RESUMO
Circulating tumor cell clusters (CTC clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Using mouse models with tagged mammary tumors, we demonstrate that CTC clusters arise from oligoclonal tumor cell groupings and not from intravascular aggregation events. Although rare in the circulation compared with single CTCs, CTC clusters have 23- to 50-fold increased metastatic potential. In patients with breast cancer, single-cell resolution RNA sequencing of CTC clusters and single CTCs, matched within individual blood samples, identifies the cell junction component plakoglobin as highly differentially expressed. In mouse models, knockdown of plakoglobin abrogates CTC cluster formation and suppresses lung metastases. In breast cancer patients, both abundance of CTC clusters and high tumor plakoglobin levels denote adverse outcomes. Thus, CTC clusters are derived from multicellular groupings of primary tumor cells held together through plakoglobin-dependent intercellular adhesion, and though rare, they greatly contribute to the metastatic spread of cancer.
Assuntos
Neoplasias da Mama/patologia , Metástase Neoplásica/patologia , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Análise de Sequência de RNA , Análise de Célula Única , gama Catenina/metabolismoRESUMO
Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases. Although these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device for efficient capture of CTCs from an endogenous mouse pancreatic cancer model and subjected CTCs to single-molecule RNA sequencing, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of WNT2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of MAP3K7 (also known as TAK1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple WNT genes, and pancreatic CTCs revealed enrichment for WNT signalling in 5 out of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Metástase Neoplásica/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Animais , Sobrevivência Celular , Inibição de Contato , Modelos Animais de Doenças , Genes Neoplásicos/genética , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Camundongos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Análise de Sequência de RNA , Proteínas Wnt/genética , Proteína Wnt2/genética , Proteína Wnt2/metabolismoRESUMO
Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture.
Assuntos
Centrômero/genética , DNA Satélite/genética , Neoplasias/genética , Sequências Repetitivas de Ácido Nucleico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Hibridização de Ácido Nucleico , RNA/genéticaRESUMO
WTX encodes a tumor suppressor implicated in the pediatric kidney cancer Wilms tumor and in mesenchymal differentiation with potentially distinct functions in the cytoplasm, at the plasma membrane, and in the nucleus. Although modulating components of the WNT signaling pathway is a proposed function for cytoplasmic and membrane-bound WTX, its nuclear properties are not well understood. Here we report that the transcriptional corepressor TRIM28 is the major binding partner for nuclear WTX. WTX interacted with the coiled coil domain of TRIM28 required for its binding to Krüppel-associated box domains of transcription factors and for its chromatin recruitment through its own coiled coil and proline-rich domains. Knockdown of endogenous WTX reduced the recruitment of TRIM28 to a chromatinized reporter sequence and its ability to repress a target transcript. In mouse embryonic stem cells where TRIM28 plays a major role in repressing endogenous retroviruses and long interspersed elements, knockdown of either TRIM28 or WTX combined with single molecule RNA sequencing revealed a highly significant shared set of differentially regulated transcripts, including derepression of non-coding repetitive sequences and their neighboring protein encoding genes (p < 1e-20). In mesenchymal precursor cells, depletion of WTX and TRIM28 resulted in analogous ß-catenin-independent defects in adipogenic and osteogenic differentiation, and knockdown of WTX reduced TRIM28 binding to Pparγ promoter. Together, the physical and functional interaction between WTX and TRIM28 suggests that the nuclear fraction of WTX plays a role in epigenetic silencing, an effect that may contribute to its function as a regulator of cellular differentiation and tumorigenesis.
Assuntos
Células-Tronco Embrionárias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Adipogenia , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Camundongos , Proteínas Nucleares/genética , Mapas de Interação de Proteínas , Proteínas Repressoras/genética , Ativação Transcricional , Proteína 28 com Motivo Tripartido , Proteínas Supressoras de Tumor/genética , Tumor de Wilms/genética , Tumor de Wilms/metabolismoRESUMO
Somatic activating mutations in EGFR identify a subset of non-small cell lung cancer that respond to tyrosine kinase inhibitors. Acquisition of drug resistance is linked to a specific secondary somatic mutation, EGFR T790M. Here we describe a family with multiple cases of non-small cell lung cancer associated with germline transmission of this mutation. Four of six tumors analyzed showed a secondary somatic activating EGFR mutation, arising in cis with the germline EGFR mutation T790M. These observations implicate altered EGFR signaling in genetic susceptibility to lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Pulmonares/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Masculino , Metionina/genética , Pessoa de Meia-Idade , Linhagem , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Treonina/genéticaRESUMO
Generating reliable expression profiles from minute cell quantities is critical for scientific discovery and potential clinical applications. Here we present low-quantity digital gene expression (LQ-DGE), an amplification-free approach involving capture of poly(A)(+) RNAs from cellular lysates onto poly(dT)-coated sequencing surfaces, followed by on-surface reverse transcription and sequencing. We applied LQ-DGE to profile malignant and nonmalignant mouse and human cells, demonstrating its quantitative power and potential applicability to archival specimens.
Assuntos
Perfilação da Expressão Gênica/métodos , Animais , Linhagem Celular , Humanos , Camundongos , Análise em Microsséries/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Poli T/metabolismo , RNA Mensageiro/metabolismo , Transcrição Reversa , Análise de Sequência de DNARESUMO
PURPOSE: Somatic mutations in the epidermal growth factor receptor (EGFR) gene occur in a subset of non-small-cell lung cancer (NSCLC) and are highly predictive of the clinical response to selective EGFR kinase inhibitors. The prevalence of EGFR-mutant NSCLC is appreciably higher in females than in males and in East Asian than in Caucasian populations. We hypothesized that these disparate frequencies may be attributable to underlying genetic modifiers. Given the coincident differences in sex and ethnic origin, we tested allozymatic variants of enzymes involved in estrogen biosynthesis and metabolism, encoded by polymorphic alleles known to differ in frequency between Caucasian and Asian populations, as modifying alleles. EXPERIMENTAL DESIGN: We genotyped nine polymorphisms in the CYP1A1, CYP17A1, CYP19, HSD17B1, COMT, GSTM1, and GSTT1 genes, in a series of 100 Japanese NSCLCs, selected for equal representation of EGFR wild-type (wt) and EGFR-mutant cases, as well as male and female cases. Associations between polymorphic variants and the EGFR genotype and sex of NSCLC cases were examined using Fisher's exact test of significance. RESULTS: Only CYP1A1 2C showed a difference in allele frequency that approached statistical significance. Heterozygotes were underrepresented among EGFR-mutant cases compared with EGFR-wt cases (27% versus 47%, P = 0.08), with a concurrent trend toward overrepresentation of CYP1A1 2C(Ile/Ile) homozygotes among EGFR-mutant cases as compared with EGFR-wt cases (69% versus 51%, P = 0.13). CONCLUSION: Within the power of this study, our findings suggest that the selected polymorphic variants in the estrogen biosynthesis and metabolism pathways are unlikely to be major genetic modifiers of the prevalence of EGFR-mutant NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Estrogênios/metabolismo , Neoplasias Pulmonares/genética , Mutação , Polimorfismo Genético , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/etnologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Frequência do Gene , Genótipo , Heterozigoto , Humanos , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/etnologia , Neoplasias Pulmonares/metabolismo , Masculino , Prevalência , Fatores SexuaisRESUMO
Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the epidermal growth factor receptor (EGFR) kinase domain tend to respond well to the tyrosine kinase inhibitors, gefitinib and erlotinib. However, following clinical response, these patients typically relapse within a year of treatment. In many cases, resistance is caused by an acquired secondary EGFR kinase domain mutation, T790M. In vitro studies have shown that a new class of EGFR-irreversible inhibitors could overcome the resistance conferred by T790M. Clinical trials are under way to examine the efficacy of one of these inhibitors, HKI-272, in patients with NSCLC who initially responded to gefitinib/erlotinib and subsequently relapsed. To anticipate the possibility that patients who respond to irreversible inhibitors will develop secondary resistance to such inhibitors, as has been seen in other similar settings, we modeled acquired resistance to the dual EGFR/HER2-irreversible tyrosine kinase inhibitor HKI-272 in a NSCLC cell culture model. We found that HKI-272-resistant clones fall into two biochemical groups based on the retention of EGFR phosphorylation in the presence of the drug. Cells that retain phosphorylated EGFR have acquired the secondary mutation T790M. Moreover, HKI-272 can overcome T790M resistance only at suprapharmacologic concentrations. We further model mutations at EGFR C797 as a mechanism of resistance to irreversible EGFR inhibitors and show that although these mutants are resistant to the irreversible inhibitor, they retain erlotinib sensitivity. Our findings suggest that HKI-272 treatment at maximally tolerated dosing may lead to the emergence of T790M-mediated resistance, whereas treatment with a more potent irreversible inhibitor could yield a resistance mutation at EGFR C797.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Mutação/genética , Quinolinas/farmacologia , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Células Cultivadas , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib , Gefitinibe , Humanos , Immunoblotting , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologiaRESUMO
We describe two non-small-cell lung cancer (NSCLC) patients in which treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKIs) gefitinib produced a prolonged control of bone disease. In the first patient, a 48-year-old male with adenocarcinoma (ADC) of the lung and multiple bone metastases, the bone scan became completely negative following treatment with gefitinib for 9 months. The patient remained alive and with no evidence of bone metastases for 20 months, despite two local recurrences that were surgically removed. Similarly, the bone scan of the second patient, a 49-year-old male with ADC of the lung and bone metastases, became negative after 6 months on gefitinib. The molecular mechanisms potentially involved in this phenomenon are discussed.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/secundário , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Quinazolinas/administração & dosagem , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Seguimentos , Gefitinibe , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/cirurgia , Fatores de TempoRESUMO
BACKGROUND: Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS: We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS: Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS: A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Genes erbB-1 , Neoplasias Pulmonares/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Quinazolinas/uso terapêutico , Deleção de Sequência , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Análise Mutacional de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Heterozigoto , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Mutação , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismoRESUMO
PURPOSE: Specific activating mutations within the epidermal growth factor receptor (EGFR) identify a subset of non-small cell lung cancers with dramatic sensitivity to the specific tyrosine kinase inhibitors (TKI), gefitinib and erlotinib. Despite the abundant expression of EGFR protein in a broad range of epithelial cancers, EGFR mutations have not been reported in a substantial fraction of other cancers. Given recent reports of TKI-responsive cases of esophageal and pancreatic cancer, this study was designed to determine the prevalence of EGFR mutations in these gastrointestinal cancers. EXPERIMENTAL DESIGN: We sequenced exons 18 to 21 of EGFR from 21 cases of Barrett's esophagus, 5 cases of high-grade esophageal dysplasia, 17 cases of esophageal adenocarcinoma, and 55 cases of pancreatic adenocarcinoma. Subsets of esophageal (n = 7) and pancreatic cancer cases (n = 5) were obtained from patients who were subsequently treated with gefitinib or erlotinib-capecitabine, respectively. RESULTS: Mutations of EGFR were identified in two esophageal cancers (11.7%), three cases of Barrett's esophagus (14.2%), and two pancreatic cancers (3.6%). The mutations consisted of the recurrent missense L858R and in-frame deletion delE746-A750, previously characterized as activating EGFR mutations in non-small cell lung cancer. We also identified the TKI drug resistance-associated EGFR T790M mutation in an untreated case of Barrett's esophagus and the corresponding adenocarcinoma. CONCLUSION: The presence of activating mutations within EGFR in both esophageal and pancreatic adenocarcinomas defines a previously unrecognized subset of gastrointestinal tumors in which EGFR signaling may play an important biological role. EGFR mutations in premalignant lesions of Barrett's esophagus also point to these as an early event in transformation of the esophageal epithelium. The role of genotype-directed TKI therapy should be tested in prospective clinical trials.
Assuntos
Adenocarcinoma/genética , Receptores ErbB/química , Receptores ErbB/genética , Neoplasias Esofágicas/genética , Mutação , Neoplasias Pancreáticas/genética , Adenocarcinoma/metabolismo , Antineoplásicos/farmacologia , Transformação Celular Neoplásica , Análise Mutacional de DNA , Éxons , Genótipo , Humanos , Estrutura Terciária de ProteínaRESUMO
PURPOSE: Most cases of non-small-cell lung cancer (NSCLC) with dramatic responses to gefitinib have specific activating mutations in the epidermal growth factor receptor (EGFR), but the predictive value of these mutations has not been defined in large clinical trials. The goal of this study was to determine the contribution of molecular alterations in EGFR to response and survival within the phase II (IDEAL) and phase III (INTACT) trials of gefitinib. PATIENTS AND METHODS: We analyzed the frequency of EGFR mutations in lung cancer specimens from both the IDEAL and INTACT trials and compared it with EGFR gene amplification, another genetic abnormality in NSCLC. RESULTS: EGFR mutations correlated with previously identified clinical features of gefitinib response, including adenocarcinoma histology, absence of smoking history, female sex, and Asian ethnicity. No such association was seen in patients whose tumors had EGFR amplification, suggesting that these molecular markers identify different biologic subsets of NSCLC. In the IDEAL trials, responses to gefitinib were seen in six of 13 tumors (46%) with an EGFR mutation, two of seven tumors (29%) with amplification, and five of 56 tumors (9%) with neither mutation nor amplification (P = .001 for either EGFR mutation or amplification v neither abnormality). Analysis of the INTACT trials did not show a statistically significant difference in response to gefitinib plus chemotherapy according to EGFR genotype. CONCLUSION: EGFR mutations and, to a lesser extent, amplification appear to identify distinct subsets of NSCLC with an increased response to gefitinib. The combination of gefitinib with chemotherapy does not improve survival in patients with these molecular markers.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Amplificação de Genes , Neoplasias Pulmonares/genética , Mutação , Quinazolinas/uso terapêutico , Adulto , Idoso , Antineoplásicos , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Feminino , Gefitinibe , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Taxa de SobrevidaRESUMO
PURPOSE: Small-molecule tyrosine kinase inhibitors (TKI) of the epidermal growth factor receptor (EGFR) have shown modest yet reproducible response rates in patients with squamous cell carcinoma of the head and neck (SCCHN). Somatic mutations in EGFR have recently been shown to be predictive of a clinical response in patients with non-small cell lung cancer (NSCLC) treated with these inhibitors. The objective of this study was to determine if such mutations, or recently reported mutations in ERBB2, also underlie EGFR-TKI responsiveness in SCCHN patients. EXPERIMENTAL DESIGN: We sequenced the kinase domain of EGFR and exon 20 of ERBB2 in tumor specimens from eight responsive patients. In addition, mutational analysis was done on tumor specimens from nine gefitinib nonresponders and 65 unselected cases of SCCHN. RESULTS: None of eight TKI-responsive specimens had mutations within the kinase domain of EGFR. EGFR amplification was also not associated with drug responsiveness. However, a single responsive case had a somatic missense mutation within exon 20 of ERBB2. CONCLUSION: Our data indicate that unlike NSCLC, EGFR kinase mutations are rare in unselected cases of SCCHN within the United States and are not linked to gefitinib or erlotinib responses in SCCHN. Alternative mechanisms, including ERBB2 mutations, may underlie responsiveness in this tumor type.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Receptores ErbB/genética , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Mutação/genética , Inibidores de Proteínas Quinases/uso terapêutico , Receptor ErbB-2/genética , Idoso , Antineoplásicos/uso terapêutico , Sequência de Bases , Carcinoma de Células Escamosas/genética , Análise Mutacional de DNA , Receptores ErbB/antagonistas & inibidores , Feminino , Gefitinibe , Neoplasias de Cabeça e Pescoço/genética , Humanos , Masculino , Pessoa de Meia-Idade , Quinazolinas/uso terapêutico , Resultado do TratamentoRESUMO
Prostate cancer is initially responsive to androgen deprivation, but the effectiveness of androgen receptor (AR) inhibitors in recurrent disease is variable. Biopsy of bone metastases is challenging; hence, sampling circulating tumor cells (CTCs) may reveal drug-resistance mechanisms. We established single-cell RNA-sequencing (RNA-Seq) profiles of 77 intact CTCs isolated from 13 patients (mean six CTCs per patient), by using microfluidic enrichment. Single CTCs from each individual display considerable heterogeneity, including expression of AR gene mutations and splicing variants. Retrospective analysis of CTCs from patients progressing under treatment with an AR inhibitor, compared with untreated cases, indicates activation of noncanonical Wnt signaling (P = 0.0064). Ectopic expression of Wnt5a in prostate cancer cells attenuates the antiproliferative effect of AR inhibition, whereas its suppression in drug-resistant cells restores partial sensitivity, a correlation also evident in an established mouse model. Thus, single-cell analysis of prostate CTCs reveals heterogeneity in signaling pathways that could contribute to treatment failure.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Células Neoplásicas Circulantes/metabolismo , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Proteínas Wnt/metabolismo , Antagonistas de Androgênios/farmacologia , Animais , Benzamidas , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Células Neoplásicas Circulantes/efeitos dos fármacos , Nitrilas , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Splicing de RNA , Análise de Sequência de RNA/métodos , Transdução de Sinais , Análise de Célula Única/métodos , Transcriptoma , Proteínas Wnt/genética , Proteína Wnt-5a , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Melanoma is an invasive malignancy with a high frequency of blood-borne metastases, but circulating tumor cells (CTCs) have not been readily isolated. We adapted microfluidic CTC capture to a tamoxifen-driven B-RAF/PTEN mouse melanoma model. CTCs were detected in all tumor-bearing mice and rapidly declined after B-RAF inhibitor treatment. CTCs were shed early from localized tumors, and a short course of B-RAF inhibition following surgical resection was sufficient to dramatically suppress distant metastases. The large number of CTCs in melanoma-bearing mice enabled a comparison of RNA-sequencing profiles with matched primary tumors. A mouse melanoma CTC-derived signature correlated with invasiveness and cellular motility in human melanoma. CTCs were detected in smaller numbers in patients with metastatic melanoma and declined with successful B-RAF-targeted therapy. Together, the capture and molecular characterization of CTCs provide insight into the hematogenous spread of melanoma.
Assuntos
Células Neoplásicas Circulantes/metabolismo , Animais , Antineoplásicos Hormonais/farmacologia , Linhagem Celular Tumoral , Separação Celular , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Técnicas Analíticas Microfluídicas , Metástase Neoplásica , Células Neoplásicas Circulantes/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/metabolismo , Análise de Sequência de RNA , Tamoxifeno/farmacologia , Transcriptoma/efeitos dos fármacosRESUMO
Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.
Assuntos
Matriz Extracelular/genética , Regulação Neoplásica da Expressão Gênica , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/patologia , Família Aldeído Desidrogenase 1 , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Movimento Celular , Matriz Extracelular/metabolismo , Humanos , Camundongos , Osteonectina/antagonistas & inibidores , Osteonectina/genética , Osteonectina/metabolismo , Neoplasias Pancreáticas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Análise de Sequência de RNA , Células Tumorais CultivadasRESUMO
UNLABELLED: Androgen deprivation therapy (ADT) is initially effective in treating metastatic prostate cancer, and secondary hormonal therapies are being tested to suppress androgen receptor (AR) reactivation in castration-resistant prostate cancer (CRPC). Despite variable responses to AR pathway inhibitors in CRPC, there are no reliable biomarkers to guide their application. Here, we used microfluidic capture of circulating tumor cells (CTC) to measure AR signaling readouts before and after therapeutic interventions. Single-cell immunofluorescence analysis revealed predominantly "AR-on" CTC signatures in untreated patients, compared with heterogeneous ("AR-on, AR-off, and AR-mixed") CTC populations in patients with CRPC. Initiation of first-line ADT induced a profound switch from "AR-on" to "AR-off" CTCs, whereas secondary hormonal therapy in CRPC resulted in variable responses. Presence of "AR-mixed" CTCs and increasing "AR-on" cells despite treatment with abiraterone acetate were associated with an adverse treatment outcome. Measuring treatment-induced signaling responses within CTCs may help guide therapy in prostate cancer. SIGNIFICANCE: Acquired resistance to first-line hormonal therapy in prostate cancer is heterogeneous in the extent of AR pathway reactivation. Measurement of pre- and posttreatment AR signaling within CTCs may help target such treatments to patients most likely to respond to second-line therapies.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Hormônio-Dependentes/metabolismo , Células Neoplásicas Circulantes/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias Hormônio-Dependentes/sangue , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/sangue , Neoplasias da Próstata/patologia , Receptores Androgênicos/genética , Transdução de SinaisRESUMO
Satellite repeats in heterochromatin are transcribed into noncoding RNAs that have been linked to gene silencing and maintenance of chromosomal integrity. Using digital gene expression analysis, we showed that these transcripts are greatly overexpressed in mouse and human epithelial cancers. In 8 of 10 mouse pancreatic ductal adenocarcinomas (PDACs), pericentromeric satellites accounted for a mean 12% (range 1 to 50%) of all cellular transcripts, a mean 40-fold increase over that in normal tissue. In 15 of 15 human PDACs, alpha satellite transcripts were most abundant and HSATII transcripts were highly specific for cancer. Similar patterns were observed in cancers of the lung, kidney, ovary, colon, and prostate. Derepression of satellite transcripts correlated with overexpression of the long interspersed nuclear element 1 (LINE-1) retrotransposon and with aberrant expression of neuroendocrine-associated genes proximal to LINE-1 insertions. The overexpression of satellite transcripts in cancer may reflect global alterations in heterochromatin silencing and could potentially be useful as a biomarker for cancer detection.
Assuntos
DNA Satélite/genética , Neoplasias/genética , Neoplasias Pancreáticas/genética , RNA Neoplásico/genética , RNA não Traduzido/genética , Animais , Carcinoma in Situ/genética , Carcinoma in Situ/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Metilação de DNA , DNA de Neoplasias/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Heterocromatina/química , Heterocromatina/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Nus , Neoplasias/patologia , Sistemas Neurossecretores/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Neoplásico/metabolismo , RNA não Traduzido/metabolismo , Transcrição GênicaRESUMO
In a genome-wide screen of 684 cancer cell lines, we identified homozygous intragenic microdeletions involving genes encoding components of the apical-basal cell polarity complexes. Among these, PARD3 is disrupted in cell lines and primary tumors from squamous carcinomas and glioblastomas. Reconstituting PARD3 expression in both cell types restores tight junctions and retards contact-dependent proliferation. Searching specifically for small intragenic microdeletions using high-resolution genomic arrays may be complementary to other genomic deletion screens and resequencing efforts in identifying new tumor suppressor genes.
Assuntos
Deleção de Genes , Neoplasias/genética , Proteínas Adaptadoras de Transdução de Sinal , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Genoma Humano , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hibridização in Situ Fluorescente , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Junções Íntimas/genética , Junções Íntimas/metabolismo , Junções Íntimas/patologiaRESUMO
Mutational inactivation of genes controlling the DNA-damage response contributes to cancer susceptibility within families and within the general population as well as to sporadic tumorigenesis. Claspin (CLSPN) encodes a recently recognized mediator protein essential for the ATR and CHK1-dependent checkpoint elicited by replicative stress or the presence of ssDNA. Here, we describe a study to determine whether mutational disruption of CLSPN contributes to cancer susceptibility and sporadic tumorigenesis. We resequenced CLSPN from the germline of selected cancer families with a history of breast cancer (n = 25) or a multicancer phenotype (n = 46) as well as from a panel of sporadic cancer cell lines (n = 52) derived from a variety of tumor types. Eight nonsynonymous variants, including a recurrent mutation, were identified from the germline of two cancer-prone individuals and five cancer cell lines of breast, ovarian, and hematopoietic origin. None of the variants was present within population controls. In contrast, mutations were rare within genes encoding the CLSPN-interacting protein ATR and its binding partner ATRIP. One variant of CLSPN, encoding the I783S missense mutation, was defective in its ability to mediate CHK1 phosphorylation following DNA damage and was unable to rescue sensitivity to replicative stress in CLSPN-depleted cells. Taken together, these observations raise the possibility that CLSPN may encode a component of the DNA-damage response pathway that is targeted by mutations in human cancers, suggesting the need for larger population-based studies to investigate whether CLSPN variants contribute to cancer susceptibility.