Despite clear evidence that exosomal microRNAs (miRNAs) are able to modulate the cellular microenvironment and that exosomal RNA cargo selection is deregulated in pathological conditions, the ...mechanisms controlling specific RNA sorting into extracellular vesicles are still poorly understood. Here, we identified the RNA binding protein SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein; also known as hnRNP-Q or NSAP1) as a component of the hepatocyte exosomal miRNA sorting machinery. SYNCRIP knockdown impairs sorting of miRNAs in exosomes. Furthermore, SYNCRIP directly binds to specific miRNAs enriched in exosomes sharing a common extra-seed sequence (hEXO motif). The hEXO motif has a role in the regulation of miRNA localization, since embedment of this motif into a poorly exported miRNA enhances its loading into exosomes. This evidence provides insights into the mechanisms of miRNA exosomal sorting process. Moreover, these findings open the way for the possible selective modification of the miRNAs exosomal cargo.
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•SYNCRIP interacts specifically with selected miRNAs•SYNCRIP has a functional role in exosomal sorting of specific miRNAs•A hEXO miRNA extra-seed motif determines exosomal loading
Santangelo et al. shed light on the mechanism that allows a cell to load exosomes with a specific repertoire of miRNAs, showing a functional role of a specific miRNA motif and a protein that specifically interacts with it, the RNA-binding protein SYNCRIP.
Melanoma aggressiveness determines its growth and metastatic potential. This study aimed at identifying new molecular pathways controlling melanoma cell malignancy.
Ten metastatic melanoma cell lines ...were characterized by their proliferation, migration and invasion capabilities. The most representative cells were also characterized by spheroid formation assay, gene- and protein- expression profiling as well as cytokines secretion and the most relevant pathways identified through bioinformatic analysis were tested by in silico transcriptomic validation on datasets generated from biopsies specimens of melanoma patients. Further, matrix metalloproteases (MMPs) activity was tested by zymography assays and TNF-alpha role was validated by anti-TNF cell-treatment.
An aggressiveness score (here named Melanoma AGgressiveness Score: MAGS) was calculated by measuring proliferation, migration, invasion and cell-doubling time in10human melanoma cell lines which were clustered in two distinct groups, according to the corresponding MAGS. SK-MEL-28 and A375 cell lines were selected as representative models for the less and the most aggressive phenotype, respectively. Gene-expression and protein expression data were collected for SK-MEL-28 and A375 cells by Illumina-, multiplex x-MAP-and mass-spectrometry technology. The collected data were subjected to an integrated Ingenuity Pathway Analysis, which highlighted that cytokine/chemokine secretion, as well as Cell-To-Cell Signaling and Interaction functions as well as matrix metalloproteases activity were significantly different in these two cell types. The key role of these pathways was then confirmed by functional validation. TNF role was confirmed by exposing cells to the anti-TNF Infliximab antibody. Upon such treatment melanoma cells aggressiveness was strongly reduced. Metalloproteases activity was assayed, and their role was confirmed by comparing transcriptomic data from cutaneous melanoma patients (n = 45) and benign nevi (n = 18).
Inflammatory signals such as TNF and MMP-2 activity are key intrinsic players to determine melanoma cells aggressiveness suggesting new venue sin the identification of novel molecular targets with potential therapeutic relevance.
Abstract
Background
Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In ...breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L.
Methods
We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes.
Results
Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine–protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells.
Conclusions
Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.
The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, ...transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERβ) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease.
Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERβ in gene regulation, we identify AGO2 as a novel partner of ERβ in human BC cells. ERβ-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERβ binding sites, and total and nascent RNA-Seq in ERβ + vs ERβ - cells, and before and after AGO2 knock-down in ERβ + cells, reveals a widespread involvement of this factor in ERβ-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERβ-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability.
These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERβ, indicating that both factors are endowed with multiple roles in the control of key cellular functions.
Hyperactivation of the phosphatydil-inositol-3' phosphate kinase (PI3K)/AKT pathway is observed in most NSCLCs, promoting proliferation, migration, invasion and resistance to therapy. AKT can be ...activated through several mechanisms that include loss of the negative regulator PTEN, activating mutations of the catalytic subunit of PI3K (PIK3CA) and/or mutations of AKT1 itself. However, number and identity of downstream targets of activated PI3K/AKT pathway are poorly defined. To identify the genes that are targets of constitutive PI3K/AKT signalling in lung cancer cells, we performed a comparative transcriptomic analysis of human lung epithelial cells (BEAS-2B) expressing active mutant AKT1 (AKT1-E17K), active mutant PIK3CA (PIK3CA-E545K) or that are silenced for PTEN. We found that, altogether, aberrant PI3K/AKT signalling in lung epithelial cells regulated the expression of 1,960/20,436 genes (9%), though only 30 differentially expressed genes (DEGs) (15 up-regulated, 12 down-regulated and 3 discordant) out of 20,436 that were common among BEAS-AKT1-E17K, BEAS-PIK3CA-E545K and BEAS-shPTEN cells (0.1%). Conversely, DEGs specific for mutant AKT1 were 133 (85 up-regulated; 48 down-regulated), DEGs specific for mutant PIK3CA were 502 (280 up-regulated; 222 down-regulated) and DEGs specific for PTEN loss were 1549 (799 up-regulated, 750 down-regulated). The results obtained from array analysis were confirmed by quantitative RT-PCR on selected up- and down-regulated genes (n = 10). Treatment of BEAS-C cells and the corresponding derivatives with pharmacological inhibitors of AKT (MK2206) or PI3K (LY294002) further validated the significance of our findings. Moreover, mRNA expression of selected DEGs (SGK1, IGFBP3, PEG10, GDF15, PTGES, S100P, respectively) correlated with the activation status of the PI3K/AKT pathway assessed by S473 phosphorylation in NSCLC cell lines (n = 6). Finally, we made use of Ingenuity Pathway Analysis (IPA) to investigate the relevant BioFunctions enriched by the costitutive activation of AKT1-, PI3K- or PTEN-dependent signalling in lung epithelial cells. Expectedly, the analysis of the DEGs common to all three alterations highlighted a group of BioFunctions that included Cell Proliferation of tumor cell lines (14 DEGs), Invasion of cells (10 DEGs) and Migration of tumour cell lines (10 DEGs), with a common core of 5 genes (ATF3, CDKN1A, GDF15, HBEGF and LCN2) that likely represent downstream effectors of the pro-oncogenic activities of PI3K/AKT signalling. Conversely, IPA analysis of exclusive DEGs led to the identification of different downstream effectors that are modulated by mutant AKT1 (TGFBR2, CTSZ, EMP1), mutant PIK3CA (CCND2, CDK2, IGFBP2, TRIB1) and PTEN loss (ASNS, FHL2). These findings not only shed light on the molecular mechanisms that are activated by aberrant signalling through the PI3K/AKT pathway in lung epithelial cells, but also contribute to the identification of previously unrecognised molecules whose regulation takes part in the development of lung cancer.
The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in ...exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.
Epithelial ovarian carcinoma (EOC) is the most lethal gynecological tumor, that almost inevitably relapses and develops chemo-resistance. A better understanding of molecular events underlying the ...biological behavior of this tumor, as well as identification of new biomarkers and therapeutic targets are the prerequisite to improve its clinical management. ZNF521 gene amplifications are present in >6% of OCs and its overexpression is associated with poor prognosis, suggesting that it may play an important role in OC. Increased ZNF521 expression resulted in an enhancement of OC HeyA8 and ES-2 cell growth and motility. Analysis of RNA isolated from transduced cells by RNA-Seq and qRT-PCR revealed that several genes involved in growth, proliferation, migration and tumor invasiveness are differentially expressed following increased ZNF521 expression. The data illustrate a novel biological role of ZNF521 in OC that, thanks to the early and easy detection by RNA-Seq, can be used as biomarker for identification and treatment of OC patients.
Abstract
Background
Neuroendocrine neoplasms (NENs) represent a heterogeneous class of rare tumors with increasing incidence. They are characterized by the ability to secrete peptide hormones and ...biogenic amines but other reliable biomarkers are lacking, making diagnosis and identification of the primary site very challenging. While in some NENs, such as the pancreatic ones, next generation sequencing technologies allowed the identification of new molecular hallmarks, our knowledge of the molecular profile of NENs from other anatomical sites is still poor.
Methods
Starting from the concept that NENs from different organs may be clinically and genetically correlated, we applied a multi-omics approach by combining multigene panel testing, CGH-array, transcriptome and miRNome profiling and computational analyses, with the aim to highlight common molecular and functional signatures of gastroenteropancreatic (GEP)-NENs and medullary thyroid carcinomas (MTCs) that could aid diagnosis, prognosis and therapy.
Results
By comparing genomic and transcriptional profiles, ATM-dependent signaling emerged among the most significant pathways at multiple levels, involving gene variations and miRNA-mediated regulation, thus representing a novel putative druggable pathway in these cancer types. Moreover, a set of circulating miRNAs was also selected as possible diagnostic/prognostic biomarkers useful for clinical management of NENs.
Conclusions
These findings depict a complex molecular and functional landscape of NENs, shedding light on novel therapeutic targets and disease biomarkers to be exploited.
Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, ...particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.
Integrins are cell-extracellular matrix adhesion molecules whose expression level undergoes quantitative changes upon neoplastic transformation and are considered functionally related to the ...development of cancer metastasis. We analyzed the largest mRNA-seq dataset available to determine the expression pattern of integrin family subunits in papillary thyroid carcinomas (PTC). ITGA2, 3, 6, V, and ITGB1 integrin subunits were overexpressed in PTC compared to normal thyroid tissue. The PTC histology variants “classical” and “tall cell” displayed a similar integrin expression profile with a higher level of ITGA3, ITGAV, and ITGB1, which differed from that of the “follicular” variant. Interestingly, compared to RAS mutations, BRAFV600E mutation was associated with a significantly higher expression of integrins. Some integrin subunits were associated with advanced disease stage, lymph node metastasis, extrathyroidal extension, and high-risk groups. Among them, ITGA3 expression displayed the highest correlation with advanced disease and was associated with a negative prognosis. In vitro scratch assay and Matrigel invasion assay in two different PTC cell lines confirmed α3β1 role in cell motility and invasion, supporting its involvement during tumor progression. These results demonstrate the existence of a PTC-specific integrin expression signature correlated to histopathology, specific driver gene mutations, and aggressiveness of the disease.
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