Physical association of functionally antagonistic enzymes: KDM5A interacts with MLLs to regulate gene expression in a promoter specific manner facilitating EMT and pluripotency

Differential expression of genes involved in physiological processes are a collaborative outcome of interactions among signalling molecules, downstream effectors and epigenetic modifiers, which together dictate the regulation of genes in response to specific stimuli. MLLs and KDM5A are functionally antagonistic proteins as one acts as writer and the other as eraser of the active chromatin mark, i.e., H3K4me3. KDM5A promotes EMT by occupying promoters of both epithelial and mesenchymal markers. Through this work, it is illustrated that when bound to E-cadherin promoter, KDM5A acts as a classical repressor by demethylating H3K4me3, but on mesenchymal marker promoters, it acts as a transcriptional activator by inhibiting the activity of HDACs and increasing H3K18ac. Further it is demonstrated that KDM5A occupancy enhances either MLL1 or MLL2 by physically interacting with them and that signalling pathways regulate the enzymatic activity of KDM5A probably by phosphorylation. When not active, KDM5A signals for MLL occupancy, a mechanism that can be called epigenetic signalling.

Monophasic Synovial Sarcoma of the Left Ventricle of the Heart: An Extremely Rare Case and Literature Review

BACKGROUND: Cardiac sarcomas account for <25% of all cardiac tumors. Of these, angiosarcomas are the most frequent. Synovial sarcomas (SS) are exceedingly rare. We present a case of primary left ventricle (LV) SS, a form of sarcoma particularly rare in the heart. CASE DESCRIPTION: A 19-year-old male was referred for further investigation of a LV tumor, presented with a 3-month history of exertional dyspnea and palpitations. He also experienced several syncopal episodes. The radiologic examination confirmed a mass in the LV, suspected for myxoma of the LV. Histopathologic examination revealed a malignant tumor with spindle cell components, suggesting leiomyosarcoma with differential diagnosis of monophasic SS. Immunohistochemistry demonstrated reactivity of the spindle cell component with the mesenchymal marker vimentin and BCL2 protein, while the smooth muscle marker, desmin, was negative, confirming the diagnosis of monophasic SS. CONCLUSIONS: Monophasic SS in the heart is diagnostically challenging since it shares the broad list of differential diagnoses of spindle cell tumors. Immunostaining is helpful to differentiate those entities to obtain a definitive diagnosis and proper treatment.

Single cell sequencing reveals endothelial plasticity with transient mesenchymal activation after myocardial infarction

After myocardial infarct (MI), followed by ischemia and scar formation, interstitial cells play key roles in the adaptation to injury. Endothelial cells (ECs), for instance, can clonally expand, migrate into the infarct area and facilitate crucial functions promoting revascularization, reestablishment of oxygen supply and secretion of paracrine factors. Moreover, ECs can transiently undergo changes towards a mesenchymal phenotype (Endothelial-to-mesenchymal transition; EndMT). Whether this process contributes to long-term cardiac fibrosis or helps to facilitate post-ischemic vessel growth remains controversial. Here, we aim to delineate kinetics and characteristics of phenotypic changes in ECs with single cell RNA-sequencing (scRNA-seq). We performed a time course (homeostasis or 0 day (d), 1d, 3d, 5d, 7d, 14d, 28d post-MI) in mice and isolated the non-cardiomyocyte fraction for scRNA-seq (n=35,312 cells). Pecam1/Cdh5 double positive ECs showed expression of apoptosis, hypoxia and inflammation markers at 3d. Bioinformatic cell cycle analysis predicted high association with proliferative capacities at 3d, indicative of EC turnover post-MI. Metabolism, recently linked to regulate EndMT, was altered. We found genes of the glycolysis and the TCA-cycle pathway upregulated at 1d to 3d, and a decrease of fatty acid signaling genes. At 3d, mesenchymal markers Fn1, Vim, S100a4, Serpine1 transiently increased compared to homeostasis (&gt;1.6-fold, p&lt;0.05) together with a reduction of EC genes such as Pecam1. Interestingly, mesenchymal transition was transient and returned to baseline levels at 28d after MI. Cell fate trajectory analysis confirmed these findings by identifying an EC state characterized by high proliferation and mesenchymal but low EC properties. At 3d to 7d the majority of the ECs were assigned to this state, based on their transcriptomic profile. We additionally used Cdh5-CreERT2; R26-mT/mG mice followed by scRNA-seq to trace the fate of ECs. Bioinformatic analysis of GFP-positive ECs confirmed the gain in mesenchymal marker but revealed no full transition to the mesenchymal state at later timepoints. This suggests a transient mesenchymal activation of ECs rather than a complete lineage transition. We further induced EndMT with TGF-β2 in ECs in vitro and observed reversibility of the phenotype after withdrawal of the stimulus. After treatment, ECs upregulated various mesenchymal marker genes. Withdrawal of TGF-β2 at 3d or 7d, reverted expression to baseline levels. We further determined DNA methylation of EndMT gene loci to assess if TGF-β2 leads to a true fate change but did not observe changes after TGF-β2 stimulation and withdrawal. Taken together, our data suggests that ECs undergo a transient mesenchymal activation concomitant with a metabolic adaptation early after MI but do not acquire a long-term mesenchymal fate. This activation may facilitate EC migration and clonal expansion to regenerate the vascular network. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Center of Cardiovascular Research (DZHK), Deutsche Forschungsgemeinschaft (DFG) CRC1366 Project B4

STEM-11. CD146/MCAM REGULATES MESENCHYMAL PROPERTIES, STEMNESS, RADIO-RESISTANCE AND YAP ACTIVITY IN GLIOBLASTOMA

Glioblastoma (GBM), a highly malignant and lethal brain tumor, is characterized by diffuse invasion into the brain and chemoradiotherapy resistance resulting in recurrences and poor prognosis. In this study we examined if the cell adhesion molecule CD146/ MCAM is contributing to the malignant properties of GBM, as it is known to have multiple protumorigenic functions in other tumor types. TCGA GBM database analyses revealed enhanced levels of CD146 in GBM compared to normal brain. A panel of patient-derived GBM spheroids, enriched for GBM stem cells (GSC), displayed variable and modest expression of CD146 that was strongly enhanced by cell adherence mediated by serum differentiation or ECM-coating. TGF-β-induced mesenchymal transition in U87 cells was accompanied by induction of CD146 expression, whereas ectopic overexpression of CD146/GFP in GG16 spheroids increased mesenchymal marker expression and cell invasion. Conversely, CRISPR/Cas9-generated CD146 knockouts in GSC23 spheroids led to reduced mesenchymal marker expression and invasion. These results were confirmed by applying embryonic stem cell-derived brain organoids as models to study invasion. Hence, GBM spheroid - brain organoid fusions demonstrated a stimulatory function of CD146 on invasion. Moreover, stem cell marker expression and clonogenic assays showed that CD146 increases the stem cell potential of GBM cells. The CD146 ectopic overexpression and knockout models also demonstrated involvement of CD146 in resistance to radiation that could be mechanistically linked with CD146-dependent suppression of p53 accumulation and activation of NF-kB. Interestingly, exploration of additional mechanisms potentially involved in CD146 functioning, led to the discovery that the oncogenic protein Yes-associated protein (YAP) is also regulated by CD146. Together, our findings demonstrate the importance of CD146 in controlling tumor aggressiveness and radioresistance in GBM cells.

The Epigenetic Factor KDM2B Regulates EMT and Small GTPases in Colon Tumor Cells

Background/Aims: The epigenetic factor KDM2B is a histone demethylase expressed in various tumors. Recently, we have shown that KDM2B regulates actin cytoskeleton organization, small Rho GTPases signaling, cell-cell adhesion and migration of prostate tumor cells. In the present study, we addressed its role in regulating EMT and small GTPases expression in colon tumor cells. Methods: We used RT-PCR for the transcriptional analysis of various genes, Western blotting for the assessment of protein expression and immunofluorescence microscopy for visualization of fluorescently labeled proteins. Results: We report here that KDM2B regulates EZH2 and BMI1 in HCT116 colon tumor cells. Knockdown of this epigenetic factor induced potent up-regulation of the protein levels of the epithelial markers E-cadherin and ZO-1, while the mesenchymal marker N-cadherin was downregulated. On the other hand, KDM2B overexpression downregulated the levels of both epithelial markers and upregulated the mesenchymal marker, suggesting control of EMT by KDM2B. In addition, RhoA, RhoB and RhoC protein levels diminished upon KDM2B-knockdown, while all three small GTPases became upregulated in KDM2B-overexpressing HCT116 cell clones. Interestingly, Rac1 GTPase level increased upon KDM2B-knockdown and diminished in KDM2B-overexpressing HCT116 colon tumor- and DU-145 prostate cancer cells. Conclusions: These results establish a clear functional role of the epigenetic factor KDM2B in the regulation of EMT and small-GTPases expression in colon tumor cells and further support the recently postulated oncogenic role of this histone demethylase in various tumors.