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AIMS: Tumour necrosis and/or increased mitoses define high-grade papillary thyroid carcinoma (PTC). It is unclear whether angioinvasion is prognostic for PTC. Cut-offs at five or more mitoses/2 mm2 and four or more angioinvasive foci have been empirically defined based upon data from all forms of aggressive non-anaplastic thyroid carcinomas. Performance of tumour necrosis, mitoses and vascular invasion in predicting distant metastases when specifically applied to PTC is undefined. METHODS: We analysed 50 consecutive PTC cases with distant metastases (DM-PTC): 16 synchronous and 34 metachronous. A total of 108 non-metastatic PTC (N-DM-PTC, 15.0-year median follow-up) were used as controls. Invasive encapsulated follicular variant PTC was excluded. Necrosis, mitoses and angioinvasion were quantified. Receiver operating characteristics (ROC) and area under the curve (AUC) analyses determined best sensitivity and specificity cut-offs predictive of distant metastases. RESULTS: Metastases correlated with necrosis (any extent = 43.8% all DM-PTC, 53.1% metachronous DM-PTC versus 5% N-DM-PTC; P < 0.001), mitoses (P < 0.001) and angioinvasion (P < 0.001). Mitoses at five or more per 2 mm2 was the best cut-off correlating with distant metastases: sensitivity/specificity 42.9%/97.2% all DM-PTC (AUC = 0.78), 18.8%/97.2% synchronous DM-PTC (AUC = 0.63), 54.6%/97.2% metachronous DM-PTC (AUC = 0.85). Angioinvasive foci at five or more was the best cut-off correlating with distant metastases: sensitivity/specificity 36.2%/91.7% all DM-PTC (AUC = 0.75), 25%/91.7% synchronous DM-PTC (AUC = 0.79) and 41.9%/91.7% metachronous DM-PTC (AUC = 0.73). Positive/negative predictive values (PPV/NPV) were: necrosis 22.6%/98.2%; five or more mitoses 32.3%/98.2%; five or more angioinvasive foci 11.8%/97.9%. After multivariable analysis, only necrosis and mitotic activity remained associated with DM-PTC. CONCLUSION: Our data strongly support PTC grading, statistically validating World Health Organisation (WHO) criteria to identify poor prognosis PTC. Angioinvasion is not an independent predictor of DM-PTC.
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Necrose , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Masculino , Neoplasias da Glândula Tireoide/patologia , Feminino , Pessoa de Meia-Idade , Câncer Papilífero da Tireoide/patologia , Adulto , Prognóstico , Estudos de Casos e Controles , Idoso , Organização Mundial da Saúde , Invasividade Neoplásica , Carcinoma Papilar/patologia , Mitose , Adulto JovemRESUMO
Multiple myeloma (MM) is a dreadful disease, marked by the uncontrolled proliferation of clonal plasma cells (PCs) within the bone marrow (BM). MM is characterized by a highly heterogeneous clinical and molecular background, supported by severe genomic alterations. Important deregulation of long non-coding RNAs (lncRNAs) expression has been reported in MM patients, influencing progression and therapy resistance. NEAT1 is a lncRNA essential for nuclear paraspeckles and involved in gene expression regulation. We showed that NEAT1 supports MM proliferation making this lncRNA an attractive therapeutic candidate. Here, we used a combinatorial strategy integrating transcriptomic and computational approaches with functional high-throughput drug screening, to identify compounds that synergize with NEAT1 inhibition in restraining MM cells growth. AUKA inhibitors were identified as top-scoring drugs in these analyses. We showed that the combination of NEAT1 silencing and AURKA inhibitors in MM profoundly impairs microtubule organization and mitotic spindle assembly, finally leading to cell death. Analysis of the large publicly CoMMpass dataset showed that in MM patients AURKA expression is strongly associated with reduced progression-free (p < 0.0001) and overall survival probability (p < 0.0001) and patients displaying high expression levels of both NEAT1 and AURKA have a worse clinical outcome. Finally, using RNA-sequencing data from NEAT1 knockdown (KD) MM cells, we identified the AURKA allosteric regulator TPX2 as a new NEAT1 target in MM and as a mediator of the interplay between AURKA and NEAT1, therefore providing a possible explanation of the synergistic activity observed upon their combinatorial inhibition.
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The primary function of 25(OH)Vitamin D (VitD) is to control calcium; however, recent evidence associated serum VitD deficiency to high aggressiveness and worse outcome in different type of malignancies including lymphomas, and the reasons of such effect are to be defined. In this study, we investigated the association of VitD blood levels with gene expression in a retrospective cohort of 181 lymphomas (104 diffuse large B-cell lymphomas [DLBCLs] and 77 classical Hodgkin's lymphomas [cHLs]) of whom 116 with available gene expression profiles (52 DLBCLs and 64 cHLs, respectively). In DLBCL, VitD deficiency did not cause significant alteration in gene expression suggesting different mechanisms of action including a possible systemic effect or an effect on pharmacokinetics. By contrast, in cHLs, VitD deficiency induced profound changes in the transcriptional program leading to the NF-κB-mediated activation of stress-protective and pro-survival pathways. Coherently, VitD signaling defined by vitamin D Receptor (VDR) expression analysis, resulted highly activated in cHLs but not in DLBCLs. Even if preliminary, these data represent the first evidence of a direct role of VitD in the biology of cHL and suggest a multimodality and disease-specific activity of this vitamin in lymphomas.
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Doença de Hodgkin/tratamento farmacológico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Vitamina D/uso terapêutico , Adulto , Humanos , Transcriptoma , Vitamina D/sangue , Vitamina D/farmacologiaRESUMO
Aberrant reactivation of embryonic pathways is a common feature of cancer. RUNX2 is a transcription factor crucial during embryogenesis that is aberrantly reactivated in many tumors, including thyroid and breast cancer, where it promotes aggressiveness and metastatic spreading. Currently, the mechanisms driving RUNX2 expression in cancer are still largely unknown. Here we showed that RUNX2 transcription in thyroid and breast cancer requires the cooperation of three distantly located enhancers (ENHs) brought together by chromatin three-dimensional looping. We showed that BRD4 controls RUNX2 by binding to the newly identified ENHs and we demonstrated that the anti-proliferative effects of bromodomain inhibitors (BETi) is associated with RUNX2 transcriptional repression. We demonstrated that each RUNX2 ENH is potentially controlled by a distinct set of TFs and we identified c-JUN as the principal pivot of this regulatory platform. We also observed that accumulation of genetic mutations within these elements correlates with metastatic behavior in human thyroid tumors. Finally, we identified RAINs, a novel family of ENH-associated long non-coding RNAs, transcribed from the identified RUNX2 regulatory unit. Our data provide a new model to explain how RUNX2 expression is reactivated in thyroid and breast cancer and how cancer-driving signaling pathways converge on the regulation of this gene.
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Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-jun/genética , Fatores de Transcrição/genética , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos/genética , Humanos , Células MCF-7 , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/metabolismoRESUMO
The identification of molecules that can reliably detect the presence of a tumor or predict its behavior is one of the biggest challenges of research in cancer biology. Biological fluids are intriguing mediums, containing many molecules that express the individual health status and, accordingly, may be useful in establishing the potential risk of cancer, defining differential diagnosis and prognosis, predicting the response to treatment, and monitoring the disease progression. The existence of circulating soluble growth factor receptors (sGFRs) deriving from their membrane counterparts has stimulated the interest of researchers to investigate the use of such molecules as potential cancer biomarkers. But what are the origins of circulating sGFRs? Are they naturally occurring molecules or tumor-derived products? Among these, the epidermal growth factor receptor (EGFR) is a cell-surface molecule significantly involved in cancer development and progression; it can be processed into biological active soluble isoforms (sEGFR). We have carried out an extensive review of the currently available literature on the sEGFRs and their mechanisms of regulation and biological function, with the intent to clarify the role of these molecules in cancer (and other pathological conditions) and, on the basis of the retrieved evidences, speculate about their potential use in the clinical setting.
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Biomarcadores Tumorais/sangue , Receptores ErbB/sangue , Neoplasias/diagnóstico , Processamento Alternativo , Receptores ErbB/fisiologia , Humanos , Modelos Biológicos , Neoplasias/sangue , Neoplasias/patologia , Medicina de Precisão , Prognóstico , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Endometrial cancer (EC) is the most common gynecologic tumor and the world's fourth most common cancer in women. Most patients respond to first-line treatments and have a low risk of recurrence, but refractory patients, and those with metastatic cancer at diagnosis, remain with no treatment options. Drug repurposing aims to discover new clinical indications for existing drugs with known safety profiles. It provides ready-to-use new therapeutic options for highly aggressive tumors for which standard protocols are ineffective, such as high-risk EC. METHODS: Here, we aimed at defining new therapeutic opportunities for high-risk EC using an innovative and integrated computational drug repurposing approach. RESULTS: We compared gene-expression profiles, from publicly available databases, of metastatic and non-metastatic EC patients being metastatization the most severe feature of EC aggressiveness. A comprehensive analysis of transcriptomic data through a two-arm approach was applied to obtain a robust prediction of drug candidates. CONCLUSIONS: Some of the identified therapeutic agents are already successfully used in clinical practice to treat other types of tumors. This highlights the potential to repurpose them for EC and, therefore, the reliability of the proposed approach.
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Reposicionamento de Medicamentos , Neoplasias do Endométrio , Humanos , Feminino , Reposicionamento de Medicamentos/métodos , Reprodutibilidade dos Testes , Neoplasias do Endométrio/patologia , Perfilação da Expressão Gênica , TranscriptomaRESUMO
Mortality from vmelanoma is associated with metastatic disease, but the mechanisms leading to spreading of the cancer cells remain obscure. Spatial profiling revealed that melanoma is characterized by a high degree of heterogeneity, which is established by the ability of melanoma cells to switch between different phenotypical stages. This plasticity, likely a heritage from embryonic pathways, accounts for a relevant part of the metastatic potential of these lesions, and requires the rapid and efficient reorganization of the transcriptional landscape of melanoma cells. A large part of the non-coding genome cooperates to control gene expression, specifically through the activity of enhancers (ENHs). In this study, we aimed to identify ex vivo the network of active ENHs and to outline their cooperative interactions in supporting transcriptional adaptation during melanoma metastatic progression. We conducted a genome-wide analysis to map active ENHs distribution in a retrospective cohort of 39 melanoma patients, comparing the profiles obtained in primary (N = 19) and metastatic (N = 20) melanoma lesions. Unsupervised clustering showed that the profile for acetylated histone H3 at lysine 27 (H3K27ac) efficiently segregates lesions into three different clusters corresponding to progressive stages of the disease. We reconstructed the map of super-ENHs (SEs) and cooperative ENHs that associate with metastatic progression in melanoma, which showed that cooperation among regulatory elements is a mandatory requirement for transcriptional plasticity. We also showed that these elements carry out specialized and non-redundant functions, and indicated the existence of a hierarchical organization, with SEs on top as masterminds of the entire transcriptional program and classical ENHs as executors. By providing an innovative vision of how the chromatin landscape of melanoma works during metastatic spreading, our data also point out the need to integrate functional profiling in the analysis of cancer lesions to increase definition and improve interpretation of tumor heterogeneity.
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Melanoma , Humanos , Melanoma/genética , Melanoma/metabolismo , Estudos Retrospectivos , Histonas/metabolismo , CromatinaRESUMO
Anaplastic Thyroid Cancer (ATC) is the most aggressive and de-differentiated subtype of thyroid cancer. Many studies hypothesized that ATC derives from Differentiated Thyroid Carcinoma (DTC) through a de-differentiation process triggered by specific molecular events still largely unknown. E2F7 is an atypical member of the E2F family. Known as cell cycle inhibitor and keeper of genomic stability, in specific contexts its function is oncogenic, guiding cancer progression. We performed a meta-analysis on 279 gene expression profiles, from 8 Gene Expression Omnibus patient samples datasets, to explore the causal relationship between DTC and ATC. We defined 3 specific gene signatures describing the evolution from normal thyroid tissue to DTC and ATC and validated them in a cohort of human surgically resected ATCs collected in our Institution. We identified E2F7 as a key player in the DTC-ATC transition and showed in vitro that its down-regulation reduced ATC cells' aggressiveness features. RNA-seq and ChIP-seq profiling allowed the identification of the E2F7 specific gene program, which is mainly related to cell cycle progression and DNA repair ability. Overall, this study identified a signature describing DTC de-differentiation toward ATC subtype and unveiled an E2F7-dependent transcriptional program supporting this process.
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Adenocarcinoma , Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Humanos , Carcinoma Anaplásico da Tireoide/genética , Carcinoma Anaplásico da Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma/genética , Diferenciação Celular/genética , Oncogenes/genética , Fator de Transcrição E2F7/genéticaRESUMO
The CCAAT/enhancer binding protein α (C/EBPα) is a transcription factor required for differentiation of myeloid progenitors. In acute myeloid leukemia (AML) cells expressing the constitutively active FLT3-ITD receptor tyrosine kinase, MAP kinase-dependent phosphorylation of serine 21 (S21) inhibits the ability of C/EBPα to induce granulocytic differentiation. To assess whether this post-translational modification also modulates the activity of C/EBPα in BCR/ABL-expressing cells, we tested the biological effects of wild-type and mutant C/EBPα mimicking phosphorylated or non-phosphorylatable serine 21 (S21D and S21A, respectively) in K562 cells ectopically expressing tamoxifen-regulated C/EBPα-ER chimeric proteins. We show here that S21D C/EBPα-ER induced terminal granulocytic differentiation of K562 cells almost as well as wild-type C/EBPα-ER, while S21A C/EBPα-ER was less efficient. Furthermore, wild-type C/EBPα suppressed the proliferation and colony formation of K562 cells vigorously, while S21D and S21A C/EBPα mutants had more modest anti-proliferative effects. Both mutants were less effective than wild-type C/EBPα in suppressing endogenous E2F-dependent transactivation and bound less E2F-2 and/or E2F-3 proteins in anti-C/EBPα immunoprecipitates. Together, these findings suggest that mutation of S21 more than its phosphorylation inhibits the anti-proliferative effects of C/EBPα due to reduced interaction with or impaired regulation of the activity of E2F proteins. By contrast, phosphorylation of serine 21 appears to have a modest role in modulating the differentiation-inducing effects of C/EBPα in K562 cells.
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Proteína alfa Estimuladora de Ligação a CCAAT/química , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Benzamidas/farmacologia , Proteína alfa Estimuladora de Ligação a CCAAT/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Proliferação de Células , Primers do DNA/genética , Fatores de Transcrição E2F/metabolismo , Granulócitos/metabolismo , Granulócitos/patologia , Humanos , Células K562 , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Fosforilação , Regiões Promotoras Genéticas , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/química , Ensaio Tumoral de Célula-TroncoRESUMO
BACKGROUND: Anaplastic Thyroid Cancer (ATC) is an undifferentiated and aggressive tumor that often originates from well-Differentiated Thyroid Carcinoma (DTC) through a trans-differentiation process. Epithelial-to-Mesenchymal Transition (EMT) is recognized as one of the major players of this process. OVOL2 is a transcription factor (TF) that promotes epithelial differentiation and restrains EMT during embryonic development. OVOL2 loss in some types of cancers is linked to aggressiveness and poor prognosis. Here, we aim to clarify the unexplored role of OVOL2 in ATC. METHODS: Gene expression analysis in thyroid cancer patients and cell lines showed that OVOL2 is mainly associated with epithelial features and its expression is deeply impaired in ATC. To assess OVOL2 function, we established an OVOL2-overexpression model in ATC cell lines and evaluated its effects by analyzing gene expression, proliferation, invasion and migration abilities, cell cycle, specific protein localization through immunofluorescence staining. RNA-seq profiling showed that OVOL2 controls a complex network of genes converging on cell cycle and mitosis regulation and Chromatin Immunoprecipitation identified new OVOL2 target genes. RESULTS: Coherently with its reported function, OVOL2 re-expression restrained EMT and aggressiveness in ATC cells. Unexpectedly, we observed that it caused G2/M block, a consequent reduction in cell proliferation and an increase in cell death. This phenotype was associated to generalized abnormalities in the mitotic spindle structure and cytoskeletal organization. By RNA-seq experiments, we showed that many pathways related to cytoskeleton and migration, cell cycle and mitosis are profoundly affected by OVOL2 expression, in particular the RHO-GTPase pathway resulted as the most interesting. We demonstrated that RHO GTPase pathway is the central hub of OVOL2-mediated program in ATC and that OVOL2 transcriptionally inhibits RhoU and RhoJ. Silencing of RhoU recapitulated the OVOL2-driven phenotype pointing to this protein as a crucial target of OVOL2 in ATC. CONCLUSIONS: Collectively, these data describe the role of OVOL2 in ATC and uncover a novel function of this TF in inhibiting the RHO GTPase pathway interlacing its effects on EMT, cytoskeleton dynamics and mitosis.
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Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Mitose , Gravidez , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Malignant pleural mesothelioma (MPM) is a rare and incurable cancer, which incidence is increasing in many countries. MPM escapes the classical genetic model of cancer evolution, lacking a distinctive genetic fingerprint. Omics profiling revealed extensive heterogeneity failing to identify major vulnerabilities and restraining development of MPM-oriented therapies. Here, we performed a multilayered analysis based on a functional genome-wide CRISPR/Cas9 screening integrated with patients molecular and clinical data, to identify new non-genetic vulnerabilities of MPM. We identified a core of 18 functionally-related genes as essential for MPM cells. The chromatin reader KAP1 emerged as a dependency of MPM. We showed that KAP1 supports cell growth by orchestrating the expression of a G2/M-specific program, ensuring mitosis correct execution. Targeting KAP1 transcriptional function, by using CDK9 inhibitors resulted in a dramatic loss of MPM cells viability and shutdown of the KAP1-mediated program. Validation analysis on two independent MPM-patients sets, including a consecutive, retrospective cohort of 97 MPM, confirmed KAP1 as new non-genetic dependency of MPM and proved the association of its dependent gene program with reduced patients' survival probability. Overall these data: provided new insights into the biology of MPM delineating KAP1 and its target genes as building blocks of its clinical aggressiveness.
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The transcription factor C/EBPα is more potent than C/EBPß in inducing granulocitic differentiation and inhibiting BCR/ABL-expressing cells. We took a "domain swapping" approach to assess biological effects, modulation of gene expression, and binding to C/EBPα-regulated promoters by wild-type and chimeric C/EBPα/C/EBPß proteins. Wild-type and N-C/EBPα+ C/EBPß-DBD induced transcription of the granulocyte-colony stimulating factor receptor (G-CSFR) gene, promoted differentiation, and suppressed proliferation of K562 cells vigorously; instead, wild-type C/EBPß and N-C/EBPß+C/EBPα-DBD had modest effects, although they bound the G-CSFR promoter like wild-type C/EBPα and N-C/EBPα+C/EBPß-DBD. Chimeric proteins consisting of the TAD of VP16 and the DBD of C/EBPα or C/EBPß inhibited proliferation and induced differentiation of K562 cells as effectively as wild-type C/EBPα. Gene expression profiles induced by C/EBPα resembled those modulated by N-C/EBPα+C/EBPß-DBD, whereas C/EBPß induced a pattern similar to that of N-C/EBPß+C/EBPα-DBD. C/EBPα activation induced changes in the expression of more cell cycle- and apoptosis-related genes than the other proteins and enhanced Imatinib-induced apoptosis of K562 cells. Expression of FOXO3a, a novel C/EBPα-regulated gene, was required for apoptosis but not for differentiation induction or proliferation inhibition of K562 cells.
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Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Ciclo Celular , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Transcrição Gênica , Apoptose/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Diferenciação Celular/genética , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fator Estimulador de Colônias de Granulócitos/biossíntese , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Células K562 , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Objective: Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, crucial prognostic factors are no gross residual disease and centralization of cases. To evaluate the centralization of EOC patients, we report the results of a survey that shows the daily management of EOC patients in Italy. Methods: A 49-items electronic unblinded survey assessing demographics, practice characteristics, current opinions and approach to managing advanced EOC at first diagnosis was sent both to general gynecologists (GG) and gynecologic oncologists (GO). Differences in frequency distribution of answers between gynecologists with different expertise were evaluated using Fisher exact test. Multivariable analyses were performed applying generalized linear models. Results: 84/192 (44%) GG and 108/192 (56%) GO from all Italian regions answered to our survey. GOs declared to perform fertility sparing surgery in early EOC more frequently than GG (p=0.002). GOs can perform a frozen section and have both a gynecopathologist and a dedicated general surgeon. 89% of GOs consider as "optimal debulking" no gross residual disease and 81% achieve this at upfront cytoreduction in more than 40% of patients. Use of neoadjuvant chemotherapy decreases in higher volume centers (p<0.001) while it is lower in the group of GOs than in the GGs group (p<0.001). Conclusions: EOC patients are still treated by GGs. GOs perform more upfront surgery and achieve optimal debulking in a greater percentage of patients than GGs. In Italy an adequate centralization of cases has not yet been achieved, and this may have detrimental effects on the quality of treatment.
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Deregulation of chromatin modifiers, including DNA helicases, is emerging as one of the mechanisms underlying the transformation of anaplastic lymphoma kinase negative (ALK-) anaplastic large cell lymphoma (ALCL). We recently identified the DNA-helicase HELLS as central for proficient ALK-ALCL proliferation and progression. Here we assessed in detail its function by performing RNA-sequencing profiling coupled with bioinformatic prediction to identify HELLS targets and transcriptional cooperators. We demonstrated that HELLS, together with the transcription factor YY1, contributes to an appropriate cytokinesis via the transcriptional regulation of genes involved in cleavage furrow regulation. Binding target promoters, HELLS primes YY1 recruitment and transcriptional activation of cytoskeleton genes including the small GTPases RhoA and RhoU and their effector kinase Pak2. Single or multiple knockdowns of these genes reveal that RhoA and RhoU mediate HELLS effects on cell proliferation and cell division of ALK-ALCLs. Collectively, our work demonstrates the transcriptional role of HELLS in orchestrating a complex transcriptional program sustaining neoplastic features of ALK-ALCL.
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Citocinese/genética , DNA Helicases/metabolismo , Linfoma Anaplásico de Células Grandes/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , DNA Helicases/genética , Humanos , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Ativação Transcricional , TransfecçãoRESUMO
Background: Papillary thyroid cancers (PTCs) are common, usually indolent malignancies. Still, a small but significant percentage of patients have aggressive tumors and develop distant metastases leading to death. Currently, it is not possible to discriminate aggressive lesions due to lack of prognostic markers. Long noncoding RNAs (lncRNAs), which are selectively expressed in a context-dependent manner, are expected to represent a new landscape to search for molecular discriminants. Transforming growth factor ß (TGFß) is a multifunctional cytokine that fosters epithelial-to-mesenchymal transition and metastatic spreading. In PTCs, it triggers the expression of the metastatic marker Cadherin 6 (CDH6). Here, we investigated the TGFß-dependent lncRNAs that may cooperate to potentiate PTC aggressiveness. Methods: We used a genome-wide approach to map enhancer (ENH)-associated lncRNAs under TGFß control. Linc00941 was selected and validated using functional in vitro assays. A combined approach using bioinformatic analyses of the thyroid cancer (THCA)-the cancer genome atlas (TCGA) dataset and RNA-seq analysis was used to identify the processes in which linc00941 was involved in and the genes under its regulation. Correlation with clinical data was performed to evaluate the potential of this lncRNA and its targets as prognostic markers in THCA. Results: Linc00941 was identified as transcribed starting from one of the TGFß-induced ENHs. Linc00941 expression was significantly higher in aggressive cancer both in the TCGA dataset and in a separate validation cohort from our institution. Loss of function assays for linc00941 showed that it promotes response to stimuli and invasiveness while restraining proliferation in PTC cells, a typical phenotype of metastatic cells. From the integration of TCGA data and linc00941 knockdown RNA-seq profiling, we identified 77 genes under the regulation of this lncRNA. Among these, we found the prometastatic gene CDH6. Linc00941 knockdown partially recapitulates the effects observed upon CDH6 silencing, promoting cell cytoskeleton and membrane adhesions rearrangements and autophagy. The combined expression of CDH6 and linc00941 is a distinctive feature of highly aggressive PTC lesions. Conclusions: Our data provide new insights into the biology driving metastasis in PTCs and highlight how lncRNAs cooperate with coding transcripts to sustain these processes.
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Caderinas/metabolismo , Movimento Celular , RNA Longo não Codificante/metabolismo , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Proliferação de Células , Bases de Dados Genéticas , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Invasividade Neoplásica , RNA Longo não Codificante/genética , Transdução de Sinais , Câncer Papilífero da Tireoide/genética , Câncer Papilífero da Tireoide/secundário , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Fator de Crescimento Transformador beta/genéticaRESUMO
According to our systematic literature review (PRISMA guidelines), only 37 vulvar squamous cell carcinomas (VSCCs) were diagnosed during pregnancy (age range: 17-41 years). The tumor size range was 0.3-15 cm. The treatment was performed after (14/37, 38%), before (10/37, 27%), or before-and-after delivery (11/37, 30%). We found that 21/37 (57%) cases were stage I, 2 II (5%), 11 III (30%), and 3 IVB (8%). HPV-related features (condylomas/warts; HPV infection; high-grade squamous intraepithelial lesion) were reported in 11/37 (30%) cases. We also found that 9/37 (24%) patients had inflammatory conditions (lichen sclerosus/planus, psoriasis, chronic dermatitis). The time-to-recurrence/progression (12/37, 32%) ranged from 0 to 36 (mean 9) months. Eight women died of disease (22%) 2.5-48 months after diagnosis, 2 (5%) were alive with disease, and 23 (62%) were disease-free at the end of follow-up. Pregnant patients must be followed-up. Even if they are small, newly arising vulvar lesions should be biopsied, especially in women with risk factors (HPV, dermatosis, etc.). The treatment of VSCCs diagnosed in late third trimester might be delayed until postpartum. Elective cesarean section may prevent vulvar wound dehiscence. In the few reported cases, pregnancy/fetal outcomes seemed to not be affected by invasive treatments during pregnancy. However, clinicians must be careful; larger cohorts should define the best treatment. Definite guidelines are lacking, so a multidisciplinary approach and discussion with patients are mandatory.
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The molecular mechanisms leading to the transformation of anaplastic lymphoma kinase negative (ALK-) anaplastic large cell lymphoma (ALCL) have been only in part elucidated. To identify new culprits which promote and drive ALCL, we performed a total transcriptome sequencing and discovered 1208 previously unknown intergenic long noncoding RNAs (lncRNAs), including 18 lncRNAs preferentially expressed in ALCL. We selected an unknown lncRNA, BlackMamba, with an ALK- ALCL preferential expression, for molecular and functional studies. BlackMamba is a chromatin-associated lncRNA regulated by STAT3 via a canonical transcriptional signaling pathway. Knockdown experiments demonstrated that BlackMamba contributes to the pathogenesis of ALCL regulating cell growth and cell morphology. Mechanistically, BlackMamba interacts with the DNA helicase HELLS controlling its recruitment to the promoter regions of cell-architecture-related genes, fostering their expression. Collectively, these findings provide evidence of a previously unknown tumorigenic role of STAT3 via a lncRNA-DNA helicase axis and reveal an undiscovered role for lncRNA in the maintenance of the neoplastic phenotype of ALK-ALCL.
Assuntos
Quinase do Linfoma Anaplásico/deficiência , DNA Helicases/genética , Regulação Neoplásica da Expressão Gênica , Linfoma Anaplásico de Células Grandes/genética , Linfoma Anaplásico de Células Grandes/patologia , Fenótipo , RNA Longo não Codificante , Biópsia , Linhagem Celular Tumoral , Proliferação de Células , Evolução Clonal , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , MicroRNAs/genética , Modelos Biológicos , Regiões Promotoras Genéticas , Interferência de RNARESUMO
Histone DeACetylases (HDACs) are enzymes that remove acetyl groups from histones and other proteins, regulating the expression of target genes. Pharmacological inhibition of these enzymes re-shapes chromatin acetylation status, confusing boundaries between transcriptionally active and quiescent chromatin. This results in reinducing expression of silent genes while repressing highly transcribed genes. Bromodomain and Extraterminal domain (BET) proteins are readers of acetylated chromatin status and accumulate on transcriptionally active regulatory elements where they serve as scaffold for the building of transcription-promoting complexes. The expression of many well-known oncogenes relies on BET proteins function, indicating BET inhibition as a strategy to counteract their activity. BETi and HDACi share many common targets and affect similar cellular processes to the point that combined inhibition of both these classes of proteins is regarded as a strategy to improve the effectiveness of these drugs in cancer. In this work, we aim to discuss the molecular basis of the interplay between HDAC and BET proteins, pointing at chromatin acetylation as a crucial node of their functional interaction. We will also describe the state of the art of their dual inhibition in cancer therapy. Finally, starting from their mechanism of action we will provide a speculative perspective on how these drugs may be employed in combination with standard therapies to improve effectiveness and/or overcome resistance.
RESUMO
BACKGROUND: RUNX2 is a Runt-related transcription factor required during embryogenesis for skeletal development and morphogenesis of other organs including thyroid and breast gland. Consistent evidence indicates that RUNX2 expression is aberrantly reactivated in cancer and supports tumor progression. The mechanisms leading to RUNX2 expression in cancer has only recently began to emerge. Previously, we showed that suppressing the activity of the epigenetic regulators HDACs significantly represses RUNX2 expression highlighting a role for these enzymes in RUNX2 reactivation in cancer. However, the molecular mechanisms by which HDACs control RUNX2 are still largely unexplored. Here, to fill this gap, we investigated the role of different HDACs in RUNX2 expression regulation in breast and thyroid cancer, tumors that majorly rely on RUNX2 for their development and progression. METHODS: Proliferation assays and evaluation of RUNX2 mRNA levels by qRT-PCR were used to evaluate the effect of several HDACi and specific siRNAs on a panel of cancer cell lines. Moreover, ChIP and co-IP assays were performed to elucidate the molecular mechanism underneath the RUNX2 transcriptional regulation. Finally, RNA-sequencing unveiled a new subset of genes whose transcription is regulated by the complex RUNX2-HDAC6. RESULTS: In this study, we showed that Class I HDACs and in particular HDAC1 are required for RUNX2 efficient transcription in cancer. Furthermore, we found an additional and cell-specific function of HDAC6 in driving RUNX2 expression in thyroid cancer cells. In this model, HDAC6 likely stabilizes the assembly of the transcriptional complex, which includes HDAC1, on the RUNX2 P2 promoter potentiating its transcription. Since a functional interplay between RUNX2 and HDAC6 has been suggested, we used RNA-Seq profiling to consolidate this evidence in thyroid cancer and to extend the knowledge on this cooperation in a setting in which HDAC6 also controls RUNX2 expression. CONCLUSIONS: Overall, our data provide new insights into the molecular mechanisms controlling RUNX2 in cancer and consolidate the rationale for the use of HDACi as potential pharmacological strategy to counteract the pro-oncogenic program controlled by RUNX2 in cancer cells.
Assuntos
Comunicação Celular , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Regulação Neoplásica da Expressão Gênica , Histona Desacetilases/metabolismo , Neoplasias/etiologia , Neoplasias/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Modelos Biológicos , Complexos Multiproteicos , Neoplasias/patologia , Ligação Proteica , RNA Interferente Pequeno/genética , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Transcrição GênicaRESUMO
PURPOSE: Pulmonary sarcomatoid carcinoma (PSC) is a rare and aggressive form of NSCLC. Rarity and poor characterization have limited the development of PSC-tailored treatment protocols, leaving patients with inadequate therapeutic options. In this study, we investigated the gene expression profile of PSCs, with the aim to characterize the molecular mechanisms responsible for their evolution and to identify new drugs for their treatment. EXPERIMENTAL DESIGN: A training set of 17 biphasic PSCs was selected and tested for the expression of a large panel of 770 genes related to cancer progression using NanoString technology. Computational analyses were used to characterize a PSCs-gene specific signature from which pathways and drivers of PSC evolution were identified and validated using functional assays in vitro. This signature was validated in a separate set of 15 PSCs and 8 differentiated NSCLC and used to interrogate the cMAP database searching for FDA-approved small molecules able to counteract PSC phenotype. RESULTS: We demonstrated that the transcriptional activation of an epithelial mesenchymal transition (EMT) program drives PSC phylogeny in vivo. We showed that loss of the epithelial-associated transcription factor (TF) OVOL2 characterizes the transition to sarcomatoid phenotype triggering the expression of EMT promoting TFs, including TWIST and ZEB and the expression of the membrane kinase DDR2. Finally, using a drug repurposing approach, we identified dasatinib as potential inhibitor of the PSC-gene expression signature and we confirmed in vitro that this drug efficiently restrains proliferation and reverts the sarcomatoid-associated phenotype. CONCLUSIONS: Our data provide new insights into PSC evolution and provide the rationale for further clinical studies with dasatinib.