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BACKGROUND: Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are oncogenic drivers. Using the Auria Biobank in Finland, we aimed to identify and characterize patients with these gene fusions, and describe their clinical and tumor characteristics, treatments received, and outcomes. MATERIAL AND METHODS: We evaluated pediatrics with any solid tumor type and adults with colorectal cancer (CRC), non-small cell lung cancer (NSCLC), sarcoma, or salivary gland cancer. We determined tropomyosin receptor kinase (TRK) protein expression by pan-TRK immunohistochemistry (IHC) staining of tumor samples from the Auria Biobank, scored by a certified pathologist. NTRK gene fusion was confirmed by next generation sequencing (NGS). All 2,059 patients were followed-up starting 1 year before their cancer diagnosis. RESULTS: Frequency of NTRK gene fusion tumors was 3.1% (4/127) in pediatrics, 0.7% (8/1,151) for CRC, 0.3% (1/288) for NSCLC, 0.9% (1/114) for salivary gland cancer, and 0% (0/379) for sarcoma. Among pediatrics there was one case each of fibrosarcoma (TPM3::NTRK1), Ewing's sarcoma (LPPR1::NTRK2), primitive neuroectodermal tumor (DAB2IP::NTRK2), and papillary thyroid carcinoma (RAD51B::NTRK3). Among CRC patients, six harbored tumors with NTRK1 fusions (three fused with TPM3), one harbored a NTRK3::GABRG1 fusion, and the other a NTRK2::FXN/LPPR1 fusion. Microsatellite instability was higher in CRC patients with NTRK gene fusion tumors versus wild-type tumors (50.0% vs. 4.4%). Other detected fusions were SGCZ::NTRK3 (NSCLC) and ETV6::NTRK3 (salivary gland cancer). Four patients (three CRC, one NSCLC) received chemotherapy; one patient (with CRC) received radiotherapy. CONCLUSION: NTRK gene fusions are rare in adult CRC, NSCLC, salivary tumors, sarcoma, and pediatric solid tumors.
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Receptor trkA , Receptor trkC , Humanos , Finlândia/epidemiologia , Masculino , Criança , Feminino , Adulto , Pessoa de Meia-Idade , Adolescente , Receptor trkA/genética , Pré-Escolar , Adulto Jovem , Receptor trkC/genética , Idoso , Bancos de Espécimes Biológicos , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Fusão Gênica , Sarcoma/genética , Sarcoma/patologia , Neoplasias das Glândulas Salivares/genética , Neoplasias das Glândulas Salivares/patologia , Receptor trkB/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Lactente , Proteínas de Fusão Oncogênica/genética , Neoplasias/genética , Neoplasias/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Glicoproteínas de MembranaRESUMO
The study of marine natural products for their bioactive potential has gained strength in recent years. Oceans harbor a vast variety of organisms that offer a biological and chemical diversity with metabolic abilities unrivalled in terrestrial systems, which makes them an attractive target for bioprospecting as an almost untapped resource of biotechnological applications. Among them, there is no doubt that microalgae could become genuine "cell factories" for the biological synthesis of bioactive substances. Thus, in the course of inter-laboratory collaboration sponsored by the European Union (7th FP) into the MAREX Project focused on the discovery of novel bioactive compounds of marine origin for the European industry, a bioprospecting study on 33 microalgae strains was carried out. The strains were cultured at laboratory scale. Two extracts were prepared for each one (biomass and cell free culture medium) and, thus, screened to provide information on the antimicrobial, the anti-proliferative, and the apoptotic potential of the studied extracts. The outcome of this study provides additional scientific data for the selection of Alexadrium tamarensis WE, Gambierdiscus australes, Prorocentrum arenarium, Prorocentrum hoffmannianum, and Prorocentrum reticulatum (Pr-3) for further investigation and offers support for the continued research of new potential drugs for human therapeutics from cultured microalgae.
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Antibacterianos/farmacologia , Fatores Biológicos/farmacologia , Bioprospecção , Descoberta de Drogas , Microalgas/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/metabolismo , Apoptose/efeitos dos fármacos , Fatores Biológicos/isolamento & purificação , Fatores Biológicos/metabolismo , Biotecnologia/métodos , Proliferação de Células/efeitos dos fármacos , Oceanos e MaresRESUMO
MicroRNAs (miRNAs) are endogenous non-coding RNAs, which play an essential role in the regulation of gene expression during carcinogenesis. The role of miRNAs in breast cancer has been thoroughly investigated, and although many miRNAs are identified as cancer related, little is known about their involvement in benign tumors. In this study, we investigated miRNA expression profiles in the two most common types of human benign tumors (fibroadenoma/fibroadenomatosis) and in malignant breast tumors and explored their role as oncomirs and tumor suppressor miRNAs. Here, we identified 33 miRNAs with similar deregulated expression in both benign and malignant tumors compared with the expression levels of those in normal tissue, including breast cancer-related miRNAs such as let-7, miR-21 and miR-155. Additionally, messenger RNA (mRNA) expression profiles were obtained for some of the same samples. Using integrated mRNA/miRNA expression analysis, we observed that overexpression of certain miRNAs co-occurred with a significant downregulation of their candidate target mRNAs in both benign and malignant tumors. In support of these findings, in vitro functional screening of the downregulated miRNAs in non-malignant and breast cancer cell lines identified several possible tumor suppressor miRNAs, including miR-193b, miR-193a-3p, miR-126, miR-134, miR-132, miR-486-5p, miR-886-3p, miR-195 and miR-497, showing reduced growth when re-expressed in cancer cells. The finding of deregulated expression of oncomirs and tumor suppressor miRNAs in benign breast tumors is intriguing, indicating that they may play a role in proliferation. A role of cancer-related miRNAs in the early phases of carcinogenesis and malignant transformation can, therefore, not be ruled out.
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Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Feminino , Genes Supressores de Tumor , Humanos , RNA Mensageiro , Valores de ReferênciaRESUMO
Selective tropomyosin receptor kinase (TRK) inhibitors are approved targeted therapies for patients with solid tumors harboring a neurotrophic tyrosine receptor kinase (NTRK) gene fusion. Country-specific estimates of NTRK gene fusion frequency, and knowledge on the characteristics of affected patients, are limited. We identified patients with histologically-confirmed papillary thyroid cancer (PTC) from Finland's Auria Biobank. TRK protein expression was determined by pan-TRK immunohistochemistry. Immuno-stained tumor samples were scored by a certified pathologist. Gene fusions and other co-occurring gene alterations were identified by next generation sequencing. Patient characteristics and vital status were determined from linked hospital electronic health records (EHRs). Patients were followed from 1 year before PTC diagnosis until death. 6/389 (1.5%) PTC patients had an NTRK gene fusion (all NTRK3); mean age 43.8 years (and none had comorbidities) at PTC diagnosis. Gene fusion partners were EML4 (n = 3), ETV6 (n = 2), and RBPMS (n = 1). Of 3/6 patients with complete EHRs, all received radioactive iodine ablation only and were alive at end of follow-up (median observation, 9.12 years). In conclusion, NTRK gene fusion is infrequent in patients with PTC. Linkage of biobank samples to EHRs is feasible in describing the characteristics and outcomes of patients with PTC and potentially other cancer types.
Assuntos
Bancos de Espécimes Biológicos , Receptores de Aminoácido , Neoplasias da Glândula Tireoide , Humanos , Adulto , Câncer Papilífero da Tireoide/genética , Finlândia , Radioisótopos do Iodo , Neoplasias da Glândula Tireoide/genética , Fusão GênicaRESUMO
Identification of protein targets for microRNAs (miRNAs) is a significant challenge due to the complexity of miRNA-mediated regulation. We have previously demonstrated that miR-193b targets estrogen receptor-α (ERα) and inhibits estrogen-induced growth of breast cancer cells. Here, we applied a high-throughput strategy using quantitative iTRAQ (isobaric tag for relative and absolute quantitation) reagents to identify other target proteins regulated by miR-193b in breast cancer cells. iTRAQ analysis of pre-miR-193b transfected MCF-7 cells resulted in identification of 743 unique proteins, of which 39 were down-regulated and 44 up-regulated as compared with negative control transfected cells. Computationally predicted targets of miR-193b were highly enriched (sevenfold) among the proteins whose level of expression decreased after miR-193b transfection. Only a minority of these (13%) showed similar effect at the mRNA level illustrating the importance of post-transcriptional regulation. The most significantly repressed proteins were selected for validation experiments. These data confirmed 14-3-3ζ (YWHAZ), serine hydroxyl transferase (SHMT2), and aldo-keto reductase family 1, member C2 (AKR1C2) as direct, previously uncharacterized, targets of miR-193b. Functional RNAi assays demonstrated that specific combinations of knockdowns of these target genes by siRNAs inhibited growth of MCF-7 cells, mimicking the effects of the miR-193b overexpression. Interestingly, the data imply that besides targeting ERα, the miR-193b effects include suppression of the local production of estrogens and other steroid hormones mediated by the AKR1C2 gene, thus provoking two separate molecular mechanisms inhibiting steroid-dependent growth of breast cancer cells. In conclusion, we present here a proteomic screen to identify targets of miR-193b, and a systems biological approach to mimic its effects at the level of cellular phenotypes. This led to the identification of multiple genes whose combinatorial knock-down likely mediates the strong anti-cancer effects observed for miR-193b in breast cancer cells.
Assuntos
Neoplasias da Mama/genética , MicroRNAs/metabolismo , Sequência de Bases , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Genes Reporter , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Humanos , Hidroliases , Hidroxiesteroide Desidrogenases/genética , Hidroxiesteroide Desidrogenases/metabolismo , Luciferases de Renilla/biossíntese , Luciferases de Renilla/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/genética , Modelos Genéticos , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteoma/genética , Proteoma/metabolismo , Interferência de RNA , Biologia de Sistemas , Tirosina 3-Mono-Oxigenase/genética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
BACKGROUND: Castration-resistant prostate cancer (CRPC) represents a therapeutic challenge for current medications. METHODS: In order to explore the molecular mechanisms involved in CRPC progression and to identify new therapeutic targets, we analyzed a unique sample set of 11 CRPCs and 7 advanced tumors by array-CGH and gene expression microarrays. The genome-wide DNA and RNA data were integrated to identify genes whose overexpression was driven by their amplification. To assess the functional role of these genes, their expression was analyzed in a transcriptional data set of 329 clinical prostate cancers and the corresponding gene products were silenced using RNA interference in prostate cancer cells. RESULTS: Six recurrent genetic targets were identified in the CRPCs; ATP1B1, AR, FAM110B, LAS1L, MYC, and YIPF6. In addition to AR and MYC, FAM110B emerged as a potential key gene involved in CRPC progression in a subset of the tumors. FAM110B was able to regulate AR signaling in prostate cancer cells and FAM110B itself was regulated by androgens. FAM110B siRNA inhibited the growth of prostate cancer cells in vitro, and this effect was substantially enhanced in androgen deficient conditions. Ectopic FAM110B expression in non-cancerous epithelial prostate cells induced aneuploidy and impaired antigen presentation. CONCLUSIONS: The DNA/RNA gene outlier detection combined with siRNA cell proliferation assay identified FAM110B as a potential growth promoting key gene for CRPC. FAM110B appears to have a key role in the androgen signaling and progression of CRPC impacting multiple cancer hallmarks and therefore highlighting a potential therapeutic target.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/metabolismo , Genômica , Neoplasias da Próstata/metabolismo , Interferência de RNA , Transcriptoma , Aneuploidia , Apresentação de Antígeno , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas Nucleares/genética , Orquiectomia , Próstata/imunologia , Próstata/metabolismo , Neoplasias da Próstata/imunologia , Proteínas Proto-Oncogênicas c-myc/genética , Receptores Androgênicos/genética , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The arachidonic acid and prostaglandin pathway has been implicated in prostate carcinogenesis, but comprehensive studies of the individual members in this key pathway are lacking. Here, we first conducted a systematic bioinformatic study of the expression of 36 arachidonic acid pathway genes across 9783 human tissue samples. The results showed that the PLA2G7, HPGD, EPHX2, and CYP4F8 genes are highly expressed in prostate cancer. Functional studies using RNA interference in prostate cancer cells indicated that all four genes are also essential for cell growth and survival. Clinical validation confirmed high PLA2G7 expression, especially in ERG oncogene-positive prostate cancers, and its silencing sensitized ERG-positive prostate cancer cells to oxidative stress. HPGD was highly expressed in androgen receptor (AR)-overexpressing advanced tumors, as well as in metastatic prostate cancers. EPHX2 mRNA correlated with AR in primary prostate cancers, and its inhibition in vitro reduced AR signaling and potentiated the effect of antiandrogen flutamide in cultured prostate cancer cells. In summary, we identified four novel putative therapeutic targets with biomarker potential for different subtypes of prostate cancer. In addition, our results indicate that inhibition of these enzymes may be particularly powerful when combined with other treatments, such as androgen deprivation or induction of oxidative stress.
Assuntos
Ácido Araquidônico/metabolismo , Terapia de Alvo Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Transdução de Sinais/genética , 1-Alquil-2-acetilglicerofosfocolina Esterase , Idoso , Idoso de 80 Anos ou mais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Proliferação de Células/efeitos dos fármacos , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/genética , Epóxido Hidrolases/metabolismo , Flutamida/farmacologia , Flutamida/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Humanos , Hidroxiprostaglandina Desidrogenases/genética , Hidroxiprostaglandina Desidrogenases/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Inibidores de Fosfolipase A2 , Fosfolipases A2/genética , Fosfolipases A2/metabolismo , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Detailed characterization of early pathophysiological changes in preclinical Alzheimer's disease (AD) is necessary to enable development of correctly targeted and timed disease-modifying treatments. ASIC-E4 study ("Beta-Amyloid, Synaptic loss, Inflammation and Cognition in healthy APOE ε4 carriers") combines state-of-the-art neuroimaging and fluid-based biomarker measurements to study the early interplay of three key pathological features of AD, i.e., beta-amyloid (Aß) deposition, neuroinflammation and synaptic dysfunction and loss in cognitively normal volunteers with three different levels of genetic (APOE-related) risk for late-onset AD. OBJECTIVE: Here, our objective is to describe the study design, used protocols and baseline demographics of the ASIC-E4 study. METHODS/DESIGN: ASIC-E4 is a prospective observational multimodal imaging study performed in Turku PET Centre in collaboration with University of Gothenburg. Cognitively normal 60-75-year-old-individuals with known APOE ε4/ε4 genotype were recruited via local Auria Biobank (Turku, Finland). Recruitment of the project has been completed in July 2020 and 63 individuals were enrolled to three study groups (Group 1: APOE ε4/ε4, N = 19; Group 2: APOE ε4/ε3, N = 22; Group 3: APOE ε3/ε3, N = 22). At baseline, all participants will undergo positron emission tomography imaging with tracers targeted against Aß deposition (11C-PIB), activated glia (11C-PK11195) and synaptic vesicle glycoprotein 2A (11C-UCB-J), two brain magnetic resonance imaging scans, and extensive cognitive testing. In addition, blood samples are collected for various laboratory measurements and blood biomarker analysis and cerebrospinal fluid samples are collected from a subset of participants based on additional voluntary informed consent. To evaluate the predictive value of the early neuroimaging findings, neuropsychological evaluation and blood biomarker measurements will be repeated after a 4-year follow-up period. DISCUSSION: Results of the ASIC-E4 project will bridge the gap related to limited knowledge of the synaptic and inflammatory changes and their association with each other and Aß in "at-risk" individuals. Thorough in vivo characterization of the biomarker profiles in this population will produce valuable information for diagnostic purposes and future drug development, where the field has already started to look beyond Aß.
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OBJECTIVES: To recontact biobank participants and collect cognitive, behavioural and lifestyle information via a secure online platform. DESIGN: Biobank-based recontacting pilot study. SETTING: Three Finnish biobanks (Helsinki, Auria, Tampere) recruiting participants from February 2021 to July 2021. PARTICIPANTS: All eligible invitees were enrolled in FinnGen by their biobanks (Helsinki, Auria, Tampere), had available genetic data and were >18 years old. Individuals with severe neuropsychiatric disease or cognitive or physical disabilities were excluded. Lastly, 5995 participants were selected based on their polygenic score for cognitive abilities and invited to the study. Among invitees, 1115 had successfully participated and completed the study questionnaire(s). OUTCOME MEASURES: The primary outcome was the participation rate among study invitees. Secondary outcomes included questionnaire completion rate, quality of data collected and comparison of participation rate boosting strategies. RESULTS: The overall participation rate was 18.6% among all invitees and 23.1% among individuals aged 18-69. A second reminder letter yielded an additional 9.7% participation rate in those who did not respond to the first invitation. Recontacting participants via an online healthcare portal yielded lower participation than recontacting via physical letter. The completion rate of the questionnaire and cognitive tests was high (92% and 85%, respectively), and measurements were overall reliable among participants. For example, the correlation (r) between self-reported body mass index and that collected by the biobanks was 0.92. CONCLUSION: In summary, this pilot suggests that recontacting FinnGen participants with the goal to collect a wide range of cognitive, behavioural and lifestyle information without additional engagement results in a low participation rate, but with reliable data. We suggest that such information be collected at enrolment, if possible, rather than via post hoc recontacting.
Assuntos
Bancos de Espécimes Biológicos , Dever de Recontatar , Adolescente , Cognição , Humanos , Estilo de Vida , Projetos Piloto , Inquéritos e QuestionáriosRESUMO
BACKGROUND: T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the alpha-cytoplasmic tail of alpha1beta1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. METHODS: We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRbeta and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. RESULTS: From the screen we identified six TCPTP agonists. Two compounds competed with alpha1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to alpha1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRbeta and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. CONCLUSIONS: In this study we showed that small molecules mimicking TCPTP-alpha1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening to identify small molecule PTP activators that could function as RTK antagonists in cells.
Assuntos
Proteína Tirosina Fosfatase não Receptora Tipo 2/química , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Endotélio Vascular/metabolismo , Receptores ErbB/metabolismo , Células HeLa , Humanos , Integrina alfa1beta1/metabolismo , Camundongos , Mitoxantrona/farmacologia , Neovascularização Patológica , Fosforilação , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Transdução de Sinais , Espermidina/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND: MET has emerged as target in head and neck squamous cell carcinoma (HNSCC). However, clinical data on MET inhibition in HNSCC are limited. METHODS: HNSCC biopsies and cell lines were tested for MET activity. The response of cell lines to BAY-853474 was tested in proliferation assays. The prognostic value of MET expression was also analyzed. RESULTS: HNSCC cell lines do not respond to MET inhibition. MET-dependent gastric cancer cell lines have much higher levels of MET expression and phosphorylation than HNSCC cell lines. Clinical samples of HNSCC contain much less MET than responsive models. CONCLUSIONS: No clinical response to MET inhibitors in monotherapy may be expected in unselected cases of HNSCC. Only selected patients with MET amplifications should be treated with MET inhibitors. Patients with increased MET immunoreactivity have shorter overall survival. MET might be useful as marker for the detection of patients with more aggressive types of HNSCC.
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Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/genética , Humanos , Proteínas Proto-Oncogênicas c-met/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genéticaRESUMO
Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2-160 microg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound.
Assuntos
Flavonoides/farmacologia , Mitose/efeitos dos fármacos , Fuso Acromático/metabolismo , Aurora Quinase B , Aurora Quinases , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Ativação Enzimática , Flavonóis , Humanos , Cinetocoros/efeitos dos fármacos , Cinetocoros/fisiologia , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/efeitos dos fármacosRESUMO
BACKGROUND: Approximately 15%-20% of all diagnosed breast cancers are characterized by amplified and overexpressed HER2 (= ErbB2). These breast cancers are aggressive and have a poor prognosis. Although improvements in treatment have been achieved after the introduction of trastuzumab and lapatinib, many patients do not benefit from these drugs. Therefore, in-depth understanding of the mechanisms behind the treatment responses is essential to find alternative therapeutic strategies. MATERIALS AND METHODS: Thirteen HER2 positive breast cancer cell lines were screened with 22 commercially available compounds, mainly targeting proteins in the ErbB2-signaling pathway, and molecular mechanisms related to treatment sensitivity were sought. Cell viability was measured, and treatment responses between the cell lines were compared. To search for response predictors and genomic and transcriptomic profiling, PIK3CA mutations and PTEN status were explored and molecular features associated with drug sensitivity sought. RESULTS: The cell lines were divided into three groups according to the growth-retarding effect induced by trastuzumab and lapatinib. Interestingly, two cell lines insensitive to trastuzumab (KPL4 and SUM190PT) showed sensitivity to an Akt1/2 kinase inhibitor. These cell lines had mutation in PIK3CA and loss of PTEN, suggesting an activated and druggable Akt-signaling pathway. Expression levels of five genes (CDC42, MAPK8, PLCG1, PTK6, and PAK6) were suggested as predictors for the Akt1/2 kinase-inhibitor response. CONCLUSION: Targeting the Akt-signaling pathway shows promise in cell lines that do not respond to trastuzumab. In addition, our results indicate that several molecular features determine the growth-retarding effects induced by the drugs, suggesting that parameters other than HER2 amplification/expression should be included as markers for therapy decisions.
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Marine organisms constitute approximately one-half of the total global biodiversity, being rich reservoirs of structurally diverse biofunctional components. The potential of cyanobacteria, micro- and macroalgae as sources of antimicrobial, antitumoral, anti-inflammatory, and anticoagulant compounds has been reported extensively. Nonetheless, biological activities of marine fauna and flora of the Aegean Sea have remained poorly studied when in comparison to other areas of the Mediterranean Sea. In this study, we screened the antimicrobial, antifouling, anti-inflammatory and anticancer potential of in total 98 specimens collected from the Aegean Sea. Ethanol extract of diatom Amphora cf capitellata showed the most promising antimicrobial results against Candida albicans while the extract of diatom Nitzschia communis showed effective results against Gram-positive bacterium, S. aureus. Extracts from the red alga Laurencia papillosa and from three Cystoseira species exhibited selective antiproliferative activity against cancer cell lines and an extract from the brown alga Dilophus fasciola showed the highest anti-inflammatory activity as measured in primary microglial and astrocyte cell cultures as well as by the reduction of proinflammatory cytokines. In summary, our study demonstrates that the Aegean Sea is a rich source of species that possess interesting potential for developing industrial applications.
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Biotecnologia/métodos , Cianobactérias/metabolismo , Microalgas/metabolismo , Oceanos e Mares , Alga Marinha/metabolismo , Animais , Antibacterianos/farmacologia , Anti-Inflamatórios/farmacologia , Antifúngicos/farmacologia , Antineoplásicos/farmacologia , Incrustação Biológica , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Ratos WistarRESUMO
Mounting in vitro, in vivo and clinical evidence suggest an important role for filopodia in driving cancer cell invasion. Using a high-throughput microscopic-based drug screen, we identify FDA-approved calcium channel blockers (CCBs) as potent inhibitors of filopodia formation in cancer cells. Unexpectedly, we discover that L-type calcium channels are functional and frequently expressed in cancer cells suggesting a previously unappreciated role for these channels during tumorigenesis. We further demonstrate that, at filopodia, L-type calcium channels are activated by integrin inside-out signalling, integrin activation and Src. Moreover, L-type calcium channels promote filopodia stability and maturation into talin-rich adhesions through the spatially restricted regulation of calcium entry and subsequent activation of the protease calpain-1. Altogether we uncover a novel and clinically relevant signalling pathway that regulates filopodia formation in cancer cells and propose that cycles of filopodia stabilization, followed by maturation into focal adhesions, directs cancer cell migration and invasion.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Integrinas/fisiologia , Invasividade Neoplásica , Pseudópodes/fisiologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Canais de Cálcio Tipo L/genética , Calpaína/genética , Calpaína/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Miosinas/genética , Miosinas/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Systematic approaches to functionally identify key players in microRNA (miRNA)-target networks regulating prostate cancer (PCa) proliferation are still missing. OBJECTIVE: To comprehensively map miRNA regulation of genes relevant for PCa proliferation through phenotypic screening and tumor expression data. DESIGN, SETTING, AND PARTICIPANTS: Gain-of-function screening with 1129 miRNA molecules was performed in five PCa cell lines, measuring proliferation, viability, and apoptosis. These results were integrated with changes in miRNA expression from two cohorts of human PCa (188 tumors in total). For resulting miRNAs, the predicted targets were collected and analyzed for patterns with gene set enrichment analysis, and for their association with biochemical recurrence free survival. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Rank product statistical analysis was used to evaluate miRNA effects in phenotypic screening and for expression differences in the prostate tumor cohorts. Expression data were analyzed using the significance analysis of microarrays (SAM) method and the patient material was subjected to Kaplan-Meier statistics. RESULTS AND LIMITATIONS: Functional screening identified 25 miRNAs increasing and 48 miRNAs decreasing cell viability. Data integration resulted in 14 miRNAs, with aberrant expression and effect on proliferation. These miRNAs are predicted to regulate >3700 genes, of which 28 were found up-regulated and 127 down-regulated in PCa compared with benign tissue. Seven genes, FLNC, MSRB3, PARVA, PCDH7, PRNP, RAB34, and SORBS1, showed an inverse association to their predicted miRNA, and were identified to significantly correlate with biochemical recurrence free survival in PCa patients. CONCLUSIONS: A systematic in vitro screening approach combined with in vivo expression and gene set enrichment analysis provide unbiased means for revealing novel miRNA-target links, possibly driving the oncogenic processes in PCa. PATIENT SUMMARY: This study identified novel regulatory molecules, which impact on PCa proliferation and are aberrantly expressed in clinical tumors. Thus, our study reveals regulatory nodes with potential for therapy.
Assuntos
Regulação da Expressão Gênica , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias da Próstata/genética , Apoptose/genética , Caderinas/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Intervalo Livre de Doença , Regulação para Baixo , Filaminas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metionina Sulfóxido Redutases/genética , Proteínas dos Microfilamentos/genética , Proteínas Nucleares , Proteínas Priônicas/genética , Antígeno Prostático Específico/sangue , Protocaderinas , Regulação para Cima , Proteínas rab de Ligação ao GTP/genéticaRESUMO
RET, BRAF and other protein kinases have been identified as major molecular players in thyroid cancer. To identify novel kinases required for the viability of thyroid carcinoma cells, we performed a RNA interference screening in the RET/PTC1(CCDC6-RET)-positive papillary thyroid cancer cell line TPC1 using a library of synthetic small interfering RNAs (siRNAs) targeting the human kinome and related proteins. We identified 14 hits whose silencing was able to significantly reduce the viability and the proliferation of TPC1 cells; most of them were active also in BRAF-mutant BCPAP (papillary thyroid cancer) and 8505C (anaplastic thyroid cancer) and in RAS-mutant CAL62 (anaplastic thyroid cancer) cells. These included members of EPH receptor tyrosine kinase family as well as SRC and MAPK (mitogen activated protein kinases) families. Importantly, silencing of the identified hits did not affect significantly the viability of Nthy-ori 3-1 (hereafter referred to as NTHY) cells derived from normal thyroid tissue, suggesting cancer cell specificity. The identified proteins are worth exploring as potential novel druggable thyroid cancer targets.
Assuntos
Proliferação de Células/genética , Proteínas Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologiaRESUMO
MicroRNAs (miRNAs) regulate a wide range of cellular signaling pathways and biological processes in both physiological and pathological states such as cancer. We have previously identified miR-135b as a direct regulator of androgen receptor (AR) protein level in prostate cancer (PCa). We wanted to further explore the relationship of miR-135b to hormonal receptors, particularly estrogen receptor α (ERα). Here we show that miR-135b expression is lower in ERα-positive breast tumors as compared to ERα-negative samples in two independent breast cancer (BCa) patient cohorts (101 and 1302 samples). Additionally, the miR-135b expression is higher in AR-low PCa patient samples (47 samples). We identify ERα as a novel miR-135b target by demonstrating miR-135b binding to the 3'UTR of the ERα and decreased ERα protein and mRNA level upon miR-135b overexpression in BCa cells. MiR-135b reduces proliferation of ERα-positive BCa cells MCF-7 and BT-474 as well as AR-positive PCa cells LNCaP and 22Rv1 when grown in 2D. To identify other genes regulated by miR-135b we performed gene expression studies and found a link to the hypoxia inducible factor 1α (HIF1α) pathway. We show that miR-135b influences the protein level of the inhibitor for hypoxia inducible factor 1α (HIF1AN) and is able to bind to HIF1AN 3'UTR. Our study demonstrates that miR-135b regulates ERα, AR and HIF1AN protein levels through interaction with their 3'UTR regions, and proliferation in ERα-positive BCa and AR-positive PCa cells.
Assuntos
Neoplasias da Mama/patologia , Proliferação de Células/fisiologia , Receptor alfa de Estrogênio/metabolismo , MicroRNAs/fisiologia , Oxigenases de Função Mista/metabolismo , Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Regiões 3' não Traduzidas , Neoplasias da Mama/metabolismo , Regulação para Baixo , Receptor alfa de Estrogênio/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Oxigenases de Função Mista/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Proteínas Repressoras/genéticaRESUMO
The antioxidant properties of hydroxymatairesinol (HM-3000) were studied in vitro in lipid peroxidation, superoxide and peroxyl radical scavenging, and LDL-oxidation models in comparison with the known synthetic antioxidants Trolox (a water-soluble vitamin E derivative), butylated hydroxyanisol (BHA) and butylated hydroxytoluene (BHT). On a molar basis HM-3000 was a more effective antioxidant than Trolox in all assays and more effective than BHT or BHA in lipid peroxidation and superoxide scavenging test. The in vivo antioxidative effect (evaluated as the weight gain of C57BL/6J mice fed an alpha-tocopherol-deficient diet) of HM-3000 (500 mg/kg per day) was comparable to that of DL-alpha-tocopherol (766 mg/kg per day). The antitumor activity of HM-3000 was studied in dimethylbenz[a]anthracene (DMBA)-induced rat mammary cancer. HM-3000 had a statistically significant inhibitory effect on tumor growth. Prevention of tumor formation was also evaluated in the Apc(Min) mice model, which develops intestinal polyps spontaneously. HM-3000 was given in diet at 30 mg/kg per day and decreased the formation of polyps and prevented beta-catenin accumulation into the nucleus, the pathophysiological hallmark of polyp formation in this mouse model. In short-term toxicity studies (up to 28 days) HM-3000 was essentially non-toxic when given p.o. to rats and dogs (daily doses up to 2000 and 665 mg/kg, respectively); HM-3000 was shown to be well absorbed (> 50% of the dose) and rapidly eliminated. In human studies HM-3000 has been given in single doses up to 1350 mg to healthy male volunteers without treatment-related adverse events. Rapid absorption from the gastrointestinal tract and partial metabolism to enterolactone in humans was demonstrated. In summary, HM-3000 is a safe, novel enterolactone precursor lignan with antioxidant and antitumor properties.
Assuntos
Antioxidantes/farmacologia , Lignanas/farmacologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Extratos Vegetais/farmacologia , Animais , Antioxidantes/farmacocinética , Disponibilidade Biológica , Modelos Animais de Doenças , Feminino , Lignanas/farmacocinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
MicroRNAs (miRNAs) are non-coding RNAs regulating gene expression post-transcriptionally. We have characterized the role of miRNAs in regulating the human epidermal growth factor receptor 2 (HER2)-pathway in breast cancer. We performed miRNA gain-of-function assays by screening two HER2 amplified cell lines (KPL-4 and JIMT-1) with a miRNA mimic library consisting of 810 human miRNAs. The levels of HER2, phospho-AKT, phospho-ERK1/2, cell proliferation (Ki67) and apoptosis (cPARP) were analyzed with reverse-phase protein arrays. Rank product analyses identified 38 miRNAs (q < 0.05) as inhibitors of HER2 signaling and cell growth, the most effective being miR-491-5p, miR-634, miR-637 and miR-342-5p. We also characterized miRNAs directly targeting HER2 and identified seven novel miRNAs (miR-552, miR-541, miR-193a-5p, miR-453, miR-134, miR-498, and miR-331-3p) as direct regulators of the HER2 3'UTR. We demonstrated the clinical relevance of the miRNAs and identified miR-342-5p and miR-744* as significantly down-regulated in HER2-positive breast tumors as compared to HER2-negative tumors from two cohorts of breast cancer patients (101 and 1302 cases). miR-342-5p specifically inhibited HER2-positive cell growth, as it had no effect on the growth of HER2-negative control cells in vitro. Furthermore, higher expression of miR-342-5p was associated with better survival in both breast cancer patient cohorts. In conclusion, we have identified miRNAs which are efficient negative regulators of the HER2 pathway that may play a role in vivo during breast cancer progression. These results give mechanistic insights in HER2 regulation which may open potential new strategies towards prevention and therapeutic inhibition of HER2-positive breast cancer.