Sequential or Concomitant Inhibition of Cyclin-Dependent Kinase 4/6 Before mTOR Pathway in Hormone-Positive HER2 Negative Breast Cancer: Biological Insights and Clinical Implications.

About 75% of all breast cancers are hormone receptor-positive (HR+). However, the efficacy of endocrine therapy is limited due to the high rate of either pre-existing or acquired resistance. In this work we reconstructed the pathways around estrogen receptor (ER), mTOR, and cyclin D in order to compare the effects of CDK4/6 and PI3K/AKT/mTOR inhibitors. A positive feedback loop links mTOR and ER that support each other. We subsequently considered whether a combined or sequential inhibition of CDK4/6 and PI3K/AKT/mTOR could ensure better results. Studies indicate that inhibition of CDK4/6 activates mTOR as an escape mechanism to ensure cell proliferation. In literature, the little evidence dealing with this topic suggests that pre-treatment with mTOR pathway inhibitors could prevent or delay the onset of CDK4/6 inhibitor resistance. Additional studies are needed in order to find biomarkers that can identify patients who will develop this resistance and in whom the sensitivity to CDK4/6 inhibitors can be restored.

Different Cardiotoxicity of Palbociclib and Ribociclib in Breast Cancer: Gene Expression and Pharmacological Data Analyses, Biological Basis, and Therapeutic Implications.

Breast cancer is the most frequent tumor in women. The recent advent of cyclin-dependent kinase (CDK) 4/6 inhibitors palbociclib and ribociclib has represented a major step forward for patients with hormone receptor-positive breast cancer. These two agents have showed similar efficacy in terms of breast cancer outcome but different cardiotoxic effects. In particular, ribociclib, but not palbociclib, has been associated with QT interval prolongation, and the mechanisms underlying this event are still unclear. In order to clarify such difference, we matched the candidate genes associated with QT interval prolongation with genes whose expression is altered following palbociclib or ribociclib treatment. We also investigated whether pharmacokinetic and pharmacodynamic characteristics, such as IC50 (hERG) [concentration of drug producing 50% inhibition (human ether-à-go-go related gene)] and maximum concentration (Cmax), could justify the different effects on QT interval prolongation. Our results show that ribociclib, but not palbociclib, could act by down-regulating the expression of KCNH2 (encoding for potassium channel hERG) and up-regulating SCN5A and SNTA1 (encoding for sodium channels Nav1.5 and syntrophin-α1, respectively), three genes associated with long QT syndrome. Consistent with the cardiotoxicity induced by ribociclib, its IC50 (hERG)/free concentration (Cmax free) ratio is closer to the safety threshold than that of palbociclib. In summary, we hypothesize that the different cardiotoxicity associated with ribociclib and palbociclib could be due to the alteration of potassium and sodium channels induced by ribociclib. A better comprehension of the mechanisms of cardiac channelopathies and drug-induced QT interval prolongation will be fundamental to avoid serious and potentially lethal adverse events and, as a consequence, optimize the management of breast cancer patients.

CXCL12 and Its Isoforms: Different Roles in Pancreatic Cancer?

CXCL12 is a chemokine that acts through CXCR4 and ACKR3 receptors and plays a physiological role in embryogenesis and haematopoiesis. It has an important role also in tumor development, since it is released by stromal cells of tumor microenvironment and alters the behavior of cancer cells. Many studies investigated the roles of CXCL12 in order to understand if it has an anti- or protumor role. In particular, it seems to promote tumor invasion, proliferation, angiogenesis, epithelial to mesenchymal transition (EMT), and metastasis in pancreatic cancer. Nevertheless, some evidence shows opposite functions; therefore research on CXCL12 is still ongoing. These discrepancies could be due to the presence of at least six CXCL12 splicing isoforms, each with different roles. Interestingly, three out of six variants have the highest levels of expression in the pancreas. Here, we report the current knowledge about the functions of this chemokine and then focus on pancreatic cancer. Moreover, we discuss the methods applied in recent studies in order to understand if they took into account the existence of the CXCL12 isoforms.

Clinical impact of different exosomes' protein expression in pancreatic ductal carcinoma patients treated with standard first line palliative chemotherapy.

INTRODUCTION: Pancreatic ductal adenocarcinoma is associated to dismal prognosis despite the use of palliative chemotherapy, partly due to the lack of knowledge of biological processes underlying disease progression. Exosomes have been identified as biomarkers sources in different cancer types. Aim of the study was to analyse the contents of circulating exosomes in patients with pancreatic cancer who received palliative chemotherapy. PATIENTS AND METHODS: Patients were submitted to blood sample collection before chemotherapy (T0) and after 3 months (T3). We quantified by an ELISA-based technique specific proteins of cancer-derived exosomes (CD44,CD44v6,EpCAM,CD9,CD81,Tspan8,Integrin α6,Integrin ß4,CD24,CXCR4). We correlated the baseline levels of these factors and changes between T3 and T0 and survival outcomes. Survival analyses were performed by Kaplan-Meier method. Correlation was assessed by log-rank test and level of statistical significance was set at 0.05. Multivariate analysis was performed by logistic regression analysis. RESULTS: Nineteen patients were enrolled. EpCAM T0 levels and increased EpCAM levels from T0 to T3 were those mostly associated with differences in survival. Patients having higher EpCAM had median progression free survival (PFS) of 3.18vs7.31 months (HR:2.82,95%CI:1.03-7.73,p = 0.01). Overall survival (OS) was shorter for patients having higher EpCAM (5.83vs16.45 months,HR:6.16,95%CI:1.93-19.58,p = 0.0001) and also response rates (RR) were worse (20%vs87%,p = 0.015). EpCAM increase during treatment was associated with better median PFS (2.88vs7.31 months,HR:0.24,95%CI:0.04-1.22,p = 0.003). OS was also better (8.75vs11.04 months, HR:0.77,95%CI:0.21-2.73,p = 0.66) and RR were 60%vs20% (p = 0.28). Among clinical factors that might determine changes on PFS and OS, only ECOG PS was associated to significantly worse PFS and OS (p = 0.0137and<0.001 respectively).Multivariate analysis confirmed EpCAM T0 levels and EpCAM T0/T3 changes as independent prognostic factors for PFS. CONCLUSIONS: Pancreatic cancer patients exosomes express EpCAM, whose levels change during treatment. This represents a useful prognostic factor and also suggests that future treatment modalities who target EpCAM should be tested in pancreatic cancer patients selected by exosome EpCAM expression.

KRIT1 Loss-Of-Function Associated with Cerebral Cavernous Malformation Disease Leads to Enhanced S-Glutathionylation of Distinct Structural and Regulatory Proteins.

Loss-of-function mutations in the KRIT1 gene are associated with the pathogenesis of cerebral cavernous malformations (CCMs), a major cerebrovascular disease still awaiting therapies. Accumulating evidence demonstrates that KRIT1 plays an important role in major redox-sensitive mechanisms, including transcriptional pathways and autophagy, which play major roles in cellular homeostasis and defense against oxidative stress, raising the possibility that KRIT1 loss has pleiotropic effects on multiple redox-sensitive systems. Using previously established cellular models, we found that KRIT1 loss-of-function affects the glutathione (GSH) redox system, causing a significant decrease in total GSH levels and increase in oxidized glutathione disulfide (GSSG), with a consequent deficit in the GSH/GSSG redox ratio and GSH-mediated antioxidant capacity. Redox proteomic analyses showed that these effects are associated with increased S-glutathionylation of distinct proteins involved in adaptive responses to oxidative stress, including redox-sensitive chaperonins, metabolic enzymes, and cytoskeletal proteins, suggesting a novel molecular signature of KRIT1 loss-of-function. Besides providing further insights into the emerging pleiotropic functions of KRIT1, these findings point definitively to KRIT1 as a major player in redox biology, shedding new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell sensitivity to oxidative stress, which may eventually lead to cellular dysfunctions and CCM disease pathogenesis.

Glutathione compartmentalization and its role in glutathionylation and other regulatory processes of cellular pathways.

Glutathione is considered the major non-protein low molecular weight modulator of redox processes and the most important thiol reducing agent of the cell. The biosynthesis of glutathione occurs in the cytosol from its constituent amino acids, but this tripeptide is also present in the most important cellular districts, such as mitochondria, nucleus, and endoplasmic reticulum, thus playing a central role in several metabolic pathways and cytoprotection mechanisms. Indeed, glutathione is involved in the modulation of various cellular processes and, not by chance, it is a ubiquitous determinant for redox signaling, xenobiotic detoxification, and regulation of cell cycle and death programs. The balance between its concentration and redox state is due to a complex series of interactions between biosynthesis, utilization, degradation, and transport. All these factors are of great importance to understand the significance of cellular redox balance and its relationship with physiological responses and pathological conditions. The purpose of this review is to give an overview on glutathione cellular compartmentalization. Information on its subcellular distribution provides a deeper understanding of glutathione-dependent processes and reflects the importance of compartmentalization in the regulation of specific cellular pathways. © 2018 BioFactors, 45(2):152-168, 2019.

Emerging Biomarkers in Bladder Cancer Identified by Network Analysis of Transcriptomic Data.

Bladder cancer is a very common malignancy. Although new treatment strategies have been developed, the identification of new therapeutic targets and reliable diagnostic/prognostic biomarkers for bladder cancer remains a priority. Generally, they are found among differentially expressed genes between patients and healthy subjects or among patients with different tumor stages. However, the classical approach includes processing these data taking into consideration only the expression of each single gene regardless of the expression of other genes. These complex gene interaction networks can be revealed by a recently developed systems biology approach called Weighted Gene Co-expression Network Analysis (WGCNA). It takes into account the expression of all genes assessed in an experiment in order to reveal the clusters of co-expressed genes (modules) that, very probably, are also co-regulated. If some genes are co-expressed in controls but not in pathological samples, it can be hypothesized that a regulatory mechanism was altered and that it could be the cause or the effect of the disease. Therefore, genes within these modules could play a role in cancer and thus be considered as potential therapeutic targets or diagnostic/prognostic biomarkers. Here, we have reviewed all the studies where WGCNA has been applied to gene expression data from bladder cancer patients. We have shown the importance of this new approach in identifying candidate biomarkers and therapeutic targets. They include both genes and miRNAs and some of them have already been identified in the literature to have a role in bladder cancer initiation, progression, metastasis, and patient survival.

LncRNA co-expression network analysis reveals novel biomarkers for pancreatic cancer.

High mortality and low survival rates for pancreatic ductal adenocarcinoma (PDAC) mainly result from the delay in diagnosis and treatment. Therefore, there is an urgent need to identify early PDAC biomarkers and new therapeutic targets. In this study, we applied a commonly used systems biology approach, the weighted gene co-expression network analysis (WGCNA), on lncRNA expression data. Eleven lncRNAs, namely A2M-AS1, DLEU2, LINC01133, LINC00675, MIR155HG, SLC25A25-AS1, LINC01857, LOC642852 (LINC00205), ITGB2-AS1, TSPOAP1-AS1 and PSMB8-AS1 have been identified and validated on an independent PDAC expression dataset. Furthermore, we characterized them by functional and pathway enrichment analysis and identified which lncRNAs showed differential expression, differential promoter methylation levels and copy number alterations between normal and PDAC samples. Finally, we also performed a survival analysis and identified A2M-AS1, LINC01133, LINC00205 and TSPOAP1-AS1 as prognostic biomarkers for PDAC. Interestingly, although only a few cancer-associated lncRNAs have been functionally characterized, LINC00675 and LINC01133 lncRNAs have already been demonstrated to be involved in PDAC development and progression. Therefore, our results provide new potential diagnostic/prognostic biomarkers and therapeutic targets for PDAC that deserve to be further investigated. Moreover, these lncRNAs may improve the understanding about molecular pathogenesis of PDAC.

A possible S-glutathionylation of specific proteins by glyoxalase II: An in vitro and in silico study.

Glyoxalase II, the second of 2 enzymes in the glyoxalase system, is a hydroxyacylglutathione hydrolase that catalyses the hydrolysis of S-d-lactoylglutathione to form d-lactic acid and glutathione, which is released from the active site. The tripeptide glutathione is the major sulfhydryl antioxidant and has been shown to control several functions, including S-glutathionylation of proteins. S-Glutathionylation is a way for the cells to store reduced glutathione during oxidative stress, or to protect protein thiol groups from irreversible oxidation, and few enzymes involved in protein S-glutathionylation have been found to date. In this work, the enzyme glyoxalase II and its substrate S-d-lactoylglutathione were incubated with malate dehydrogenase or with actin, resulting in a glutathionylation reaction. Glyoxalase II was also submitted to docking studies. Computational data presented a high propensity of the enzyme to interact with malate dehydrogenase or actin through its catalytic site and further in silico investigation showed a high folding stability of glyoxalase II toward its own reaction product glutathione both protonated and unprotonated. This study suggests that glyoxalase II, through a specific interaction of its catalytic site with target proteins, could be able to perform a rapid and specific protein S-glutathionylation using its natural substrate S-d-lactoylglutathione. SIGNIFICANCE: This article reports for the first time a possible additional role of Glo2 that, after interacting with a target protein, is able to promote S-glutathionylation using its natural substrate SLG, a glutathione derived compound. In this perspective, Glo2 can play a new important regulatory role inS-glutathionylation, acquiring further significance in cellular post-translational modifications of proteins.

Weighted gene co-expression network analysis reveals key genes involved in pancreatic ductal adenocarcinoma development.

PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy. Up till now, the patient's prognosis remains poor which, among others, is due to the paucity of reliable early diagnostic biomarkers. In the past, candidate diagnostic biomarkers and therapeutic targets have been delineated from genes that were found to be differentially expressed in normal versus tumour samples. Recently, new systems biology approaches have been developed to analyse gene expression data, which may yield new biomarkers. As of yet, the weighted gene co-expression network analysis (WGCNA) tool has not been applied to PDAC microarray-based gene expression data. METHODS: PDAC microarray-based gene expression datasets, listed in the Gene Expression Omnibus (GEO) database, were analysed. After pre-processing of the data, we built two final datasets, Normal and PDAC, encompassing 104 and 129 patient samples, respectively. Next, we constructed a weighted gene co-expression network and identified modules of co-expressed genes distinguishing normal from disease conditions. Functional annotations of the genes in these modules were carried out to highlight PDAC-associated molecular pathways and common regulatory mechanisms. Finally, overall survival analyses were carried out to assess the suitability of the genes identified as prognostic biomarkers. RESULTS: Using WGCNA, we identified several key genes that may play important roles in PDAC. These genes are mainly related to either endoplasmic reticulum, mitochondrion or membrane functions, exhibit transferase or hydrolase activities and are involved in biological processes such as lipid metabolism or transmembrane transport. As a validation of the applied method, we found that some of the identified key genes (CEACAM1, MCU, VDAC1, CYCS, C15ORF52, TMEM51, LARP1 and ERLIN2) have previously been reported by others as potential PDAC biomarkers. Using overall survival analyses, we found that several of the newly identified genes may serve as biomarkers to stratify PDAC patients into low- and high-risk groups. CONCLUSIONS: Using this new systems biology approach, we identified several genes that appear to be critical to PDAC development. As such, they may represent potential diagnostic biomarkers as well as therapeutic targets with clinical utility.

Epithelial to Mesenchymal Transition in Renal Cell Carcinoma: Implications for Cancer Therapy.

Epithelial-to-mesenchymal transition (EMT) is a developmentally vital reversible process by which fully differentiated cells lose their epithelial features and acquire a migratory mesenchymal phenotype. EMT contributes to the metastatic potential of tumors. The expression profile and other biological properties of EMT suggest potential targets for cancer therapy, including in renal-cell carcinoma (RCC). The preclinical and clinical results have substantiated the promises that dysregulated elements leading to EMT can be a potential target in RCC patients. In this study, we illustrated the pathogenic and prognostic role of EMT in RCC. In addition, we reconstructed, by literature analysis, the different pathways implicated in the EMT process, thus supporting the rational for future EMT-directed therapeutic approaches for RCC patients.

Computational analysis of the mutations in BAP1, PBRM1 and SETD2 genes reveals the impaired molecular processes in renal cell carcinoma.

Clear cell Renal Cell Carcinoma (ccRCC) is due to loss of von Hippel-Lindau (VHL) gene and at least one out of three chromatin regulating genes BRCA1-associated protein-1 (BAP1), Polybromo-1 (PBRM1) and Set domain-containing 2 (SETD2). More than 350, 700 and 500 mutations are known respectively for BAP1, PBRM1 and SETD2 genes. Each variation damages these genes with different severity levels. Unfortunately for most of these mutations the molecular effect is unknown, so precluding a severity classification. Moreover, the huge number of these gene mutations does not allow to perform experimental assays for each of them. By bioinformatic tools, we performed predictions of the molecular effects of all mutations lying in BAP1, PBRM1 and SETD2 genes. Our results allow to distinguish whether a mutation alters protein function directly or by splicing pattern destruction and how much severely. This classification could be useful to reveal correlation with patients' outcome, to guide experiments, to select the variations that are worth to be included in translational/association studies, and to direct gene therapies.

BAP1, PBRM1 and SETD2 in clear-cell renal cell carcinoma: molecular diagnostics and possible targets for personalized therapies.

Several novel recurrent mutations of histone modifying and chromatin remodeling genes have been identified in renal cell carcinoma. These mutations cause loss of function of several genes located in close proximity to VHL and include PBRM1, BAP1 and SETD2. PBRM1 encodes for BAF180, a component of the SWI/SNF chromatin remodeling complex, and is inactivated in, on average, 36% of clear cell renal cell carcinoma (ccRCC). Mutations of BAP1 encode for the histone deubiquitinase BRCA1 associated protein-1, and are present in 10% of ccRCCs. They are largely mutually exclusive with PBRM1 mutations. Mutations to SETD2, a histone methyltransferase, occur in 10% of ccRCC. BAP1- or SETD2-mutated ccRCCs have been associated with poor overall survival, while PBRM1 mutations seem to identify a favorable group of ccRCC tumors. This review describes the roles of PBRM1, BAP1 and SETD2 in the development and progression of ccRCC and their potential for future personalized approaches.

KRAS mutation status is associated with specific pattern of genes expression in pancreatic adenocarcinoma.

AIMS: To evaluate potential differences at a molecular level between KRAS mutant tumors (MT) and KRAS wild-type (WT) pancreatic tumors and the biological and prognostic significance of different KRAS mutations. MATERIALS & METHODS: Expression of a panel of 29 genes was analyzed in KRAS WT and MT tumors. Effects of KRAS mutation and gene expression levels were assessed on patients' survival. RESULTS: MUC6 (p = 0.009), HGF (p = 0.011), VEGFR-2 (p = 0.020) and VEGFB (p = 0.026) were significantly more expressed and SMAD4 was less suppressed (p = 0.003) in WT KRAS. Contrariwise, SHH (p = 0.012) and IHH (p = 0.031) were more expressed in MT KRAS patients. No OS difference was found between WT and MT KRAS tumors. CONCLUSION: KRAS mutation status seems to identify two different subtypes of pancreatic ductal adenocarcinoma with similar outcome but distinct molecular features and probably different therapeutic targets.

Investigational therapies targeting signal transducer and activator of transcription 3 for the treatment of cancer.

INTRODUCTION: Signal transducer and activator of transcription 3 (STAT3) mediates the expression of a variety of genes in response to cell stimuli and thus plays a key role in several cellular processes such as cell growth and apoptosis. Deregulation of the STAT3 activity has been shown in many malignancies, including breast, head and neck, prostate, pancreas, ovarian and brain cancers and melanoma. Thus, STAT3 may represent an ideal target for cancer therapy. AREAS COVERED: The authors review recent data on the role of STAT3 in tumor initiation and progression, as well as the ongoing clinical trials in cancer patients. The content includes information derived from trial databases, regulatory authorities and scientific literature. EXPERT OPINION: Targeting STAT3 activation leads to the inhibition of tumor growth and metastasis both in vitro and in vivo without affecting normal cells; this suggests that STAT3 could be a valid molecular target for cancer therapy. Extensive clinical research is trying to find anti-STAT3 agents with high single-agent activity. The identification and development of novel drugs that can target deregulated STAT3 activation effectively is both a scientific and clinical challenge that needs to be addressed in the near future.

Lgr5 expression, cancer stem cells and pancreatic cancer: results from biological and computational analyses.

AIMS: To determine the relationship between Lgr5 and other stemness markers and pathologic features in pancreatic ductal adenocarcinoma (PDAC) samples. MATERIALS & METHODS: In 69 samples, Lgr5 was analyzed by qRT-PCR together with a panel of 29 genes. Bioinformatic analysis was carried out to identify a possible pathway regulating Lgr5 expression in PDAC. RESULTS: Lgr5 expression was not associated with the expression of tested cancer stem cell markers. Moreover, it was not an independent predictor of survival neither at univariate analysis (p = 0.21) nor at multivariate analysis (p = 0.225). CONCLUSION: Based on the lack of correlation between Lgr5 and tested cancer stem cell markers, Lgr5 does not seem to be a potential stemness marker or prognostic factor in PDAC.

ExportAid: database of RNA elements regulating nuclear RNA export in mammals.

MOTIVATION: Regulation of nuclear mRNA export or retention is carried out by RNA elements but the mechanism is not yet well understood. To understand the mRNA export process, it is important to collect all the involved RNA elements and their trans-acting factors. RESULTS: By hand-curated literature screening we collected, in ExportAid database, experimentally assessed data about RNA elements regulating nuclear export or retention of endogenous, heterologous or artificial RNAs in mammalian cells. This database could help to understand the RNA export language and to study the possible export efficiency alterations owing to mutations or polymorphisms. Currently, ExportAid stores 235 and 96 RNA elements, respectively, increasing and decreasing export efficiency, and 98 neutral assessed sequences. AVAILABILITY AND IMPLEMENTATION: Freely accessible without registration at http://www.introni.it/ExportAid/ExportAid.html. Database and web interface are implemented in Perl, MySQL, Apache and JavaScript with all major browsers supported.

How much do we know about the coupling of G-proteins to serotonin receptors?

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis.

Strawberry intake increases blood fluid, erythrocyte and mononuclear cell defenses against oxidative challenge.

The health promoting effects of a regular consumption of strawberries deserve attention, and a direct or indirect antioxidant role of strawberry bioactive compounds is among the most probable mechanisms underlying their beneficial properties. In the present study, we evaluated the overall effects of a 2-week daily consumption of strawberries on plasma antioxidant status, membrane lipid susceptibility to ex vivo-induced oxidation, and erythrocyte and mononuclear cell resistance to oxidative damage in apparently healthy volunteers. After strawberry intake, a moderate increase in fasting plasma antioxidant capacity and vitamin C was observed, together with a significant increase in the lag phase preceding plasma lipid oxidation. A significantly enhanced resistance to oxidative hemolysis was confirmed in red blood cells, while no significant changes were found in the extent of their membrane lipid peroxidation. For the first time, increased intake of strawberries for only 2weeks was shown to be sufficient to attenuate mononuclear cell mortality after ex vivo exposure to a single acuteoxidative challenge, but the analysis of DNA oxidative damage gave conflicting results. These findings suggest that a regular consumption of strawberries may enhance body defences against oxidative challenges.

Oncogenic H-Ras up-regulates acid ß-hexosaminidase by a mechanism dependent on the autophagy regulator TFEB.

The expression of constitutively active H-RasV12 oncogene has been described to induce proliferative arrest and premature senescence in many cell models. There are a number of studies indicating an association between senescence and lysosomal enzyme alterations, e.g. lysosomal ß-galactosidase is the most widely used biomarker to detect senescence in cultured cells and we previously reported that H-RasV12 up-regulates lysosomal glycohydrolases enzymatic activity in human fibroblasts. Here we investigated the molecular mechanisms underlying lysosomal glycohydrolase ß-hexosaminidase up-regulation in human fibroblasts expressing the constitutively active H-RasV12. We demonstrated that H-Ras activation increases ß-hexosaminidase expression and secretion by a Raf/extracellular signal-regulated protein kinase dependent pathway, through a mechanism that relies on the activity of the transcription factor EB (TFEB). Because of the pivotal role of TFEB in the regulation of lysosomal system biogenesis and function, our results suggest that this could be a general mechanism to enhance lysosomal enzymes activity during oncogene-induced senescence.