Yeast-Based Screen to Identify Natural Compounds with a Potential Therapeutic Effect in Hailey-Hailey Disease

The term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene ATP2C1 the orthologous of the yeast gene PMR1. We observed that K. lactis cells defective for PMR1 gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with PMR1 deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in K. lactis. These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target NQO1. In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD

DNA damage stress: Cui prodest?

DNA is an entity shielded by mechanisms that maintain genomic stability and are essential for living cells; however, DNA is constantly subject to assaults from the environment throughout the cellular life span, making the genome susceptible to mutation and irreparable damage. Cells are prepared to mend such events through cell death as an extrema ratio to solve those threats from a multicellular perspective. However, in cells under various stress conditions, checkpoint mechanisms are activated to allow cells to have enough time to repair the damaged DNA. In yeast, entry into the cell cycle when damage is not completely repaired represents an adaptive mechanism to cope with stressful conditions. In multicellular organisms, entry into cell cycle with damaged DNA is strictly forbidden. However, in cancer development, individual cells undergo checkpoint adaptation, in which most cells die, but some survive acquiring advantageous mutations and selfishly evolve a conflictual behavior. In this review, we focus on how, in cancer development, cells rely on checkpoint adaptation to escape DNA stress and ultimately to cell death

The epigenetic factor BORIS/CTCFL regulates the NOTCH3 gene expression in cancer cells.

Aberrant upregulation of NOTCH3 gene plays a critical role in cancer pathogenesis. However, the underlying mechanisms are still unknown. We tested here the hypothesis that aberrant epigenetic modifications in the NOTCH3 promoter region might account for its upregulation in cancer cells. We compared DNA and histone methylation status of NOTCH3 promoter region in human normal blood cells and T cell acute lymphoblastic leukemia (T-ALL) cell lines, differentially expressing NOTCH3. We found that histone methylation, rather than DNA hypomethylation, contributes towards establishing an active chromatin status of NOTCH3 promoter in NOTCH3 overexpressing cancer cells. We discovered that the chromatin regulator protein BORIS/CTCFL plays an important role in regulating NOTCH3 gene expression. We observed that BORIS is present in T-ALL cell lines as well as in cell lines derived from several solid tumors overexpressing NOTCH3. Moreover, BORIS targets NOTCH3 promoter in cancer cells and it is able to induce and to maintain a permissive/active chromatin conformation. Importantly, the association between NOTCH3 overexpression and BORIS presence was confirmed in primary T-ALL samples from patients at the onset of the disease. Overall, our results provide novel insights into the determinants of NOTCH3 overexpression in cancer cells, by revealing a key role for BORIS as the main mediator of transcriptional deregulation of NOTCH3. Copyright © 2014 Elsevier B.V. All rights reserved

Synergistic Post-Transcriptional Regulation of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) by miR-101 and miR-494 Specific Binding

microRNAs (miRNAs) are a class of regulatory small non-coding molecules that control gene expression at post-transcriptional level. Deregulation of miRNA functions affects a variety of biological processes also involved in the etiology of several human mendelian and complex diseases. Recently, aberrant miRNA expression has been observed in Cystic Fibrosis (CF), an autosomal-recessive genetic disorder caused by mutations in the CFTR gene, in which a genotype-phenotype correlation is not always found. In order to determine miRNA role in CFTR post-transcriptional regulation, we searched for miR-responsive elements in the CFTR 3′-UTR. In silico analysis, performed using different computational on-line programs, identified some putative miRNAs. Both miR-101 and miR-494 synthetic mimics significantly inhibited the expression of a reporter construct containing the 3′-UTR of CFTR in luciferase assays. Interestingly, miR-101/miR-494 combination was able to markedly suppress CFTR activity by approximately 80% (p<0.001). This is one of the first in vitro studies implicating microRNAs as negative regulators of the CFTR gene expression. miRNA aberrant expression and function might explain the wide phenotypic variability observed among CF patients

Functional cooperation between ASK1 and p21Waf1/Cip1 in the balance of cell-cycle arrest, cell death and tumorigenesis of stressed keratinocytes

Both CDKN1A (p21 Waf1/Cip1) and Apoptosis signal-regulating kinase 1 (ASK1) play important roles in tumorigenesis. The role of p21 Waf1/Cip1 in attenuating ASK1-induced apoptosis by various stress conditions is well established. However, how ASK1 and p21 Waf1/Cip1 functionally interact during tumorigenesis is still unclear. To address this aspect, we crossed ASK1 knockout (ASK1KO) mice with p21 Waf1/Cip1 knockout (p21KO) mice to compare single and double-mutant mice. We observed that deletion of p21 Waf1/Cip1 leads to increased keratinocyte proliferation but also increased cell death. This is mechanistically linked to the ASK1 axis-induced apoptosis, including p38 and PARP. Indeed, deletion of ASK1 does not alter the proliferation but decreases the apoptosis of p21KO keratinocytes. To analyze as this interaction might affect skin carcinogenesis, we investigated the response of ASK1KO and p21KO mice to DMBA/TPA-induced tumorigenesis. Here we show that while endogenous ASK1 is dispensable for skin homeostasis, ASK1KO mice are resistant to DMBA/TPA-induced tumorigenesis. However, we found that epidermis lacking both p21 and ASK1 reacquires increased sensitivity to DMBA/TPA-induced tumorigenesis. We demonstrate that apoptosis and cell-cycle progression in p21KO keratinocytes are uncoupled in the absence of ASK1. These data support the model that a critical event ensuring the balance between cell death, cell-cycle arrest, and successful divisions in keratinocytes during stress conditions is the p21-dependent ASK1 inactivation

The epigenetic factor BORIS/CTCFL regulates the NOTCH3 gene expression in cancer cells.

International audience; Aberrant upregulation of NOTCH3 gene plays a critical role in cancer pathogenesis. However, the underlying mechanisms are still unknown. We tested here the hypothesis that aberrant epigenetic modifications in the NOTCH3 promoter region might account for its upregulation in cancer cells. We compared DNA and histone methylation status of NOTCH3 promoter region in human normal blood cells and T cell acute lymphoblastic leukemia (T-ALL) cell lines, differentially expressing NOTCH3. We found that histone methylation, rather than DNA hypomethylation, contributes towards establishing an active chromatin status of NOTCH3 promoter in NOTCH3 overexpressing cancer cells. We discovered that the chromatin regulator protein BORIS/CTCFL plays an important role in regulating NOTCH3 gene expression. We observed that BORIS is present in T-ALL cell lines as well as in cell lines derived from several solid tumors overexpressing NOTCH3. Moreover, BORIS targets NOTCH3 promoter in cancer cells and it is able to induce and to maintain a permissive/active chromatin conformation. Importantly, the association between NOTCH3 overexpression and BORIS presence was confirmed in primary T-ALL samples from patients at the onset of the disease. Overall, our results provide novel insights into the determinants of NOTCH3 overexpression in cancer cells, by revealing a key role for BORIS as the main mediator of transcriptional deregulation of NOTCH3

Notch3/Jagged1 Circuitry Reinforces Notch Signaling and Sustains T-ALL

AbstractDeregulated Notch signaling has been extensively linked to T-cell acute lymphoblastic leukemia (T-ALL). Here, we show a direct relationship between Notch3 receptor and Jagged1 ligand in human cell lines and in a mouse model of T-ALL. We provide evidence that Notch-specific ligand Jagged1 is a new Notch3 signaling target gene. This essential event justifies an aberrant Notch3/Jagged1 cis-expression inside the same cell. Moreover, we demonstrate in Notch3-IC–overexpressing T lymphoma cells that Jagged1 undergoes a raft-associated constitutive processing. The proteolytic cleavage allows the Jagged1 intracellular domain to empower Notch signaling activity and to increase the transcriptional activation of Jagged1 itself (autocrine effect). On the other hand, the release of the soluble Jagged1 extracellular domain has a positive impact on activating Notch signaling in adjacent cells (paracrine effect), finally giving rise to a Notch3/Jagged1 auto-sustaining loop that supports the survival, proliferation, and invasion of lymphoma cells and contributes to the development and progression of Notch-dependent T-ALL. These observations are also supported by a study conducted on a cohort of patients in which Jagged1 expression is associated to adverse prognosis

A sketch of known and novel MYCN-associated miRNA networks in neuroblastoma

Neuroblastoma (NB) originates from neural crest-derived precursors and represents the most common childhood extracranial solid tumour. MicroRNAs (miRNAs), a class of small non-coding RNAs that participate in a wide variety of biological processes by regulating gene expression, appear to play an essential role within the NB context. High-throughput next generation sequencing (NGS) was applied to study the miRNA transcriptome in a cohort of NB tumours with and without MYCN-amplification (MNA and MNnA, respectively) and in dorsal root ganglia (DRG), as a control. Out of the 128 miRNAs differentially expressed in the NB vs. DRG comparison, 47 were expressed at higher levels, while 81 were expressed at lower levels in the NB tumours. We also found that 23 miRNAs were differentially expressed in NB with or without MYCN-amplification, with 17 miRNAs being upregulated and 6 being downregulated in the MNA subtypes. Functional annotation analysis of the target genes of these differentially expressed miRNAs demonstrated that many mRNAs were involved in cancer-related pathways, such as DNA-repair and apoptosis as well as FGFR and EGFR signalling. In particular, we found that miR-628-3p negatively affects MYCN gene expression. Furthermore, we identified a novel miRNA candidate with variable expression in MNA vs. MNnA tumours, whose putative target genes are implicated in the mTOR pathway. The present study provides further insight into the molecular mechanisms that correlate miRNA dysregulation to NB development and progression

The Translation Factor eIF6 Is a Notch-Dependent Regulator of Cell Migration and Invasion

A growing body of evidence indicates that protein factors controlling translation play an important role in tumorigenesis. The protein known as eIF6 is a ribosome anti-association factor that has been implicated in translational initiation and in ribosome synthesis. Over-expression of eIF6 is observed in many natural tumours, and causes developmental and differentiation defects in certain animal models. Here we show that the transcription of the gene encoding eIF6 is modulated by the receptor Notch-1, a protein involved in embryonic development and cell differentiation, as well as in many neoplasms. Inhibition of Notch-1 signalling by γ-secretase inhibitors slowed down cell-cycle progression and reduced the amount of eIF6 in lymphoblastoid and ovarian cancer cell lines. Cultured ovarian cancer cell lines engineered to stably over-expressing eIF6 did not show significant changes in proliferation rate, but displayed an enhanced motility and invasive capacity. Inhibition of Notch-1 signalling in the cells over-expressing eIF6 was effective in slowing down the cell cycle, but did not reduce cell migration and invasion. On the whole, the results suggest that eIF6 is one of the downstream effectors of Notch-1 in the pathway that controls cell motility and invasiveness