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Tubulointerstitial fibrosis is observed in diabetic nephropathy. It is still debated whether tubular cells, undergoing epithelial-mesenchymal transition (EMT) in high glucose (HG) conditions, may contribute to interstitial fibrosis development. In this study, we investigated the phenotypic and molecular EMT-like changes and the alteration of inflammatory and fibrogenic secretome induced by HG in human primary tubular cell cultures. Taking advantage of this in vitro cell model composed of proximal and distal tubular cells, we showed that HG-treated tubular cells acquired a fibroblast-like morphology with increased cytoplasmic stress fibers, maintaining the expression of the epithelial markers specific of proximal and distal tubular cells. HG increased Snail1, miRNA210 and Vimentin mesenchymal markers, decreased N-cadherin expression and migration ability of primary tubular cells, while E-cadherin expression and focal adhesion distribution were not affected. Furthermore, HG treatment of tubular cells altered the inflammatory cytokine secretion creating a secretome able to enhance the proliferation and migration of fibroblasts. Our findings show that HG promotes an activated state of partial EMT in human tubular primary cells and induces a pro-inflammatory and pro-fibrogenic microenvironment, supporting the active role of tubular cells in diabetic nephropathy onset.

Simple Summary Acute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. In recent years, broad application of NGS technologies enabled the discovery of novel genomically defined ALL. In this study, as a proof-of-principle, we applied RNA-seq technology to comprehensively profile the transcriptional landscape of a collection of 10 childhood BCP-ALL cases, and performed a deep bioinformatics analysis including several publicly available datasets, in order to characterize their full spectrum of transcriptional events. The paired-end RNA sequencing of our BCP-ALL pediatric cohort revealed a total of 9001 raw fusion events, which, after filtering, resulted in 245 candidate fusions. Overall, 235 out of 245 events were intra-chromosomal fusions, among which 229 involved two contiguous or overlapping genes, also known as conjoined genes (CGs). Among them, we identified a subset of 14 CGs (6.1%) exclusively expressed in leukemic cases but neither in solid cancers nor in normal samples. These events could be suggestive of a novel mechanism of transcriptional regulation in childhood leukemia and may represent novel potential leukemia-specific biomarkers. Acute lymphoblastic leukemia (ALL) is the most frequent childhood cancer. For the last three decades, conventional cytogenetic and molecular approaches allowed the identification of genetic abnormalities having prognostic and therapeutic relevance. Although the current cure rate in pediatric B cell acute leukemia is approximately 90%, it remains one of the leading causes of mortality in childhood. Furthermore, in the contemporary protocols, chemotherapy intensity was raised to the maximal levels of tolerability, and further improvements in the outcome will depend on the characterization and reclassification of the disease, as well as on the development of new targeted drugs. The recent technological advances in genome-wide profiling techniques have allowed the exploration of the molecular heterogeneity of this

Background: Glioblastoma multiforme (GBM) is a heterogeneous CNS neoplasm which causes significant morbidity and mortality. One reason for the poor prognostic outcome of GBM is attributed to the presence of cancer stem cells (CSC) which confer resistance against standard chemo- and radiotherapeutics modalities. Two types of GBM-associated CSC were isolated from the same patient: tumor core- (c-CSC) and peritumor tissue-derived cancer stem cells (p-CSC). Our experiments are focused on glioblastoma–IDH-wild type, and no disease-defining alterations were present in histone, BRAF or other genes. Methods: In the present study, potential differences in genetic variants between c-CSC versus p-CSC derived from four GBM patients were investigated with the aims of (1) comparing the exome sequences between all the c-CSC or p-CSC to identify the common variants; (2) identifying the variants affecting the function of genes known to be involved in cancer origin and development. Results: By comparative analyses, we identified common gene single nucleotide variants (SNV) in all GBM c-CSC and p-CSC, a potentially deleterious variant was a frameshift deletion at Gln461fs in the MLLT1 gene, that was encountered only in p-CSC samples with different allelic frequency. Conclusions: We discovered a potentially harmful frameshift deletion at Gln461fs in the MLLT1 gene. Further investigation is required to confirm the presence of the identified mutations in patient tissue samples, as well as the significance of the frameshift mutation in the MLLT1 gene on GBM biology and response to therapy based on genomic functional experiments.

Background Rewiring of metabolism induced by oncogenicK-Rasin cancer cells involves both glucose and glutamine utilization sustaining enhanced, unrestricted growth. The development of effective anti-cancer treatments targeting metabolism may be facilitated by the identification and rational combinatorial targeting of metabolic pathways. Methods We performed mass spectrometric metabolomics analysis in vitro and in vivo experiments to evaluate the efficacy of drugs and identify metabolic connectivity. Results We show thatK-Ras-mutant lung and colon cancer cells exhibit a distinct metabolic rewiring, the latter being more dependent on respiration. Combined treatment with the glutaminase inhibitor CB-839 and the PI3K/aldolase inhibitor NVP-BKM120 more consistently reduces cell growth of tumor xenografts. Maximal growth inhibition correlates with the disruption of redox homeostasis, involving loss of reduced glutathione regeneration, redox cofactors, and a decreased connectivity among metabolites primarily involved in nucleic acid metabolism. Conclusions Our findings open the way to develop metabolic connectivity profiling as a tool for a selective strategy of combined drug repositioning in precision oncology.

The adipocyte-like morphology of clear cell renal cell carcinoma (ccRCC) cells results from a grade-dependent neutral lipid accumulation; however, the molecular mechanism and role in renal cancer progression have yet to be clarified. ccRCC shows a gene expression signature consistent with adipogenesis, and the phospholipid-binding protein annexin A3 (AnxA3), a negative regulator of adipocyte differentiation, is down-regulated in RCC and shows a differential expression pattern for two isoforms of 36 and 33 kDa. Using primary cell cultures and cell lines, we investigated the involvement of AnxA3 isoforms in lipid storage modulation of ccRCC cells. We found that the increased accumulation of lipids into ccRCC cells correlated with a decrease of the 36/33 isoform ratio. Treatment with adipogenic medium induced a significant increment of lipid storage in ccRCC cells that had a low 36-kDa AnxA3 expression and 36/33 ratio. The 36-kDa AnxA3 silencing in ccRCC cells increased lipid storage induced by adipogenic medium. These data suggest that 36-kDa AnxA3 negatively modulates the response to adipogenic treatment and may act as negative regulator of lipid storage in ccRCC cells. The subcellular distribution of AnxA3 in the cellular endocytic compartment suggests its involvement in modulation of vesicular trafficking, and it might serve as a putative mechanism of lipid storage regulation in ccRCC cells, opening novel translational outcomes.

Clear cell renal cell carcinoma (ccRCC) has a poor prognosis despite novel biological targeted therapies. Tumor aggressiveness and poor survival may correlate with tumor grade at diagnosis and with complex metabolic alterations, also involving glucose and lipid metabolism. However, currently no grade-specific metabolic therapy addresses these alterations. Here we used primary cell cultures from ccRCC of low- and high-grade to investigate the effect on energy state and reduced pyridine nucleotide level, and on viability and proliferation, of specific inhibition of glycolysis with 2-deoxy-D-glucose (2DG), or fatty acid oxidation with Etomoxir. Our primary cultures retained the tissue grade-dependent modulation of lipid and glycogen storage and aerobic glycolysis (Warburg effect). 2DG affected lactate production, energy state and reduced pyridine nucleotide level in high-grade ccRCC cultures, but the energy state only in low-grade. Rather, Etomoxir affected energy state in high-grade and reduced pyridine nucleotide level in low-grade cultures. Energy state and reduced pyridine nucleotide level were evaluated by ATP and reduced 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) dye quantification, respectively. 2DG treatment impaired cell proliferation and viability of low-grade ccRCC and normal cortex cultures, whereas Etomoxir showed a cytostatic and cytotoxic effect only in high-grade ccRCC cultures. Our data indicate that in ccRCC the Warburg effect is a grade-dependent feature, and fatty acid oxidation can be activated for different grade-dependent metabolic needs. A possible grade-dependent metabolic therapeutic approach in ccRCC is also highlighted

Oxidative stress, pulmonary and systemic inflammation, endothelial cell dysfunction, atherosclerosis and cardiac autonomic dysfunction have been linked to urban particulate matter exposure. The chemical composition of airborne pollutants in Milano is similar to those of other European cities though with a higher PM2.5 fraction. Milano winter fine particles (PM2.5win) are characterized by the presence of nitrate, organic carbon fraction, with high amount of polycyclic aromatic hydrocarbons and elements such as Pb, Al, Zn, V, Fe, Cr and others, with a negligible endotoxin presence. In BALB/c mice, we examined, at biochemical and transcriptomic levels, the adverse effects of repeated Milano PM2.5win exposure in lung and heart. We found that ET-1, Hsp70, Cyp1A1, Cyp1B1 and Hsp-70, HO-1, MPO respectively increased within lung and heart of PM2.5win-treated mice. The PM2.5win exposure had a strong impact on global gene expression of heart tissue (181 upregulated and 178 down-regulated genes) but a lesser impact on lung tissue (14 up-regulated genes and 43 downregulated genes). Focusing on modulated genes, in lung we found two- to three-fold changes of those genes related to polycyclic aromatic hydrocarbons exposure and calcium signalling. Within heart the most striking aspect is the twofold to threefold increase in collagen and laminin related genes as well as in genes involved in calcium signaling. The current study extends our previous findings, showing that repeated instillations of PM2.5win trigger systemic adverse effects. PM2.5win thus likely poses an acute threat primarily to susceptible people, such as the elderly and those with unrecognized coronary artery or structural heart disease. The study of genomic responses will improve understanding of disease mechanisms and enable future clinical testing of interventions against the toxic effects of air pollutant

Particulate matter (PM) exposure is related to diverse health effects, ranging from respiratory and cardiovascular diseases, to cancer induction; the determination of the elicited effects can be consequence of PM characteristics. We have previously reported (Longhin et al., 2013) that Milan PM composition is related to the sampling season and the particles size, and these properties are responsible for different in vitro effects. Briefly, PM2.5 is dominated by combustion derived particles, consisting of a carbonaceous core with organic and inorganic compounds adsorbed on its surface. In this fraction, organic components are mostly present in winter season, and as a consequence, winter PM2.5 induces a high level of ROS formation and a DNA damaging potential. PM10 derives from abrasion and resuspension processes, and is enriched in crustal material (metals) and biological components (endotoxins), especially during summer season; thus human cells exposed to summer PM10 have a strong pro-inflammatory response. We have now investigated the global gene and microRNA expression changes by GeneChip technology in human bronchial epithelial cells (BEAS-2B) exposed to single dose of Milan winter PM2.5 or summer PM10 for 24 h PM2.5 and PM10 respectively modulated 162 and 144 genes, with 2 fold values respect to the control. Gene prioritization analysis by Toppgene revealed that PM exposure modulates molecular pathways such as TNF and NFKB signalling. Moreover season of sampling and PM size fraction have a specific impact in the regulation of miRNAs expression associated to inflammation and cancer.

Background Clear cell renal cell carcinoma (ccRCC) is characterized by recurrent copy number alterations (CNAs) and loss of heterozygosity (LOH), which may have potential diagnostic and prognostic applications. Here, we explored whether ccRCC primary cultures, established from surgical tumor specimens, maintain the DNA profile of parental tumor tissues allowing a more confident CNAs and LOH discrimination with respect to the original tissues. Methods We established a collection of 9 phenotypically well-characterized ccRCC primary cell cultures. Using the Affymetrix SNP array technology, we performed the genome-wide copy number (CN) profiling of both cultures and corresponding tumor tissues. Global concordance for each culture/tissue pair was assayed evaluating the correlations between whole-genome CN profiles and SNP allelic calls. CN analysis was performed using the two CNAG v3.0 and Partek software, and comparing results returned by two different algorithms (Hidden Markov Model and Genomic Segmentation). Results A very good overlap between the CNAs of each culture and corresponding tissue was observed. The finding, reinforced by high whole-genome CN correlations and SNP call concordances, provided evidence that each culture was derived from its corresponding tissue and maintained the genomic alterations of parental tumor. In addition, primary culture DNA profile remained stable for at least 3 weeks, till to third passage. These cultures showed a greater cell homogeneity and enrichment in tumor component than original tissues, thus enabling a better discrimination of CNAs and LOH. Especially for hemizygous deletions, primary cultures presented more evident CN losses, typically accompanied by LOH; differently, in original tissues the intensity of these deletions was weaken by normal cell contamination and LOH calls were missed. Conclusions ccRCC primary cultures are a reliable in vitro model, well-reproducing original tumor genetics and phenotype, potentially useful

Primary cell cultures from renal cell carcinoma (RCC) and normal renal cortex tissue of 60 patients have been established, with high efficiency (more than 70%) and reproducibility, and extensively characterized. These cultures composed of more than 90% of normal or tumor tubular cells have been instrumental for molecular characterization of Annexin A3 (AnxA3), never extensively studied before in RCC cells although AnxA3 has a prognostic relevance in some cancer and it has been suggested to be involved in the hypoxia-inducible factor-1 pathway. Western blot analysis of 20 matched cortex/RCC culture lysates showed two AnxA3 protein bands of 36 and 33 kDa, and two-dimensional Western blot evidenced several specific protein spots. In RCC cultures the 36-kDa isoform was significantly down-regulated and the 33-kDa isoform up-regulated. Furthermore, the inversion of the quantitative expression pattern of two AnxA3 isoforms in tumor cultures correlate with hypoxia-inducible factor-1alpha expression. The total AnxA3 protein is down-regulated in RCC cultures as confirmed also in tissues by tissue microarray. Two AnxA3 transcripts that differ for alternative splicing of exon III have been also detected. Real-time PCR quantification in 19 matched cortex/RCC cultures confirms the down-regulation of longer isoform in RCC cells. The characteristic expression pattern of AnxA3 in normal and tumor renal cells, documented in our primary cultures, may open new insight in RCC management

The integration of high-throughput genomic data represents an opportunity for deciphering the interplay between structural and functional organization of genomes and for discovering novel biomarkers. However, the development of integrative approaches to complement gene expression (GE) data with other types of gene information, such as copy number (CN) and chromosomal localization, still represents a computational challenge in the genomic arena. This work presents a computational procedure that directly integrates CN and GE profiles at genome-wide level. When applied to DNA/RNA paired data, this approach leads to the identification of Significant Overlaps of Differentially Expressed and Genomic Imbalanced Regions (SODEGIR). This goal is accomplished in three steps. The first step extends to CN a method for detecting regional imbalances in GE. The second part provides the integration of CN and GE data and identifies chromosomal regions with concordantly altered genomic and transcriptional status in a tumor sample. The last step elevates the single-sample analysis to an entire dataset of tumor specimens. When applied to study chromosomal aberrations in a collection of astrocytoma and renal carcinoma samples, the procedure proved to be effective in identifying discrete chromosomal regions of coordinated CN alterations and changes in transcriptional levels. © 2009 The Author(s).

BACKGROUND: Clear cell renal carcinoma (RCC) is the most common and invasive adult renal cancer. For the purpose of identifying RCC biomarkers, we investigated chromosomal regions and individual genes modulated in RCC pathology. We applied the dual strategy of assessing and integrating genomic and transcriptomic data, today considered the most effective approach for understanding genetic mechanisms of cancer and the most sensitive for identifying cancer-related genes. RESULTS: We performed the first integrated analysis of DNA and RNA profiles of RCC samples using Affymetrix technology. Using 100K SNP mapping arrays, we assembled a genome-wide map of DNA copy number alterations and LOH areas. We thus confirmed the typical genetic signature of RCC but also identified other amplified regions (e.g. on chr. 4, 11, 12), deleted regions (chr. 1, 9, 22) and LOH areas (chr. 1, 2, 9, 13). Simultaneously, using HG-U133 Plus 2.0 arrays, we identified differentially expressed genes (DEGs) in tumor vs. normal samples. Combining genomic and transcriptomic data, we identified 71 DEGs in aberrant chromosomal regions and observed, in amplified regions, a predominance of up-regulated genes (27 of 37 DEGs) and a trend to clustering. Functional annotation of these genes revealed some already implicated in RCC pathology and other cancers, as well as others that may be novel tumor biomarkers. CONCLUSION: By combining genomic and transcriptomic profiles from a collection of RCC samples, we identified specific genomic regions with concordant alterations in DNA and RNA profiles and focused on regions with increased DNA copy number. Since the transcriptional modulation of up-regulated genes in amplified regions may be attributed to the genomic alterations characteristic of RCC, these genes may encode novel RCC biomarkers actively involved in tumor initiation and progression and useful in clinical applications

Caveolin-1 and flotillin-1 belong to plasma membrane microdomains. They are characterized by peculiar lipid and protein composition and are involved in fundamental cellular events such as: signal transduction, cell adhesion, lipid/protein sorting, and human cancer. We addressed caveolin-1 and flotillin-1 expression in 30 human renal cell carcinoma (RCC) and adjacent normal kidney (ANK) samples by SDS-PAGE and immunoblotting with specific antibodies. Significant caveolin-1 and flotillin-1 over-expression was found in RCC tissues compared to ANK, and was confirmed by immunohistochemistry. Caveolin-1 and flotillin-1 protein levels were found by 1-D, 2-DE, and MS to be increased also in RCC microdomain-enriched subcellular fractions purified from paired RCC and ANK samples.

The systematic integration of expression profiles and other types of gene information, such as copy number, chromosomal localization, and sequence characteristics, still represents a challenge in the genomic arena. In particular, the integrative analysis of genomic and transcriptional data in context of the physical location of genes in a genome appears promising in detecting chromosomal regions with structural and transcriptional imbalances often characterizing cancer. A computational framework based on locally adaptive statistical procedures (Global Smoothing Copy Number, GLSCN, and Locally Adaptive Statistical Procedure, LAP), which incorporate genomic and transcriptional data with structural information for the identification of imbalanced chromosomal regions, is described. Both GLSCN and LAP accounts for variations in the distance between genes and in gene density by smoothing standard statistics on gene position before testing the significance of copy number and gene expression signals. The application of GLSCN and LAP to the integrative analysis of a human metastatic clear cell renal carcinoma cell line (Caki-1) allowed identifying chromosomal regions that are directly involved in known chromosomal aberrations characteristic of tumors

The application of genome-wide approaches to the molecular characterization of cancer was investigated, identifying footprints that can potentially assist in the subclassification of tumors in order to contribute to diagnosis and clinical management of patients. High resolution DNA copy number analysis by single nucleotide polymorphism mapping array technology has been widely applied to study copy number aberrations and to distinguish among different loss of heterozigosity mechanisms associated with or without copy number changes in tumors. However, assessment of statistically significant common aberrations across the whole data set or a subset of tumor samples is still an open problem. Therefore, we adapted the recently developed STAC algorithm, previously applied to comparative genomic hybridization data, to identify common copy number aberrations in renal carcinoma samples using Affymetrix 100K SNP arrays. SNP copy number data were processed by a homebrew pipeline implemented in R and analyzed using STAC.