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Insulin-like growth factor binding protein 6 (IGFBP6) is a secreted protein with a controversial role in human malignancies, being downregulated in most types of human cancer, but upregulated in selected tumors. Ovarian cancer (OC) is a human malignancy characterized by IGFBP6 downregulation; however, the significance of its low expression during ovarian carcinogenesis is still poorly understood. In the present study, IGFBP6 expression and activation of its associated signaling pathway were evaluated in two matched OC cell lines derived from a high-grade serous OC before and after platinum resistance (PEA1 and PEA2 cells, respectively). A whole genome gene expression analysis was comparatively performed in both cell lines upon IGFBP6 stimulation using Illumina technology. IGFBP6 gene expression data from human OC cases were obtained from public datasets. Gene expression data from public datasets confirmed the downregulation of IGFBP6 in primary and metastatic OC tissues compared with in normal ovarian tissues. The comparative analysis of platinum-sensitive (PEA1) and platinum-resistant (PEA2) cell lines showed quantitative and qualitative differences in the activation of IGFBP6 signaling. Notably, IGFBP6 enhanced ERK1/2 phosphorylation only in PEA1 cells, and induced more evident and significant gene expression reprogramming in PEA1 cells compared with in PEA2 cells. Furthermore, the analysis of selected genes modulated by IGFBP6 (i.e.

Metabolic rewiring fuels rapid cancer cell proliferation by promoting adjustments in energetic resources, and increasing glucose uptake and its conversion into lactate, even in the presence of oxygen. Furthermore, solid tumors often contain hypoxic areas and can rapidly adapt to low oxygen conditions by activating hypoxia inducible factor (HIF)-1α and several downstream pathways, thus sustaining cell survival and metabolic reprogramming. Since TNF receptor-associated protein 1 (TRAP1) is a HSP90 molecular chaperone upregulated in several human malignancies and is involved in cancer cell adaptation to unfavorable environments and metabolic reprogramming, in the present study, its role was investigated in the adaptive response to hypoxia in human colorectal cancer (CRC) cells and organoids. In the present study, glucose uptake, lactate production and the expression of key metabolic genes were evaluated in TRAP1-silenced CRC cell models under conditions of hypoxia/normoxia. Whole genome gene expression profiling was performed in TRAP1-silenced HCT116 cells exposed to hypoxia to establish the role of TRAP1 in adaptive responses to oxygen deprivation. The results revealed that TRAP1 was involved in regulating hypoxia-induced HIF-1α stabilization and glycolytic metabolism and that glucose transporter 1 expression, glucose uptake and lactate production were partially impaired in TRAP1-silenced CRC cells under hypoxic conditions. At the transcriptional level, the gene expression reprogramming of cancer cells driven by HIF-1α was partially inhibited in TRAP1-silenced CRC cells and organoids exposed to hypoxia. Moreover, Gene Set Enrichment Analysis of TRAP1-silenced HCT116 cells exposed to hypoxia demonstrated that TRAP1 was involved in the regulation of ribosome biogenesis and this occurred with the inhibition of the mTOR pathway. Therefore, as demonstrated herein, TRAP1 is a key factor in maintaining HIF-1α-induced genetic/metabolic program under hypoxic conditions and may represent a promising target for novel metabolic therapies.

Wnt/&beta, Catenin signaling is involved in embryonic development, regeneration, and cellular differentiation and is responsible for cancer stemness maintenance. The HSP90 molecular chaperone TRAP1 is upregulated in 60&ndash, 70% of human colorectal carcinomas (CRCs) and favors stem cells maintenance, modulating the Wnt/&beta, Catenin pathway and preventing &beta, Catenin phosphorylation/degradation. The role of TRAP1 in the regulation of Wnt/&beta, Catenin signaling was further investigated in human CRC cell lines, patient-derived spheroids, and CRC specimens. TRAP1 relevance in the activation of Wnt/&beta, Catenin signaling was highlighted by a TCF/LEF Cignal Reporter Assay in Wnt-off HEK293T and CRC HCT116 cell lines. Of note, this regulation occurs through the modulation of Wnt ligand receptors LRP5 and LRP6 that are both downregulated in TRAP1-silenced cell lines. However, while LRP5 mRNA is significantly downregulated upon TRAP1 silencing, LRP6 mRNA is unchanged, suggesting independent mechanisms of regulation by TRAP1. Indeed, LRP5 is regulated upon promoter methylation in CRC cell lines and human CRCs, whereas LRP6 is controlled at post-translational level by protein ubiquitination/degradation. Consistently, human CRCs with high TRAP1 expression are characterized by the co-upregulation of active &beta, Catenin, LRP5 and LRP6. Altogether, these data suggest that Wnt/&beta, Catenin signaling is modulated at multiple levels by TRAP1.

Background. Epigenetic remodeling is responsible for tumor progression and drug resistance in human colorectal carcinoma (CRC). A subgroup of human CRCs exhibits the CIMP status, with extensive hypermethylation events in promoter regions of several genes, even though the prognostic significance of CIMP is controversial. This study addressed the hypothesis that DNA methylation profiling may identify metastatic CRC (mCRC) subtypes with different clinical behavior. Methods. Global methylation profile was comparatively analyzed between 24 first-line primary-resistant and 12 drug-sensitive mCRCs (in-house cohort), two subgroups of tumors with significantly different outcome. Methylation and gene expression data from 33 mCRCs of the TCGA COAD dataset (TCGA COAD cohort) were used to identify, among differentially methylated genes, a prognostic signature of functionally methylated genes. Clusters of mCRCs with different methylation patterns were further characterized for DNA mutational load, gene copy number and gene expression profiles. Human CRC HT29 and HCT116 cell lines were adapted to growth in presence of oxaliplatin and irinotecan and used as in vitro model to validate gene expression data.Results. Twelve functionally methylated genes yielded a hierarchical clustering of patients in two well-defined clusters with hypermethylated tumors characterized by a significantly worse relapse-free and overall survival compared to hypomethylated cancers and this was reproduced in both the in-house and the TCGA COAD cohorts. Interestingly, the hypermethylated poor prognosis cluster was enriched of CIMP-high and MSI-like cases. Furthermore, methylation events were enriched in genes located on q-arm of chromosomes 13 and 20, two chromosomal regions with gain/loss alterations strongly associated with adenoma-to-carcinoma progression. Finally, the expression of the 12-genes signature and MSI-enriching genes was confirmed in two independent oxaliplatin- and irinotecan-resistant CRC cell lines. Conclusions. These data represent the proof of concept that the hypermethylation of specific sets of genes may provide prognostic information being able to identify a subgroup of mCRCs with poor prognosis.

Here, we show that TRAP1 modulates glycolytic metabolism by regulating PFK1 activity/stability. In a high TRAP1 background, TRAP1 inhibits cellular respiration and interacts with PFK1 on the ER and this enables PFK1 glycolytic activity preventing its ubiquitination/degradation. In a low TRAP1 background, cellular respiration is upregulated and PFK1 activity reduced due to increased ubiquitination/degradation and this results in loss of TRAP1 control on glycolytic cascade. The increased levels of citrate, observed in conditions of enhanced cellular respiration, are responsible for the inhibition of PFK1 activity, and this results in enhancement of PFK1 ubiquitination/degradation., Metabolic rewiring is a mechanism of adaptation to unfavorable environmental conditions and tumor progression. TRAP1 is an HSP90 molecular chaperone upregulated in human colorectal carcinomas (CRCs) and responsible for downregulation of oxidative phosphorylation (OXPHOS) and adaptation to metabolic stress. The mechanism by which TRAP1 regulates glycolytic metabolism and the relevance of this regulation in resistance to EGFR inhibitors were investigated in patient‐derived CRC spheres, human CRC cells, samples, and patients. A linear correlation was observed between TRAP1 levels and 18F‐fluoro‐2‐deoxy‐glucose (18F‐FDG) uptake upon PET scan or GLUT1 expression in human CRCs. Consistently, TRAP1 enhances GLUT1 expression, glucose uptake, and lactate production and downregulates OXPHOS in CRC patient‐derived spheroids and cell lines. Mechanistically, TRAP1 maximizes lactate production to balance low OXPHOS through the regulation of the glycolytic enzyme phosphofructokinase‐1 (PFK1); this depends on the interaction between TRAP1 and PFK1, which favors PFK1 glycolytic activity and prevents its ubiquitination/degradation. By contrast, TRAP1/PFK1 interaction is lost in conditions of enhanced OXPHOS, which results in loss of TRAP1 regulation of PFK1 activity and lactate production. Notably, TRAP1 regulation of glycolysis is involved in resistance of RAS‐wild‐type CRCs to EGFR monoclonals. Indeed, either TRAP1 upregulation or high glycolytic metabolism impairs cetuximab activity in vitro, whereas TRAP1 targeting and/or inhibition of glycolytic pathway enhances cell response to cetuximab. Finally, a linear correlation between 18F‐FDG PET uptake and poor response to cetuximab in first‐line therapy in human metastatic CRCs was observed. These results suggest that TRAP1 is a key determinant of CRC metabolic rewiring and favors resistance to EGFR inhibitors through regulation of glycolytic metabolism.

Cholangiocarcinoma is a primary malignancy of the biliary tract characterized by late and unspecific symptoms, unfavorable prognosis, and few treatment options. The advent of next-generation sequencing has revealed potential targetable or actionable molecular alterations in biliary tumors. Among several identified genetic alterations, the IDH1 mutation is arousing interest due to its role in epigenetic and metabolic remodeling. Indeed, some IDH1 point mutations induce widespread epigenetic alterations by means of a gain-of-function of the enzyme, which becomes able to produce the oncometabolite 2-hydroxyglutarate, with inhibitory activity on α-ketoglutarate-dependent enzymes, such as DNA and histone demethylases. Thus, its accumulation produces changes in the expression of several key genes involved in cell differentiation and survival. At present, small-molecule inhibitors of IDH1 mutated enzyme are under investigation in preclinical and clinical phases as promising innovative treatments for IDH1-mutated intrahepatic cholangiocarcinomas. This review examines the molecular rationale and the results of preclinical and early-phase studies on novel pharmacological agents targeting mutant IDH1 in cholangiocarcinoma patients. Contextually, it will offer a starting point for discussion on combined therapies with metabolic and epigenetic drugs, to provide molecular support to target the interplay between metabolism and epigenetics, two hallmarks of cancer onset and progression.

Cancer stem cells (CSCs) are a subpopulation of tumor cells with unlimited self-renewal capability, multilineage differentiation potential and long-term tumor repopulation capacity. CSCs reside in anatomically distinct regions within the tumor microenvironment, called niches, and this favors the maintenance of CSC properties and preserves their phenotypic plasticity. Indeed, CSCs are characterized by a flexible state based on their capacity to interconvert between a differentiated and a stem-like phenotype, and this depends on the activation of adaptive mechanisms in response to different environmental conditions. Heat Shock Proteins (HSPs) are molecular chaperones, upregulated upon cell exposure to several stress conditions and are responsible for normal maturation, localization and activity of intra and extracellular proteins. Noteworthy, HSPs play a central role in several cellular processes involved in tumor initiation and progression (i.e. cell viability, resistance to apoptosis, stress conditions and drug therapy, EMT, bioenergetics, invasiveness, metastasis formation) and, thus, are widely considered potential molecular targets. Furthermore, much evidence suggests a key regulatory function for HSPs in CSC maintenance and their upregulation has been proposed as a mechanism used by CSCs to adapt to unfavorable environmental conditions, such as nutrient deprivation, hypoxia, inflammation. This review discusses the relevance of HSPs in CSC biology, highlighting their role as novel potential molecular targets to develop anticancer strategies aimed at CSC targeting.

Cancer has been considered, for a long time, a genetic disease where mutations in key regulatory genes drive tumor initiation, growth, metastasis, and drug resistance. Instead, the advent of high-throughput technologies has revolutionized cancer research, allowing to investigate molecular alterations at multiple levels, including genome, epigenome, transcriptome, proteome, and metabolome and showing the multifaceted aspects of this disease. The multi-omics approaches revealed an intricate molecular landscape where different cellular functions are interconnected and cooperatively contribute to shaping the malignant phenotype. Recent evidence has brought to light how metabolism and epigenetics are highly intertwined, and their aberrant crosstalk can contribute to tumorigenesis. The oncogene-driven metabolic plasticity of tumor cells supports the energetic and anabolic demands of proliferative tumor programs and secondary can alter the epigenetic landscape via modulating the production and/or the activity of epigenetic metabolites. Conversely, epigenetic mechanisms can regulate the expression of metabolic genes, thereby altering the metabolome, eliciting adaptive responses to rapidly changing environmental conditions, and sustaining malignant cell survival and progression in hostile niches. Thus, cancer cells take advantage of the epigenetics-metabolism crosstalk to acquire aggressive traits, promote cell proliferation, metastasis, and pluripotency, and shape tumor microenvironment. Understanding this bidirectional relationship is crucial to identify potential novel molecular targets for the implementation of robust anti-cancer therapeutic strategies.

Simple Summary The global methylation profile of two human metastatic colorectal carcinoma subgroups with significantly different outcomes (primary-resistant versus drug-sensitive tumors) was analyzed and compared with the gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) metastatic colorectal carcinoma dataset with the aim to identify a prognostic signature of functionally methylated genes. A novel epigenetic eight-gene signature, with hypermethylation of the promoter regions, was identified and validated for its capacity to predict poor outcome, which had a CpG-island methylator phenotype (CIMP)-high status and microsatellite instability (MSI)-like phenotype. Abstract Epigenetics is involved in tumor progression and drug resistance in human colorectal carcinoma (CRC). This study addressed the hypothesis that the DNA methylation profiling may predict the clinical behavior of metastatic CRCs (mCRCs). The global methylation profile of two human mCRC subgroups with significantly different outcome was analyzed and compared with gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) and the NCBI GENE expression Omnibus repository (GEO) GSE48684 mCRCs datasets to identify a prognostic signature of functionally methylated genes. A novel epigenetic signature of eight hypermethylated genes was characterized that was able to identify mCRCs with poor prognosis, which had a CpG-island methylator phenotype (CIMP)-high and microsatellite instability (MSI)-like phenotype. Interestingly, methylation events were enriched in genes located on the q-arm of chromosomes 13 and 20, two chromosomal regions with gain/loss alterations associated with adenoma-to-carcinoma progression. Finally, the expression of the eight-genes signature and MSI-enriching genes was confirmed in oxaliplatin- and irinotecan-resistant CRC cell lines. These data reveal that the hypermethylation of specific genes may provide prognostic information that is able to identify a subgroup of mCRCs with poor prognosis.

The aim of this work was to evaluate the possibility of using capillary electrophoresis for rapid and sensitive detection of microorganisms, such as yeasts, in order to propose it as an alternative to traditional microbiology methods both in manufacturing and research areas. After optimization of condition for the electrophoretic separation, three Saccharomyces cerevisiae strains, cultured in YPD medium, were analyzed by capillary electrophoresis at different growth times (3, 7, 24 and 48 h). The analysis was carried out on undamaged and non-pretreated cells. For all the strains, the electrophoretic profile was characterised by two distinct peaks, at each time of growth. Nevertheless some differences in the electropherograms of each strain were evident, probably in consequence of changing in the surface charge of yeasts at various stage of growth. The results demonstrated the high efficacy of capillary electrophoresis for intact cells separation of yeast and support the application of this technique for the evaluation of the viability of cultured cells.

Despite initial chemotherapy response, ovarian cancer is the deadliest gynecologic cancer, due to frequent relapse and onset of drug resistance. To date, there is no affordable diagnostic/prognostic biomarker for early detection of the disease. However, it has been recently shown that high grade serous ovarian cancers show peculiar oxidative metabolism, which is in turn responsible for inflammatory response and drug resistance. The molecular chaperone TRAP1 plays pivotal roles in such metabolic adaptations, due to the involvement in the regulation of mitochondrial respiration. Here, we show that platinum-resistant ovarian cancer cells also show reduced cholesterol biosynthesis, and mostly rely on the uptake of exogenous cholesterol for their needs. Expression of FDPS and OSC, enzymes involved in cholesterol synthesis, are decreased both in drug-resistant cells and upon TRAP1 silencing, whereas the expression of LDL receptor, the main mediator of extracellular cholesterol uptake, is increased. Strikingly, treatment with statins to inhibit cholesterol synthesis reduces cisplatin-induced apoptosis, whereas silencing of LIPG, an enzyme involved in lipid metabolism, or withdrawal of lipids from the culture medium, increases sensitivity to the drug. These results suggest caveats for the use of statins in ovarian cancer patients and highlights the importance of lipid metabolism in ovarian cancer treatment. This research was funded by POR CAMPANIA FESR 2014/2020 [project “SATIN” (Sviluppo di Approcci Terapeutici INnovativi per patologie neoplastiche resistenti ai trattamenti)]

Thyroid cancer is the most common endocrine malignancy, with well-differentiated subtypes characterized by an excellent prognosis due to their optimal sensitivity to standard therapies whereas poorly differentiated and anaplastic tumours by chemo/radio-resistance and unfavourable outcome. Heat Shock Proteins (HSPs) are molecular chaperones overexpressed in thyroid malignancies and involved in crucial functions responsible for thyroid carcinogenesis, as protection from apoptosis, drug resistance and cell migration. Thus, HSPs inhibitors have been proposed as novel therapeutic agents in thyroid cancer to revert molecular mechanisms of tumour progression. In this review, we report an overview on the biological role of HSPs, and specifically HSP90s, in thyroid cancer and their potential involvement as biomarkers. We discuss the rationale to evaluate HSPs inhibitors as innovative anticancer agents in specific subtypes of thyroid cancer characterized by poor response to therapies with the objective to target single family chaperones to reduce, simultaneously, the expression/stability of multiple client proteins.

The Kirsten rat sarcoma viral oncogene homolog (RAS)/v-raf-1 murine leukemia viral oncogene homolog 1 (RAF)/mitogen-activated protein kinase 1 (MAPK) signaling cascade is the most important oncogenic pathway in human cancers. Tumors leading mutations in the gene encoding for v-raf murine sarcoma viral oncogene homolog B (BRAF) serine-threonine kinase are reliant on the MAPK signaling pathway for their growth and survival. Indeed, the constitutive activation of MAPK pathway results in continuous stimulation of cell proliferation, enhancement of the apoptotic threshold and induction of a migratory and metastatic phenotype. In a clinical perspective, this scenario opens to the possibility of targeting BRAF pathway for therapy. Thyroid carcinomas (TCs) bearing BRAF mutations represent approximately 29–83% of human thyroid malignancies and, differently from melanomas, are less sensitive to BRAF inhibitors and develop primary or acquired resistance due to mutational events or activation of alternative signaling pathways able to reactivate ERK signaling. In this review, we provide an overview on the current knowledge concerning the mechanisms leading to resistance to BRAF inhibitors in human thyroid carcinomas and discuss the potential therapeutic strategies, including combinations of BRAF inhibitors with other targeted agents, which might be employed to overcome drug resistance and potentiate the activity of single agent BRAF inhibitors.

AIMS This study aimed to investigate the dynamics and physiological heterogeneity of Oenococcus oeni under different conditions, cell membrane fluidity and permeability variations, and assessment of changes in cell surface charging rates. METHODS AND RESULTS Flow cytometry, membrane fatty acid analysis and capillary electrophoresis were performed to study ethanol-induced variations. Different physiological states were assessed, revealing cell subpopulations able to adapt and withstand to environmental stress, in order to recover their functionality. Moreover, total results demonstrated changes in cell surface and membrane fatty acid redistribution with a saturation degree and an unsaturated/saturated fatty acid ratio fairly steady in control and in different ethanol stresses. CONCLUSIONS This study revealed a great variability among O. oeni strains and the importance to investigate the mechanisms by a multiparametric approach based on the structural and physiological bacterial adjustments in different stresses tolerance. SIGNIFICANCE AND IMPACT OF THE STUDY Intermediate physiological state assessment in O. oeni with recovery possibility could be an important criterion for potential starter culture application. The flow cytometry application with changes in monitoring membrane fatty acid composition and in surface charging rates allowed the characterization of sorted subpopulations that may contribute to further understanding of diversity and heterogeneity in physiology of bacterial populations.

A Laccase-based biosensor for the determination of phenolic compounds was developed by using Matrix Assisted Pulsed Laser Evaporation as an innovative enzyme immobilization technique. and the deriving biosensor was characterized and applied for the first time. Laccase was immobilized onto different substrates including screen printed carbon electrodes and spectroscopic, morphologic and electrochemical characterizations were carried out. A linear range from 1 to 60 μM was achieved working at 5.5 pH and −0.2 V detection potential vs Ag pseudoreference. The limits of detection and quantification were found to be 1 and 5 μM, respectively. A good fabrication reproducibility, stability of response and selectivity toward interferents were also found The potential of the developed biosensor was tested in the determination of total polyphenol content in real matrices (tea infusion, ethanolic extract from Muscari comosum bulbs and aqueous solution of a food supplement from black radish root and artichoke leaves) and the results were compared with those obtained by using the Folin–Ciocalteu method.

MMP-9, human matrix metalloproteinase 9, belongs to the family of zinc-dependent peptide-bond hydrolases and is involved in the degradation of the extracellular matrix (ECM). In clinics, it is well known that elevated MMP-9 serum levels are associated with cardiovascular dysfunctions, several aspects of the physiology and pathology of the central nervous system, neuropsychiatric disorders and degenerative diseases related to brain tumors, and excitotoxic/neuroinflammatory processes. Due to the large interest of diagnostics in this protein, efforts to set up sensitive methods to detect MMP-9 for early diagnosis of a number of metabolic alterations are rapidly increasing. In this panorama, biosensors could play a key role; therefore we explored for the first time the development of an aptamer-based piezoelectric biosensor for a sensitive, label free, and real time detection of MMP-9. The detecting strategy involved two different aptamers in a sandwich-like approach able to detect down to 100 pg mL(-1) (1.2 pM) of MMP-9 as detection limit in standard solution. As proof of principle, commercial serum was investigated in terms of possible interferents, their identification and role in MMP-9 detection. The estimated detection limit for MMP-9 is about 560 pg mL(-1) (6.8 pM) in untreated serum.

Lysine quantification in cheese by a novel, highly selective amperometric biosensor is reported. Based on l-lysine-α-oxidase immobilized by co-crosslinking onto Platinum (Pt) electrodes modified by overoxidized polypyrrole, the sensor proved almost specific to lysine, sensitive and stable. The pure enzymatic nature of current signals was confirmed by a control electrode modified without enzyme. The precision of the method showed relative standard deviations of 4.7% and 9.2% respectively for Parmigiano Reggiano and Grana Padano cheese (n=5). The recovery data on various cheese, spiked with lysine at 50-100% of the measured content, ranged from 85% to 99%. Different types of cheese were analysed showing lysine concentrations related to the ripening time and the manufacture technology, in agreement with literature data. Within dairy products, no appreciable lysine was detected in yogurts. The method adopted revealed suited to satisfy the demands for precise and sensitive detection of lysine with minimal sample preparation and clean-up.