Publications by authors named "Alessandro Weisz"

88 Publications

Identification of Antiestrogen-Bound Estrogen Receptor α Interactomes in Hormone-Responsive Human Breast Cancer Cell Nuclei.

Proteomics 2020 10 16;20(19-20):e2000135. Epub 2020 Sep 16.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Scuola Medica Salernitana', University of Salerno, Baronissi, Salerno, 84081, Italy.

Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor which mediates estrogen actions in hormone-responsive tumors and is targeted by effective anticancer therapies based on the ERα antagonist ligands, selective estrogen receptor modulators (such as Tamoxifen/TAM) or disruptors (such as Fulvestrant/ICI). Despite its importance for cancer therapy, including acquired resistance to endocrine therapy, the molecular basis of ERα response to different ligands is not fully known to date. Interaction proteomics shows great potential to identify and characterize molecular mechanisms of disease based on physical and functional protein-protein interaction networks. Tandem affinity purification coupled to mass spectrometry is applied here for mapping in hormone-responsive breast cancer cells nuclei, the ERα interactomes, induced by each of the two classes of antiestrogens. The results provide new insights on the molecular bases for antiestrogen-mediated control of ERα function and reveal new potential ways to overcome endocrine therapy resistance in cancer.
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http://dx.doi.org/10.1002/pmic.202000135DOI Listing
October 2020

Global View of Candidate Therapeutic Target Genes in Hormone-Responsive Breast Cancer.

Int J Mol Sci 2020 Jun 6;21(11). Epub 2020 Jun 6.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Scuola Medica Salernitana', University of Salerno, 84081 Baronissi (SA), Italy.

Breast cancer (BC) is a heterogeneous disease characterized by different biopathological features, differential response to therapy and substantial variability in long-term-survival. BC heterogeneity recapitulates genetic and epigenetic alterations affecting transformed cell behavior. The estrogen receptor alpha positive (ERα+) is the most common BC subtype, generally associated with a better prognosis and improved long-term survival, when compared to ERα-tumors. This is mainly due to the efficacy of endocrine therapy, that interfering with estrogen biosynthesis and actions blocks ER-mediated cell proliferation and tumor spread. Acquired resistance to endocrine therapy, however, represents a great challenge in the clinical management of ERα+ BC, causing tumor growth and recurrence irrespective of estrogen blockade. Improving overall survival in such cases requires new and effective anticancer drugs, allowing adjuvant treatments able to overcome resistance to first-line endocrine therapy. To date, several studies focus on the application of loss-of-function genome-wide screenings to identify key (hub) "fitness" genes essential for BC progression and representing candidate drug targets to overcome lack of response, or acquired resistance, to current therapies. Here, we review the biological significance of essential genes and relative functional pathways affected in ERα+ BC, most of which are strictly interconnected with each other and represent potential effective targets for novel molecular therapies.
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http://dx.doi.org/10.3390/ijms21114068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312026PMC
June 2020

Insights into the Role of Estrogen Receptor β in Triple-Negative Breast Cancer.

Cancers (Basel) 2020 Jun 5;12(6). Epub 2020 Jun 5.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Scuola Medica Salernitana', University of Salerno, 84081 Baronissi, Italy.

Estrogen receptors (ERα and ERβ) are ligand-activated transcription factors that play different roles in gene regulation and show both overlapping and specific tissue distribution patterns. ERβ, contrary to the oncogenic ERα, has been shown to act as an oncosuppressor in several instances. However, while the tumor-promoting actions of ERα are well-known, the exact role of ERβ in carcinogenesis and tumor progression is not yet fully understood. Indeed, to date, highly variable and even opposite effects have been ascribed to ERβ in cancer, including for example both proliferative and growth-inhibitory actions. Recently ERβ has been proposed as a potential target for cancer therapy, since it is expressed in a variety of breast cancers (BCs), including triple-negative ones (TNBCs). Because of the dependence of TNBCs on active cellular signaling, numerous studies have attempted to unravel the mechanism(s) behind ERβ-regulated gene expression programs but the scenario has not been fully revealed. We comprehensively reviewed the current state of knowledge concerning ERβ role in TNBC biology, focusing on the different signaling pathways and cellular processes regulated by this transcription factor, as they could be useful in identifying new diagnostic and therapeutic approaches for TNBC.
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http://dx.doi.org/10.3390/cancers12061477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7353068PMC
June 2020

An Overview of Candidate Therapeutic Target Genes in Ovarian Cancer.

Cancers (Basel) 2020 Jun 4;12(6). Epub 2020 Jun 4.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitan", University of Salerno, 84081 Baronissi, Italy.

Ovarian cancer (OC) shows the highest mortality rate among gynecological malignancies and, because of the absence of specific symptoms, it is frequently diagnosed at an advanced stage, mainly due to the lack of specific and early biomarkers, such as those based on cancer molecular signature identification. Indeed, although significant progress has been made toward improving the clinical outcome of other cancers, rates of mortality for OC are essentially unchanged since 1980, suggesting the need of new approaches to identify and characterize the molecular mechanisms underlying pathogenesis and progression of these malignancies. In addition, due to the low response rate and the high frequency of resistance to current treatments, emerging therapeutic strategies against OC focus on targeting single factors and pathways specifically involved in tumor growth and metastasis. To date, loss-of-function screenings are extensively applied to identify key drug targets in cancer, seeking for more effective, disease-tailored treatments to overcome lack of response or resistance to current therapies. We review here the information relative to essential genes and functional pathways recently discovered in OC, often strictly interconnected with each other and representing promising biomarkers and molecular targets to treat these malignancies.
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http://dx.doi.org/10.3390/cancers12061470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352306PMC
June 2020

Loss of Spry1 reduces growth of BRAF-mutant cutaneous melanoma and improves response to targeted therapy.

Cell Death Dis 2020 05 22;11(5):392. Epub 2020 May 22.

Immunopathology and Cancer Biomarkers, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, Aviano, Italy.

Mitogen-activated protein kinase (MAPK) pathway activation is a central step in BRAF-mutant cutaneous melanoma (CM) pathogenesis. In the last years, Spry1 has been frequently described as an upstream regulator of MAPK signaling pathway. However, its specific role in BRAF-mutant CM is still poorly defined. Here, we report that Spry1 knockdown (Spry1) in three BRAF-mutant CM cell lines markedly induced cell cycle arrest and apoptosis, repressed cell proliferation in vitro, and impaired tumor growth in vivo. Furthermore, our findings indicated that Spry1 reduced the expression of several markers of epithelial-mesenchymal transition, such as MMP-2 both in vitro and in vivo. These effects were associated with a sustained and deleterious phosphorylation of ERK1/2. In addition, p38 activation along with an increase in basal ROS levels were found in Spry1 clones compared to parental CM cell lines, suggesting that BRAF-mutant CM may restrain the activity of Spry1 to avoid oncogenic stress and to enable tumor growth. Consistent with this hypothesis, treatment with the BRAF inhibitor (BRAFi) vemurafenib down-regulated Spry1 levels in parental CM cell lines, indicating that Spry1 expression is sustained by the MAPK/ERK signaling pathway in a positive feedback loop that safeguards cells from the potentially toxic effects of ERK1/2 hyperactivation. Disruption of this feedback loop rendered Spry1 cells more susceptible to apoptosis and markedly improved response to BRAFi both in vitro and in vivo, as a consequence of the detrimental effect of ERK1/2 hyperactivation observed upon Spry1 abrogation. Therefore, targeting Spry1 might offer a treatment strategy for BRAF-mutant CM by inducing the toxic effects of ERK-mediated signaling.
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http://dx.doi.org/10.1038/s41419-020-2585-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7244546PMC
May 2020

Atrial myxomas arise from multipotent cardiac stem cells.

Eur Heart J 2020 12;41(45):4332-4345

Department of Experimental and Clinical Medicine, Molecular and Cellular Cardiology, Magna Graecia University, Viale Europa, 88100 Catanzaro, Italy.

Aims: Cardiac myxomas usually develop in the atria and consist of an acid-mucopolysaccharide-rich myxoid matrix with polygonal stromal cells scattered throughout. These human benign tumours are a valuable research model because of the rarity of cardiac tumours, their clinical presentation and uncertain origin. Here, we assessed whether multipotent cardiac stem/progenitor cells (CSCs) give rise to atrial myxoma tissue.

Methods And Results: Twenty-three myxomas were collected and analysed for the presence of multipotent CSCs. We detected myxoma cells positive for c-kit (c-kitpos) but very rare Isl-1 positive cells. Most of the c-kitpos cells were blood lineage-committed CD45pos/CD31pos cells. However, c-kitpos/CD45neg/CD31neg cardiac myxoma cells expressed stemness and cardiac progenitor cell transcription factors. Approximately ≤10% of the c-kitpos/CD45neg/CD31neg myxoma cells also expressed calretinin, a characteristic of myxoma stromal cells. In vitro, the c-kitpos/CD45neg/CD31neg myxoma cells secrete chondroitin-6-sulfate and hyaluronic acid, which are the main components of gelatinous myxoma matrix in vivo. In vitro, c-kitpos/CD45neg/CD31neg myxoma cells have stem cell properties being clonogenic, self-renewing, and sphere forming while exhibiting an abortive cardiac differentiation potential. Myxoma-derived CSCs possess a mRNA and microRNA transcriptome overall similar to normal myocardium-derived c-kitpos/CD45neg/CD31negCSCs , yet showing a relatively small and relevant fraction of dysregulated mRNA/miRNAs (miR-126-3p and miR-335-5p, in particular). Importantly, myxoma-derived CSCs but not normal myocardium-derived CSCs, seed human myxoma tumours in xenograft's in immunodeficient NOD/SCID mice.

Conclusion: Myxoma-derived c-kitpos/CD45neg/CD31neg CSCs fulfill the criteria expected of atrial myxoma-initiating stem cells. The transcriptome of these cells indicates that they belong to or are derived from the same lineage as the atrial multipotent c-kitpos/CD45neg/CD31neg CSCs. Taken together the data presented here suggest that human myxomas could be the first-described CSC-related human heart disease.
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http://dx.doi.org/10.1093/eurheartj/ehaa156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7735815PMC
December 2020

Small Non-Coding RNA Profiling Identifies miR-181a-5p as a Mediator of Estrogen Receptor Beta-Induced Inhibition of Cholesterol Biosynthesis in Triple-Negative Breast Cancer.

Cells 2020 04 3;9(4). Epub 2020 Apr 3.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Scuola Medica Salernitana', University of Salerno, 84081 Baronissi, Italy.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease, representing the most aggressive breast cancer (BC) subtype with limited treatment options due to a lack of estrogen receptor alpha (ERα), progesterone receptor (PR), and Erb-B2 receptor tyrosine kinase 2 (HER2/neu) expression. Estrogen receptor beta (ERβ) is present in a fraction of TNBC patients, where its expression correlates with improved patient outcomes, supported by the fact that it exerts oncosuppressive effects in TNBC cell models in vitro. ERβ is involved in microRNA-mediated regulation of gene expression in hormone-responsive BC cells and could mediate its actions through small noncoding RNAs (sncRNAs) in TNBCs also. To verify this possibility, smallRNA sequencing was performed on three ERβ-expressing cell lines from different TNBC molecular subtypes. Several sncRNAs resulted modulated by ERβ, with a subset being regulated in a tumor subtype-independent manner. Interestingly, sncRNA profiling of 12 ERβ+and 32 ERβ- primary TNBC biopsies identified 7 microRNAs, 1 PIWI-interacting RNA (piRNA), and 1 transfer RNA (tRNA) differentially expressed in ERβ+ compared to ERβ- tumors and cell lines. Among them, miR-181a-5p was found to be overexpressed in ERβ+ tumors and predicted target key components of the cholesterol biosynthesis pathway previously found to be inhibited by ERβ in TNBC cells.
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http://dx.doi.org/10.3390/cells9040874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226848PMC
April 2020

Interaction Proteomics Identifies ERbeta Association with Chromatin Repressive Complexes to Inhibit Cholesterol Biosynthesis and Exert An Oncosuppressive Role in Triple-negative Breast Cancer.

Mol Cell Proteomics 2020 02 2;19(2):245-260. Epub 2019 Dec 2.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Scuola Medica Salernitana', University of Salerno, Baronissi (SA), 84081, Italy. Electronic address:

Triple-negative breast cancer (TNBC) is characterized by poor response to therapy and low overall patient survival. Recently, Estrogen Receptor beta (ERβ) has been found to be expressed in a fraction of TNBCs where, because of its oncosuppressive actions on the genome, it represents a potential therapeutic target, provided a better understanding of its actions in these tumors becomes available. To this end, the cell lines Hs 578T, MDA-MB-468 and HCC1806, representing the claudin-low, basal-like 1 and 2 TNBC molecular subtypes respectively, were engineered to express ERβ under the control of a Tetracycline-inducible promoter and used to investigate the effects of this transcription factor on gene activity. The antiproliferative effects of ERβ in these cells were confirmed by multiple functional approaches, including transcriptome profiling and global mapping of receptor binding sites in the genome, that revealed direct negative regulation by ERβ of genes, encoding for key components of cellular pathways associated to TNBC aggressiveness representing novel therapeutic targets such as angiogenesis, invasion, metastasis and cholesterol biosynthesis. Supporting these results, interaction proteomics by immunoprecipitation coupled to nano LC-MS/MS mass spectrometry revealed ERβ association with several potential nuclear protein partners, including key components of regulatory complexes known to control chromatin remodeling, transcriptional and post-transcriptional gene regulation and RNA splicing. Among these, ERβ association with the Polycomb Repressor Complexes 1 and 2 (PRC1/2), known for their central role in gene regulation in cancer cells, was confirmed in all three TNBC subtypes investigated, suggesting its occurrence independently from the cellular context. These results demonstrate a significant impact of ERβ in TNBC genome activity mediated by its cooperation with regulatory multiprotein chromatin remodeling complexes, providing novel ground to devise new strategies for the treatment of these diseases based on ligands affecting the activity of this nuclear receptor or some of its protein partners.
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http://dx.doi.org/10.1074/mcp.RA119.001817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7000115PMC
February 2020

Small-bowel carcinomas associated with celiac disease: transcriptomic profiling shows predominance of microsatellite instability-immune and mesenchymal subtypes.

Virchows Arch 2020 May 6;476(5):711-723. Epub 2019 Nov 6.

Unit of Pathology, Dept. of Medicine and Surgery and Research Center for the Study of Hereditary and Familial Tumors, University of Insubria, Via Rossi 9, 21100, Varese, Italy.

Celiac disease (CD) is a risk factor for developing small-bowel carcinoma with a 14-fold higher risk compared with general population. As small-bowel carcinomas associated with CD (CD-SBCs) are extremely rare, very few molecular data are available about their pathogenesis, and information about their transcriptomic profiling is lacking. We generated RNA-seq data on 13 CD-SBCs, taken from the largest well-characterized series published so far, collected by the Small Bowel Cancer Italian Consortium, and compared the tumor transcriptional signatures with the four Consensus Molecular Subtypes (CMS) of colorectal carcinoma by applying the "CMS classifier." CpG Island Methylator Phenotype (CIMP) was evaluated using methylation-sensitive multiple ligation-dependent probe amplification. Up to 12 of 13 cancers fell within the two main subtypes exhibiting high immune and inflammatory signatures, i.e., subtypes 1 and 4. The first and predominant subset was commonly microsatellite unstable, and exhibited CIMP and high CD3+ and CD8+ T cell infiltration. Moreover, it showed increased expression of genes associated with T helper 1 and natural killer cell infiltration, as well as upregulation of apoptosis, cell cycle progression, and proteasome pathways. By contrast, cancers falling in subtype 4 showed prominent transforming growth factor-β activation and were characterized by complement-associated inflammation, matrix remodeling, cancer-associated stroma production, and angiogenesis. Parallel histologic and histochemical analysis confirmed such tumor subtyping. In conclusion, two molecular subtypes have been consistently identified in our series of CD-SBCs, a microsatellite instability-immune and a mesenchymal subtype, the former likely associated with an indolent and the latter with a worse tumor behavior.
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http://dx.doi.org/10.1007/s00428-019-02675-wDOI Listing
May 2020

Molecular and Functional Characterization of the Somatic PIWIL1/piRNA Pathway in Colorectal Cancer Cells.

Cells 2019 11 5;8(11). Epub 2019 Nov 5.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, 84081 Baronissi (SA), Italy.

(PIWIL) proteins and small non-coding piRNAs, involved in genome regulation in germline cells, are found aberrantly expressed in human tumors. Gene expression data from The Cancer Genome Atlas (TCGA), the Genotype-Tissue Expression (GTEx) project, and the European Genome-Phenome Archive (EGA) indicate that the gene is ectopically activated in a significant fraction of colorectal cancers (CRCs), where this is accompanied by promoter demethylation, together with germline factors required for piRNA production. Starting from this observation, the PIWIL/piRNA pathway was studied in detail in COLO 205 CRC cells, which express significant levels of this protein, to investigate role and significance of ectopic expression in human tumors. RNA sequencing and cell and computational biology led to the demonstration that PIWIL1 localizes in a nuage-like structure located in the perinuclear region of the cell and that a significant fraction of the piRNAs expressed in these cells are methylated, and, therefore, present in an active form. This was further supported by RNA immunoprecipitation, which revealed how several piRNAs can be found loaded into PIWIL1 to form complexes also comprising their target mRNAs. The mature transcripts associated with the PIWIL-piRNA complex encode key regulatory proteins involved in the molecular mechanisms sustaining colorectal carcinogenesis, suggesting that the PIWI/piRNA pathway may actively contribute to the establishment and/or maintenance of clinico-pathological features of CRCs.
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http://dx.doi.org/10.3390/cells8111390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912267PMC
November 2019

The Histone Methyltransferase DOT1L Is a Functional Component of Estrogen Receptor Alpha Signaling in Ovarian Cancer Cells.

Cancers (Basel) 2019 Nov 4;11(11). Epub 2019 Nov 4.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, 84081 Baronissi (SA), Italy.

Although a large fraction of high-grade serous epithelial ovarian cancers (OCs) expresses Estrogen Receptor alpha (ERα), anti-estrogen-based therapies are still not widely used against these tumors due to a lack of sufficient evidence. The histone methyltransferase Disruptor of telomeric silencing-1-like (DOT1L), which is a modulator of ERα transcriptional activity in breast cancer, controls chromatin functions involved in tumor initiation and progression and has been proposed as a prognostic OC biomarker. As molecular and clinico-pathological data from TCGA suggest a correlation between ERα and DOT1L expression and OC prognosis, the presence and significance of ERα/DOT1L association was investigated in chemotherapy-sensitive and chemotherapy-resistant ER+ OC cells. RNA sequencing before and after inhibition of these factors showed that their activity is implicated in OC cell proliferation and that they functionally cooperate with each other to control the transcription of genes involved in key cancer cell features, such as the cell cycle, epithelial-mesenchymal transition (EMT), drug metabolism, and cell-to-cell signaling, as well as expression of the ERα gene itself. Together with evidence from loss-of-function genetic screens showing that ERα and DOT1L behave as core fitness factors in OC cells, these results suggest that combined inhibition of their activity might be effective against ERα-expressing, chemotherapy-resistant ovarian tumors.
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http://dx.doi.org/10.3390/cancers11111720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895927PMC
November 2019

The RNA-mediated estrogen receptor α interactome of hormone-dependent human breast cancer cell nuclei.

Sci Data 2019 09 16;6(1):173. Epub 2019 Sep 16.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, 84081, Baronissi, SA, Italy.

Estrogen Receptor alpha (ERα) is a ligand-inducible transcription factor that mediates estrogen signaling in hormone-responsive cells, where it controls key cellular functions by assembling in gene-regulatory multiprotein complexes. For this reason, interaction proteomics has been shown to represent a useful tool to investigate the molecular mechanisms underlying ERα action in target cells. RNAs have emerged as bridging molecules, involved in both assembly and activity of transcription regulatory protein complexes. By applying Tandem Affinity Purification (TAP) coupled to mass spectrometry (MS) before and after RNase digestion in vitro, we generated a dataset of nuclear ERα molecular partners whose association with the receptor involves RNAs. These data provide a useful resource to elucidate the combined role of nuclear RNAs and the proteins identified here in ERα signaling to the genome in breast cancer and other cell types.
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http://dx.doi.org/10.1038/s41597-019-0179-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746822PMC
September 2019

DNA methylation dynamic of bone marrow hematopoietic stem cells after allogeneic transplantation.

Stem Cell Res Ther 2019 05 20;10(1):138. Epub 2019 May 20.

Laboratory of Preclinical and Translational Research, IRCCS - Referral Cancer Center of Basilicata (CROB), 85028, Rionero in Vulture, Italy.

Background: Allogeneic hematopoietic stem cell transplantation (AHSCT) is a curative therapeutic approach for different hematological malignancies (HMs), and epigenetic modifications, including DNA methylation, play a role in the reconstitution of the hematopoietic system after AHSCT. This study aimed to explore global DNA methylation dynamic of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) from donors and their respective recipients affected by acute myeloid leukemia (AML), acute lymphoid leukemia (ALL) and Hodgkin lymphoma (HL) during the first year after transplant.

Methods: We measured DNA methylation profile by Illumina HumanMethylationEPIC in BM HSPC of 10 donors (t0) and their matched recipients at different time points after AHSCT, at day + 30 (t1), + 60 (t2), + 120 (t3), + 180 (t4), and + 365 (t5). Differential methylation analysis was performed by using R software and CRAN/Bioconductor packages. Gene set enrichment analysis was carried out on promoter area of significantly differentially methylated genes by clusterProfiler package and the mSigDB genes sets.

Results: Results show significant differences in the global methylation profile between HL and acute leukemias, and between patients with mixed and complete chimerism, with a strong methylation change, with prevailing hyper-methylation, occurring 30 days after AHSCT. Functional analysis of promoter methylation changes identified genes involved in hematopoietic cell activation, differentiation, shaping, and movement. This could be a consequence of donor cell "adaptation" in recipient BM niche. Interestingly, this epigenetic remodeling was reversible, since methylation returns similar to that of donor HSPCs after 1 year. Only for a pool of genes, mainly involved in dynamic shaping and trafficking, the DNA methylation changes acquired after 30 days were maintained for up to 1 year post-transplant. Finally, preliminary data suggest that the methylation profile could be used as predictor of relapse in ALL.

Conclusions: Overall, these data provide insights into the DNA methylation changes of HSPCs after transplantation and a new framework to investigate epigenetics of AHSCT and its outcomes.
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http://dx.doi.org/10.1186/s13287-019-1245-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528331PMC
May 2019

Inhibition of histone methyltransferase DOT1L silences ERα gene and blocks proliferation of antiestrogen-resistant breast cancer cells.

Sci Adv 2019 02 6;5(2):eaav5590. Epub 2019 Feb 6.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry, "Scuola Medica Salernitana", University of Salerno, Baronissi, SA, Italy.

Breast cancer (BC) resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor α (ERα) signaling, and ways to block ERα pathway in these tumors are sought after. We identified the H3K79 methyltransferase DOT1L as a novel cofactor of ERα in BC cell chromatin, where the two proteins colocalize to regulate estrogen target gene transcription. DOT1L blockade reduces proliferation of hormone-responsive BC cells in vivo and in vitro, consequent to cell cycle arrest and apoptotic cell death, with widespread effects on ER-dependent gene transcription, including ERα and FOXA1 gene silencing. Antiestrogen-resistant BC cells respond to DOT1L inhibition also in mouse xenografts, with reduction in ERα levels, H3K79 methylation, and tumor growth. These results indicate that DOT1L is an exploitable epigenetic target for treatment of endocrine therapy-resistant ERα-positive BCs.
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http://dx.doi.org/10.1126/sciadv.aav5590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365116PMC
February 2019

TNF-alpha and metalloproteases as key players in melanoma cells aggressiveness.

J Exp Clin Cancer Res 2018 Dec 28;37(1):326. Epub 2018 Dec 28.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, ISS, viale Regina Elena 299, 00161, Rome, Italy.

Background: Melanoma aggressiveness determines its growth and metastatic potential. This study aimed at identifying new molecular pathways controlling melanoma cell malignancy.

Methods: 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.

Results: 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).

Conclusions: 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.
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http://dx.doi.org/10.1186/s13046-018-0982-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309098PMC
December 2018

Identification of long non‑coding RNA expression patterns useful for molecular‑based classification of type I endometrial cancers.

Oncol Rep 2019 Feb 21;41(2):1209-1217. Epub 2018 Nov 21.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry ῾Scuola Medica Salernitana᾽, University of Salerno, I‑84081 Baronissi (SA), Italy.

Endometrial cancer is the most frequently diagnosed gynecologic malignant disease. Although several genetic alterations have been associated with the increased risk of endometrial cancer, to date, the diagnosis and prognosis still rely on morphological features of the tumor, such as histological type, grading and invasiveness. As molecular‑based classification is desirable for optimal treatment and prognosis of these cancers, we explored the potential of lncRNAs as molecular biomarkers. To this end, we first identified by RNA sequencing (RNA‑Seq) a set of lncRNAs differentially expressed in cancer vs. normal endometrial tissues, a result confirmed also by analysis of normal and cancerous endometrium RNA‑Seq data from TCGA (The Cancer Genome Atlas). A significant association of a subset of these differentially expressed lncRNAs with tumor grade was then determined in 405 TCGA endometrial cancer profiles. Integrating endometrial cancer‑specific expression profiles of long and small non‑coding RNAs, a functional association network was then identified. These results describe for the first time a functional ῾core᾽ network, comprising small and long RNAs, whose deregulation is associated with endometrial neoplastic transformation, representing a set of cancer biomarkers that can be monitored and targeted for diagnosis, follow‑up and therapy of these tumors.
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http://dx.doi.org/10.3892/or.2018.6880DOI Listing
February 2019

miRNA Regulation of the Hyperproliferative Phenotype of Vascular Smooth Muscle Cells in Diabetes.

Diabetes 2018 12 26;67(12):2554-2568. Epub 2018 Sep 26.

Cardiovascular Institute, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy.

Harnessing the mechanisms underlying the exacerbated vascular remodeling in diabetes mellitus (DM) is pivotal to prevent the high toll of vascular diseases in patients with DM. miRNA regulates vascular smooth muscle cell (VSMC) phenotypic switch. However, miRNA modulation of the detrimental diabetic VSMC phenotype is underexplored. Streptozotocin-induced type 1 DM (T1DM) Wistar rats and type 2 DM (T2DM) Zucker rats underwent right carotid artery experimental angioplasty, and global miRNA/mRNA expression profiling was obtained by RNA sequencing (RNA-Seq). Two days after injury, a set of six miRNAs were found to be uniquely downregulated or upregulated in VSMCs both in T1DM and T2DM. Among these miRNAs, miR-29c and miR-204 were the most significantly misregulated in atherosclerotic plaques from patients with DM. miR-29c overexpression and miR-204 inhibition per se attenuated VSMC phenotypic switch in DM. Concomitant miR-29c overexpression and miR-204 inhibition fostered an additive reduction in VSMC proliferation. Epithelial membrane protein 2 () and Caveolin-1 () mRNAs were identified as direct targets of miR-29c and miR-204, respectively. Importantly, contemporary miR-29c overexpression and miR-204 inhibition in the injured artery robustly reduced arterial stenosis in DM rats. Thus, contemporaneous miR-29c activation and miR-204 inhibition in DM arterial tissues is necessary and sufficient to prevent the exaggerated VSMC growth upon injury.
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http://dx.doi.org/10.2337/db17-1434DOI Listing
December 2018

miR-196a Is Able to Restore the Aggressive Phenotype of Annexin A1 Knock-Out in Pancreatic Cancer Cells by CRISPR/Cas9 Genome Editing.

Int J Mol Sci 2018 Jul 6;19(7). Epub 2018 Jul 6.

Department of Pharmacy, University of Salerno, via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy.

Annexin A1 (ANXA1) is a Ca-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.
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http://dx.doi.org/10.3390/ijms19071967DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6073506PMC
July 2018

Small non-coding RNA landscape is modified by GPAT2 silencing in MDA-MB-231 cells.

Oncotarget 2018 Jun 15;9(46):28141-28154. Epub 2018 Jun 15.

Instituto de Investigaciones Bioquímicas de La Plata, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de La Plata, La Plata, Argentina.

Glycerol-3-phosphate acyltransferase-2 is a member of "cancer-testis gene" family. Initially linked to lipid metabolism, this gene has been recently found involved also in PIWI-interacting RNAs biogenesis in germline stem cells. To investigate its role in piRNA metabolism in cancer, the gene was silenced in MDA-MB-231 breast cancer cells and small RNA sequencing was applied. PIWI-interacting RNAs and tRNA-derived fragments expression profiles showed changes following GPAT2 silencing. Interestingly, a marked shift in length distribution for both small RNAs was detected in GPAT2-silenced cells. Most downregulated PIWI-interacting RNAs are single copy in the genome, intragenic, hosted in snoRNAs and previously found to be upregulated in cancer cells. Putative targets of these PIWI-interacting RNAs are linked to lipid metabolism. Downregulated tRNA derived fragments derived from, so-called 'differentiation tRNAs', whereas upregulated ones derived from proliferation-linked tRNAs. miRNA amounts decrease after Glycerol-3-phosphate acyltransferase-2 silencing and functional enrichment analysis of deregulated miRNA putative targets point to mitochondrial biogenesis, IGF1R signaling and oxidative metabolism of lipids and lipoproteins. In addition, miRNAs known to be overexpressed in breast cancer tumors with poor prognosis where found downregulated in GPAT2-silenced cells. In conclusion, GPAT2 silencing quantitatively and qualitatively affects the population of PIWI-interacting RNAs, tRNA derived fragments and miRNAs which, in combination, result in a more differentiated cancer cell phenotype.
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http://dx.doi.org/10.18632/oncotarget.25582DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6021339PMC
June 2018

Relations of gut liver axis components and gut microbiota in obese children with fatty liver: A pilot study.

Clin Res Hepatol Gastroenterol 2018 09 10;42(4):387-390. Epub 2018 Jul 10.

Pediatrics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, Via Allende, 84081 Baronissi (Sa), Italy; University Hospital "San Giovanni di Dio e Ruggi d'Aragona", 84131 Salerno, Italy; European Laboratory of Food Induced Intestinal Diseases (ELFID), University of Naples Federico II, 80100 Naples, Italy.

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http://dx.doi.org/10.1016/j.clinre.2018.03.015DOI Listing
September 2018

Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.

FASEB J 2018 08 7;32(8):4343-4355. Epub 2018 Mar 7.

Department of Pharmacy, Health, and Nutritional Sciences, University of Calabria, Rende, Italy.

Adipose tissue is a metabolic and endocrine organ that secretes bioactive molecules called adipocytokines. Among these, adiponectin has a crucial role in obesity-associated breast cancer. The key molecule of adiponectin signaling is AMPK, which is mainly activated by liver kinase B1 (LKB1). Here, we demonstrated that estrogen receptor-α (ERα)/LKB1 interaction may negatively interfere with the LKB1 capability to phosphorylate AMPK and inhibit its downstream signaling TSC2/mTOR/p70S6k. In adiponectin-treated MCF-7 cells, AMPK signaling was not working, resulting in its downstream target acetyl-CoA carboxylase (ACC) being still active. In contrast, in MDA-MB-231 cells, AMPK and ACC phosphorylation was enhanced by adiponectin, inhibiting lipogenesis and cell growth. Upon adiponectin, ERα signaling switched the energy balance of breast cancer cells toward a lipogenic phenotype. Therefore, adiponectin played an inhibitory role on ERα-negative cell growth and progression in vitro and in vivo. In contrast, low adiponectin levels, similar to those circulating in obese patients, acted on ERα-positive cells as a growth factor, stimulating proliferation. The latter effect was blunted in vivo by high adiponectin concentration. All this may have translational relevance, addressing how the handling of adiponectin, as a therapeutic tool in breast cancer treatment, needs to be carefully considered in ERα-positive obese patients, where circulating levels of this adipocytokine are relatively low. In other words, in ERα-positive breast cancer obese patients, higher adiponectin doses should be administered with respect to ERα-negative breast cancer, also opportunely combined with antiestrogen therapy. -Mauro, L., Naimo, G. D., Gelsomino, L., Malivindi, R., Bruno, L., Pellegrino, M., Tarallo, R., Memoli, D., Weisz, A., Panno, M. L., Andò, S. Uncoupling effects of estrogen receptor α on LKB1/AMPK interaction upon adiponectin exposure in breast cancer.
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http://dx.doi.org/10.1096/fj.201701315RDOI Listing
August 2018

Quantitative mapping of RNA-mediated nuclear estrogen receptor β interactome in human breast cancer cells.

Sci Data 2018 03 6;5:180031. Epub 2018 Mar 6.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, 84081 Baronissi (SA), Italy.

The nuclear receptor estrogen receptor 2 (ESR2, ERβ) modulates cancer cell proliferation and tumor growth, exerting an oncosuppressive role in breast cancer (BC). Interaction proteomics by tandem affinity purification coupled to mass spectrometry was previously applied in BC cells to identify proteins acting in concert with ERβ to control key cellular functions, including gene transcription, RNA splicing and post-transcriptional mRNA regulation. These studies revealed an involvement of RNA in ERβ interactome assembly and functions. By applying native protein complex purification followed by nano LC-MS/MS before and after in vitro RNA removal, we generated a large dataset of newly identified nuclear ERβ interactors, including a subset associating with the receptor via RNA bridging. These datasets will be useful to investigate further the role of ERβ, nuclear RNAs and the other proteins identified here in BC and other cell types.
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http://dx.doi.org/10.1038/sdata.2018.31DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5839158PMC
March 2018

Identification of a novel truncating mutation in PALB2 gene by a multigene sequencing panel for mutational screening of breast cancer risk-associated and related genes.

J Clin Lab Anal 2018 Jul 27;32(6):e22418. Epub 2018 Feb 27.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry, 'Scuola Medica Salernitana', University of Salerno, Baronissi, Italy.

Background: Breast cancer (BC) is the most common neoplasm in women, with 5%-10% patients showing a familial predisposition, where germline mutations in BRCA1/BRCA2 genes are found in -20% of cases. Next-generation sequencing (NGS) is among the best available options for genetic screening, providing several benefits that include enhanced sensitivity and unbiased mutation detection. PALB2 (partner and localizer of BRCA2) is a cancer predisposing gene recently described that encodes a protein partner of BRCA2 involved in DNA double-strand break repair and cell cycle control. The DNA damage response represents a key cellular event, targeted by innovative anticancer therapies, including those based on poly (ADP-ribose) polymerase (PARP) inhibitors targeting PARP1 and PARP2 enzymes, activated by DNA damage and involved in single-strand break and base excision repair.

Methods: Genomic DNA was isolated from 34 patient samples and four BC cell lines, as controls, and 27 breast cancer predisposing genes belonging to the BRCA1/BRCA2 and PARP pathways were sequenced by NGS.

Results: The panel described here allowed identification of several sequence variations in most investigated genes, among which we found a novel truncating mutation in PALB2.

Conclusions: The NGS-based strategy designed here for molecular analysis of a customized panel of BC predisposing and related genes was found to perform effectively, providing a comprehensive exploration of all genomic sequences of the investigated genes. It is thus useful for BC molecular diagnosis, in particular for familiar cases where alterations in routinely investigated genes, such as BRCAs, result to be absent.
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http://dx.doi.org/10.1002/jcla.22418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817177PMC
July 2018

Splicing of platelet resident pre-mRNAs upon activation by physiological stimuli results in functionally relevant proteome modifications.

Sci Rep 2018 01 11;8(1):498. Epub 2018 Jan 11.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Scuola Medica Salernitana", University of Salerno, Baronissi, (SA), Italy.

Platelet activation triggers thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies still fail to prevent thrombotic events in numerous patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how megakaryocyte-derived mRNAs are regulated in these anucleate cell fragments. Proteogenomics was applied here to investigate this phenomeon in platelets activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining proteomics and transcriptomics allowed in depth platelet proteome characterization, revealing a significant effect of either stimulus on proteome composition. In silico analysis revealed the presence of resident immature RNAs in resting platelets, characterized by retained introns, while unbiased proteogenomics correlated intron removal by RNA splicing with changes on proteome composition upon activation. This allowed identification of a set of transcripts undergoing maturation by intron removal during activation and resulting in accumulation of the corresponding peptides at exon-exon junctions. These results indicate that RNA splicing events occur in platelets during activation and that maturation of specific pre-mRNAs is part of the activation cascade, contributing to a dynamic fine-tuning of the transcriptome.
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http://dx.doi.org/10.1038/s41598-017-18985-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765118PMC
January 2018

A Large Set of miRNAs Is Dysregulated from the Earliest Steps of Human Hepatocellular Carcinoma Development.

Am J Pathol 2018 03 15;188(3):785-794. Epub 2017 Dec 15.

Pathology Unit, Humanitas Clinical and Research Center, and Department of Biomedical Sciences, Humanitas University, Milan, Italy.

Hepatocellular carcinoma (HCC) typically results from a stepwise process characterized by the development of premalignant lesions, such as low- or high-grade dysplastic nodules (LGDNs and HGDNs, respectively), in a cirrhotic setting. MicroRNAs (miRNAs) are small noncoding RNAs involved in post-transcriptional regulation of gene expression that can act as oncogenes or tumor suppressors. Whether and which miRNAs are involved in the early stages of HCC development remains elusive. Here, small-RNA sequencing was applied to profile miRNA expression in 55 samples (cirrhotic nodules; CNs), LGDNs, HGDNs, early HCCs, and small progressed HCCs, obtained from 17 patients bearing HCCs of different etiologies. An miRNA expression signature of 62 miRNAs distinguishing small progressed HCCs from matched CNs was identified. Interestingly, 52 of these miRNAs discriminated CNs from LGDNs/HGDNs, regardless of etiology, and remained modified along the tumorigenic process. Functional analysis of the predicted mRNA targets of deregulated miRNAs identified common modifications between the early and late stages of HCC development likely involved in the stepwise process of HCC development. Our results demonstrate that miRNA deregulation happens very early in HCC in humans, implying their crucial role in the tumorigenic process. The identification of miRNAs discriminating CNs from neoplastic nodules may have relevant translational implications in early diagnosis.
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http://dx.doi.org/10.1016/j.ajpath.2017.10.024DOI Listing
March 2018

The nuclear receptor ERβ engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading.

Genome Biol 2017 10 6;18(1):189. Epub 2017 Oct 6.

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry "Schola Medica Salernitana", University of Salerno, via S. Allende, 1, 84081, Baronissi, SA, Italy.

Background: 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.

Results: 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.

Conclusions: 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.
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http://dx.doi.org/10.1186/s13059-017-1321-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634881PMC
October 2017

Specific gene expression signatures induced by the multiple oncogenic alterations that occur within the PTEN/PI3K/AKT pathway in lung cancer.

PLoS One 2017 29;12(6):e0178865. Epub 2017 Jun 29.

Dipartimento di Medicina Sperimentale e Clinica, Università "Magna Graecia", Catanzaro, Italia.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178865PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491004PMC
September 2017

iSmaRT: a toolkit for a comprehensive analysis of small RNA-Seq data.

Bioinformatics 2017 03;33(6):938-940

Laboratory of Molecular Medicine and Genomics, Department of Medicine, Surgery and Dentistry 'Scuola Medica Salernitana', University of Salerno, Baronissi, SA, Italy.

Summary: The interest in investigating the biological roles of small non-coding RNAs (sncRNAs) is increasing, due to the pleiotropic effects of these molecules exert in many biological contexts. While several methods and tools are available to study microRNAs (miRNAs), only few focus on novel classes of sncRNAs, in particular PIWI-interacting RNAs (piRNAs). To overcome these limitations, we implemented iSmaRT ( i ntegrative Sm all R NA T ool-kit), an automated pipeline to analyze smallRNA-Seq data.

Availability And Implementation: iSmaRT is a collection of bioinformatics tools and own algorithms, interconnected through a Graphical User Interface (GUI). In addition to performing comprehensive analyses on miRNAs, it implements specific computational modules to analyze piRNAs, predicting novel ones and identifying their RNA targets. A smallRNA-Seq dataset generated from brain samples of Huntington's Disease patients was used here to illustrate iSmaRT performances, demonstrating how the pipeline can provide, in a rapid and user friendly way, a comprehensive analysis of different classes of sncRNAs. iSmaRT is freely available on the web at ftp://labmedmolge-1.unisa.it (User: iSmart - Password: password).

Contact: aweisz@unisa.it or ggiurato@unisa.it.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btw734DOI Listing
March 2017