Cholesterol biosynthetic pathway induces cellular senescence through ERRα.

Cellular senescence is a cell program induced by various stresses that leads to a stable proliferation arrest and to a senescence-associated secretory phenotype. Accumulation of senescent cells during age-related diseases participates in these pathologies and regulates healthy lifespan. Recent evidences point out a global dysregulated intracellular metabolism associated to senescence phenotype. Nonetheless, the functional contribution of metabolic homeostasis in regulating senescence is barely understood. In this work, we describe how the mevalonate pathway, an anabolic pathway leading to the endogenous biosynthesis of poly-isoprenoids, such as cholesterol, acts as a positive regulator of cellular senescence in normal human cells. Mechanistically, this mevalonate pathway-induced senescence is partly mediated by the downstream cholesterol biosynthetic pathway. This pathway promotes the transcriptional activity of ERRα that could lead to dysfunctional mitochondria, ROS production, DNA damage and a p53-dependent senescence. Supporting the relevance of these observations, increase of senescence in liver due to a high-fat diet regimen is abrogated in ERRα knockout mouse. Overall, this work unravels the role of cholesterol biosynthesis or level in the induction of an ERRα-dependent mitochondrial program leading to cellular senescence and related pathological alterations.

In Vivo Characterization of the Toxicological Properties of DPhP, One of the Main Degradation Products of Aryl Phosphate Esters.

BACKGROUND: Aryl phosphate esters (APEs) are widely used and commonly present in the environment. Health hazards associated with these compounds remain largely unknown and the effects of diphenyl phosphate (DPhP), one of their most frequent derivatives, are poorly characterized. OBJECTIVE: Our aim was to investigate whether DPhP per se may represent a more relevant marker of exposure to APEs than direct assessment of their concentration and determine its potential deleterious biological effects in chronically exposed mice. METHODS: Conventional animals (FVB mice) were acutely or chronically exposed to relevant doses of DPhP or to triphenyl phosphate (TPhP), one of its main precursors. Both molecules were measured in blood and other tissues by liquid chromatography-mass spectrometry (LC-MS). Effects of chronic DPhP exposure were addressed through liver multi-omics analysis to determine the corresponding metabolic profile. Deep statistical exploration was performed to extract correlated information, guiding further physiological analyses. RESULTS: Multi-omics analysis confirmed the existence of biological effects of DPhP, even at a very low dose of 0.1mg/mL in drinking water. Chemical structural homology and pathway mapping demonstrated a clear reduction of the fatty acid catabolic processes centered on acylcarnitine and mitochondrial ß-oxidation in mice exposed to DPhP in comparison with those treated with vehicle. An interesting finding was that in mice exposed to DPhP, mRNA, expression of genes involved in lipid catabolic processes and regulated by peroxisome proliferator-activated receptor alpha (PPARα) was lower than that in vehicle-treated mice. Immunohistochemistry analysis showed a specific down-regulation of HMGCS2, a kernel target gene of PPARα. Overall, DPhP absorption disrupted body weight-gain processes. CONCLUSIONS: Our results suggest that in mice, the effects of chronic exposure to DPhP, even at a low dose, are not negligible. Fatty acid metabolism in the liver is essential for controlling fast and feast periods, with adverse consequences on the overall physiology. Therefore, the impact of DPhP on circulating fat, cardiovascular pathologies and metabolic disease incidence deserves, in light of our results, further investigations. https://doi.org/10.1289/EHP6826.

Combined nanomedicines targeting colorectal cancer stem cells and cancer cells.

The study aims to combine the delivery of two anticancer drugs to target both proliferating cancer cells and dormant cancer stem cells (CSCs) present in colorectal cancer. Two drugs were selected and encapsulated in lipid nanocapsules: SN38, the active form of irinotecan, which is unstable in the plasma but active against replicating cells, and salinomycin, a highly toxic ionophore active against cancer stem cells that is not suitable for clinical use. Using an engineered medium that enhanced the ratio of CSCs in HCT116 cell cultures, we demonstrated by clonogenicity tests and in sphere assays that Salinomycin acts mainly on CSCs, while SN38 acts mainly on proliferating cancer cells. In a preclinical murine CRC model, encapsulation of both drugs in lipid nanocapsules reduced their toxicity, including hemolysis, and led to a higher survival than what was observed following treatment with single drugs or non-encapsulated drugs. Nanoparticles loaded with an anticancer drug and salinomycin were effective against the therapy-resistant dormant CSCs and cancer cells.

Assessing Cell Activities rather than Identities to Interpret Intra-Tumor Phenotypic Diversity and Its Dynamics.

Despite advances in single-cell and molecular techniques, it is still unclear how to best quantify phenotypic heterogeneity in cancer cells that evolved beyond normal, known classifications. We present an approach to phenotypically characterize cells based on their activities rather than static classifications. We validated the detectability of specific activities (epithelial-mesenchymal transition, glycolysis) in single cells, using targeted RT-qPCR analyses and in vitro inductions. We analyzed 50 established activity signatures as a basis for phenotypic description in public data and computed cell-cell distances in 28,513 cells from 85 patients and 8 public datasets. Despite not relying on any classification, our measure correlated with standard diversity indices in populations of known structure. We identified bottlenecks as phenotypic diversity reduced upon colorectal cancer initiation. This suggests that focusing on what cancer cells do rather than what they are can quantify phenotypic diversity in universal fashion, to better understand and predict intra-tumor heterogeneity dynamics.

Combinational drug-loaded lipid nanocapsules for the treatment of cancer.

The purpose of this study was to investigate the feasibility of an intravenously administered combinational therapy using lipid nanocapsules (LNCs) as a drug delivery carrier for the treatment of different cancers. Therefore, we encapsulated 6 anticancer drugs within LNCs. Their size was approximately 50 nm. Except for oxaliplatin, their encapsulation efficiency, which was measured by different analytical methods, varied between 75% for SN38 to 100% for regorafenib. The in vitro studies showed a nonsignificant difference between the cytotoxicity of free and encapsulated drugs and a significant decrease in haemolysis by encapsulation in LNCs. Finally, the in vivo experiment showed that a combinational regimen of SN38-LNCs and regorafenib-LNCs abates CT26 murine colorectal cancer growth and increases median survival time.

Metabolomics and cytotoxicity of monomethylhydrazine (MMH) and (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), two liquid propellants.

Hydrazine-based liquid propellants are routinely used for space rocket propulsion, in particular monomethylhydrazine (MMH), although such compounds are highly hazardous. For several years, great efforts were devoted to developing a less hazardous molecule. To explore the toxicological effects of an alternative compound, namely (E)-1,1,4,4-tetramethyl-2-tetrazene (TMTZ), we exposed various cellular animal and human models to this compound and to the reference compound MMH. We observed no cytotoxic effects following exposure to TMTZ in animal, as well as human models. However, although the three animal models were unaffected by MMH, exposure of the human hepatic HepaRG cell model revealed that apoptotic cytotoxic effects were only detectable in proliferative human hepatic HepaRG cells and not in differentiated cells, although major biochemical modifications were uncovered in the latter. The present findings indicate that the metabolic mechanisms of MMH toxicity is close to those described for hydrazine with numerous biochemical alterations induced by mitochondrial disruption, production of radical species, and aminotransferase inhibition. The alternative TMTZ molecule had little impact on cellular viability and proliferation of rodent and human dermic and hepatic cell models. TMTZ did not produce any metabolomic effects and appears to be a promising putative industrial alternative to MMH.

Longitudinal serum metabolomics evaluation of trastuzumab and everolimus combination as pre-operative treatment for HER-2 positive breast cancer patients.

The mammalian target of rapamycin complex 1 (mTORC1) is an attractive target for HER-2 positive breast cancer therapy because of its key role in protein translation regulation, cell growth and metabolism. We present here a metabolomic investigation exploring the impact of mTOR inhibition on serum metabolic profiles from patients with non-metastatic breast cancer overexpressing HER-2. Baseline, treatment-related and post-treatment serum samples were analyzed for 79 patients participating in the French clinical trial RADHER, in which randomized patients with HER-2 positive breast cancer received either trastuzumab alone (arm T) or a trastuzumab and everolimus combination (arm T+E). Longitudinal series of NMR serum metabolic profiles were exploited to investigate treatment effects on the patients metabolism over time, in both group. Trastuzumab and everolimus combination induces faster changes in patients metabolism than trastuzumab alone, visible after only one week of treatment as well as a residual effect detectable up to three weeks after ending the treatment. These metabolic fingerprints highlight the involvement of several metabolic pathways reflecting a systemic effect, particularly on the liver and visceral fat. Comparison of serum metabolic profiles between the two arms shows that everolimus, an mTORC1 inhibitor, is responsible for host metabolism modifications observed in arm T+E. In HER-2 positive breast cancer, our metabolomic approach confirms a fast and persistent host metabolism modification caused by mTOR inhibition.

Destabilization of the TWIST1/E12 complex dimerization following the R154P point-mutation of TWIST1: an in silico approach.

BACKGROUND: The bHLH transcription factor TWIST1 plays a key role in the embryonic development and in tumorigenesis. Some loss-of-function mutations of the TWIST1 gene have been shown to cause an autosomal dominant craniosynostosis, known as the Saethre-Chotzen syndrome (SCS). Although the functional impacts of many TWIST1 mutations have been experimentally reported, little is known on the molecular mechanisms underlying their loss-of-function. In a previous study, we highlighted the predictive value of in silico molecular dynamics (MD) simulations in deciphering the molecular function of TWIST1 residues. RESULTS: Here, since the substitution of the arginine 154 amino acid by a glycine residue (R154G) is responsible for the SCS phenotype and the substitution of arginine 154 by a proline experimentally decreases the dimerizing ability of TWIST1, we investigated the molecular impact of this point mutation using MD approaches. Consistently, MD simulations highlighted a clear decrease in the stability of the α-helix during the dimerization of the mutated R154P TWIST1/E12 dimer compared to the wild-type TE complex, which was further confirmed in vitro using immunoassays. CONCLUSIONS: Our study demonstrates that MD simulations provide a structural explanation for the loss-of-function associated with the SCS TWIST1 mutation and provides a proof of concept of the predictive value of these MD simulations. This in silico methodology could be used to determine reliable pharmacophore sites, leading to the application of docking approaches in order to identify specific inhibitors of TWIST1 complexes.

A stemness-related ZEB1-MSRB3 axis governs cellular pliancy and breast cancer genome stability.

Chromosomal instability (CIN), a feature of most adult neoplasms from their early stages onward, is a driver of tumorigenesis. However, several malignancy subtypes, including some triple-negative breast cancers, display a paucity of genomic aberrations, thus suggesting that tumor development may occur in the absence of CIN. Here we show that the differentiation status of normal human mammary epithelial cells dictates cell behavior after an oncogenic event and predetermines the genetic routes toward malignancy. Whereas oncogene induction in differentiated cells induces massive DNA damage, mammary stem cells are resistant, owing to a preemptive program driven by the transcription factor ZEB1 and the methionine sulfoxide reductase MSRB3. The prevention of oncogene-induced DNA damage precludes induction of the oncosuppressive p53-dependent DNA-damage response, thereby increasing stem cells' intrinsic susceptibility to malignant transformation. In accord with this model, a subclass of breast neoplasms exhibit unique pathological features, including high ZEB1 expression, a low frequency of TP53 mutations and low CIN.

Deciphering the molecular mechanisms underlying the binding of the TWIST1/E12 complex to regulatory E-box sequences.

The TWIST1 bHLH transcription factor controls embryonic development and cancer processes. Although molecular and genetic analyses have provided a wealth of data on the role of bHLH transcription factors, very little is known on the molecular mechanisms underlying their binding affinity to the E-box sequence of the promoter. Here, we used an in silico model of the TWIST1/E12 (TE) heterocomplex and performed molecular dynamics (MD) simulations of its binding to specific (TE-box) and modified E-box sequences. We focused on (i) active E-box and inactive E-box sequences, on (ii) modified active E-box sequences, as well as on (iii) two box sequences with modified adjacent bases the AT- and TA-boxes. Our in silico models were supported by functional in vitro binding assays. This exploration highlighted the predominant role of protein side-chain residues, close to the heart of the complex, at anchoring the dimer to DNA sequences, and unveiled a shift towards adjacent ((-1) and (-1*)) bases and conserved bases of modified E-box sequences. In conclusion, our study provides proof of the predictive value of these MD simulations, which may contribute to the characterization of specific inhibitors by docking approaches, and their use in pharmacological therapies by blocking the tumoral TWIST1/E12 function in cancers.

Ubiquitin-specific peptidase 42 (USP42) functions to deubiquitylate histones and regulate transcriptional activity.

Ubiquitin-specific peptidase 42 (USP42) is a deubiquitylating enzyme that can target p53 and contribute to the stabilization of p53 in response to stress. We now show that USP42 can also regulate transcription independently of p53. USP42 co-localized with RNA polymerase II (RNA Pol II) in nuclear foci, bound to histone H2B, and deubiquitylated H2B. Depletion of USP42 increased H2B ubiquitylation at a model promoter and decreased both basal and induced transcription from a number of promoters. These results are consistent with a role for USP42 in regulating transcription by deubiquitylating histones.

Conventional techniques to monitor mitochondrial oxygen consumption.

Following several key discoveries on hypoxia-inducible factors, we have observed an explosion of studies investigating how the hypoxic microenvironment provokes bioenergetic alterations. This is particularly relevant for cancer cells, as they are often exposed to hypoxic conditions in the course of tumor progression. Thus, interest in the measurement of oxygen consumption at the tissue, cell, or mitochondrion level has been revived. Here, we describe the basic principles of cellular respiration and survey some of the conventional methods for measuring O2 consumption in intact or permeabilized cells.

An indirect role for ASPP1 in limiting p53-dependent p21 expression and cellular senescence.

In addition to acting as a transcriptional cofactor for p53, ASPP1 has been shown to function in the cytoplasm to regulate the nuclear localization and activity of YAP/TAZ. We show here that the ability of ASPP1 to activate YAP results in the decreased expression of LATS2, which lowers the ability of p53 to induce p21, cell-cycle arrest and senescence. ASPP1 expression peaks in S-phase, and down-regulation of ASPP1 leads to a reduction in DNA synthesis and enhanced senescence in response to drugs that impede DNA replication. These activities of cytoplasmic ASPP1 in opposing p53-mediated p21 expression are in contrast to the role of nuclear ASPP1 in cooperating with p53 to induce the expression of apoptotic target genes, and may help to dampen p53 activity in normal cells.

Regulation of p53 stability and function by the deubiquitinating enzyme USP42.

The p53 tumour suppressor protein is a transcription factor that prevents oncogenic progression by activating the expression of apoptosis and cell-cycle arrest genes in stressed cells. The stability of p53 is tightly regulated by ubiquitin-dependent degradation, driven mainly by the ubiquitin ligase MDM2. In this study, we have identified USP42 as a DUB that interacts with and deubiquitinates p53. USP42 forms a direct complex with p53 and controls level of ubiquitination during the early phase of the response to a range of stress signals. Although we do not find a clear role for USP42 in controlling either the basal or fully activated levels of p53, the function of USP42 is required to allow the rapid activation of p53-dependent transcription and a p53-dependent cell-cycle arrest in response to stress. These functions of USP42 are likely to contribute to the repair and recovery of cells from mild or transient damage.

MDM2 promotes SUMO-2/3 modification of p53 to modulate transcriptional activity.

The tumor suppressor p53 is extensively regulated by post-translational modification, including modification by the small ubiquitin-related modifier SUMO. We show here that MDM2, previously shown to promote ubiquitin, Nedd8 and SUMO-1 modification of p53, can also enhance conjugation of endogenous SUMO-2/3 to p53. Sumoylation activity requires p53-MDM2 binding but does not depend on an intact RING finger. Both ARF and L11 can promote SUMO-2/3 conjugation of p53. However, unlike the previously described SUMO-1 conjugation of p53 by an MDM2-ARF complex, this activity does not depend on the ability of MDM2 to relocalize to the nucleolus. Interestingly, the SUMO consensus is not conserved in mouse p53, which is therefore not modified by SUMO-2/3. Finally, we show that conjugation of SUMO-2/3 to p53 correlates with a reduction of both activation and repression of a subset of p53-target genes.

Cytoplasmic ASPP1 inhibits apoptosis through the control of YAP.

The ASPP (apoptosis-stimulating protein of p53) family of proteins can function in the nucleus to modulate the transcriptional activity of p53, with ASPP1 and ASPP2 contributing to the expression of apoptotic target genes. In this study, we describe a new function for cytoplasmic ASPP1 in controlling YAP (Yes-associated protein)/TAZ. ASPP1 can inhibit the interaction of YAP with LATS1 (large tumor suppressor 1), a kinase that phosphorylates YAP/TAZ and promotes cytoplasmic sequestration and protein degradation. This function of ASPP1 therefore enhances nuclear accumulation of YAP/TAZ and YAP/TAZ-dependent transcriptional regulation. The consequence of YAP/TAZ activation by ASPP1 is to inhibit apoptosis, in part through the down-regulation of Bim expression, leading to resistance to anoikis and enhanced cell migration. These results reveal a potential oncogenic role for cytoplasmic ASPP1, in contrast to the tumor-suppressive activity described previously for nuclear ASPP1.

p53, ROS and senescence in the control of aging.

In addition to its function as a tumour suppressor, p53 is also involved in an increasing number of pathology associated with aging. Several activities of p53 appear contribute to its role in aging; one function that might be particularly relevant in this context is the regulation of senescence. The control of ROS and senescence by p53 may help to explain how p53 can function to both restrain and promote aging.

Regulation of the Aurora-A gene following topoisomerase I inhibition: implication of the Myc transcription factor.

During the G2 phase of the cell cycle, the Aurora-A kinase plays an important role in centrosome maturation and progression to mitosis. In this study, we show in colorectal cell lines that Aurora-A expression is downregulated in response to topoisomerase I inhibition. Using chromatin immunoprecipitation assays, we have observed that the Myc transcription factor and its Max binding partner are associated with the Aurora-A promoter during the G2 phase of the cell cycle. RNA interference experiments indicated that Myc is involved in the regulation of the Aurora-A gene. Following topoisomerase I inhibition, the expression of Myc decreased whereas Mad was upregulated, and the association of Myc and Max with the promoter of the kinase was inhibited. In parallel, an increased association of Mad and Miz-1 was detected on DNA, associated with an inhibition of the recruitment of transcriptional coactivators. Interestingly, a gain of H3K9 trimethylation and HP1gamma recruitment was observed on the Aurora-A promoter following sn38 treatment, suggesting that this promoter is located within SAHF foci following genotoxic treatment. Since Aurora-A is involved in centrosome maturation, we observed as expected that topoisomerase I inhibition prevented centrosome separation but did not affect their duplication. As a consequence, this led to G2 arrest and senescence induction.These results suggest a model by which the Aurora-A gene is inactivated by the G2 checkpoint following topoisomerase I inhibition. We therefore propose the hypothesis that the coordinated overexpression of Myc and Aurora-A, together with a downregulation of Mad and Miz-1 should be tested as a prognosis signature of poor responses to topoisomerase I inhibitors.

The cdk5 kinase regulates the STAT3 transcription factor to prevent DNA damage upon topoisomerase I inhibition.

The STAT3 transcription factors are cytoplasmic proteins that induce gene activation in response to growth factor stimulation. Following tyrosine phosphorylation, STAT3 proteins dimerize, translocate to the nucleus, and activate specific target genes involved in cell-cycle progression. Despite its importance in cancer cells, the molecular mechanisms by which this protein is regulated in response to DNA damage remain to be characterized. In this study, we show that STAT3 is activated in response to topoisomerase I inhibition. Following treatment, STAT3 is phosphorylated on its C-terminal serine 727 residue but not on its tyrosine 705 site. We also show that topoisomerase I inhibition induced the up-regulation of the cdk5 kinase, a protein initially described in neuronal stress responses. In co-immunoprecipitations, cdk5 was found to associate with STAT3, and pulldown experiments indicated that it associates with the C-terminal activation domain of STAT3 upon DNA damage. Importantly, the cdk5-STAT3 pathway reduced DNA damage in response to topoisomerase I inhibition through the up-regulation of Eme1, an endonuclease involved in DNA repair. ChIP experiments indicated that STAT3 can be found associated with the Eme1 promoter when phosphorylated only on its serine 727 residue and not on tyrosine 705. We therefore propose that the cdk5-STAT3 oncogenic pathway plays an important role in the expression of DNA repair genes and that these proteins could be used as predictive markers of tumors that will fail to respond to chemotherapy.