Accumulation of 8-oxodG within the human mitochondrial genome positively associates with transcription.

Mitochondrial DNA (mtDNA) can be subject to internal and environmental stressors that lead to oxidatively generated damage and the formation of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxodG). The accumulation of 8-oxodG has been linked to degenerative diseases and aging, as well as cancer. Despite the well-described implications of 8-oxodG in mtDNA for mitochondrial function, there have been no reports of mapping of 8-oxodG across the mitochondrial genome. To address this, we used OxiDIP-Seq and mapped 8-oxodG levels in the mitochondrial genome of human MCF10A cells. Our findings indicated that, under steady-state conditions, 8-oxodG is non-uniformly distributed along the mitochondrial genome, and that the longer non-coding region appeared to be more protected from 8-oxodG accumulation compared with the coding region. However, when the cells have been exposed to oxidative stress, 8-oxodG preferentially accumulated in the coding region which is highly transcribed as H1 transcript. Our data suggest that 8-oxodG accumulation in the mitochondrial genome is positively associated with mitochondrial transcription.

EGR1 drives cell proliferation by directly stimulating TFEB transcription in response to starvation.

The stress-responsive transcription factor EB (TFEB) is a master controller of lysosomal biogenesis and autophagy and plays a major role in several cancer-associated diseases. TFEB is regulated at the posttranslational level by the nutrient-sensitive kinase complex mTORC1. However, little is known about the regulation of TFEB transcription. Here, through integrative genomic approaches, we identify the immediate-early gene EGR1 as a positive transcriptional regulator of TFEB expression in human cells and demonstrate that, in the absence of EGR1, TFEB-mediated transcriptional response to starvation is impaired. Remarkably, both genetic and pharmacological inhibition of EGR1, using the MEK1/2 inhibitor Trametinib, significantly reduced the proliferation of 2D and 3D cultures of cells displaying constitutive activation of TFEB, including those from a patient with Birt-Hogg-Dubé (BHD) syndrome, a TFEB-driven inherited cancer condition. Overall, we uncover an additional layer of TFEB regulation consisting in modulating its transcription via EGR1 and propose that interfering with the EGR1-TFEB axis may represent a therapeutic strategy to counteract constitutive TFEB activation in cancer-associated conditions.

Autophagy Roles in Genome Maintenance.

In recent years, a considerable correlation has emerged between autophagy and genome integrity. A range of mechanisms appear to be involved where autophagy participates in preventing genomic instability, as well as in DNA damage response and cell fate decision. These initial findings have attracted particular attention in the context of malignancy; however, the crosstalk between autophagy and DNA damage response is just beginning to be explored and key questions remain that need to be addressed, to move this area of research forward and illuminate the overall consequence of targeting this process in human therapies. Here we present current knowledge on the complex crosstalk between autophagy and genome integrity and discuss its implications for cancer cell survival and response to therapy.

Inhibition of lysine-specific demethylase LSD1 induces senescence in Glioblastoma cells through a HIF-1α-dependent pathway.

Senescence is a stress-responsive cellular program that leads to cell cycle arrest. In cancer cells, senescence has profound implications for tumor aggressiveness and clinical outcome, but the molecular events that provoke cancer cells to undergo senescence remain unclear. Herein, we provide evidence that the histone demethylase LSD1/KDM1A supports the growth of Glioblastoma tumor cells and its inhibition triggers senescence response. LSD1 is a histone modifier that participates in key aspects of gene transcription as well as in the regulation of methylation dynamics of non-histone proteins. We found that down-regulation of LSD1 inhibits Glioblastoma cell growth, impairs mTOR pathway and cell migration and induces senescence. At mechanistic level, we found that LSD1 regulates HIF-1α protein stability. Pharmacological inhibition or siRNA-mediated silencing of LSD1 expression effectively reduces HIF-1α protein levels, which suffices for the induction of senescence. Our findings elucidate a mechanism whereby LSD1 controls senescence in Glioblastoma tumor cells through the regulation of HIF-1α, and we propose the novel defined LSD1/HIF-1α axis as a new target for the therapy of Glioblastoma tumors.

Histone methyl-transferases and demethylases in the autophagy regulatory network: the emerging role of KDM1A/LSD1 demethylase.

Macroautophagy/autophagy is a physiological mechanism that is essential for the maintenance of cellular homeostasis and stress adaptation. Defective autophagy is associated with many human diseases, including cancer and neurodegenerative disorders. The emerging implication of epigenetic events in the control of the autophagic process opens new avenues of investigation and offers the opportunity to develop novel therapeutic strategies in diseases associated with dysfunctional autophagy-lysosomal pathways. Accumulating evidence reveals that several methyltransferases and demethylases are essential regulators of autophagy, and recent studies have led to the identification of the lysine demethylase KDM1A/LSD1 as a promising drug target. KDM1A/LSD1 modulates autophagy at multiple levels, participating in the transcriptional control of several downstream effectors. This review summarizes our current understanding of the role of KDM1A/LSD1 in the autophagy regulatory network.

Targeting Histone Demethylase LSD1/KDM1a in Neurodegenerative Diseases.

The autophagy-lysosome pathway sustains cellular homeostasis and is a protective mechanism against neurodegenerative diseases. Recent findings highlight the role of the histone demethylases LSD1/LDM1A as a pivotal regulator of autophagy process, by controlling the mTORC1 cascade, in neuroblastoma cells. LSD1 binds to the promoter region of the SESN2 gene, where LSD1-mediated demethylation leads to the accumulation of repressive histone marks that maintain SESN2 expression at low levels. LSD1 depletion results in enhanced SESN2 expression and consequently mTORC1 inhibition, thereby triggering the induction of autophagy. Our study provides important insight into neuroepigenetic mechanisms regulating the autophagic process, offering additional opportunities for the development of novel therapeutic strategies in diseases associated with dysfunctional autophagy-lysosomal pathway.

Epigenetic regulation of epithelial to mesenchymal transition by the Lysine-specific demethylase LSD1/KDM1A.

The Lysine-specific demethylase 1, KDM1A/LSD1, plays a central role in the regulation of Pol II transcription through the removal of the activation mark (mono- and dimethyl lysine 4 of histone H3). LSD1 is often deregulated in human cancers, and it is frequently overexpressed in human solid cancers and leukemia. LSD1 regulates the epithelial mesenchymal transition (EMT) in epithelial cells, i.e., the ability to transition into mesenchymal cells, to lose homotypic adhesion and to acquire migratory capacity. From its initial discovery as a component of the Snail complex, multiple studies highlighted the causative role of LSD1 in cell invasiveness and EMT, describing its direct involvement in different molecular processes through the interaction with specific partners. Here we present an overview of the role of LSD1 in the EMT process, summarizing recent findings on its emerging functions in cell migration and invasion in cancer.

LSD1 mediates MYCN control of epithelial-mesenchymal transition through silencing of metastatic suppressor NDRG1 gene.

Neuroblastoma (NB) with MYCN amplification is a highly aggressive and metastatic tumor in children. The high recurrence rate and resistance of NB cells to drugs urgently demands a better therapy for this disease. We have recently found that MYCN interacts with the lysine-specific demethylase 1 (LSD1), a histone modifier that participates in key aspects of gene transcription. In cancer cells, LSD1 contributes to the genetic reprogramming that underlies to Epithelial-Mesenchymal Transition (EMT) and tumor metastasis. Here, we show that LSD1 affects motility and invasiveness of NB cells by modulating the transcription of the metastasis suppressor NDRG1 (N-Myc Downstream-Regulated Gene 1). At mechanistic level, we found that LSD1 co-localizes with MYCN at the promoter region of the NDRG1 gene and inhibits its expression. Pharmacological inhibition of LSD1 relieves repression of NDRG1 by MYCN and affects motility and invasiveness of NB cells. These effects were reversed by overexpressing NDRG1. In NB tissues, high levels of LSD1 correlate with low levels of NDRG1 and reduced patients survival. Collectively, our findings elucidate a mechanism of how MYCN/LSD1 control motility and invasiveness of NB cells through transcription regulation of NDRG1 expression and suggest that pharmacological targeting of LSD1 represents a valuable approach for NB therapy.

Lysine-specific demethylase (LSD1/KDM1A) and MYCN cooperatively repress tumor suppressor genes in neuroblastoma.

The chromatin-modifying enzyme lysine-specific demethylase 1, KDM1A/LSD1 is involved in maintaining the undifferentiated, malignant phenotype of neuroblastoma cells and its overexpression correlated with aggressive disease, poor differentiation and infaust outcome. Here, we show that LSD1 physically binds MYCN both in vitro and in vivo and that such an interaction requires the MYCN BoxIII. We found that LSD1 co-localizes with MYCN on promoter regions of CDKN1A/p21 and Clusterin (CLU) suppressor genes and cooperates with MYCN to repress the expression of these genes. KDM1A needs to engage with MYCN in order to associate with the CDKN1A and CLU promoters. The expression of CLU and CDKN1A can be restored in MYCN-amplified cells by pharmacological inhibition of LSD1 activity or knockdown of its expression. Combined pharmacological inhibition of MYCN and LSD1 through the use of small molecule inhibitors synergistically reduces MYCN-amplified Neuroblastoma cell viability in vitro. These findings demonstrate that LSD1 is a critical co-factor of the MYCN repressive function, and suggest that combination of LSD1 and MYCN inhibitors may have strong therapeutic relevance to counteract MYCN-driven oncogenesis.

MYC impairs resolution of site-specific DNA double-strand breaks repair.

Although it is established that when overexpressed, the MYC family proteins can cause DNA double-stand breaks (DSBs) and genome instability, the mechanisms involved remain unclear. MYC induced genetic instability may result from increased DNA damage and/or reduced DNA repair. Here we show that when overexpressed, MYC proteins induce a sustained DNA damage response (DDR) and reduce the wave of DSBs repair. We used a cell-based DSBs system whereby, upon induction of an inducible restriction enzyme AsiSI, hundreds of site-specific DSBs are generated across the genome to investigate the role of MYC proteins on DSB. We found that high levels of MYC do not block accumulation of γH2AX at AsiSI sites, but delay its clearance, indicating an inefficient repair, while the initial recognition of DNA damage is largely unaffected. Repair of both homologous and nonhomologous repair-prone segments, characterized by high or low levels of recruited RAD51, respectively, was delayed. Collectively, these data indicate that high levels of MYC proteins delay the resolution of DNA lesions engineered to occur in cell cultures.

Sequence-specific double strand breaks trigger P-TEFb-dependent Rpb1-CTD hyperphosphorylation.

Double strand DNA breaks (DSBs) are one of the most challenging forms of DNA damage which, if left unrepaired, can trigger cellular death and can contribute to cancer. A number of studies have been focused on DNA-damage response (DDR) mechanisms, and most of them rely on the induction of DSBs triggered by chemical compounds or radiations. However, genotoxic drugs and radiation treatments of cultured cell lines induce random DSBs throughout the genome, thus heterogeneously across the cell population, leading to variability of the cellular response. To overcome this aspect, we used here a recently described cell-based DSBs system whereby, upon induction of an inducible restriction enzyme, hundreds of site-specific DSBs are generated across the genome. We show here that sequence-specific DSBs are sufficient to activate the positive transcription elongation factor b (P-TEFb), to trigger hyperphosphorylation of the largest RNA polymerase II carboxyl-terminal-domain (Rpb1-CTD) and to induce activation of p53-transcriptional axis resulting in cell cycle arrest.