Proteotoxic stress-induced apoptosis in cancer cells: understanding the susceptibility and enhancing the potency.

Leiomyosarcoma (LMS) is aggressive cancer with few therapeutic options. LMS cells are more sensitive to proteotoxic stress compared to normal smooth muscle cells. We used small compound 2c to induce proteotoxic stress and compare the transcriptomic adaptations of immortalized human uterine smooth muscle cells (HUtSMC) and LMS cells SK-UT-1. We found that the expression of the heat shock proteins (HSPs) gene family is upregulated with higher efficiency in normal cells. In contrast, the upregulation of BH3-only proteins is higher in LMS cells. HSF1, the master regulator of HSP transcription, is sequestered into transcriptionally incompetent nuclear foci only in LMS cells, which explains the lower HSP upregulation. We also found that several compounds can enhance the cell death response to proteotoxic stress. Specifically, when low doses were used, an inhibitor of salt-inducible kinases (SIKs) and the inhibitor of IRE1α, a key element of the unfolded protein response (UPR), support proteotoxic-induced cell death with strength in LMS cells and without effects on the survival of normal cells. Overall, our data provide an explanation for the higher susceptibility of LMS cells to proteotoxic stress and suggest a potential option for co-treatment strategies.

hnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression.

Recent studies have proven that the genetic landscape of pancreatic cancer is dominated by the KRAS oncogene. Its transcription is controlled by a G-rich motif (called 32R) located immediately upstream of the TSS. 32R may fold into a G-quadruplex (G4) in equilibrium between two G4 conformers: G9T (T M = 61.2 °C) and G25T (T M = 54.7 °C). We found that both G4s bind to hnRNPA1 and its proteolytic fragment UP1, promoting several contacts with the RRM protein domains. 1D NMR analysis of DNA imino protons shows that, upon binding to UP1, G25T is readily unfolded at both 5' and 3' tetrads, while G9T is only partially unfolded. The impact of hnRNPA1 on KRAS expression was determined by comparing Panc-1 cells with two Panc-1 knockout cell lines in which hnRNPA1 was deleted by the CRISPR/Cas9 technology. The results showed that the expression of KRAS is inhibited in the knockout cell lines, indicating that hnRNPA1 is essential for the transcription of KRAS. In addition, the knockout cell lines, compared to normal Panc-1 cells, show a dramatic decrease in cell growth and capacity of colony formation. Pull-down and Western blot experiments indicate that conformer G25T is a better platform than conformer G9T for the assembly of the transcription preinitiation complex with PARP1, Ku70, MAZ, and hnRNPA1. Together, our data prove that hnRNPA1, being a key transcription factor for the activation of KRAS, can be a new therapeutic target for the rational design of anticancer strategies.

HDAC4 degradation during senescence unleashes an epigenetic program driven by AP-1/p300 at selected enhancers and super-enhancers.

BACKGROUND: Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined. RESULTS: Enhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program. CONCLUSIONS: Overall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.

Enhancing Proteotoxic Stress in Leiomyosarcoma Cells Triggers Mitochondrial Dysfunctions, Cell Death, and Antitumor Activity in vivo.

Leiomyosarcomas are rare and aggressive tumors characterized by a complex karyotype. Surgical resection with or without radiotherapy and chemotherapy is the standard curative treatment. Unfortunately, a high percentage of leiomyosarcomas recurs and metastasizes. In these cases, doxorubicin and ifosfamide represent the standard treatment but with low response rates. Here, we evaluated the induction of proteotoxic stress as a possible strategy to kill leiomyosarcoma cells in a therapeutic perspective. We show that aggressive leiomyosarcomas coexist with high levels of proteotoxic stress. As a consequence, we hypothesized that leiomyosarcoma cells are vulnerable to further increases of proteotoxic stress. The small compound 2c is a strong inducer of proteotoxic stress. In leiomyosarcoma cells, it triggers cell death coupled to a profound reorganization of the mitochondrial network. By using stimulated emission depletion microscopy, we have unveiled the existence of DIABLO/SMAC clusters that are modulated by 2c. Finally, we have engineered a new version of 2c linked to polyethylene glycol though a short peptide, named 2cPP. This new prodrug is specifically activated by proteases present in the tumor microenvironment. 2cPP shows a strong antitumor activity in vivo against leiomyosarcomas and no toxicity against normal cells.

Different class IIa HDACs repressive complexes regulate specific epigenetic responses related to cell survival in leiomyosarcoma cells.

Transcriptional networks supervising class IIa HDAC expression are poorly defined. Here we demonstrate that MEF2D is the key factor controlling HDAC9 transcription. This control, which is part of a negative feed-back loop during muscle differentiation, is hijacked in cancer. In leiomyosarcomas the MEF2D/HDAC9 vicious circuit sustains proliferation and cell survival, through the repression of the death receptor FAS. Comprehensive genome-wide studies demonstrate that HDAC4 and HDAC9 control different genetic programs and show both specific and common genomic binding sites. Although the number of MEF2-target genes commonly regulated is similar, only HDAC4 represses many additional genes that are not MEF2D targets. As expected, HDAC4-/- and HDAC9-/- cells increase H3K27ac levels around the TSS of the respective repressed genes. However, these genes rarely show binding of the HDACs at their promoters. Frequently HDAC4 and HDAC9 bind intergenic regions. We demonstrate that these regions, recognized by MEF2D/HDAC4/HDAC9 repressive complexes, show the features of active enhancers. In these regions HDAC4 and HDAC9 can differentially influence H3K27 acetylation. Our studies describe new layers of class IIa HDACs regulation, including a dominant positional effect, and can contribute to explain the pleiotropic actions of MEF2 TFs.

Genetic Programs Driving Oncogenic Transformation: Lessons from in Vitro Models.

Cancer complexity relies on the intracellular pleiotropy of oncogenes/tumor suppressors and in the strong interplay between tumors and micro- and macro-environments. Here we followed a reductionist approach, by analyzing the transcriptional adaptations induced by three oncogenes (RAS, MYC, and HDAC4) in an isogenic transformation process. Common pathways, in place of common genes became dysregulated. From our analysis it emerges that, during the process of transformation, tumor cells cultured in vitro prime some signaling pathways suitable for coping with the blood supply restriction, metabolic adaptations, infiltration of immune cells, and for acquiring the morphological plasticity needed during the metastatic phase. Finally, we identified two signatures of genes commonly regulated by the three oncogenes that successfully predict the outcome of patients affected by different cancer types. These results emphasize that, in spite of the heterogeneous mutational burden among different cancers and even within the same tumor, some common hubs do exist. Their location, at the intersection of the various signaling pathways, makes a therapeutic approach exploitable.

HDAC7-mediated control of tumour microenvironment maintains proliferative and stemness competence of human mammary epithelial cells.

HDAC7 is a pleiotropic transcriptional coregulator that controls different cellular fates. Here, we demonstrate that in human mammary epithelial cells, HDAC7 sustains cell proliferation and favours a population of stem-like cells, by maintaining a proficient microenvironment. In particular, HDAC7 represses a repertoire of cytokines and other environmental factors, including elements of the insulin-like growth factor signalling pathway, IGFBP6 and IGFBP7. This HDAC7-regulated secretome signature predicts negative prognosis for luminal A breast cancers. ChIP-seq experiments revealed that HDAC7 binds locally to the genome, more frequently distal from the transcription start site. HDAC7 can colocalize with H3K27-acetylated domains and its deletion further increases H3K27ac at transcriptionally active regions. HDAC7 levels are increased in RAS-transformed cells, in which this protein was required not only for proliferation and cancer stem-like cell growth, but also for invasive features. We show that an important direct target of HDAC7 is IL24, which is sufficient to suppress the growth of cancer stem-like cells.

Mitochondrial Adaptations in Elderly and Young Men Skeletal Muscle Following 2 Weeks of Bed Rest and Rehabilitation.

The aim of the study was to evaluate the expression levels of proteins related to mitochondrial biogenesis regulation and bioenergetics in vastus lateralis muscle biopsies from 16 elderly and 7 young people subjected to 14 days of bed-rest, causing atrophy, and subsequent 14 days of exercise training. Based on quantitative immunoblot analyses, in both groups a reduction of two key regulators of mitochondrial biogenesis/remodeling and activity, namely PGC-1α and Sirt3, was revealed during bed-rest, with a subsequent up-regulation after rehabilitation, indicating an involvement of PGC-1α-Sirt3 axis in response to the treatments. A difference was observed comparing the young and elderly subjects as, for both proteins, the abundance in the elderly was more affected by immobility and less responsive to exercise. The expression levels of TOM20 and Citrate Synthase, assayed as markers of outer mitochondrial membrane and mitochondrial mass, showed a noticeable sensitivity in the elderly group, where they were affected by bed-rest and rehabilitation recalling the pattern of PGC-1α. TOM20 and CS remained unchanged in young subjects. Single OXPHOS complexes showed peculiar patterns, which were in some cases dissimilar from PGC-1α, and suggest different influences on protein biogenesis and degradation. Overall, exercise was capable to counteract the effect of immobility, when present, except for complex V, which was markedly downregulated by bed-rest, but remained unaffected after rehabilitation, maybe as result of greater extent of degradation processes over biogenesis. Phosphorylation extent of AMPK, and its upstream activator LKB1, did not change after bed-rest and rehabilitation in either young or elderly subjects, suggesting that the activation of energy-sensing LKB1-AMPK signaling pathway was "missed" due to its transient nature, or was not triggered under our conditions. Our study demonstrates that, as far as the expression of various proteins related to mitochondrial biogenesis/remodeling, adaptations to bed-rest and rehabilitation in the two populations were different. The impact of bed-rest was greater in the elderly subjects, where the pattern (decrease after bed rest and recovery following rehabilitation) was accompanied by changes of mitochondrial mass. Modifications of protein abundance were matched with data obtained from gene expression analyses of four public human datasets focusing on related genes.

MicroRNA therapeutics: design of single-stranded miR-216b mimics to target KRAS in pancreatic cancer cells.

Datasets reporting microRNA expression profiles in normal and cancer cells show that miR-216b is aberrantly downregulated in pancreatic ductal adenocarcinoma (PDAC). We found that KRAS, whose mutant G12D allele drives the pathogenesis of PDAC, is a target of miR-216b. To suppress oncogenic KRAS in PDAC cells, we designed single-stranded (ss) miR-216b mimics with unlocked nucleic acid (UNA) modifications to enhance their nuclease resistance. We prepared variants of ss-miR-216b mimics with and without a 5' phosphate group. Both variants strongly suppressed oncogenic KRAS in PDAC cells and inhibited colony formation in pancreatic cancer cells. We observed that the designed ss-miR-216b mimics engaged AGO2 to promote the silencing of KRAS. We also tested a new delivery strategy based on the use of palmityl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with ss-miR-216b conjugated with two palmityl chains and a lipid-modified cell penetrating peptide (TAT). These versatile nanoparticles suppressed oncogenic KRAS in PDAC cells.

The co-existence of transcriptional activator and transcriptional repressor MEF2 complexes influences tumor aggressiveness.

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS.

The isopeptidase inhibitor 2cPE triggers proteotoxic stress and ATM activation in chronic lymphocytic leukemia cells.

Relapse after treatment is a common and unresolved problem for patients suffering of the B-cell chronic lymphocytic leukemia (B-CLL). Here we investigated the ability of the isopeptidase inhibitor 2cPE to trigger apoptosis in leukemia cells in comparison with bortezomib, another inhibitor of the ubiquitin-proteasome system (UPS). Both inhibitors trigger apoptosis in CLL B cells and gene expression profiles studies denoted how a substantial part of genes up-regulated by these compounds are elements of adaptive responses, aimed to sustain cell survival. 2cPE treatment elicits the up-regulation of chaperones, proteasomal subunits and elements of the anti-oxidant response. Selective inhibition of these responses augments apoptosis in response to 2cPE treatment. We have also observed that the product of the ataxia telangiectasia mutated gene (ATM) is activated in 2cPE treated cells. Stimulation of ATM signaling is possibly dependent on the alteration of the redox homeostasis. Importantly ATM inhibition, mutations or down-modulation increase cell death in response to 2cPE. Overall this work suggests that 2cPE could offer new opportunities for the treatment of B-CLL.

The MEF2-HDAC axis controls proliferation of mammary epithelial cells and acini formation in vitro.

The myocyte enhancer factor 2 and histone deacetylase (MEF2-HDAC) axis is a master regulator of different developmental programs and adaptive responses in adults. In this paper, we have investigated the contribution of the axis to the regulation of epithelial morphogenesis, using 3D organotypic cultures of MCF10A cells as a model. We have demonstrated that MEF2 transcriptional activity is upregulated during acini formation, which coincides with exit from the proliferative phase. Upregulation of the transcription of MEF2 proteins is coupled to downregulation of HDAC7, which occurs independently from changes in mRNA levels, and proteasome- or autophagy-mediated degradation. During acini formation, the MEF2-HDAC axis contributes to the promotion of cell cycle exit, through the engagement of the CDK inhibitor CDKN1A. Only in proliferating cells can HDAC7 bind to the first intron of the CDKN1A gene, a region characterized by epigenetic markers of active promoters and enhancers. In cells transformed by the oncogene HER2 (ERBB2), acini morphogenesis is altered, MEF2 transcription is repressed and HDAC7 is continuously expressed. Importantly, reactivation of MEF2 transcriptional activity in these cells, through the use of a HER2 inhibitor or by enhancing MEF2 function, corrected the proliferative defect and re-established normal acini morphogenesis.

Synthesis, characterization, and optimization for in vivo delivery of a nonselective isopeptidase inhibitor as new antineoplastic agent.

Bis-arylidenecycloalkanones structurally related to the nonselective isopeptidase inhibitor G5 were synthesized and tested for cytotoxic activity against glioblastoma cells. Cytotoxicities correlate well with Hammett σ constants for substituted arylidene groups, confirming the proposed inhibition mechanism. A new inhibitor (2c) based on the 4-hydroxycyclohexanone scaffold, which favors apoptosis over necrosis, was selected for further development. 2c inhibited representative deubiquitinases with micromolar IC50, and its proapoptotic activity was studied on several cancer cell lines. Inhibitor 2c was conjugated to PEG via dicarbamate and diester linkers. While the dicarbamate was inactive, the diester (2cPE) behaves like a prodrug and is converted into the active species 2c by secreted esterase activities. Finally, 2cPE was also tested in vivo on A549 lung carcinoma xenografts generated in mice. Intravenous treatment with 2cPE led to a significant reduction in primary tumor growth, without appreciable toxicity to mice.

Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain.

Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

Next-generation sequencing analysis of miRNA expression in control and FSHD myogenesis.

Emerging evidence has demonstrated that miRNA sequences can regulate skeletal myogenesis by controlling the process of myoblast proliferation and differentiation. However, at present a deep analysis of miRNA expression in control and FSHD myoblasts during differentiation has not yet been derived. To close this gap, we used a next-generation sequencing (NGS) approach applied to in vitro myogenesis. Furthermore, to minimize sample genetic heterogeneity and muscle-type specific patterns of gene expression, miRNA profiling from NGS data was filtered with FC ≥ 4 (log(2)FC ≥ 2) and p-value<0.05, and its validation was derived by qRT-PCR on myoblasts from seven muscle districts. In particular, control myogenesis showed the modulation of 38 miRNAs, the majority of which (34 out 38) were up-regulated, including myomiRs (miR-1, -133a, -133b and -206). Approximately one third of the modulated miRNAs were not previously reported to be involved in muscle differentiation, and interestingly some of these (i.e. miR-874, -1290, -95 and -146a) were previously shown to regulate cell proliferation and differentiation. FSHD myogenesis evidenced a reduced number of modulated miRNAs than healthy muscle cells. The two processes shared nine miRNAs, including myomiRs, although with FC values lower in FSHD than in control cells. In addition, FSHD cells showed the modulation of six miRNAs (miR-1268, -1268b, -1908, 4258, -4508- and -4516) not evidenced in control cells and that therefore could be considered FSHD-specific, likewise three novel miRNAs that seem to be specifically expressed in FSHD myotubes. These data further clarify the impact of miRNA regulation during control myogenesis and strongly suggest that a complex dysregulation of miRNA expression characterizes FSHD, impairing two important features of myogenesis: cell cycle and muscle development. The derived miRNA profiling could represent a novel molecular signature for FSHD that includes diagnostic biomarkers and possibly therapeutic targets.

VAMP subfamilies identified by specific R-SNARE motifs.

In eukaryotes, interactions among the alpha-helical coiled-coil domains (CCDs) of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a pivotal role in mediating the fusion among vesicles and target membranes. Surface residues of such CCDs are major candidates to regulate the specificity of membrane fusion, as they may alter local charge at the interaction layers and surface of the fusion complex, possibly modulating its formation and/or the binding of non-SNARE regulatory factors. Based on alternate patterns in surface residues, we have identified two motifs which group vesicular SNAREs in two novel subfamilies: RG-SNAREs and RD-SNAREs. The RG-SNARE CCD is common to all members of the widely conserved family of long VAMPs or longins and to yeast and non-neuronal VAMPs, possibly mediating "basic" fusion mechanisms; instead, only synaptobrevins from Bilateria share an RD-SNARE CCD, which is likely to mediate interactions to specific, yet unknown, regulatory factors and/or be the landmark of rapid fusion reactions like that mediating the release of neurotransmitters.