in SLE, directly impaired the induction of miR-146a. We also observed additive effects of the risk alleles of miR-146a and ETS1. Our data identified and confirmed an association between a functional promoter variant of miR-146a and SLE. This risk allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients.

Cancer-related deregulation of miRNA biogenesis has been suggested, but the underlying mechanisms remain elusive. Here we report a previously unrecognized effect of hypoxia in the downregulation of Drosha and Dicer in cancer cells that leads to dysregulation of miRNA biogenesis and increased tumour progression. We show that hypoxia-mediated downregulation of Drosha is dependent on ETS1/ELK1 transcription factors. Moreover, mature miRNA array and deep sequencing studies reveal altered miRNA maturation in cells under hypoxic conditions. At a functional level, this phenomenon results in increased cancer progression in vitro and in vivo , and data from patient samples are suggestive of miRNA biogenesis downregulation in hypoxic tumours. Rescue of Drosha by siRNAs targeting ETS1/ELK1 in vivo results in significant tumour regression. These findings provide a new link in the mechanistic understanding of global miRNA downregulation in the tumour microenvironment. MicroRNAs play important roles in the maintenance of cellular homeostasis through the post-transcriptional regulation of gene expression. Here, the authors implicate loss of the miRNA biogenesis factor Drosha and altered miRNA maturation in tumour progression under hypoxic conditions.

Increasing evidence has revealed that miR‐199a‐5p is actively involved in tumor invasion and metastasis as well as in the decline of breast cancer tissues. In this research, overexpression of miR‐199a‐5p weakened motility and invasion of breast cancer cells MCF‐7 and MDA‐MB‐231. Upregulation of Ets‐1 increased breast cancer cell invasion, but the mechanism by which miR‐199a‐5p modulates activation of Ets‐1 in breast cancer was not clarified. We investigated the relationship between miR‐199a‐5p and Ets‐1 on the basis of 158 primary breast cancer case specimens, and the results showed that Ets‐1 expression was inversely correlated with endogenous miR‐199a‐5p. Overexpression of miR‐199a‐5p reduced the mRNA and protein levels of Ets‐1 in MCF‐7 and MDA‐MB‐231 cells, whereas anti‐miR‐199a‐5p elevated Ets‐1. siRNA‐mediated Ets‐1 knockdown phenocopied the inhibition invasion of miR‐199a‐5p in vitro. Moreover, luciferase reporter assay revealed that miR‐199a‐5p directly targeted 3′‐UTR of Ets‐1 mRNA. This research revealed that miR‐199a‐5p could descend the levels of β1 integrin by targeting 3′‐UTR of Ets‐1 to alleviate the invasion of breast cancer via FAK/Src/Akt/mTOR signaling pathway. Our results provide insight into the regulation of β1 integrin through miR‐199a‐5p‐mediated Ets‐1 silence and will help in designing new therapeutic strategies to inhibit signal pathways induced by miR‐199a‐5p in breast cancer invasion. Our results provide insight into the regulation of β1 integrin through miR‐199a‐5p‐mediated Ets‐1 silence and help in designing new therapeutic strategies to inhibit signal pathways induced by miR‐199a‐5p in breast cancer invasion.