To metabolomics and beyond: a technological portfolio to investigate cancer metabolism.

Tumour cells have exquisite flexibility in reprogramming their metabolism in order to support tumour initiation, progression, metastasis and resistance to therapies. These reprogrammed activities include a complete rewiring of the bioenergetic, biosynthetic and redox status to sustain the increased energetic demand of the cells. Over the last decades, the cancer metabolism field has seen an explosion of new biochemical technologies giving more tools than ever before to navigate this complexity.

3-Bromo-Isoxazoline Derivatives Inhibit GAPDH Enzyme in PDAC Cells Triggering Autophagy and Apoptotic Cell Death.

A growing interest in the study of aerobic glycolysis as a key pathway for cancer-cell energetic metabolism, favouring tumour progression and invasion, has led to consider GAPDH as an effective drug target to specifically hit cancer cells. In this study, we have investigated a panel of 3-bromo-isoxazoline derivatives based on previously identified inhibitors of GAPDH (GAPDH). The compounds are active, to a different extent, as inhibitors of human-recombinant GAPDH.

Tumor Suppressor Role of Wild-Type P53-Dependent Secretome and Its Proteomic Identification in PDAC.

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space.

Redox Sensitive Cysteine Residues as Crucial Regulators of Wild-Type and Mutant p53 Isoforms.

The wild-type protein p53 plays a key role in preventing the formation of neoplasms by controlling cell growth. However, in more than a half of all cancers, the gene has missense mutations that appear during tumorigenesis. In most cases, the mutated gene encodes a full-length protein with the substitution of a single amino acid, resulting in structural and functional changes and acquiring an oncogenic role.

Mutant p53-Associated Molecular Mechanisms of ROS Regulation in Cancer Cells.

The tumor suppressor gene is the most frequently altered gene in tumors and an increasing number of studies highlight that mutant p53 proteins can acquire oncogenic properties, referred to as gain-of-function (GOF). Reactive oxygen species (ROS) play critical roles as intracellular messengers, regulating numerous signaling pathways linked to metabolism and cell growth. Tumor cells frequently display higher ROS levels compared to healthy cells as a result of their increased metabolism as well as serving as an oncogenic agent because of its damaging and mutational properties.

Mutant p53 induces SIRT3/MnSOD axis to moderate ROS production in melanoma cells.

The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 isoforms can acquire oncogenic properties referred to as gain-of-function (GOF). In this study, we used wild-type (A375) and mutant p53 (MeWo) melanoma cell lines to assess the regulation of the mitochondrial antioxidant manganese superoxide dismutase (MnSOD) by mutant p53. The effects of mutant p53 were evaluated by qPCR, immunoblotting, enzyme activity assay, cell proliferation assay, reactive oxygen species (ROS) assay after cellular transfection.

Regulation of succinate dehydrogenase and role of succinate in cancer.

Succinate dehydrogenase (SDH) has been classically considered a mitochondrial enzyme with the unique property to participate in both the citric acid cycle and the electron transport chain. However, in recent years, several studies have highlighted the role of the SDH substrate, i.e.

Regulation of Autophagy by Nuclear GAPDH and Its Aggregates in Cancer and Neurodegenerative Disorders.

Several studies indicate that the cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has pleiotropic functions independent of its canonical role in glycolysis. The GAPDH functional diversity is mainly due to post-translational modifications in different amino acid residues or due to protein-protein interactions altering its localization from cytosol to nucleus, mitochondria or extracellular microenvironment. Non-glycolytic functions of GAPDH include the regulation of cell death, autophagy, DNA repair and RNA export, and they are observed in physiological and pathological conditions as cancer and neurodegenerative disorders.

Oncometabolites in cancer aggressiveness and tumour repopulation.

Tumour repopulation is recognized as a crucial event in tumour relapse where therapy-sensitive dying cancer cells influence the tumour microenvironment to sustain therapy-resistant cancer cell growth. Recent studies highlight the role of the oncometabolites succinate, fumarate, and 2-hydroxyglutarate in the aggressiveness of cancer cells and in the worsening of the patient's clinical outcome. These oncometabolites can be produced and secreted by cancer and/or surrounding cells, modifying the tumour microenvironment and sustaining an invasive neoplastic phenotype.

Mutant p53 blocks SESN1/AMPK/PGC-1α/UCP2 axis increasing mitochondrial O· production in cancer cells.

Background: The TP53 tumor suppressor gene is the most frequently altered gene in tumors and mutant p53 gain-of-function isoforms actively promote cancer malignancy.

Methods: A panel of wild-type and mutant p53 cancer cell lines of different tissues, including pancreas, breast, skin, and lung were used, as well as chronic lymphocytic leukemia (CLL) patients with different TP53 gene status. The effects of mutant p53 were evaluated by confocal microscopy, reactive oxygen species production assay, immunoblotting, and quantitative reverse transcription polymerase chain reaction after cellular transfection.

Mutant p53 prevents GAPDH nuclear translocation in pancreatic cancer cells favoring glycolysis and 2-deoxyglucose sensitivity.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and devastating human malignancies. In about 70% of PDACs the tumor suppressor gene TP53 is mutated generally resulting in conformational changes of mutant p53 (mutp53) proteins, which acquire oncogenic functions triggering aggressiveness of cancers and alteration of energetic metabolism. Here, we demonstrate that mutant p53 prevents the nuclear translocation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) stabilizing its cytoplasmic localization, thus supporting glycolysis of cancer cells and inhibiting cell death mechanisms mediated by nuclear GAPDH.

Trichostatin A alters cytoskeleton and energy metabolism of pancreatic adenocarcinoma cells: An in depth proteomic study.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal of all human cancers with a high mortality rate. Resistance to conventional treatments and chemotherapeutics is a typical feature of PDAC. To investigate the causes of drug resistance it is essential to deeply investigate the mechanism of action of chemotherapeutics.

UCP2 inhibition induces ROS/Akt/mTOR axis: Role of GAPDH nuclear translocation in genipin/everolimus anticancer synergism.

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus.

Autocrine mechanisms of cancer chemoresistance.

An ever-increasing number of studies highlight the role of cancer secretome in the modification of tumour microenvironment and in the acquisition of cancer cell resistance to therapeutic drugs. The knowledge of the mechanisms underlying the relationship between cancer cell-secreted factors and chemoresistance is becoming fundamental for the identification of novel anticancer therapeutic strategies overcoming drug resistance and novel prognostic secreted biomarkers. In this review, we summarize the novel findings concerning the regulation of secreted molecules by cancer cells compromising drug sensitivity.

The antioxidant uncoupling protein 2 stimulates hnRNPA2/B1, GLUT1 and PKM2 expression and sensitizes pancreas cancer cells to glycolysis inhibition.

Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose.

Molecular interplay between mutant p53 proteins and autophagy in cancer cells.

An increasing number of studies highlight the role of mutant p53 proteins in cancer cell growth and in the worsening of cancer patients' clinical outcome. Autophagy has been widely recognized as a main biological event involved in both the regulation of cancer cell proliferation and in the response of several anticancer drugs. A thorough analysis of scientific literature underlines the reciprocal interplay between mutant p53 proteins and autophagy regulation.

Mutant p53 proteins alter cancer cell secretome and tumour microenvironment: Involvement in cancer invasion and metastasis.

An ever-increasing number of studies highlight the role of mutant p53 proteins in the alteration of cancer cell secretome and in the modification of tumour microenvironment, sustaining an invasive phenotype of cancer cell. The knowledge of the molecular mechanisms underlying the interplay between mutant p53 proteins and the microenvironment is becoming fundamental for the identification of both efficient anticancer therapeutic strategies and novel serum biomarkers. In this review, we summarize the novel findings concerning the regulation of secreted molecules by cancer cells bearing mutant TP53 gene.

Combining immunofluorescence with in situ proximity ligation assay: a novel imaging approach to monitor protein-protein interactions in relation to subcellular localization.

The in situ Proximity Ligation Assay (PLA) is suited for visualizing protein-protein interactions and post-translational protein modifications in both tissue sections and in vitro cell cultures. Accurate identification and quantification of protein-protein interactions are critical for in vitro cell analysis, especially when studying the dynamic involvement of proteins in various processes, including cell proliferation, differentiation, and apoptosis. Here, we monitored the interactions between protein kinase-Cζ (PKCζ) and Bcl10 protein in untreated and etoposide (VP-16)-treated C4-I cells by means of a new combined morphological approach and validated it by taking stock of our previous proteomic and biochemical work (Chiarini et al.

The Aβ peptides-activated calcium-sensing receptor stimulates the production and secretion of vascular endothelial growth factor-A by normoxic adult human cortical astrocytes.

The excess vascular endothelial growth factor (VEGF) produced in the Alzheimer's disease (AD) brain can harm neurons, blood vessels, and other components of the neurovascular units (NVUs). But could astrocytes partaking in networks of astrocyte-neuron teams and connected to blood vessels of NVUs contribute to VEGF production? We have shown with cultured cerebral cortical normal (i.e.

Do astrocytes collaborate with neurons in spreading the "infectious" aβ and Tau drivers of Alzheimer's disease?

Evidence has begun emerging for the "contagious" and destructive Aβ42 (amyloid-beta42) oligomers and phosphorylated Tau oligomers as drivers of sporadic Alzheimer's disease (AD), which advances along a pathway starting from the brainstem or entorhinal cortex and leading to cognition-related upper cerebral cortex regions. Seemingly, Aβ42 oligomers trigger the events generating the neurotoxic Tau oligomers, which may even by themselves spread the characteristic AD neuropathology. It has been assumed that only neurons make and spread these toxic drivers, whereas their associated astrocytes are just janitorial bystanders/scavengers.

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25-35 in human cortical astrocytes and neurons-therapeutic relevance to Alzheimer's disease.

The "amyloid-β (Aβ) hypothesis" posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25-35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes.

Alzheimer's disease: an update of the roles of receptors, astrocytes and primary cilia (review).

The pathophysiological mechanisms underlying the onset and inexorable progression of the late‑onset form of Alzheimer's disease (AD) are still the object of controversy. This review takes stock of some most recent advancements of this field concerning the complex roles played by the amyloid‑β (Aβ)‑binding p75 neurotrophin receptor (p75NTR) and calcium‑sensing receptor (CaSR) and by the primary cilia in AD. Apart from their physiological roles, p75NTR is more intensely expressed in the hippocampus of human AD brains and Aβ‑bound p75NTR triggers cell death, whereas Aβ‑bound CaSR signalling induces the de novo synthesis and release of nitric oxide (NO), vascular endothelial growth factor (VEGF)‑A and Aβ peptides (Aβs), particularly on the part of normal adult human astrocytes.

The calcium-sensing receptor: a novel Alzheimer's disease crucial target?

Alzheimer's disease (AD) is the most common human neurodegenerative ailment, the most prevalent (>95%) late-onset type of which has a still uncertain etiology. The progressive decline of cognitive functions, dementia, and physical disabilities of AD is caused by synaptic losses that progressively disconnect key neuronal networks in crucial brain areas, like the hippocampus and temporoparietal cortex, and critically impair language, sensory processing, memory, and conscious thought. AD's two main hallmarks are fibrillar amyloid-β (fAβ) plaques in extracellular spaces and intracellular accumulation of fAβ peptides and neurofibrillary tangles (NFTs).