RESUMO
Activation in transformed cells of normal stem cells' self-renewal pathways might contribute to the survival life cycle of cancer stem cells and promote tumor progression. The BMI-1 oncogene-driven gene expression pathway is essential for the self-renewal of hematopoietic and neural stem cells. We applied a mouse/human comparative translational genomics approach to identify an 11-gene signature that consistently displays a stem cell-resembling expression profile in distant metastatic lesions as revealed by the analysis of metastases and primary tumors from a transgenic mouse model of prostate cancer and cancer patients. To further validate these results, we examined the prognostic power of the 11-gene signature in several independent therapy-outcome sets of clinical samples obtained from 1,153 cancer patients diagnosed with 11 different types of cancer, including 5 epithelial malignancies (prostate, breast, lung, ovarian, and bladder cancers) and 5 nonepithelial malignancies (lymphoma, mesothelioma, medulloblastoma, glioma, and acute myeloid leukemia). Kaplan-Meier analysis demonstrated that a stem cell-like expression profile of the 11-gene signature in primary tumors is a consistent powerful predictor of a short interval to disease recurrence, distant metastasis, and death after therapy in cancer patients diagnosed with 11 distinct types of cancer. These data suggest the presence of a conserved BMI-1-driven pathway, which is similarly engaged in both normal stem cells and a highly malignant subset of human cancers diagnosed in a wide range of organs and uniformly exhibiting a marked propensity toward metastatic dissemination as well as a high probability of unfavorable therapy outcome.
Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Células-Tronco Neoplásicas , Animais , Biomarcadores Tumorais/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 1 , Prognóstico , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
One of the major problems in management of prostate cancer is the lack of reliable genetic markers predicting the clinical course of the disease. We analyzed expression profiles of 12,625 transcripts in prostate tumors from patients with distinct clinical outcomes after therapy as well as metastatic human prostate cancer xenografts in nude mice. We identified small clusters of genes discriminating recurrent versus nonrecurrent disease with 90% and 75% accuracy in two independent cohorts of patients. We examined one group of samples (21 tumors) to discover the recurrence predictor genes and then validated the predictive power of these genes in a different set (79 tumors). Kaplan-Meier analysis demonstrated that recurrence predictor signatures are highly informative (P < 0.0001) in stratification of patients into subgroups with distinct relapse-free survival after therapy. A gene expression-based recurrence predictor algorithm was informative in predicting the outcome in patients with early-stage disease, with either high or low preoperative prostate-specific antigen levels and provided additional value to the outcome prediction based on Gleason sum or multiparameter nomogram. Overall, 88% of patients with recurrence of prostate cancer within 1 year after therapy were correctly classified into the poor-prognosis group. The identified algorithm provides additional predictive value over conventional markers of outcome and appears suitable for stratification of prostate cancer patients at the time of diagnosis into subgroups with distinct survival probability after therapy.
Assuntos
Perfilação da Expressão Gênica , Neoplasias da Próstata/genética , Algoritmos , Animais , Biomarcadores , Humanos , Masculino , Camundongos , Metástase Neoplásica/genética , Prognóstico , Neoplasias da Próstata/diagnóstico , Neoplasias da Próstata/metabolismo , Análise de SobrevidaRESUMO
Survival in lymph or blood is an essential prerequisite for metastasis of carcinoma cells to distant organs. Recently, we reported isolation and initial biological characterization of circulating metastatic cells in a fluorescent, orthotopic, metastatic nude-mouse model of human prostate cancer. Here we show that the metastatic human prostate carcinoma cells selected for survival in the circulation have increased resistance to anoikis, which is apoptosis induced by cell detachment. Using gene silencing and gene transfer techniques, we show that increased expression of the apoptosis inhibitory protein XIAP contributes to anoikis resistance of the circulating metastatic human prostate carcinoma cells. We also provide initial preclinical data on the antimetastatic efficacy of recently discovered small-molecule antagonists of XIAP.
Assuntos
Anoikis/fisiologia , Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/patologia , Proteínas/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Masculino , Metástase Neoplásica , Células Neoplásicas Circulantes/metabolismo , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , Proteínas/antagonistas & inibidores , Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Transfecção , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Elucidation of the mechanisms of hormone-independent metastatic prostate cancer remains a significant and highly relevant challenge. We report here that hormone-refractory human prostate carcinoma growing orthotopically efficiently deliver viable metastatic cells in the host circulation. This is in contrast to the ectopic tumors of the same lineage, which do not deliver live cells into the circulation. To investigate the malignant potential of viable circulating carcinoma cells, we developed a novel dual-color orthotopic coimplantation model of human prostate cancer metastasis in nude mice. This model is comprised of coinjection of an equivalent mixture of isolated and cultured circulating green fluorescent protein-expressing clones and parental red fluorescent protein-expressing human prostate carcinoma cells. In the dual-color model, the selected green fluorescent protein-labeled viable circulating cells have an increased metastatic propensity relative to the red fluorescent protein-labeled parental cells. The identification and isolation of highly malignant viable circulating human prostate carcinoma cells from orthotopic but not ectopic models will enable important new insights into the metastatic process including the role of the tumor microenvironment.
Assuntos
Células Neoplásicas Circulantes/patologia , Neoplasias da Próstata/patologia , Animais , Humanos , Masculino , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Metástase Neoplásica , Transplante de Neoplasias , Neoplasias da Próstata/sangue , Transplante HeterólogoRESUMO
BACKGROUND: The progression of a metastatic disease is a multistep process involving various genetic events. High-throughput technologies such as microarrays offer the unique opportunity of screening the entire gene expression profile of malignant cells. Defining consistent changes in the gene expression pattern of cancer models may lead to detection of genes essential for tumor progression and may ultimately identify new targets for therapy. MATERIALS AND METHODS: Gene expression was determined in multiple progression models of human prostate cancer cell lines derived from tumors after 1 to 5 passages as orthotopic xenografts in nude mice. Samples were analyzed using Affymetrix microarrays determining expression of 7,129 genes. Changes in gene expression were calculated referring to normal epithelial cells and confirmed by quantitative PCR. Data were validated by comparison to expression profiles of tumors induced by these cell lines and were also compared to expression data from clinical samples. RESULTS: Two hundred and fourteen genes were regulated in the same direction in all cell lines compared to RNA of normal epithelial cells. Seventy-nine % of the 214 gene consensus class of the cell culture models were also found to be differentially-regulated in orthotopic tumors induced in mice and 83% also in clinical samples. CONCLUSION: The expression pattern of these genes is consistent with their potential role in cancer progression. The results indicate the relevance of these xenograft models for expression analysis in the progression and development of metastatic cancer.
Assuntos
Neoplasias/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Progressão da Doença , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: Selection of treatment options with the highest likelihood of successful outcome for individual breast cancer patients is based to a large degree on accurate classification into subgroups with poor and good prognosis reflecting a different probability of disease recurrence and survival after therapy. Here we propose a breast cancer classification algorithm taking into account three main prognostic features determined at the time of diagnosis: estrogen receptor (ER) status; lymph node (LN) status; and gene expression signatures associated with distinct therapy outcome. EXPERIMENTAL DESIGN: Using microarray expression profiling and quantitative reverse transcription-PCR analyses, we compared expression profiles of the 70-gene breast cancer survival signature in established breast cancer cell lines and primary breast carcinomas from cancer patients. We classified 295 breast cancer patients using 14-, 13-, 6-, and 4-gene survival predictor signatures into subgroups having statistically distinct probability of therapy failure (P < 0.0001). We evaluated the prognostic power of breast cancer survival predictor signatures alone and in combination with ER and LN status using Kaplan-Meier analysis. RESULTS: The breast cancer survival predictor algorithm allowed highly accurate classification into subgroups with dramatically distinct 5- and 10-year survival after therapy of a large cohort of 295 breast cancer patients with either ER+ or ER- tumors as well as LN+ or LN- disease (P < 0.0001, log-rank test). CONCLUSIONS: Our data imply that quantitative laboratory tests measuring expression profiles of a limited set of identified small gene clusters may be useful in stratification of breast cancer patients at the time of diagnosis into subgroups with statistically distinct probability of positive outcome after therapy and assisting in selection of optimal treatment strategies. The estimated increase in survival due to the optimization of treatment protocols may reach many thousands of breast cancer survivors every year at the 10-year follow-up check point.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Algoritmos , Neoplasias da Mama/classificação , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Humanos , Metástase Linfática , Família Multigênica , Metástase Neoplásica , Prognóstico , RNA Mensageiro/metabolismo , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Despite remarkable advances in our understanding of a genetic basis of cancer, the precise molecular definition of the phenotypically relevant genetic features associated with human epithelial malignancies remains a significant and highly relevant challenge. Here we performed a systematic analysis of the chromosomal positions of cancer-associated transcripts for prostate, breast, ovarian, and colon tumors, and identified short segments of human chromosomes that appear to represent a common target for transcriptional activation in major epithelial malignancies in human. These cancer-associated transcriptomeres correspond well to the regions of transient transcriptional activity on chromosomes 1q21-q23 (144-160 Mbp), 12q13 (52-63 Mbp), 17q21 (38-50 Mbp), 17q23-q25 (72-82 Mbp), 19p13 (1-16 Mbp), and Xq28 (132-142 Mbp) during human cell cycle, suggesting a common epigenetic mechanism of transcriptional activation. Consistent with this idea, two of these transcriptomeres (12q13 and 17q21) seemed to be related to the p53-regulated transcriptional clusters, and some of the cancer-associated transcriptomeres appeared to correspond well to the recently identified regions of increased gene expression on human chromosomes.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Ativação Transcricional/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Neoplasias da Próstata/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Transcriptional co-regulation of sets of adjacent genes at the chromosomal domain level has been demonstrated recently in eukaryotes, supporting the idea that genomes are divided into regions of clustered expression of groups of adjacent genes. The mechanism underlying this phenomenon and its significance for human pathology is not yet known. Systematic analysis of the chromosomal positions of cancer-associated transcripts for prostate, breast, ovarian, and colon tumors identified short segments of human chromosomes that appear to represent a common target for transcriptional activation in major epithelial malignancies in human (1q21-q23 [140-160 Mbp]; 11q12-q13 [62-69 Mbp]; 12q13 [49-58 Mbp]; 17q21 [37-50 Mbp]; 17q23-q25 [70-81 Mbp]; 19p13 [0.2-19 Mbp]; Xq28 [147-153 Mbp]). At least some of these cancer-associated MARTAs correspond well to the regions of recurrent amplification in human cancer and seemed to be targets for DNA amplifications in established cancer cell lines.
Assuntos
Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Reação em Cadeia da Polimerase , Neoplasias da Próstata/genética , Ativação TranscricionalRESUMO
The mechanistic relevance of intergenic disease-associated genetic loci (IDAGL) containing highly statistically significant disease-linked SNPs remains unknown. Here, we present experimental and clinical evidence supporting the importantance of the role of IDAGL in human diseases. A targeted RT-PCR screen coupled with sequencing of purified PCR products detects widespread transcription at multiple IDAGL and identifies 96 small noncoding trans-regulatory RNAs of ~100-300 nt in length containing SNPs (snpRNAs) associated with 21 common disorders. Multiple independent lines of experimental evidence support functionality of snpRNAs by documenting their cell type-specific expression and evolutionary conservation of sequences, genomic coordinates and biological effects. Chromatin state signatures, expression profiling experiments and luciferase reporter assays demonstrate that many IDAGL are Polycomb-regulated long-range enhancers. Expression of snpRNAs in human and mouse cells markedly affects cellular behavior and induces allele-specific clinically relevant phenotypic changes: NLRP1-locus snpRNAs rs2670660 exert regulatory effects on monocyte/macrophage transdifferentiation, induce prostate cancer (PC) susceptibility snpRNAs and transform low-malignancy hormone-dependent human PC cells into highly malignant androgen-independent PC. Q-PCR analysis and luciferase reporter assays demonstrate that snpRNA sequences represent allele-specific "decoy" targets of microRNAs that function as SNP allele-specific modifiers of microRNA expression and activity. We demonstrate that trans-acting RNA molecules facilitating resistance to androgen depletion (RAD) in vitro and castration-resistant phenotype (CRP) in vivo of PC contain intergenic 8q24-locus SNP variants (rs1447295; rs16901979; rs6983267) that were recently linked with increased risk of PC. Q-PCR analysis of clinical samples reveals markedly increased and highly concordant (r = 0.896; p < 0.0001) snpRNA expression levels in tumor tissues compared with the adjacent normal prostate [122-fold and 45-fold in Gleason 7 tumors (p = 0.03); 370-fold and 127-fold in Gleason 8 tumors (p = 0.0001) for NLRP1-locus and 8q24-locus snpRNAs, respectively]. Our experiments indicate that RAD and CR phenotype of human PC cells can be triggered by ncRNA molecules transcribed from the NLRP1-locus intergenic enhancer at 17p13 and by downstream activation of the 8q24-locus snpRNAs. Our results define the IDAGL at 17p13 and 8q24 as candidate regulatory loci of RAD and CR phenotypes of PC, reveal previously unknown molecular links between the innate immunity/inflammasome system and development of hormone-independent PC and identify novel molecular and genetic targets with diagnostic and therapeutic potentials, exploration of which should be highly beneficial for personalized clinical management of PC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Cromossomos Humanos Par 17/genética , Cromossomos Humanos Par 8/genética , Regulação Neoplásica da Expressão Gênica/genética , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Neoplasias da Próstata/genética , Sequências Reguladoras de Ácido Ribonucleico/genética , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Biologia Computacional , DNA Intergênico/genética , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Luciferases , Masculino , Camundongos , Análise em Microsséries , Proteínas NLR , Neoplasias da Próstata/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
Meta-analysis of genomic coordinates of SNP variations identified in genome-wide association studies (GWAS) of up to 712,253 samples (comprising 221,158 disease cases, 322,862 controls, and 168,233 case/control subjects of obesity GWAS) reveals that 39% of SNPs associated with 22 common human disorders are located within intergenic regions. Chromatin-state maps based on H3K4me3-H3K36me3 signatures show that many intergenic disease-linked SNPs are located within the boundaries of the K4-K36 domains, suggesting that SNP-harboring genomic regions are transcribed. Here we report identification of 13 trans-regulatory RNAs (transRNAs) 100 to 200 nucleotides in length containing intergenic SNP sequences associated with Crohn's disease, rheumatoid arthritis, type 1 diabetes, vitiligo, hypertension and multiple types of epithelial malignancies (prostate, breast, ovarian and colorectal cancers). We demonstrate that NALP1 loci intergenic SNP sequence, rs2670660, is expressed in human cells and may contribute to clinical manifestations of autoimmune and autoimflammatory phenotypes by generating distinct allelic variants of transRNAs. Stable expression of allele-specific sense and anti-sense variants of transRNAs markedly alters cellular behavior, affect cell cycle progression, and interfere with monocyte/macrophage transdifferentiation. On a molecular level, forced expression of allele-specific sense and anti-sense variants of transRNAs asserts allele-specific genome-wide effects on abundance of hundreds microRNAs and mRNAs. Using lentiviral gene transfer, microarray and Q-RT-PCR technologies, we identify rs2670660 allele-specific gene expression signatures (GES) which appear useful for detecting the activated states of innate immunity/inflammasome pathways in approximately 700 clinical samples from 185 control subjects and 350 patients diagnosed with nine common human disorders, including Crohn's disease, ulcerative colitis, rheumatoid arthritis, Huntington disease, autism, Alzheimer disease, obesity, prostate and breast cancers. Microarray analysis of clinical samples demonstrates that rs2670660 allele-specific GES are engaged in patients' peripheral blood mononuclear cells (PBMC) which encounter pathological conditions in coherent tissues of a human body during immune surveillance and homeostasis monitoring. These data indicate that expression of transRNAs encoded by specific intergenic sequences can trigger activation of innate immunity/inflammasome pathways and contribute to clinical development of autoinflammatory and autoimmune syndromes. Documented in this work single-base substitution-driven molecular and biological antagonisms of intergenic SNP-containing transRNAs suggest a guiding mechanism of selection and retention of phenotype-compatible intergenic variations during evolution. According to this model, random genetic variations which generate transRNAs asserting antagonistic phenotype-altering effects compared to ancestral alleles will be selected and retained as SNP variants.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Polimorfismo de Nucleotídeo Único , Sequências Reguladoras de Ácido Ribonucleico/genética , Alelos , Doenças Autoimunes/genética , Sequência de Bases , Diferenciação Celular , Divisão Celular , Predisposição Genética para Doença , Genoma Humano , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Proteínas NLR , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin alphavbeta3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.
Assuntos
Neoplasias da Mama/metabolismo , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Transporte/agonistas , Proteínas de Transporte/metabolismo , Caspase 2/efeitos dos fármacos , Caspase 2/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes ras/efeitos dos fármacos , Genes ras/fisiologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Nanopartículas , Proteínas Nucleares/agonistas , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-bcl-2/agonistas , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Trombospondina 1/agonistas , Trombospondina 1/metabolismo , Tiroxina/análogos & derivados , Tiroxina/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
At present, there is no efficient curative therapy for cancer patients with advanced metastatic disease. Targeting of antiapoptotic molecules acting on the mitochondrial apoptosis pathway could potentially augment antimetastatic effect of cytotoxic drugs. Similarly to Bcl-2 family members, beta-galactoside-binding lectin galectin-3 protects cancer cells from apoptosis induced by cytotoxic drugs through the mitochondrial pathway. In this study, we tested the hypothesis that inhibiting galectin-3 antiapoptotic function using a synthetic low-molecular weight carbohydrate-based compound lactulosyl-L-leucine (Lac-L-Leu) will augment apoptosis induced in human cancer cells by paclitaxel and increase its efficacy against established metastases. Treatment with synthetic glycoamine Lac-L-Leu alone reduced the number of established MDA-MB-435Lung2 pulmonary metastases 5.5-fold (P = .032) but did not significantly affect the incidence of metastasis. Treatment with paclitaxel alone (10 mg/kg three times with 3-day intervals) had no significant effect on the incidence or on the number of MDA-MB-435Lung2 metastases. Treatment with Lac-L-Leu/paclitaxel combination decreased both the number (P = .02) and the incidence (P = .001) of pulmonary metastases, causing a five-fold increase in the number of metastasis-free animals from 14% in the control group to 70% in the combination therapy group. The median number of lung metastases dropped to 0 in the combination therapy group compared with 11 in the control (P = .02). Synergistic inhibition of clonogenic survival and induction of apoptosis in metastatic cells by Lac-L-Leu/paclitaxel combination was functionally linked with an increase in mitochondrial damage and was sufficient for the antimetastatic activity that caused a reversal and eradication of advanced metastatic disease in 56% of experimental animals.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Galactosídeos/farmacologia , Galectina 3/antagonistas & inibidores , Neoplasias Pulmonares/secundário , Paclitaxel/farmacologia , Animais , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Combinação de Medicamentos , Galectina 3/metabolismo , Humanos , Técnicas In Vitro , Leucina/química , Neoplasias Pulmonares/tratamento farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-TroncoRESUMO
The Polycomb group (PcG) gene BMI1 is required for the proliferation and self-renewal of normal and leukemic stem cells. Overexpression of Bmi1 oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia. Another PcG protein, Ezh2, was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMI1 and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors as well remains unknown. Here we demonstrate that activation of the BMI1 oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis. To characterize the functional status of the PcG pathway in metastatic prostate cancer, we utilized advanced cell- and whole animal-imaging technologies, gene and protein expression profiling, stable siRNA-gene targeting, and tissue microarray (TMA) analysis in relevant experimental and clinical settings. We demonstrate that in multiple experimental models of metastatic prostate cancer both BMI1 and Ezh2 genes are amplified and gene amplification is associated with increased expression of corresponding mRNAs and proteins. For the first time, we provide images of human prostate carcinoma metastasis precursor cells isolated from blood and shown to overexpress both BMI1 and Ezh2 oncoproteins. Consistent with the PcG pathway activation hypothesis, increased BMI1 and Ezh2 expression in metastatic cancer cells is associated with elevated levels of H2AubiK119 and H3metK27 histones. Quantitative immunofluorescence colocalization analysis and expression profiling experiments documented increased BMI1 and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMI1 and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy. Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. We conclude that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer. Our work suggests that the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides justification for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.
Assuntos
Adenocarcinoma/metabolismo , Montagem e Desmontagem da Cromatina , Inativação Gênica , Células Neoplásicas Circulantes/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Repressoras/biossíntese , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Anoikis , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Amplificação de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Células Neoplásicas Circulantes/patologia , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Complexo Repressor Polycomb 2 , Proteínas do Grupo Polycomb , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/genética , Análise Serial de Tecidos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
Color-coded imaging analysis revealed yellow fluorescent metastasis precursor cells that were readily recognized in the blood of tumor-bearing mice after mixtures of red fluorescent protein (RFP)- and green fluorescent protein (GFP)-expressing PC-3 human prostate carcinoma cells were implanted in the nude mouse prostate and metastasized. The yellow fluorescent cells were purified from the blood of nude mice to 99% homogeneity by FACS, expanded in culture, and reimplanted in the prostate of nude mice. The yellow fluorescent phenotype was heritable and stably maintained by tumor cells for many generations in vitro and in vivo. In the animals implanted with the yellow-fluorescing cells, 100% developed aggressive metastatic cancer. Lung metastases were demonstrated in 100% of the animals as early as four weeks after injection of the yellow-fluorescing cells in the mouse prostate. In contrast, when the GFP- and RFP-expressing parental cells were inoculated into the mouse prostate separately, none of the animals developed lung metastasis. All animals had almost exclusively yellow fluorescent cells in the blood and bone marrow. These results are consistent with the idea that spontaneous genetic exchange between tumor cells in vivo contributes to genomic instability and creation of highly metastatic cells.
Assuntos
Cor , Transferência Genética Horizontal/genética , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Fluorescência , Instabilidade Genômica/genética , Humanos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fenótipo , Neoplasias da Próstata/sangue , Neoplasias da Próstata/diagnósticoRESUMO
Expression analysis of 7129 transcripts was carried out in five human prostate cancer cell lines derived from orthotopic xenografts after one to five passages in nude mice and primary cultures of human normal prostate epithelial (NPE) cells. These experiments identified a consensus class of 214 genes (43 up- and 171 downregulated transcripts), expression of which was altered at least twofold in the same direction in all the cell lines relative to NPE cells. To validate the relevance of altered expression behavior of these genes for human prostate cancer, their expression pattern was evaluated in multiple additional experimental and clinical settings. Expression of 170 of these 214 genes (79%) was altered in the same direction in vivo in experimental human prostate tumors in mice. Similarly, the expression of 151 of the 214 genes (71%) was altered in the same direction in M12 cells, a variant of an SV40 large T antigen transformed normal human prostate epithelial cell line selected for increased malignancy in vivo. In clinical samples of human prostate tumors, the changes in transcript expression levels of majority of these genes (85% of downregulated and 76% of upregulated transcripts) are consistent with alterations of their expression pattern in xenograft-derived cancer cell lines. These results imply that the expression pattern of a large class of genes is consistently altered in multiple experimental models and clinical samples of human prostate cancer and underscore the potential relevance of the xenograft models and cell lines derived from them for expression analysis studies relevant to human cancer.
Assuntos
Regulação para Baixo , Neoplasias Experimentais/genética , Neoplasias da Próstata/genética , Animais , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The importance of Thomsen-Friedenreich antigen (T antigen)-galectin-3 interactions in adhesion of human breast carcinoma cells to the endothelium under conditions of flow was studied. Highly metastatic cells (MDA-MB-435) expressing high levels of both galectin-3 and T antigen demonstrated significantly increased adhesion to monolayers of endothelial cells compared with their non-metastatic counterpart (MDA-MB-468) in vitro. Within minutes of adhesion, the highly metastatic cells acquire the ability of enhanced homotypic adhesion, leading to the formation of multicellular aggregates at sites of attachment to endothelial cells in vitro. Treatment of cells with lactulosyl-l-leucine, a synthetic T antigen antagonist that targets galectin-3 by mimicking T antigen, caused a 60-80% inhibition of both homo- and heterotypic adhesion of MDA-MB-435 cells. Confocal microscopy and fluorescence-activated cell sorter analysis revealed redistribution of endothelial galectin-3 to the site of heterotypic intercellular contacts, whereas galectin-3 in MDA-MB-435 cells accumulated at sites of homotypic interaction. MDA-MB-435 cells also exhibited increased adhesion and intravascular retention within the microvessels of transplanted lung allografts in nude mice. T antigen and galectin-3-mediated interactions of metastatic cancer cells with endothelium under conditions of flow are characterized by a unique adhesion mechanism that qualitatively distinguishes their homo- and heterotypic adhesive behavior from other cell types such as leukocytes.