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1.
Genome Biol ; 25(1): 143, 2024 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-38822412

RESUMO

BACKGROUND: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. RESULTS: Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. CONCLUSIONS: Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Proteína de Leucina Linfoide-Mieloide/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Subunidade alfa 2 de Fator de Ligação ao Core/genética
2.
Pathology ; 53(7): 875-882, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34049715

RESUMO

B-cell lineage acute lymphoblastic leukaemia (B-ALL) is the most common paediatric malignancy. Transcription factor B-cell lymphoma 6 (BCL6) is essential to germinal centre formation and antibody affinity maturation and plays a major role in mature B-cell malignancies. More recently, it was shown to act as a critical downstream regulator in pre-BCR+ B-ALL. We investigated the expression of the BCL6 protein in a population-based cohort of paediatric B-ALL cases and detected moderate to strong positivity through immunohistochemistry in 7% of cases (8/117); however, only two of eight BCL6 cases (25%) co-expressed the ZAP70 protein. In light of these data, the subtype with active pre-BCR signalling constitutes a rare entity in paediatric B-ALL. In three independent larger cohorts with gene expression data, high BCL6 mRNA levels were associated with the TCF3-PBX1, Ph-like, NUTM1, MEF2D and PAX5-alt subgroups and the 'metagene' signature for pre-BCR-associated genes. However, higher-than-median BCL6 mRNA level alone was associated with favourable event free survival in the Nordic paediatric cohort, indicating that using BCL6 as a diagnostic marker requires careful design, and evaluation of protein level is needed alongside the genetic or transcriptomic data.


Assuntos
Variações do Número de Cópias de DNA , Linfoma de Células B/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linfócitos B/patologia , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Pediatria , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Prognóstico , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas c-bcl-6/genética , RNA Mensageiro/genética
3.
Genome Med ; 12(1): 99, 2020 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-33218352

RESUMO

BACKGROUND: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. METHODS: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. RESULTS: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. CONCLUSIONS: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.


Assuntos
Diferenciação Celular/genética , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Leucemia/genética , Linfócitos/fisiologia , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/genética , Medula Óssea , Linhagem Celular Tumoral , Criança , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Sistemas de Liberação de Medicamentos , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Leucemia/tratamento farmacológico , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição , Transcriptoma , Translocação Genética , Variante 6 da Proteína do Fator de Translocação ETS
4.
Cancer Res ; 79(10): 2466-2479, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30940663

RESUMO

Large collections of genome-wide data can facilitate the characterization of disease states and subtypes, permitting pan-cancer analysis of molecular phenotypes and evaluation of disease context for new therapeutic approaches. We analyzed 9,544 transcriptomes from more than 30 hematologic malignancies, normal blood cell types, and cell lines, and showed that disease types could be stratified in a data-driven manner. We then identified cluster-specific pathway activity, new biomarkers, and in silico drug target prioritization through interrogation of drug target databases. Using known vulnerabilities and available drug screens, we highlighted the importance of integrating molecular phenotype with drug target expression for in silico prediction of drug responsiveness. Our analysis implicated BCL2 expression level as an important indicator of venetoclax responsiveness and provided a rationale for its targeting in specific leukemia subtypes and multiple myeloma, linked several polycomb group proteins that could be targeted by small molecules (SFMBT1, CBX7, and EZH1) with chronic lymphocytic leukemia, and supported CDK6 as a disease-specific target in acute myeloid leukemia. Through integration with proteomics data, we characterized target protein expression for pre-B leukemia immunotherapy candidates, including DPEP1. These molecular data can be explored using our publicly available interactive resource, Hemap, for expediting therapeutic innovations in hematologic malignancies. SIGNIFICANCE: This study describes a data resource for researching derailed cellular pathways and candidate drug targets across hematologic malignancies.


Assuntos
Neoplasias Hematológicas/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Compostos Bicíclicos Heterocíclicos com Pontes/uso terapêutico , Neoplasias Hematológicas/tratamento farmacológico , Humanos , Imunoterapia/métodos , Internet , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Linfoma de Células B/tratamento farmacológico , Fenótipo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Bibliotecas de Moléculas Pequenas/uso terapêutico , Sulfonamidas/uso terapêutico , Transcriptoma/genética
5.
Cancer Discov ; 8(5): 616-631, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29496663

RESUMO

Leukemia is caused by the accumulation of multiple genomic lesions in hematopoietic precursor cells. However, how these events cooperate during oncogenic transformation remains poorly understood. We studied the cooperation between activated JAK3/STAT5 signaling and HOXA9 overexpression, two events identified as significantly co-occurring in T-cell acute lymphoblastic leukemia. Expression of mutant JAK3 and HOXA9 led to a rapid development of leukemia originating from multipotent or lymphoid-committed progenitors, with a significant decrease in disease latency compared with JAK3 or HOXA9 alone. Integrated RNA sequencing, chromatin immunoprecipitation sequencing, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that STAT5 and HOXA9 have co-occupancy across the genome, resulting in enhanced STAT5 transcriptional activity and ectopic activation of FOS/JUN (AP1). Our data suggest that oncogenic transcription factors such as HOXA9 provide a fertile ground for specific signaling pathways to thrive, explaining why JAK/STAT pathway mutations accumulate in HOXA9-expressing cells.Significance: The mechanism of oncogene cooperation in cancer development remains poorly characterized. In this study, we model the cooperation between activated JAK/STAT signaling and ectopic HOXA9 expression during T-cell leukemia development. We identify a direct cooperation between STAT5 and HOXA9 at the transcriptional level and identify PIM1 kinase as a possible drug target in mutant JAK/STAT/HOXA9-positive leukemia cases. Cancer Discov; 8(5); 616-31. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 517.


Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Homeodomínio/metabolismo , Janus Quinases/metabolismo , Leucemia/etiologia , Leucemia/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Animais , Transplante de Medula Óssea , Montagem e Desmontagem da Cromatina , Modelos Animais de Doenças , Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Janus Quinases/genética , Masculino , Camundongos , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Ligação Proteica , Fatores de Transcrição STAT/genética , Fator de Transcrição AP-1/metabolismo , Transdução Genética , Transgenes
6.
RNA Biol ; 14(7): 827-830, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28387584

RESUMO

Methodological advances that allow deeper characterization of non-coding elements in the genome have started to reveal the full spectrum of deregulation in cancer. We generated an inducible cell model to track transcriptional changes after induction of a well-known leukemia-inducing fusion gene, ETV6-RUNX1. Our data revealed widespread transcriptional alterations outside coding elements in the genome. This adds to the growing list of various alterations in the non-coding genome in cancer and pinpoints their role in diseased cellular state.


Assuntos
Regulação Leucêmica da Expressão Gênica , Genoma , Leucemia/genética , RNA não Traduzido/genética , Humanos , Proteínas de Fusão Oncogênica/metabolismo , RNA não Traduzido/metabolismo
7.
Leuk Res ; 54: 1-6, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-28063378

RESUMO

Cell signalling, which is often derailed in cancer, is a network of multiple interconnected pathways with numerous feedback mechanisms. Dynamics of cell signalling is intimately regulated by addition and removal of phosphate groups by kinases and phosphatases. We examined expression of members of the PTP4A family of phosphatases across acute leukemias. While expression of PTP4A1 and PTP4A2 remained relatively unchanged across diseases, PTP4A3 showed marked overexpression in ETV6-RUNX1 and BCR-ABL1 subtypes of precursor B cell acute lymphoblastic leukemia. We show that PTP4A3 is regulated by the ETV6-RUNX1 fusion, but noticed no marked impact on cell viability either after PTP4A3 silencing or treatment with a PTP4A3 inhibitor. Regulation of PTP4A3 expression is altered in specific subgroups of acute leukemias and this is likely brought about by expression of the aberrant fusion genes.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/genética , Proteínas de Fusão Oncogênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Tirosina Fosfatases/genética , Linhagem Celular , Sobrevivência Celular , Proteínas de Fusão bcr-abl , Humanos
8.
Genome Res ; 26(11): 1468-1477, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27620872

RESUMO

Approximately 20%-25% of childhood acute lymphoblastic leukemias carry the ETV6-RUNX1 (E/R) fusion gene, a fusion of two central hematopoietic transcription factors, ETV6 (TEL) and RUNX1 (AML1). Despite its prevalence, the exact genomic targets of E/R have remained elusive. We evaluated gene loci and enhancers targeted by E/R genome-wide in precursor B acute leukemia cells using global run-on sequencing (GRO-seq). We show that expression of the E/R fusion leads to widespread repression of RUNX1 motif-containing enhancers at its target gene loci. Moreover, multiple super-enhancers from the CD19+/CD20+-lineage were repressed, implicating a role in impediment of lineage commitment. In effect, the expression of several genes involved in B cell signaling and adhesion was down-regulated, and the repression depended on the wild-type DNA-binding Runt domain of RUNX1. We also identified a number of E/R-regulated annotated and de novo noncoding genes. The results provide a comprehensive genome-wide mapping between E/R-regulated key regulatory elements and genes in precursor B cell leukemia that disrupt normal B lymphopoiesis.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Loci Gênicos , Proteínas de Fusão Oncogênica/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Linhagem Celular Tumoral , Subunidade alfa 2 de Fator de Ligação ao Core/química , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Elife ; 52016 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-27431763

RESUMO

Progression of malignancy to overt disease requires multiple genetic hits. Activation-induced deaminase (AID) can drive lymphomagenesis by generating off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription. The first active transcriptional profiles from acute lymphoblastic leukemia (ALL) patients acquired here reveal striking similarity at structural variation (SV) sites. Specific transcriptional features, namely convergent transcription and Pol2 stalling, were detected at breakpoints. The overlap was most prominent at SV with recognition motifs for the recombination activating genes (RAG). We present signal feature analysis to detect vulnerable regions and quantified from human cells how convergent transcription contributes to R-loop generation and RNA polymerase stalling. Wide stalling regions were characterized by high DNAse hypersensitivity and unusually broad H3K4me3 signal. Based on 1382 pre-B-ALL patients, the ETV6-RUNX1 fusion positive patients had over ten-fold elevation in RAG1 while high expression of AID marked pre-B-ALL lacking common cytogenetic changes.


Assuntos
Instabilidade Genômica , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Transcrição Gênica , Citidina Desaminase/metabolismo , Humanos , RNA Polimerase II/metabolismo
10.
Exp Cell Res ; 336(1): 130-40, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26112215

RESUMO

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen-fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment.


Assuntos
Inibidores da Angiogênese/farmacologia , Carcinoma de Células Escamosas/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Endostatinas/farmacologia , Neoplasias da Língua/patologia , Microambiente Tumoral/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/irrigação sanguínea , Carcinoma de Células Escamosas/tratamento farmacológico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Mioma/irrigação sanguínea , Mioma/tratamento farmacológico , Mioma/patologia , Invasividade Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neoplasias da Língua/irrigação sanguínea , Neoplasias da Língua/tratamento farmacológico , Células Tumorais Cultivadas , Neoplasias Uterinas/irrigação sanguínea , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
11.
BMC Cancer ; 15: 25, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25633184

RESUMO

BACKGROUND: Caveolin-1 (CAV1) may be upregulated by hypoxia and acts in a tumor-dependent manner. We investigated CAV1 in tongue squamous cell carcinoma (TSCC) and its association with clinical outcomes, and studied in vitro possible ways for CAV1 accumulation in the tumor microenvironment (TME). METHODS: TSCC cases (N = 64) were immunohistochemically stained for CAV1. Scores were separately assessed in the tumor and TME and plotted for association with recurrence and survival (univariate analysis with log-rank test). In vitro studies were performed on a 3D myoma organotypic model, a mimicker of TME. Prior to monoculturing HSC-3 tongue cancer cells, the model underwent modifications in oxygenation level (1%O2 hypoxia to upregulate CAV1) and/or in the amount of natural soluble factors [deleted by 14-day rinsing (rinsed myoma, RM), to allow only HSC-3-derived factors to act]. Controls included normoxia (21%O2) and naturally occurring soluble factors (intact myoma, IM). HSC-3 cells were also co-cultured with CaDEC12 cells (fibroblasts exposed to human tongue cancer). CAV1 expression and cellular distribution were examined in different cellular components in hypoxic and rinsed myoma assays. Twist served as a marker for the process of epithelial-mesenchymal transition (EMT). Exosomes isolated from HSC-3 media were investigated for containing CAV1. RESULTS: Expression of CAV1 in TSCC had a higher score in TME than in the tumor cells and a negative impact on recurrence (p = 0.01) and survival (p = 0.003). Monocultures of HSC-3 revealed expression of CAV1 mainly in the TME-like myoma assay, similar to TSCC. CAV1+, alpha-smooth muscle actin (αSMA) + and Twist + CAF-like cells were observed surrounding the invading HSC-3, possibly reflecting EMT. RM findings were similar to IM, inferring action of HSC-3 derived factors, and no differences were seen when hypoxia was induced. HSC-3-CaDEC12 co-cultures revealed CAV1+, αSMA+ and cytokeratin-negative CAF-like cells, raising the possibility of CaDEC12 cells gaining a CAF phenotype. HSC-3-derived exosomes were loaded with CAV1. CONCLUSIONS: Accumulation of CAV1-TME in TSCC had a negative prognostic value. In vitro studies showed the presence of CAV1 in cancer cells undergoing EMT and in fibroblasts undergoing trans-differentiation to CAFs. CAV1 delivery to the TME involved cancer cell-derived exosomes.


Assuntos
Caveolina 1/metabolismo , Neoplasias da Língua/metabolismo , Neoplasias da Língua/mortalidade , Microambiente Tumoral , Adulto , Idoso , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidade , Caveolina 1/genética , Técnicas de Cultura de Células , Técnicas de Cocultura , Feminino , Seguimentos , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Neoplasias da Língua/genética , Neoplasias da Língua/patologia , Células Tumorais Cultivadas , Microambiente Tumoral/genética
12.
PLoS One ; 8(8): e70925, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23951042

RESUMO

OBJECTIVES: Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens. MATERIALS AND METHODS: OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated. RESULTS: Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively). CONCLUSIONS: Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.


Assuntos
Carcinoma de Células Escamosas/genética , Catepsina K/genética , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias da Língua/genética , Idoso , Western Blotting , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/patologia , Catepsina K/antagonistas & inibidores , Catepsina K/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Leucina/análogos & derivados , Leucina/farmacologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia , Técnicas de Cultura de Órgãos , Prognóstico , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Língua/enzimologia , Neoplasias da Língua/patologia , Microambiente Tumoral/genética
13.
Anticancer Res ; 33(1): 45-52, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23267127

RESUMO

BACKGROUND: Actin-related protein 2/3 (ARP2/3) complex is an actin nucleator responsible for actin cytoskeleton branching which is essential for efficient cell migration. MATERIALS AND METHODS: The expression of the seven ARP2/3 complex subunits was assessed in pancreatic cancer cell lines and in normal pancreatic samples by quantitative RT-PCR. siRNA-mediated silencing was used to study the contribution of each ARP2/3 complex member to pancreatic cancer cell migration. RESULTS: ARPC3 and ARPC4 were the most highly expressed complex members, while ARPC1B and ARPC2 were expressed at low levels. Silencing of the ARP2/3 complex subunits typically resulted in reduced cell migration capacity. In particular, silencing of ARPC4 significantly reduced cell migration in all studied cell lines, with a major impact on Hs700T and HPAFII migration (50% and 68% decrease, p<0.001). CONCLUSION: We offer comprehensive expression data on the ARP2/3 complex members for pancreatic cancer and normal pancreas. In addition, we show cell line-specific differences in ARP2/3 complex subunit dependency on cell migratory potential, and suggest ARPC4 to be one of the key members of the ARP2/3 complex in pancreatic cancer.


Assuntos
Citoesqueleto de Actina , Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas/genética , Actinas/metabolismo , Proteínas do Citoesqueleto , Neoplasias Pancreáticas , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno
14.
Exp Cell Res ; 319(4): 376-89, 2013 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-23262025

RESUMO

Invasion is an important hallmark of cancer involving interactions between the tumor microenvironment and the cancer cells. Hypoxia, low oxygen level, is related to increased invasion and metastasis in many cancers. The aim was to elucidate the effect of hypoxia on invasion of oral squamous cell carcinoma cells (OSCCs), and the applicability of a novel 3-dimentional myoma organotypic invasion model in hypoxia experiments. OSCC cell lines (primary oral carcinoma derived cells UT-SCC-43A, recurrent oral carcinoma cells UT-SCC-43B and aggressive tongue carcinoma cells HSC-3) were studied for their migration and invasion capabilities under normoxia, hypoxia, and in the presence a hypoxia-mimicker cobalt chloride. As expected, the recurrent UT-SCC-43B cells were significantly more aggressive than the primary tumor derived cells. In contrast to tongue carcinoma HSC-3 cells, they only mildly responded to hypoxia in the migration or invasion assays, indicating a cell line specific response of hypoxia on the invasive potential. The modification of the organotypic human tissue-derived matrix via the removal of various yet unidentified soluble factors by rinsing the tissue resulting in stripped matrix substantially changed the invasion pattern of HSC-3 cells and the outcomes of hypoxic treatments. Only in the stripped tissue hypoxia significantly increased invasion, whereas in native intact tissue the induced invasion was not observed. This demonstrates the importance of the soluble factors to the invasion pattern and to the hypoxia response. A metastasis and poor prognosis marker, hypoxia-regulated lysyl oxidase (LOX), was present in the myoma tissue, but could be removed by rinsing. The inhibition of LOX resulted in a decrease in invasion area, but only very mildly in invasion depth. Thus, it may have a role in the modulation of the invasion pattern. Another hypoxia-related poor prognosis marker carbonic anhydrase 9 (CAIX) was induced in HSC-3 cells both by the hypoxic exposure and interestingly in invading HSC-3 cells inside the tissue even in normoxic conditions. In conclusion, this suggests that the intact myoma organotypic model offers optimally hypoxic surroundings, thus being an excellent human tumor microenvironment mimicker.


Assuntos
Carcinoma de Células Escamosas/patologia , Hipóxia/patologia , Neoplasias Bucais/patologia , Microambiente Tumoral/fisiologia , Idoso , Aminopropionitrilo/farmacologia , Carcinoma de Células Escamosas/metabolismo , Ensaios de Migração Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hipóxia/complicações , Leiomioma/metabolismo , Leiomioma/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/metabolismo , Invasividade Neoplásica , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Proteína-Lisina 6-Oxidase/metabolismo , Proteína-Lisina 6-Oxidase/fisiologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/patologia , Cicatrização
15.
PLoS One ; 7(12): e51044, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23227231

RESUMO

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activity. Among these are the collagen-derived anti-angiogenic fragments, some of which are suggested to affect carcinoma cells also directly. Arresten is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of the basement membrane collagen IV α1 chain. As the mere prevention of tumor angiogenesis leads to hypoxia that can result in selection of more aggressive cell types and reduces the efficacy of chemotherapy, we aimed here to elucidate how arresten influences the aggressive human carcinoma cells. Arresten efficiently inhibited migration and invasion of HSC-3 tongue carcinoma cells in culture and in an organotypic model. Subcutaneous Arr-HSC xenografts grew markedly more slowly in nude mice and showed reduced tumor cell proliferation, vessel density and local invasiveness. In the organotypic assay, HSC-3 cells overproducing arresten (Arr-HSC) showed induction of cell death. In monolayer culture the Arr-HSC cells grew in aggregated cobblestone-like clusters and, relative to the control cells, showed increased expression and localization of epithelial marker E-cadherin in cell-cell contacts. Application of electric cell-substrate impedance sensing (ECIS) further supported our observations on altered morphology and motility of the Arr-HSC cells. Administration of a function-blocking α1 integrin antibody abolished the impedance difference between the Arr-HSC and control cells suggesting that the effect of arresten on promotion of HSC-3 cell-cell contacts and cell spreading is at least partly mediated by α1ß1 integrin. Collectively, our data suggest novel roles for arresten in the regulation of oral squamous carcinoma cell proliferation, survival, motility and invasion through the modulation of cell differentiation state and integrin signaling.


Assuntos
Inibidores da Angiogênese/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Colágeno Tipo IV/metabolismo , Colágeno/química , Neoplasias da Língua/tratamento farmacológico , Neoplasias da Língua/patologia , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Bloqueadores/farmacologia , Apoptose/efeitos dos fármacos , Caderinas/metabolismo , Carcinoma de Células Escamosas/irrigação sanguínea , Adesão Celular/efeitos dos fármacos , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/farmacologia , Colágeno Tipo IV/uso terapêutico , Impedância Elétrica , Epitélio/efeitos dos fármacos , Epitélio/patologia , Humanos , Integrina alfa1beta1/imunologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Neovascularização Patológica/tratamento farmacológico , Neoplasias da Língua/irrigação sanguínea , Ensaios Antitumorais Modelo de Xenoenxerto
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