The CAIX inhibitor SLC-0111 exerts anti-cancer activity on gastric cancer cell lines and resensitizes resistant cells to 5-Fluorouracil, taxane-derived, and platinum-based drugs.

Gastric cancer (GC) is the fifth most frequent malignancy and the fourth leading cause of worldwide cancer-related death. Despite the usage of multimodal perioperative chemotherapy (pCT), GC progressively gains chemoresistance, thereby, the identification of suitable targets to overcome drug resistance is fundamental. Amongst the potential biomarkers, carbonic anhydrase IX (CAIX) - associated with a poor prognosis of several solid cancers - has gained the most attention. In a cohort of GC patients who received perioperative FLOT (i.e., Leucovorin, 5-Fluouracil, Docetaxel, and Oxaliplatin) or FOLFOX (i.e., Leucovorin, 5-Fluouracil, and Oxaliplatin), non-responder patients showed an increased expression of tumor CAIX compared to responder group. Moreover, GC cell lines induced to be resistant to 5-Fluouracil, Paclitaxel, Cisplatin, or the combination of 5-Fluorouracil, Oxaliplatin, and Docetaxel, overexpressed CAIX compared to the control. Accordingly, CAIX-high-expressing GC cells showed increased therapy resistance compared to low-expressing cells. Notably, SLC0111 significantly improved the therapy response of both wild-type and resistant GC cells. Overall, these data suggest a correlation between CAIX and GC drug resistance highlighting the potential of SLC-0111 in re-sensitizing GC cells to pCT.

Moving beyond the Tip of the Iceberg: DJ-1 Implications in Cancer Metabolism.

DJ-1, also called Parkinson's protein 7 (PARK7), is ubiquitously expressed and plays multiple actions in different physiological and, especially, pathophysiological processes, as evidenced by its identification in neurodegenerative diseases and its high expression in different types of cancer. To date, the exact activity of DJ-1 in carcinogenesis has not been fully elucidated, however several recent studies disclosed its involvement in regulating fundamental pathways involved in cancer onset, development, and metastatization. At this purpose, we have dissected the role of DJ-1 in maintaining the transformed phenotype, survival, drug resistance, metastasis formation, and differentiation in cancer cells. Moreover, we have discussed the role of DJ-1 in controlling the redox status in cancer cells, along with the ability to attenuate reactive oxygen species (ROS)-dependent cell death, as well as to mediate ferropotosis. Finally, a mention to the development of therapeutic strategies targeting DJ-1 has been done. We have reported the most recent studies, aiming to shed light on the role played by DJ-1 in different cancer aspects and create the foundation for moving beyond the tip of the iceberg.

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.

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.