SETD8 stabilized by USP17 epigenetically activates SREBP1 pathway to drive lipogenesis and oncogenesis of ccRCC.

Recently, epigenetic modifications, including DNA methylation, histone modification and noncoding RNA (ncRNA)-associated gene silencing, have received increasing attention from the scientific community. Many studies have demonstrated that epigenetic regulation can render dynamic alterations in the transcriptional potential of a cell, which then affects the cell's biological function. The initiation and development of clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell cancer (RCC), is also closely related to genomic alterations by epigenetic modification. For ccRCC, lipid accumulation is one of the most typical characteristics. In other words, dysregulation of lipid uptake and synthesis occurs in ccRCC, which inversely promotes cancer proliferation and progression. However, the link among epigenetic alterations, lipid biosynthesis and renal cancer progression remains unclear. SETD8 is a histone methyltransferase and plays pivotal roles in cell cycle regulation and oncogenesis of various cancers, but its role in RCC is not well understood. In this study, we discovered that SETD8 was significantly overexpressed in RCC tumors, which was positively related to lipid storage and correlated with advanced tumor grade and stage and poor patient prognosis. Depletion of SETD8 by siRNAs or inhibitor UNC0379 diminished fatty acid (FA) de novo synthesis, cell proliferation and metastasis in ccRCC cells. Mechanistically, SETD8, which was posttranslationally stabilized by USP17, could transcriptionally modulate sterol regulatory element-binding protein 1 (SREBP1), a key transcription factor in fatty acid biosynthesis and lipogenesis, by monomethylating the 20th lysine of the H4 histone, elevating lipid biosynthesis and accumulation in RCC and further promoting cancer progression and metastasis. Taken together, the USP17/SETD8/SREBP1 signaling pathway plays a pivotal role in promoting RCC progression. SETD8 might be a novel biomarker and potential therapeutic target for treating RCC.

TERT promoter mutations and methylation for telomerase activation in urothelial carcinomas: New mechanistic insights and clinical significance.

Telomerase, an RNA-dependent DNA polymerase synthesizing telomeric TTAGGG sequences, is primarily silent in normal human urothelial cells (NHUCs), but widely activated in urothelial cell-derived carcinomas or urothelial carcinomas (UCs) including UC of the bladder (UCB) and upper track UC (UTUC). Telomerase activation for telomere maintenance is required for the UC development and progression, and the key underlying mechanism is the transcriptional de-repression of the telomerase reverse transcriptase (TERT), a gene encoding the rate-limiting, telomerase catalytic component. Recent mechanistic explorations have revealed important roles for TERT promoter mutations and aberrant methylation in activation of TERT transcription and telomerase in UCs. Moreover, these TERT-featured genomic and epigenetic alterations have been evaluated for their usefulness in non-invasive UC diagnostics, recurrence monitoring, outcome prediction and response to treatments such as immunotherapy. Importantly, the detection of the mutated TERT promoter and TERT mRNA as urinary biomarkers holds great promise for urine-based UC liquid biopsy. In the present article, we review recent mechanistic insights into altered TERT promoter-mediated telomerase activation in UCs and discuss potential clinical implications. Specifically, we compare differences in senescence and transformation between NHUCs and other types of epithelial cells, address the interaction between TERT promoter mutations and other factors to affect UC progression and outcomes, evaluate the impact of TERT promoter mutations and TERT-mediated activation of human endogenous retrovirus genes on UC immunotherapy including Bacillus Calmette-Guérin therapy and immune checkpoint inhibitors. Finally, we suggest the standardization of a TERT assay and evaluation system for UC clinical practice.

Intrinsic 5-lipoxygenase activity regulates migration and adherence of mantle cell lymphoma cells.

Human B-lymphocytes express 5-lipoxygenase (5-LOX) and 5-LOX activating protein (FLAP) and can convert arachidonic acid to leukotriene B4. Mantle cell lymphoma (MCL) cells contain similar amounts of 5-LOX as human neutrophils but the function and mechanism of activation of 5-LOX in MCL cells, and in normal B-lymphocytes, are unclear. Here we show that the intrinsic 5-LOX pathway in the MCL cell line JeKo-1 has an essential role in migration and adherence of the cells, which are important pathophysiological characteristics of B-cell lymphoma. Incubation of JeKo-1 with the FLAP inhibitor GSK2190915 or the 5-LOX inhibitor zileuton, at a concentration below 1 µM, prior to stimulation with the chemotactic agent CXCL12, led to a significant reduction of migration. CRISPR/Cas9 mediated deletion of ALOX5 gene in JeKo-1 cells also led to a significantly decreased migration of the cells. Furthermore, 5-LOX and FLAP inhibitors markedly decreased the adherence of JeKo-1 cells to stromal cells. In comparison, these drugs had a similar effect on adherence of JeKo-1 cells as the Bruton tyrosine kinase inhibitor ibrutinib, which has a proven anti-tumour effect. These results indicate that inhibition of 5-LOX may be a novel treatment for MCL and certain other B-cell lymphomas.

Hodgkin Lymphoma Monozygotic Triplets Reveal Divergences in DNA Methylation Signatures.

We studied DNA methylation profiles in four different cell populations from a unique constellation of monozygotic triplets in whom two had developed Hodgkin Lymphoma (HL). We detected shared differences in DNA methylation signatures when comparing the two HL-affected triplets with the non-affected triplet. The differences were observed in naïve B-cells and marginal zone-like B-cells. DNA methylation differences were also detected when comparing each of the HL-affected triplets against each other. Even though we cannot determine whether treatment and/or disease triggered the observed differences, we believe our data are important on behalf of forthcoming studies, and that it might provide important clues for a better understanding of HL pathogenesis.

JAK2 inhibition in JAK2V617F-bearing leukemia cells enriches CD34+ leukemic stem cells that are abolished by the telomerase inhibitor GRN163L.

The activating-mutation of JAK2V617F drives the development of myeloproliferative neoplasms (MPNs). Several JAK2 inhibitors such as ruxolitinib and gandotinib (LY2784544) currently in clinical trials and, provide improvements in MPNs including myelofibrosis. However, JAK2 inhibitors are non-curative and murine experiments show that JAK2 inhibitors don't eradicate MPN stem cells and it is currently unclear how they escape. We thus determined the effect of the specific JAK2V617F inhibitor LY2784544 on leukemic stem (CD34+) cells (LSCs) using the JAK2V617F-bearing erythroleukemia cell line HEL. The LY2784544 treatment caused a transient proliferation inhibition and apoptosis of HEL cells, but a recovery occurred within a week. Thereafter, the continuous LY2784544 exposure induced the accumulation of CD34+ LSCs, and the CD34+ cells increased from 2% to >90% by week 9, which was accompanied by increased clonogenic potentials. LY2784544 was capable of stimulating CD34 expression even in CD34- HEL cells, which indicated cellular de-differentiation. A significantly enhanced expression of the stem cell factor KLF4 was observed in LY2784544-treated HEL cells. Inhibiting KLF4 expression attenuated LY2784544-mediated accumulation of CD34+ LSCs. Moreover, the telomerase inhibitor GRN163L abolished the LY2784544-effect. JAK2 inhibitors thus cause enrichment of LSCs and are unlikely to cure MPN as a monotherapy. Simultaneously targeting JAK2V617F and KLF4 or telomerase may be a novel strategy for MPN therapy, which should be of significance both biologically and clinically.

VHL-HIF-2α axis-induced SMYD3 upregulation drives renal cell carcinoma progression via direct trans-activation of EGFR.

It has been well established that the von Hippel-Lindau/hypoxia-inducible factor α (VHL-HIFα) axis and epidermal growth factor receptor (EGFR) signaling pathway play a critical role in the pathogenesis and progression of renal cell carcinoma (RCC). However, few studies have addressed the relationship between the two oncogenic drivers in RCC. SET and MYND domain-containing protein 3 (SMYD3) is a histone methyltransferase involved in gene transcription and oncogenesis, but its expression and function in RCC remain unclear. In the present study, we found that SMYD3 expression was significantly elevated in RCC tumors and correlated with advanced tumor stage, histological and nuclear grade, and shorter survival. Depletion of SMYD3 inhibited RCC cell proliferation, colony numbers, and xenograft tumor formation, while promoted apoptosis. Mechanistically, SMYD3 cooperates with SP1 to transcriptionally promote EGFR expression, amplifying its downstream signaling activity. TCGA data analyses revealed a significantly increased SMYD3 expression in primary RCC tumors carrying the loss-of-function VHL mutations. We further showed that HIF-2α can directly bind to the SMYD3 promoter and subsequently induced SMYD3 transcription and expression. Taken together, we identify the VHL/HIF-2α/SMYD3 signaling cascade-mediated EGFR hyperactivity through which SMYD3 promotes RCC progression. Our study suggests that SMYD3 is a potential therapeutic target and prognostic factor in RCC.

High expression of SMYD3 indicates poor survival outcome and promotes tumour progression through an IGF-1R/AKT/E2F-1 positive feedback loop in bladder cancer.

The AKT/mTOR pathway is critical for bladder cancer (BC) pathogenesis and is hyper-activated during BC progression. In the present study, we identified a novel positive feedback loop involving oncogenic factors histone methyltransferase SMYD3, insulin-like growth factor-1 receptor (IGF-1R), AKT, and E2F-1. SMYD3 expression was significantly up-regulated in BC tumors and positively associated with histological grade, lymph node metastasis, and shorter patient survival. Depletion of SMYD3 inhibited BC cell proliferation, colony formation, migration, invasion, and xenograft tumor growth. Mechanistically, SMYD3 inhibition led to the diminished AKT/mTOR signaling activity, thereby triggering deleterious effects on BC cells. Furthermore, SMYD3 directly activates the expression of IGF-1R, a critical activator of AKT in BC, by inducing hyper-methylation of histone H3-K4 and subsequent chromatin remodeling in the IGF-1R promoter region. On the other hand, E2F-1, a downstream factor of the AKT pathway, binds to the E2F-1 binding motifs at the SMYD3 promoter and consequently induces SMYD3 transcription and expression. Thus, SMYD3/IGF-1R/AKT/E2F-1 forms a positive feedback loop leading to the hyper-activated AKT signaling. Our findings provide not only profound insights into SMYD3-mediated oncogenic activity but also present a unique avenue for treating BC by directly disrupting this signaling circuit.

TERT Promoter Hypermethylation in Gastrointestinal Cancer: A Potential Stool Biomarker.

BACKGROUND: There is a high demand for noninvasive screening tools for gastrointestinal cancer (GIC) detection, and GIC-specific markers are required for such purposes. It is established that induction of the telomerase reverse transcriptase gene (TERT) coupled with telomerase activation is essential for cancer development/progression and aberrant TERT promoter methylation of specific 5'-C-phosphate-G-3' (CpGs) has been linked to TERT induction in oncogenesis. Here we analyzed TERT promoter methylation in fecal samples from GIC patients and healthy adults and determined its value as a stool biomarker for GIC detection. MATERIALS AND METHODS: Sixty-nine GIC patients (34 colorectal carcinoma and 35 gastric cancer) and 62 healthy adults were recruited and fecal samples were collected. Paired tumors and adjacent non-cancerous tissues from 34 patients and normal mucosa tissues from 12 healthy individuals were collected. TERT promoter methylation density was determined using pyrosequencing. RESULTS: We identified two GIC-specific methylation sites at -218 (CpG site 1) and -210 (CpG site 2) in the TERT promoter in tumor tissues. Methylated TERT promoter CpG sites 1 and 2 were also detectable in patient stool, while only background levels were observed in healthy individuals. The overall sensitivity reached 52.2% (95% confidence interval [CI]: 48.3-56.0) for fecal methylated TERT promoter assays at 90% specificity, which was comparable to other known stool methylation markers for GIC detection. The combined assays of fecal TERT promoter methylation and occult blood (OB) significantly improved sensitivity and specificity in colorectal cancer (area under curves for methylation alone: 0.798, 95% CI: 0.707-0.889 vs. methylation + OB: 0.920, 95% CI: 0.859-0.981; p = .028), but not in gastric cancer. CONCLUSION: This proof-of-concept study suggests the feasibility of stool TERT promoter methylation analyses as an additional tool in noninvasive GIC screening. IMPLICATIONS FOR PRACTICE: Induction of telomerase reverse transcriptase (TERT) expression coupled with telomerase activation is essential for cancer development/progression, while aberrant TERT promoter methylation has been linked to TERT induction in oncogenesis. We identified two cancer-specific methylation sites (CpG1 and 2) in the TERT promoter in tumors from GIC patients. Methylated TERT promoter CpG sites 1 and 2 were detectable in patient stool, while only background levels were observed in healthy individuals. The sensitivity and specificity was comparable to other known stool methylation markers for GIC detection. This proof-of-concept study suggests the feasibility of stool TERT promoter methylation analyses for noninvasive screening of GIC.