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Supplementary table s1 + supplementary methods

Non-alcoholic fatty liver disease (NAFLD) is considered a relevant liver chronic disease. Variable percentages of NAFLD cases progress from steatosis to steatohepatitis (NASH), cirrhosis and, eventually, hepatocellular carcinoma (HCC). In this study, we aimed to deepen our understanding of expression levels and functional relationships between miR-182-5p and Cyld-Foxo1 in hepatic tissues from C57BL/6J mouse models of diet-induced NAFL/NASH/HCC progression. A miR-182-5p increase was detected early in livers as NAFLD damage progressed, and in tumors compared to peritumor normal tissues. An in vitro assay on HepG2 cells confirmed Cyld and Foxo1, both tumor-suppressor, as miR-182-5p target genes. According to miR-182-5p expression, decreased protein levels were observed in tumors compared to peritumor tissues. Analysis of miR-182-5p, Cyld and Foxo1 expression levels, based on datasets from human HCC samples, showed results consistent with those from our mouse models, and also highlighted the ability of miR-182-5p to distinguish between normal and tumor tissues (AUC 0.83). Overall, this study shows, for the first time, miR-182-5p overexpression and Cyld-Foxo1 downregulation in hepatic tissues and tumors from a diet-induced NAFLD/HCC mouse model. These data were confirmed by the analysis of datasets from human HCC samples, highlighting miR-182-5p diagnostic accuracy and demonstrating the need for further studies to assess its potential role as a biomarker or therapeutic target.

Purpose Human tau is a highly dynamic, multifunctional protein expressed in different isoforms and conformers, known to modulate microtubule turnover. Tau oligomers are considered pathologic forms of the protein able to initiate specific protein accumulation diseases, called tauopathies. In our study, we investigated the potential association between autophagy and tau oligomers accumulation and its role in the response of prostate cancer cells to docetaxel. Methods We evaluated in vitro the expression of tau oligomers in prostate cancer cell lines, PC3 and DU145, in presence of autophagy inhibitors and investigated the role of tau oligomers accumulation in resistance to docetaxel treatment. Results Tau protein was basally expressed in prostate cancer lines as several monomeric and oligomeric forms. The pharmacologic inhibition of autophagy induced in cancer cells the accumulation of tau protein, with a prevalent expression of oligomeric forms. Immunofluorescence analysis of untreated cells revealed that tau was visible mainly in dividing cells where it was localized on the mitotic spindle. Inhibition of autophagy determined an evident upregulation of tau signal in dividing cells and the presence of aberrant monoastral mitotic spindles. The accumulation of tau oligomers was associated with DNA DSB and increased cytotoxic effect by docetaxel. Conclusions Our data indicate that autophagy could exert a promoting role in cancer growth and during chemotherapy facilitating degradation of tau protein and thus blocking the antimitotic effect of accumulated tau oligomers. Thus, therapeutic strategies aimed at stimulating tau oligomers formation, such as autophagy inhibition, could be an effective adjuvant in cancer therapy.

Si306, a pyrazolo[3,4

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.

Simple Summary In the last ten years, the human helicase protein DDX3X turned out to be an extremely interesting target for the development of potential anticancer drugs. Herein, we discovered BA103, a novel specific inhibitor of the helicase binding site of DDX3X, which is characterized by broad-spectrum anticancer activity. BA103 revealed promising tolerability in fibroblasts and good pharmacokinetic properties. Furthermore, BA103 was able to decrease the expression of β-catenin and to reduce tumor migration. Its capability to pass the blood–brain barrier led us to investigate its potential against glioblastoma, which is a high refractory disease with poor prognosis. High efficacy was proven in both xenograft and orthotopic animal models. Abstract DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy that relies on highly heterogeneous cytogenetic alterations. Although in the last few years new agents have been developed for AML treatment, the overall survival prospects for AML patients are still gloomy and new therapeutic options are still urgently needed. Constitutive NF-κB activation has been reported in around 40% of AML patients, where it sustains AML cell survival and chemoresistance. Given the central role of NF-κB in AML, targeting the NF-κB pathway represents an attractive strategy to treat AML. This review focuses on current knowledge of NF-κB’s roles in AML pathogenesis and summarizes the main therapeutic approaches used to treat NF-κB-driven AML.

Glioblastoma (GBM) is known to be the most common and lethal primary malignant brain tumor. Therapies against this neoplasia have a high percentage of failure, associated with the survival of self-renewing glioblastoma stem cells (GSCs), which repopulate treated tumors. In addition, despite new radical surgery protocols and the introduction of new anticancer drugs, protocols for treatment, and technical advances in radiotherapy, no significant improvement in the survival rate for GBMs has been realized. Thus, novel antitarget therapies could be used in conjunction with standard radiochemotherapy approaches. Targeted therapy, indeed, may address specific targets that play an essential role in the proliferation, survival, and invasiveness of GBM cells, including numerous molecules involved in signal transduction pathways. Significant cellular heterogeneity and the hierarchy with GSCs showing a therapy-resistant phenotype could explain tumor recurrence and local invasiveness and, therefore, may be a target for new therapies. Therefore, the forced differentiation of GSCs may be a promising new approach in GBM treatment. This article provides an updated review of the current standard and experimental therapies for GBM, as well as an overview of the molecular characteristics of GSCs, the mechanisms that activate resistance to current treatments, and a new antitumor strategy for treating GSCs for use as therapy.

Mathematical models can be profitably used to establish whether our perception of the external world is accurate. Donald Hoffman and his collaborators have developed a promising mathematical framework within which this question can be addressed and which is based on an exhaustive taxonomy of the different possible relations between perceptual representations and the external world. After reformulating their framework by means of an improved formal system, we discuss their application of evolutionary game theory, which appears to show that an essentially anti-realistic perceptual strategy would in the long run biologically outcompete its rivals. We argue that their model does not take the crucial biological significance of environmental changes into due consideration and propose alternative models which do. We conclude that a partially realistic representation would be favoured in our models. Les modèles mathématiques peuvent être utilisés avec profit pour établir si notre perception du monde extérieur est précis. Donald Hoffman et ses collaborateurs ont développé un cadre mathématique prometteur dans lequel cette question peut être abordée, et qui repose sur une taxonomie exhaustive des différentes relations possibles qui peuvent tenir entre les représentations perceptuelles et le monde extérieur. Après avoir reformulé leur cadre au moyen d’un système formel amélioré, nous discutons de leur application de la théorie des jeux évolutifs, qui semble montrer qu’une stratégie perceptuelle essentiellement anti-réaliste à long terme surpasserait biologiquement ses rivaux. Nous soutenons que leur modèle ne prend pas dûment en considération la signification biologique cruciale des changements environnementaux et proposons des modèles alternatifs qui le font. Nous concluons que, dans nos modèles, une représentation partiellement réaliste serait privilégiée.

Leptin is a hormone that plays a major role as mediator of long-term regulation of energy balance, suppressing food intake, and stimulating weight loss. More recently, important physiological roles other than controlling appetite and energy expenditure have been suggested for leptin, including neuroendocrine, reparative, reproductive, and immune functions. These emerging peripheral roles let hypothesize that leptin can modulate also cancer progression. Indeed, many studies have demonstrated that elevated chronic serum concentrations of leptin, frequently seen in obese subjects, represent a stimulatory signal for tumor growth. Current knowledge indicates that also different non-tumoral cells resident in tumor microenvironment may respond to leptin creating a favorable soil for cancer cells. In addition, leptin is produced also within the tumor microenvironment creating the possibility for paracrine and autocrine action. In this review, we describe the main mechanisms that regulate peripheral leptin availability and how leptin can shape tumor microenvironment.

Src is the prototypal member of Src Family tyrosine Kinases (SFKs), a large non-receptor kinase class that controls multiple signaling pathways in animal cells. SFKs activation is necessary for the mitogenic signal from many growth factors, but also for the acquisition of migratory and invasive phenotype. Indeed, oncogenic activation of SFKs has been demonstrated to play an important role in solid cancers; promoting tumor growth and formation of distant metastases. Several drugs targeting SFKs have been developed and tested in preclinical models and many of them have successfully reached clinical use in hematologic cancers. Although in solid tumors SFKs inhibitors have consistently confirmed their ability in blocking cancer cell progression in several experimental models; their utilization in clinical trials has unveiled unexpected complications against an effective utilization in patients. In this review, we summarize basic molecular mechanisms involving SFKs in cancer spreading and metastasization; and discuss preclinical and clinical data highlighting the main challenges for their future application as therapeutic targets in solid cancer progression

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.

[Image: see text] Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-d]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. In vitro HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds.

Leptin is a hormone that plays a major role as mediator of long-term regulation of energy balance, suppressing food intake, and stimulating weight loss. More recently, important physiological roles other than controlling appetite and energy expenditure have been suggested for leptin, including neuroendocrine, reparative, reproductive, and immune functions. These emerging peripheral roles let hypothesize that leptin can modulate also cancer progression. Indeed, many studies have demonstrated that elevated chronic serum concentrations of leptin, frequently seen in obese subjects, represent a stimulatory signal for tumor growth. Current knowledge indicates that also different non-tumoral cells resident in tumor microenvironment may respond to leptin creating a favorable soil for cancer cells. In addition, leptin is produced also within the tumor microenvironment creating the possibility for paracrine and autocrine action. In this review, we describe the main mechanisms that regulate peripheral leptin availability and how leptin can shape tumor microenvironment.

Over recent years, many authors discussed the effects of different natural compounds on glioblastoma (GBM). Due to its capacity to impair survival and progression of different cancer types, saffron extract (SE), named crocetin (CCT), is particularly noteworthy. In this work, we elucidated the antitumor properties of crocetin in glioma in vivo and in vitro models for the first time. The in vitro results showed that the four tumor cell lines observed in this study (U251, U87, U138, and U373), which were treated with increasing doses of crocetin, showed antiproliferative and pro-differentiative effects as demonstrated by a significant reduction in the number of viable cells, deep changes in cell morphology, and the modulation of mesenchymal and neuronal markers. Indeed, crocetin decreased the expression of Cluster of Differentiation CD44, CD90, CXCR4, and OCT3/4 mesenchymal markers, but increased the expression of &beta, III-Tubulin and neurofilaments (NFH) neuronal linage-related markers. Epigenetic mechanisms may modulate these changes, since Histone Deacetylase, HDAC1 and HDAC3 were downmodulated in U251 and U87 cells, whereas HDAC1 expression was downmodulated in U138 and U373 cells. Western blotting analyses of Fatty Acid Synthase, FASN, and CD44 resulted in effective inhibition of these markers after CCT treatment, which was associated with important activation of the apoptosis program and reduced glioma cell movement and wound repair. The in vivo studies aligned with the results obtained in vitro. Indeed, crocetin was demonstrated to inhibit the growth of U251 and U87 cells that were subcutaneously injected into animal models. In particular, the Tumor To Progression or TTP values and Kaplan&ndash, Meier curves indicated that crocetin had more major effects than radiotherapy alone, but similar effects to temozolomide (TMZ). An intra-brain cell inoculation of a small number of luciferase-transfected U251 cells provided a model that was able to recapitulate recurrence after surgical tumor removal. The results obtained from the orthotopic intra-brain model indicated that CCT treatment increased the disease-free survival (DFS) and overall survival (OS) rates, inducing a delay in appearance of a detectable bioluminescent lesion. CCT showed greater efficacy than Radio Therapy (RT) but comparable efficacy to temozolomide in xenograft models. Therefore, we aimed to continue the study of crocetin&rsquo, s effects in glioma disease, focusing our attention on the radiosensitizing properties of the natural compound and highlighting the ways in which this was realized.

[Image: see text] Gold-nanoparticle (AuNP)-conjugated drugs represent a promising and innovative antitumor therapeutic approach. In our study, we describe the design, the synthesis, the preparation, and the characterization of AuNPs conjugated with the pyrazolo[3,4-d]pyrimidine derivative SI306, a c-Src inhibitor. AuNPs–SI306 showed a good loading efficacy (65%), optimal stability in polar media and in human plasma, and a suitable morphological profile: a ζ-potential of −43.9 mV, a nanoparticle diameter of 48.6 nm, and a 0.441 PDI value. The antitumoral activity of AuNPs–SI306 was evaluated in vitro in the glioblastoma model, by the low-density growth assay, and also in combination with radiotherapy (RT). Results demonstrated that AuNPs had a basal radiosensitization ability and that AuNPs–SI306, when used in combination with RT, were more effective in inhibiting tumor cell growth with respect to AuNPs and free SI306.

RAS mutational status in colorectal cancer (CRC) represents a predictive biomarker of response to anti-EGFR therapy, but to date it cannot be considered an appropriate biomarker of response to anti-VEGF therapy. To elucidate the function of K-Ras in promoting angiogenesis, the effect of conditioned media from KRAS mutated and wild type colon cancer cell lines on HUVECs tubule formation ability and the correspondent production of pro-angiogenic factors have been evaluated by a specific ELISA assay. Ras-activated signaling pathways were compared by western blot analysis and RTq-PCR. In addition, VEGF, IL-8, bFGF and HIF-1α expression was determined in K-RAS silenced cells. Furthermore, we conducted an observational study in a cohort of RAS mutated metastatic CRC patients, treated with first-line bevacizumab-based regimens, evaluating VEGF-A and IL-8 plasma levels at baseline, and during treatment. K-RAS promotes VEGF production by cancer cell lines. At the transcriptional level, this is reflected to a K-RAS dependent HIF-1α over-expression. Moreover, the HIF-1α, VEGF and FGF expression inhibition in KRAS knocked cells confirmed these results. Within the clinical part, no statistically significant correlation has been found between progression-free survival (PFS) and VEGF-A/IL-8 levels, but we cannot exclude that these biomarkers could be further investigated as predictive or prognostic biomarkers in this setting. Our study confirmed the direct involvement of K-Ras in promoting angiogenesis into colon cancer cell lines.

Dental pulp has been revealed as an accessible and a rich source of mesenchymal stem cells (MSCs) and its biological potential is currently under intense investigation. MSCs from dental pulp stem cells (DPSCs) have been indicated as a heterogeneous population oriented not only in repairing dentine but also in maintaining vascular and nervous homeostasis of the teeth. We sought to verify the phenotype of cells isolated from dental pulp of young donors and to investigate in vitro their role as pericyte-like cells. Specifically, we evaluated how culture conditions can modulate expression of pericyte markers in DPSCs and their capacity to stabilize endothelial tubes in vitro. DPSCs cultured in standard conditions expressed MSC markers and demonstrated to contain a population expressing the pericyte marker NG2. These DPSCs were associated with low sprouting capacity in extra-cellular (EC) Matrix and limited ability in retaining tubes formed by endothelial cells in a coculture angiogenesis model. When cultured in endothelial growth medium (EGM)-2, DPSCs significantly upregulated NG2, and partially alpha-smooth muscle actin. The resulting population conserved the stem marker CD73, but was negative for calponin and endothelial markers. EGM-2-conditioned DPSCs showed a higher sprouting ability in EC Matrix and efficient association with human umbilical vein endothelial cells allowing the partial retention of endothelial tubes for several days. Among growth factors contained in EGM-2 we identified basic fibroblast growth factor (bFGF) as mainly responsible for NG2 upregulation and long-term stabilization of endothelial tubes. According to the in vitro analysis, DPSCs represent an effective source of pericytes and the appropriate culture conditions could result in a population with a promising ability to stabilize vessels and promote vascular maturation.

14-3-3 are regulatory proteins that through protein–protein interactions (PPI) with numerous binding partners could be involved in several human diseases, including cancer, neurodegenerative disorders, and pathogens infections. Following our research interest in the development of 14-3-3 PPI inhibitors, here we exploited the privileged 4-aminoantipyrine scaffold in the design and synthesis of some derivatives endowed with antiproliferative activity against K-562 cells, and capable of binding to recombinant 14-3-3σ as evidenced by NMR spectroscopy. The binding mode was further explored by molecular modelling, while coupling confocal microscopy with intensitometric analysis showed that compound 1 was able to promote the nuclear translocation of c-Abl at low micromolar concentrations. Overall, 1 is chemically stable compared to parent 14-3-3 PPI inhibitors, and thus emerged as a confirmed hit for further development.

Leptin, a cytokine produced by the adipose tissue in response to food intake, is a key player in the regulation of energy balance and body weight control. Physiological action of leptin in modulating the metabolic adaptation of different peripheral tissues supports the hypothesis that it could also exert a direct effect on cancer cells. In vitro, treatment with leptin up-regulated HIF-1α and stimulated adhesion and invasion of prostate cancer cells cultured in hypoxia. Leptin action was effective in both low and high glycolytic cancer cell lines, and determined the up-regulation of lactate exporter MCT4 and its associated protein CD147. HIF-1α stabilization was oligomycin-independent and was associated with an important modulation of mitochondrial homeostasis. In fact, leptin treatment produced mitochondrial biogenesis, stabilization of mitochondrial membrane potential and increased uncoupled respiration through the up-regulation of UCP2. Furthermore, leptin counteracted the downmodulation of SIRT1 induced by hypoxia, and persistent high levels of SIRT1 were directly involved in HIF-1α stabilization. Leptin can sustain cancer progression in hypoxic environment and when mitochondrial respiration is impaired. Leptin signaling axis, including the new proposed intermediate SIRT1, could represent a new diagnostic and therapeutic target in prostate cancer.

Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23&ndash, 231 (recPrPC) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrPC was able to activate an intracellular signal pathway mediated by extracellular-signal-regulated kinase 1 and 2 (ERK 1/2) and protein kinase B (Akt). Moreover, in order to understand whether endogenous prion protein (PrPC) was necessary to mediate the signaling induced by recPrPC, we silenced PrPC, demonstrating that the presence of endogenous PrPC was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrPC is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrPC. Our results suggest that lipid rafts integrity play a key role in recPrPC activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and M&beta, CD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrPC. In addition, we investigated the capacity of recPrPC to induce hDPSCs neuronal differentiation process after long-term stimulation through the evaluation of typical neuronal markers expression such as B3-Tubulin, neurofilament-H (NFH) and growth associated protein 43 (GAP43). Accordingly, when we silenced endogenous PrPC, we observed the inhibition of neuronal differentiation induced by recPrPC. The combined data suggest that recPrPC plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.

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Human Dental Pulp Stem Cells (hDPSCs) represent a type of adult mesenchymal stem cells that have the ability to differentiate in vitro in several lineages such as odontoblasts, osteoblasts, chondrocytes, adipocytes and neurons. In the current work, we used hDPSCs as the experimental model to study the role of recombinant prion protein 23–231 (recPrPC) in the neuronal differentiation process, and in the signal pathway activation of ERK 1/2 and Akt. We demonstrated that recPrPC was able to activate an intracellular signal pathway mediated by extracellular-signal-regulated kinase 1 and 2 (ERK 1/2) and protein kinase B (Akt). Moreover, in order to understand whether endogenous prion protein (PrPC) was necessary to mediate the signaling induced by recPrPC, we silenced PrPC, demonstrating that the presence of endogenous PrPC was essential for ERK 1/2 and Akt phosphorylation. Since endogenous PrPC is a well-known lipid rafts component, we evaluated the role of these structures in the signal pathway induced by recPrPC. Our results suggest that lipid rafts integrity play a key role in recPrPC activity. In fact, lipid rafts inhibitors, such as fumonisin B1 and MβCD, significantly prevented ERK 1/2 and Akt phosphorylation induced by recPrPC. In addition, we investigated the capacity of recPrPC to induce hDPSCs neuronal differentiation process after long-term stimulation through the evaluation of typical neuronal markers expression such as B3-Tubulin, neurofilament-H (NFH) and growth associated protein 43 (GAP43). Accordingly, when we silenced endogenous PrPC, we observed the inhibition of neuronal differentiation induced by recPrPC. The combined data suggest that recPrPC plays a key role in the neuronal differentiation process and in the activation of specific intracellular signal pathways in hDPSCs.

Peroxisome proliferator-activated receptor alpha (PPARα) has been involved in the regulation of somatotroph tumour cells and may be targeted by different drugs, some of them are in current clinical use. The aim of this study was to investigate the expression of PPARα in additional phenotypes of pituitary adenomas (PA), the relationship between PPARα and its potential molecular partner aryl hydrocarbon receptor interacting protein (AIP) in these tumours, and the effects of PPARα agonists on lactotroph cells. Seventy-five human PA – 57 non-functioning (NFPA) and 18 prolactinomas (PRL-PA) – were characterised for PPARα and AIP expression by real time RT-PCR and/or immunohistochemistry (IHC), and the effects of fenofibrate and WY 14 643 on MMQ cells were studied in vitro. PPARα was expressed in a majority of PA. PPARα immunostaining was observed in 93.7% PRL-PA vs. 60.6% NFPA (p=0.016), the opposite being found for AIP (83.3% in NFPA vs. 43.7% in PRL-PA, p=0.003). PPARα expression was unrelated to gonadotroph differentiation in NFPA, but positively correlated with tumour volume in PRL-PA. Both drugs significantly reduced MMQ cell growth at high concentrations (100–200 μM). At the same time, despite modest stimulating effects on PRL secretion were observed, these were overcome by the reduction in cell number. In conclusion, PPARα is commonly expressed by PRL-PA and NFPA, regardless of AIP, and may represent a new target of PPARα agonists.

Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.

KRIT1 (CCM1) is a disease gene responsible for Cerebral Cavernous Malformations (CCM), a major cerebrovascular disease of proven genetic origin affecting 0.3–0.5% of the population. Previously, we demonstrated that KRIT1 loss-of-function is associated with altered redox homeostasis and abnormal activation of the redox-sensitive transcription factor c-Jun, which collectively result in pro-oxidative, pro-inflammatory and pro-angiogenic effects, suggesting a novel pathogenic mechanism for CCM disease and raising the possibility that KRIT1 loss-of-function exerts pleiotropic effects on multiple redox-sensitive mechanisms. To address this possibility, we investigated major redox-sensitive pathways and enzymatic systems that play critical roles in fundamental cytoprotective mechanisms of adaptive responses to oxidative stress, including the master Nrf2 antioxidant defense pathway and its downstream target Glyoxalase 1 (Glo1), a pivotal stress-responsive defense enzyme involved in cellular protection against glycative and oxidative stress through the metabolism of methylglyoxal (MG). This is a potent post-translational protein modifier that may either contribute to increased oxidative molecular damage and cellular susceptibility to apoptosis, or enhance the activity of major apoptosis-protective proteins, including heat shock proteins (Hsps), promoting cell survival. Experimental outcomes showed that KRIT1 loss-of-function induces a redox-sensitive sustained upregulation of Nrf2 and Glo1, and a drop in intracellular levels of MG-modified Hsp70 and Hsp27 proteins, leading to a chronic adaptive redox homeostasis that counteracts intrinsic oxidative stress but increases susceptibility to oxidative DNA damage and apoptosis, sensitizing cells to further oxidative challenges. While supporting and extending the pleiotropic functions of KRIT1, these findings shed new light on the mechanistic relationship between KRIT1 loss-of-function and enhanced cell predisposition to oxidative damage, thus providing valuable new insights into CCM pathogenesis and novel options for the development of preventive and therapeutic strategies., Graphical abstract Schematic models representing adaptive redox responses associated with KRIT1 loss-of-function. KRIT1 loss-of-function causes a persistent activation of the redox-sensitive transcription factors c-Jun and Nrf2 and consequent upregulation of downstream targets, including cycloxygenase-2 (COX-2), heme oxygenase-1 (HO-1) and glyoxalase 1 (GLO1). While the c-Jun/COX-2 axis promotes pro-oxidant and pro-inflammatory effects, the Nrf2/HO-1 and Nrf2/GLO1 pathways mediate adaptive antioxidant responses that counteract these effects by limiting ROS* and MG intracellular accumulation, thus contributing to reduce a vicious cycle of oxidative stress and providing an adaptive defense for long term cell survival. However, this sustained adaptive redox homeostasis occurs at the expense of other cytoprotective mechanisms, including the MG-dependent formation of cytoprotective AP-Hsp70 and AP-Hsp27 protein adducts, leading to enhanced cell susceptibility to oxidative DNA damage and apoptosis, and sensitizing cells to additional stressful insults. Inter-individual differences in Nrf2-mediated adaptive defense mechanisms might influence susceptibility to CCM disease onset and progression. *The generic ROS term refers to O2•−and H2O2as well as to putative secondary oxidative products that might be implicated without certainty.fx1, Highlights • KRIT1 loss causes a chronic adaptive redox response based on the JNK-Nrf2-Glo1 axis. • Phospho-JNK, Nrf2 and Glo1 are upregulated in endothelial cells lining human CCMs. • Defective autophagy contributes to the sustained upregulation of the Nrf2-Glo1 axis. • Nrf2-Glo1 upregulation causes a drop of AP-modified Hsp70 and Hsp27 proteins. • Sustained Nrf2-Glo1 activation sensitizes cells to oxidative stress and apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0061484.].