DJ-1 Proteoforms in Breast Cancer Cells: The Escape of Metabolic Epigenetic Misregulation.

Enhanced glycolysis is a hallmark of breast cancer. In cancer cells, the high glycolytic flux induces carbonyl stress, a damaging condition in which the increase of reactive carbonyl species makes DNA, proteins, and lipids more susceptible to glycation. Together with glucose, methylglyoxal (MGO), a byproduct of glycolysis, is considered the main glycating agent. MGO is highly diffusible, enters the nucleus, and can react with easily accessible lysine- and arginine-rich tails of histones. Glycation adducts on histones undergo oxidization and further rearrange to form stable species known as advanced glycation end-products (AGEs). This modification alters nucleosomes stability and chromatin architecture deconstructing the histone code. Formation of AGEs has been associated with cancer, diabetes, and several age-related diseases. Recently, DJ-1, a cancer-associated protein that protects cells from oxidative stress, has been described as a deglycase enzyme. Although its role in cell survival results still controversial, in several human tumors, its expression, localization, oxidation, and phosphorylation were found altered. This work aimed to explore the molecular mechanism that triggers the peculiar cellular compartmentalization and the specific post-translational modifications (PTM) that, occurring in breast cancer cells, influences the DJ-1 dual role. Using a proteomic approach, we identified on DJ-1 a novel threonine phosphorylation (T125) that was found, by the in-silico tool scansite 4, as part of a putative Akt consensus. Notably, this threonine is in addition to histidine 126, a key residue involved in the formation of catalytic triade (glu18-Cys106-His126) inside the glioxalase active site of DJ. Interestingly, we found that pharmacological modulation of Akt pathway induces a functional tuning of DJ-1 proteoforms, as well as their shuttle from cytosol to nucleus, pointing out that pathway as critical in the development of DJ-1 pro-tumorigenic abilities. Deglycase activity of DJ-1 on histones proteins, investigated by coupling 2D tau gel with LC-MS/MS and 2D-TAU (Triton-Acid-Urea)-Western blot, was found correlated with its phosphorylation status that, in turn, depends from Akt activation. In normal conditions, DJ-1 acts as a redox-sensitive chaperone and as an oxidative stress sensor. In cancer cells, glycolytic rewiring, inducing increased reactive oxygen species (ROS) levels, enhances AGEs products. Alongside, the moderate increase of ROS enhances Akt signaling that induces DJ-1-phosphorylation. When phosphorylated DJ-1 increases its glyoxalase activity, the level of AGEs on histones decreases. Therefore, phospho-DJ-1 prevents glycation-induced histones misregulation and its Akt-related hyperactivity represents a way to preserve the epigenome landscape sustaining proliferation of cancer cells. Together, these results shed light on an interesting mechanism that cancer cells might execute to escape the metabolic induced epigenetic misregulation that otherwise could impair their malignant proliferative potential.

Histone proteomics reveals novel post-translational modifications in breast cancer.

Histones and their variants are subjected to several post-translational modifications (PTMs). Histones PTMs play an important role in the regulation of gene expression and are critical for the development and progression of many types of cancer, including breast cancer. In this study, we used two-dimensional TAU/SDS electrophoresis, coupled with mass spectrometry for a comprehensive profiling of histone PTMs in breast cancer cell lines.Proteomic approach allowed us to identify 85 histone PTMs, seventeen of which are not reported in the UniProt database. Western blot analysis was performed to confirm a peculiar pattern of PTMs in the sporadic and hereditary breast cancer cell lines compared to normal cells. Overlapping mass spectrometry data with western blotting results, we identified, for the first time to our knowledge, a tyrosine phosphorylation on histone H1, which is significantly higher in breast cancer cells. Additionally, by inhibiting specific signaling paths, such as PI3K, PPARγ and FAK pathways, we established a correlation between their regulation and the presence of new histone PTMs. Our results may provide new insight on the possible implication of these modifications in breast cancer and may offer new perspectives for future clinical applications.

In Preclinical Model of Ovarian Cancer, the SGK1 Inhibitor SI113 Counteracts the Development of Paclitaxel Resistance and Restores Drug Sensitivity.

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)-dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.

Integration of "Omics" Strategies for Biomarkers Discovery and for the Elucidation of Molecular Mechanisms Underlying Brugada Syndrome.

PURPOSE: The Brugada syndrome (BrS) is a severe inherited cardiac disorder. Given the high genetic and phenotypic heterogeneity of this disease, three different "omics" approaches are integrated in a synergic way to elucidate the molecular mechanisms underlying the pathophysiology of BrS as well as for identifying reliable diagnostic/prognostic markers. EXPERIMENTAL DESIGN: The profiling of plasma Proteome and MiRNome is perfomed in a cohort of Brugada patients that were preliminary subjected to genomic analysis to assess a peculiar gene mutation profile. RESULTS: The integrated analysis of "omics" data unveiled a cooperative activity of mutated genes, deregulated miRNAs and proteins in orchestrating transcriptional and post-translational events that are critical determining factors for the development of the Brugada pattern. CONCLUSIONS AND CLINICAL RELEVANCE: This study provides the basis to shed light on the specific molecular fingerprints underlying BrS development and to gain further insights on the pathogenesis of this life-threatening cardiac disease.

Proteomics Analysis to Assess the Role of Mitochondria in BRCA1-Mediated Breast Tumorigenesis.

Mitochondria are the organelles deputed to energy production, but they are also involved in carcinogenesis, cancer progression, and metastasis, playing a role in altered energy metabolism in cancer cells. Mitochondrial metabolism is connected with several mitochondrial pathways such as ROS signaling, Ca2+ homeostasis, mitophagy, and mitochondrial biogenesis. These pathways are merged in an interactive super-network that seems to play a crucial role in cancer. Germline mutations of the BRCA1 gene account for 5-10% of breast cancers and confer a risk of developing the disease 10- to 20-fold much higher than in non-carriers. By considering metabolic networks that could reconcile both genetic and non-genetic causal mechanisms in BRCA1 driven tumorigenesis, we herein based our study on the hypothesis that BRCA1 haploinsufficiency might drive metabolic rewiring in breast epithelial cells, acting as a push toward malignant transformation. Using 2D-DIGE we analyzed and compared the mitochondrial proteomic profile of sporadic breast cancer cell line (MCF7) and BRCA1 mutated breast cancer cell line (HCC1937). Image analysis was carried out with Decider Software, and proteins differentially expressed were identified by LC-MS/MS on a quadrupole-orbitrap mass spectrometer Q-Exactive. Ingenuity pathways analysis software was used to analyze the fifty-three mitochondrial proteins whose expression resulted significantly altered in response to BRCA1 mutation status. Mitochondrial Dysfunction and oxidative phosphorylation, and energy production and nucleic acid metabolism were, respectively, the canonical pathway and the molecular function mainly affected. Western blotting analysis was done to validate the expression and the peculiar mitochondrial compartmentalization of specific proteins such us HSP60 and HIF-1α. Particularly intriguing is the correlation between BRCA1 mutation status and HIF-1α localization into the mitochondria in a BRCA1 dependent manner. Data obtained led us to hypothesize an interesting connection between BRCA1 and mitochondria pathways, capable to trigger metabolic changes, which, in turn, sustain the high energetic and anabolic requirements of the malignant phenotype.

Unraveling the Mechanistic Complexity of the Glomerulocystic Phenotype in Dicer Conditional KO Mice by 2D Gel Electrophoresis Coupled Mass Spectrometry.

PURPOSE: Dicer, an RNase III type endonuclease, is a key enzyme involved in miRNA biogenesis. It has been shown that this enzyme is essential for several aspects of postnatal kidney functions and homeostasis. In this study, we have examined conditional knockout (cKO) mice for Dicer in Pax8 (Paired-box gene 8) expressing cells to investigate the kidney protein profile. This specific model develops a glomerulocystic phenotype coupled with urinary concentration impairment, proteinuria, and severe renal failure. EXPERIMENTAL DESIGN: Proteomic analysis was performed on kidney tissue extracts from cKO and control (Ctr) mice by 2D Gel Electrophoresis coupled with mass spectrometry. RESULTS: The analysis highlighted 120 protein spots differentially expressed in Dicer cKO tissue compared with control; some of these proteins were validated by Western blotting. Ingenuity Pathway Analysis led to the identification of some interesting networks; among them, the one having ERK as a central hub may explain, through the modulation of the expression of a number of identified protein targets, the metabolic and structural alterations occurring during kidney cyst development in Dicer cKO mouse model. CONCLUSIONS AND CLINICAL RELEVANCE: Our results contribute to gain new insights into molecular mechanisms through which Dicer endonuclease controls kidney development and physiological functions.

Preclinical model in HCC: the SGK1 kinase inhibitor SI113 blocks tumor progression in vitro and in vivo and synergizes with radiotherapy.

The SGK1 kinase is pivotal in signal transduction pathways operating in cell transformation and tumor progression. Here, we characterize in depth a novel potent and selective pyrazolo[3,4-d]pyrimidine-based SGK1 inhibitor. This compound, named SI113, active in vitro in the sub-micromolar range, inhibits SGK1-dependent signaling in cell lines in a dose- and time-dependent manner. We recently showed that SI113 slows down tumor growth and induces cell death in colon carcinoma cells, when used in monotherapy or in combination with paclitaxel. We now demonstrate for the first time that SI113 inhibits tumour growth in hepatocarcinoma models in vitro and in vivo. SI113-dependent tumor inhibition is dose- and time-dependent. In vitro and in vivo SI113-dependent SGK1 inhibition determined a dramatic increase in apoptosis/necrosis, inhibited cell proliferation and altered the cell cycle profile of treated cells. Proteome-wide biochemical studies confirmed that SI113 down-regulates the abundance of proteins downstream of SGK1 with established roles in neoplastic transformation, e.g. MDM2, NDRG1 and RAN network members. Consistent with knock-down and over-expressing cellular models for SGK1, SI113 potentiated and synergized with radiotherapy in tumor killing. No short-term toxicity was observed in treated animals during in vivo SI113 administration. These data show that direct SGK1 inhibition can be effective in hepatic cancer therapy, either alone or in combination with radiotherapy.

Plasma Proteomic Profiling in Hereditary Breast Cancer Reveals a BRCA1-Specific Signature: Diagnostic and Functional Implications.

BACKGROUND: Breast cancer (BC) is a leading cause of death among women. Among the major risk factors, an important role is played by familial history of BC. Germ-line mutations in BRCA1/2 genes account for most of the hereditary breast and/or ovarian cancers. Gene expression profiling studies have disclosed specific molecular signatures for BRCA1/2-related breast tumors as compared to sporadic cases, which might help diagnosis and clinical follow-up. Even though, a clear hallmark of BRCA1/2-positive BC is still lacking. Many diseases are correlated with quantitative changes of proteins in body fluids. Plasma potentially carries important information whose knowledge could help to improve early disease detection, prognosis, and response to therapeutic treatments. The aim of this study was to develop a comprehensive approach finalized to improve the recovery of specific biomarkers from plasma samples of subjects affected by hereditary BC. METHODS: To perform this analysis, we used samples from patients belonging to highly homogeneous population previously reported. Depletion of high abundant plasma proteins, 2D gel analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analysis were used into an integrated approach to investigate tumor-specific changes in the plasma proteome of BC patients and healthy family members sharing the same BRCA1 gene founder mutation (5083del19), previously reported by our group, with the aim to identify specific signatures. RESULTS: The comparative analysis of the experimental results led to the identification of gelsolin as the most promising biomarker. CONCLUSIONS: Further analyses, performed using a panel of breast cancer cell lines, allowed us to further elucidate the signaling network that might modulate the expression of gelsolin in breast cancer.

Identification of prognosis-related proteins in gingival squamous cell carcinoma by twodimensional gel electrophoresis and mass spectrometry-based proteomics.

OBJECTIVES: The aim of this study was to identify differentially expressed proteins in oral squamous carcinoma cells that could be potential prognosis-related cancer biomarkers. MATERIALS AND METHODS: We compared protein expression patterns from gingival squamous cellc carcinoma (GSCC) tissues and adjacent non-cancerous matched tissues by proteomic analysis using two-dimensional gel electrophoresis coupled to mass spectrometry (2D-PAGE/MS). RESULTS: Seventeen protein spots were found to be over-expressed and eight were under-expressed in cancerous tissue compared to the normal counterpart. Of these, annexin A2 and ezrin were validated by Western blot. We also demonstrated by immunohistochemistry that POSTN is highly expressed in the neoplastic tissues examined. Among the differentially expressed proteins, we focused our attention on Chloride intracellular channel 1 (CLIC1). CONCLUSION: The 2D-PAGE/MS-based proteomics appears an efficient approach in detecting and identifying differentially expressed proteins that might function as potential biomarkers and/or molecular targets for early cancer diagnosis and prognosis and that might contribute to a innovative therapeutic strategies in GSCC. However, further validation and functional studies are needed to confirm and to support these promising, still preliminary data. KEY WORDS: Cancer biomarkers, Oral squamous cell carcinoma, Proteomics.