RESUMEN
Global changes in transcriptional regulation and RNA metabolism are crucial features of cancer development. However, little is known about the role of the core promoter in defining transcript identity and post-transcriptional fates, a potentially crucial layer of transcriptional regulation in cancer. In this study, we use CAGE-seq analysis to uncover widespread use of dual-initiation promoters in which non-canonical, first-base-cytosine (C) transcription initiation occurs alongside first-base-purine initiation across 59 human cancers and healthy tissues. C-initiation is often followed by a 5' terminal oligopyrimidine (5'TOP) sequence, dramatically increasing the range of genes potentially subjected to 5'TOP-associated post-transcriptional regulation. We show selective, dynamic switching between purine and C-initiation site usage, indicating transcription initiation-level regulation in cancers. We additionally detail global metabolic changes in C-initiation transcripts that mark differentiation status, proliferative capacity, radiosensitivity, and response to irradiation and to PI3K-Akt-mTOR and DNA damage pathway-targeted radiosensitization therapies in colorectal cancer organoids and cancer cell lines and tissues.
Asunto(s)
Fosfatidilinositol 3-Quinasas , ARN , Humanos , Sitio de Iniciación de la Transcripción , ARN/genética , Proliferación Celular , PurinasRESUMEN
Objectives: Partial or total resistance to preoperative chemoradiotherapy occurs in more than half of locally advanced rectal cancer patients. Several novel or repurposed drugs have been trialled to improve cancer cell sensitivity to radiotherapy, with limited success. We aimed to understand the mechanisms of resistance to chemoradiotherapy in rectal cancer using patient derived organoid models. Design: To understand the mechanisms underlying this resistance, we compared the pre-treatment transcriptomes of patient-derived organoids (PDO) with measured radiotherapy sensitivity to identify biological pathways involved in radiation resistance coupled with single cell sequencing, genome wide CRISPR-Cas9 and targeted drug screens. Results: RNA sequencing enrichment analysis revealed upregulation of PI3K/AKT/mTOR and epithelial mesenchymal transition pathway genes in radioresistant PDOs. Single-cell sequencing of pre & post-irradiation PDOs showed mTORC1 and PI3K/AKT upregulation, which was confirmed by a genome-wide CRSIPR-Cas9 knockout screen using irradiated colorectal cancer (CRC) cell lines. We then tested the efficiency of dual PI3K/mTOR inhibitors in improving cancer cell sensitivity to radiotherapy. After irradiation, significant AKT phosphorylation was detected (p=0.027) which was abrogated with dual PI3K/mTOR inhibitors and lead to significant radiosensitisation of the HCT116 cell line and radiation resistant PDO lines. Conclusions: The PI3K/AKT/mTOR pathway upregulation contributes to radioresistance and its targeted pharmacological inhibition leads to significant radiosensitisation in CRC organoids, making it a potential target for clinical trials.
RESUMEN
BACKGROUND: Pathological complete response (pathCR) in rectal cancer is beneficial, as up to 75% of patients do not experience regrowth of the primary tumour, but it is poorly understood. We hypothesised that the changes seen in the pre-treatment biopsies of pathCR but not seen in residual tumour after chemoradiotherapy were the determinants of responsiveness. METHODS: Two groups of patients with either complete response (pathCR group, N = 24) or no response (poor response group, N = 24) were retrieved. Pre-treatment biopsies of cancers from these patients underwent high read depth amplicon sequencing for a targeted panel, exome sequencing, methylation profiling and immunohistochemistry for DNA repair pathway proteins. RESULTS: Twenty four patients who underwent pathCR and twenty-four who underwent poor response underwent molecular characterisation. Patients in the pathCR group had significantly higher tumour mutational burden and neoantigen load, frequent copy number alterations but fewer structural variants and enrichment for driver mutations in the PI3K/AKT/mTOR signalling pathway. There were no significant differences in tumour heterogeneity as measured by MATH score. Methylation analysis demonstrated enrichment for hypomethyation in the PI3K/AKT/mTOR signalling pathway. DISCUSSION: The phenomenon of pathCR in rectal cancer may be related to immunovisibility caused by a high tumour mutational burden phenotype. Potential therapy resistance mechanisms involve the PI3K/AKT/mTOR signalling pathway, but tumour heterogeneity does not seem to play a role in resistance.
Asunto(s)
Adenocarcinoma/terapia , Quimioradioterapia , Neoplasias del Recto/terapia , Adenocarcinoma/genética , Adenocarcinoma/patología , Adulto , Estudios de Casos y Controles , Quimioradioterapia/métodos , Evolución Clonal/genética , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Regulación Neoplásica de la Expresión Génica , Genes ras/genética , Mutación de Línea Germinal , Humanos , Masculino , Terapia Neoadyuvante , Proteínas Proto-Oncogénicas c-akt/genética , Neoplasias del Recto/genética , Neoplasias del Recto/patología , Inducción de Remisión , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Radiotherapy is routinely used as a neoadjuvant, adjuvant or palliative treatment in various cancers. There is significant variation in clinical response to radiotherapy with or without traditional chemotherapy. Patients with a good response to radiotherapy demonstrate better clinical outcomes universally across different cancers. The PI3K/AKT/mTOR pathway upregulation has been linked to radiotherapy resistance. We reviewed the current literature exploring the role of inhibiting targets along this pathway, in enhancing radiotherapy response. We identified several studies using in vitro cancer cell lines, in vivo tumour xenografts and a few Phase I/II clinical trials. Most of the current evidence in this area comes from glioblastoma multiforme, non-small cell lung cancer, head and neck cancer, colorectal cancer, and prostate cancer. The biological basis for radiosensitivity following pathway inhibition was through inhibited DNA double strand break repair, inhibited cell proliferation, enhanced apoptosis and autophagy as well as tumour microenvironment changes. Dual PI3K/mTOR inhibition consistently demonstrated radiosensitisation of all types of cancer cells. Single pathway component inhibitors and other inhibitor combinations yielded variable outcomes especially within early clinical trials. There is ample evidence from preclinical studies to suggest that direct pharmacological inhibition of the PI3K/AKT/mTOR pathway components can radiosensitise different types of cancer cells. We recommend that future in vitro and in vivo research in this field should focus on dual PI3K/mTOR inhibitors. Early clinical trials are needed to assess the feasibility and efficacy of these dual inhibitors in combination with radiotherapy in brain, lung, head and neck, breast, prostate and rectal cancer patients.
RESUMEN
The exact identity of castrate-resistant (CR) cells and their relation to CR prostate cancer (CRPC) is unresolved. We use single-cell gene profiling to analyze the molecular heterogeneity in basal and luminal compartments. Within the luminal compartment, we identify a subset of cells intrinsically resistant to castration with a bi-lineage gene expression pattern. We discover LY6D as a marker of CR prostate progenitors with multipotent differentiation and enriched organoid-forming capacity. Lineage tracing further reveals that LY6D+ CR luminal cells can produce LY6D- luminal cells. In contrast, in luminal cells lacking PTEN, LY6D+ cells predominantly give rise to LY6D+ tumor cells, contributing to high-grade PIN lesions. Gene expression analyses in patients' biopsies indicate that LY6D expression correlates with early disease progression, including progression to CRPC. Our studies thus identify a subpopulation of luminal progenitors characterized by LY6D expression and intrinsic castration resistance. LY6D may serve as a prognostic maker for advanced prostate cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Madre Neoplásicas/metabolismo , Próstata/patología , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/patología , Análisis de la Célula Individual , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Linaje de la Célula , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Proteínas Ligadas a GPI/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Madre Neoplásicas/patología , Organoides/metabolismo , Organoides/patología , RegeneraciónRESUMEN
Overtreatment is a major concern in men diagnosed with prostate cancer. The aim of this study was to evaluate the combined prognostic role of three frequent molecular alterations in prostate cancer, namely relative 8q gain, ERG overexpression, and loss of PTEN expression, in a series of 136 patients with prostate cancer treated with prostatectomy and with a long follow-up. Fluorescent in situ hybridization was used to detect the relative copy number of 8q and immunohistochemistry was used for quantitative assessment of ERG and PTEN expression. During a median follow-up period of 117.8 months, 66 (49%) patients had disease recurrence. Relative 8q gain, ERG overexpression, and loss of PTEN expression were observed in 18%, 56%, and 33% of the cases, respectively. No association with patient recurrence-free survival was found for relative 8q gain or ERG overexpression on their own, whereas loss of PTEN expression was associated with worse recurrence-free survival (P=.006). Interestingly, in the subgroup of patients with normal PTEN expression, we found that the combined relative 8q gain/ERG overexpression is associated with high risk of recurrence (P=.008), suggesting that alternative mechanisms exist for progression into clinically aggressive disease. Additionally, in intermediate-risk patients with normal PTEN expression in their tumors, the combination of 8q gain/ERG overexpression was associated with a poor recurrence-free survival (P<.001), thus indicating independent prognostic value. This study shows that the combined analysis of 8q, ERG and PTEN contributes to an improved clinical outcome stratification of prostate cancer patients treated with radical prostatectomy.
RESUMEN
Prostate carcinomas harboring 8q gains are associated with poor clinical outcome, but the target genes of this genomic alteration remain to be unveiled. In this study, we aimed to identify potential 8q target genes associated with clinically aggressive prostate cancer (PCa) using fluorescence in situ hybridization (FISH), genome-wide mRNA expression, and protein expression analyses. Using FISH, we first characterized the relative copy number of 8q (assessed with MYC flanking probes) of a series of 50 radical prostatectomy specimens, with available global gene expression data and typed for E26 transformation specific (ETS) rearrangements, and then compared the gene expression profile of PCa subsets with and without 8q24 gain using Significance Analysis of Microarrays. In the subset of tumors with ERG fusion genes (ERG+), five genes were identified as significantly overexpressed (false discovery rate [FDR], ≤ 5%) in tumors with relative 8q24 gain, namely VN1R1, ZNF417, CDON, IKZF2, and NCOA2. Of these, only NCOA2 is located in 8q (8q13.3), showing a statistically higher mRNA expression in the subgroup with relative 8q gain, both in the ERG+ subgroup and in the whole series (P = 0.000152 and P = 0.008, respectively). Combining all the cases with NCOA2 overexpression, either at the mRNA or at the protein level, we identified a group of tumors with NCOA2 copy-number increase, independently of ETS status and relative 8q24 gain. Furthermore, for the first time, we detected a structural rearrangement involving NCOA2 in PCa. These findings warrant further studies with larger series to evaluate if NCOA2 relative copy-number gain presents prognostic value independently of the well-established poor prognosis associated with MYC relative copy-number gain.
Asunto(s)
Duplicación Cromosómica , Cromosomas Humanos Par 8 , Coactivador 2 del Receptor Nuclear/genética , Neoplasias de la Próstata/genética , Perfilación de la Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Masculino , Pronóstico , Neoplasias de la Próstata/fisiopatologíaRESUMEN
Gastrin-releasing peptide receptor (GRPR) is known to be overexpressed in several human malignancies, including prostate cancer, and has been implicated in multiple important neoplastic signaling pathways. We recently have shown that GRPR is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exon-level expression microarray platform. Due to its cellular localization, the relevance of its function and the availability of blocking agents, GRPR seems to be a promising candidate as therapeutic target. Our present work shows that effective knockdown of GRPR in LNCaP and VCaP cells attenuates their malignant phenotype by decreasing proliferation, invasion and anchorage-independent growth, while increasing apoptosis. Using an antibody microarray we were able to validate known and identify new targets of GRPR pathway, namely AKT1, PKCε, TYK2 and MST1. Finally, we show that overexpression of these GRPR targets is restricted to prostate carcinomas harboring ERG and/or ETV1 rearrangements, establishing their potential as therapeutic targets for these particular molecular subsets of the disease.
RESUMEN
Genomic rearrangements involving ETS transcription factors are found in 50-70% of prostate carcinomas. While the large majority of the rearrangements involve ERG, around 10% involve members of the PEA3 subfamily (ETV1, ETV4 and ETV5). Using a panel of prostate cancer cell lines we found co-overexpression of ETV1 and ETV4 in two cell line models of advanced prostate cancer (MDA-PCa-2b and PC3) and questioned whether these PEA3 family members would cooperate in the acquisition of oncogenic properties or show functional redundancy. Using shRNAs we found that ETV1 and ETV4 have partially overlapping functions, with ETV1 being more relevant for cell invasion and ETV4 for anchorage-independent growth. In vitro expression signatures revealed the regulation of both specific and shared candidate targets that may resemble cellular mechanisms in vivo by interaction with the same intermediate partners. By combining the phenotypic impact data and the gene expression profiles of in vitro models with clinico-pathological features and gene expression profiles of ETS-subtyped tumors, we identified a set of eight genes associated with advanced stage and a set of three genes associated with higher Gleason score, supporting an oncogenic role of ETV1 and ETV4 overexpression and revealing gene sets that may be useful as prognostic markers.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Biomarcadores de Tumor/genética , Proteínas de Unión al ADN/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Proteínas E1A de Adenovirus/antagonistas & inhibidores , Proteínas E1A de Adenovirus/metabolismo , Apoptosis , Biomarcadores de Tumor/metabolismo , Western Blotting , Adhesión Celular , Movimiento Celular , Proliferación Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-ets , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
Gene fusions involving the erythroblast transformation-specific (ETS) transcription factors ERG, ETV1, ETV4, ETV5, and FLI1 are a common feature of prostate carcinomas (PCas). The most common upstream fusion partner described is the androgen-regulated prostate-specific gene TMPRSS2, most frequently with ERG, but additional 5' fusion partners have been described. We performed 5' rapid amplification of cDNA ends in 18 PCas with ETV1, ETV4, or ETV5 outlier expression to identify the 5' fusion partners. We also evaluated the exon-level expression profile of these ETS genes in 14 cases. We identified and confirmed by fluorescent in situ hybridization (FISH) and reverse transcription-polymerase chain reaction the two novel chimeric genes OR51E2-ETV1 and UBTF-ETV4 in two PCas. OR51E2 encodes a G-protein-coupled receptor that is overexpressed in PCas, whereas UBTF is a ubiquitously expressed gene encoding an HMG-box DNA-binding protein involved in ribosome biogenesis. We additionally describe two novel gene fusion combinations of previously described genes, namely, SLC45A3-ETV4 and HERVK17-ETV4. Finally, we found one PCa with TMPRSS2-ETV1, one with C15orf21-ETV1, one with EST14-ETV1, and two with 14q133-q21.1-ETV1. In nine PCas (eight ETV1 and one ETV5), exhibiting ETS outlier expression and genomic rearrangement detected by FISH, no 5' fusion partner was found. Our findings contribute significantly to characterize the heterogeneous group of ETS gene fusions and indicate that all genes described as 5' fusion partners with one ETS gene can most likely be rearranged with any of the other ETS genes involved in prostate carcinogenesis.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas de Unión al ADN/genética , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Transformación Celular Neoplásica/genética , Perfilación de la Expresión Génica , Reordenamiento Génico , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas Proto-Oncogénicas c-ets , Receptores Odorantes/genéticaRESUMEN
FLI1 and ERG, the major ETS transcription factors involved in rearrangements in the Ewing's sarcoma family of tumors (ESFT) and in prostate carcinomas (PCa), respectively, belong to the same subfamily, having 98% sequence identity in the DNA binding domain. We therefore decided to investigate whether the aberrant transcription factors in both malignancies have some common downstream targets. We crossed a publicly available list of all putative EWSR1-FLI1 target genes in ESFT with our microarray expression data on 24 PCa and 6 non-malignant prostate tissues (NPT) and choose four genes among the top-most differentially expressed between PCa with (PCa ERG+) and without (PCa ETS-) ETS fusion genes (HIST1H4L, KCNN2, ECRG4 and LDOC1), as well as four well-validated direct targets of the EWSR1-FLI1 chimeric protein in ESFT (NR0B1, CAV1, IGFBP3 and TGFBR2). Using quantitative expression analysis in 16 ESFT and seven alveolar rhabdomyosarcomas (ARMS), we were able to validate the four genes previously described as direct targets of the EWSR1-FLI1 oncoprotein, showing overexpression of CAV1 and NR0B1 and underexpression of IGFBP3 and TGFBR2 in ESFT as compared to ARMS. Although none of these four genes showed significant expression differences between PCa ERG+ and PCa ETS-, CAV1, IGFBP3 and TGFBR2 were less expressed in PCa in an independent series of 56 PCa and 15 NPT, as also observed for ECRG4 and LDOC1, suggesting a role in prostate carcinogenesis in general. On the other hand, we demonstrate for the first time that both HIST1H4L and KCNN2 are significantly overexpressed in PCa ERG+ and that ERG binds to the promoter of these genes. Conversely, KCNN2 was found underexpressed in ESFT relative to ARMS, suggesting that the EWSR1-ETS oncoprotein may have the opposite effect of ERG rearrangements in PCa. We conclude that aberrant ETS transcription factors modulate target genes differently in ESFT and PCa.
Asunto(s)
Neoplasias de la Próstata , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas c-ets , Sarcoma de Ewing , Caveolina 1/genética , Caveolina 1/metabolismo , Línea Celular Tumoral , Receptor Nuclear Huérfano DAX-1/genética , Receptor Nuclear Huérfano DAX-1/metabolismo , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Análisis por Micromatrices , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína Proto-Oncogénica c-fli-1/genética , Proteína Proto-Oncogénica c-fli-1/metabolismo , Proteínas Proto-Oncogénicas c-ets/genética , Proteínas Proto-Oncogénicas c-ets/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Sarcoma de Ewing/genética , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patología , Transactivadores/genética , Transactivadores/metabolismo , Regulador Transcripcional ERGRESUMEN
This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.
Asunto(s)
Fusión de Oncogenes , Neoplasias de la Próstata/genética , Proteínas Proto-Oncogénicas c-ets/genética , Línea Celular Tumoral , Análisis por Conglomerados , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias de la Próstata/clasificación , Neoplasias de la Próstata/metabolismo , Proteínas Proto-Oncogénicas c-ets/metabolismo , Interferencia de ARN , Reproducibilidad de los Resultados , Proteínas de Unión a Tacrolimus/genética , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Regulador Transcripcional ERGRESUMEN
Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant.
Asunto(s)
Reordenamiento Génico , Leucemia Mieloide Aguda/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteínas de Fusión Oncogénica/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Proteínas de Unión al ADN/genética , Femenino , Humanos , Lactante , Cinesinas/genética , Leucemia Mieloide Aguda/diagnóstico , Masculino , Persona de Mediana Edad , Miosinas/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Pronóstico , Factores de Transcripción/genética , Factores de Elongación Transcripcional , Adulto JovenRESUMEN
To characterize the pattern of ETS rearrangements and to uncover novel ETS fusion genes, we analyzed 200 prostate carcinomas (PCa) with TaqMan low-density arrays (TLDAs), followed by selective analyses with fluorescence in situ hybridization (FISH), RT-PCR, and sequencing. Besides confirming the recurrent presence of ERG, ETV1, ETV4, and ETV5 rearrangements, we here report FLI1 as the fifth ETS transcription factor involved in fusion genes in prostate cancer. Outlier expression of the FLI1 gene was detected by TLDAs in one PCa that showed relative overexpression of FLI1 exons 4:5 as compared with FLI1 exons 2:3. A structural rearrangement was found using FISH probes flanking the FLI1 gene and RT-PCR and sequencing analyses showed fusion of SLC45A3 exon 1 with FLI1 exon 3. Interestingly, we found four cases with two different ETS rearrangements in the index tumor, thus revealing intratumor genetic heterogeneity. Correlation analysis with clinico-pathological data showed association of ERG rearrangements with locally advanced disease (pT3, P = 0.007) and MYC overexpression (P = 0.001), and association of ETV1 rearrangements with PTEN downregulation (P = 0.015). We report that FLI1 is a novel ETS transcription factor involved in gene fusions in prostate cancer and that intratumor genetic heterogeneity of ETS rearrangements can occasionally be found in index primary tumors.
Asunto(s)
Adenocarcinoma/genética , Proteínas de Transporte de Membrana/genética , Fusión de Oncogenes , Proteínas de Fusión Oncogénica/genética , Neoplasias de la Próstata/genética , Proteína Proto-Oncogénica c-fli-1/genética , Adenocarcinoma/patología , Anciano , Secuencia de Bases , Proteínas de Unión al ADN/genética , Exones , Reordenamiento Génico , Heterogeneidad Genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias de la Próstata/patología , Transactivadores/genética , Factores de Transcripción/genética , Regulador Transcripcional ERGRESUMEN
A large percentage of prostate cancers harbor TMPRSS2-ERG gene fusions, leading to aberrant overexpression of the transcription factor ERG. The target genes deregulated by this rearrangement, however, remain mostly unknown. To address this subject we performed genome-wide mRNA expression analysis on 6 non-malignant prostate samples and 24 prostate carcinomas with (nâ=â16) and without (nâ=â8) TMPRSS2-ERG fusion as determined by FISH. The top-most differentially expressed genes and their associations with ERG over-expression were technically validated by quantitative real-time PCR and biologically validated in an independent series of 200 prostate carcinomas. Several genes encoding metabolic enzymes or extracellular/transmembrane proteins involved in cell adhesion, matrix remodeling and signal transduction pathways were found to be co-expressed with ERG. Within those significantly over-expressed in fusion-positive carcinomas, CRISP3 showed more than a 50-fold increase when compared to fusion-negative carcinomas, whose expression levels were in turn similar to that of non-malignant samples. In the independent validation series, ERG and CRISP3 mRNA levels were strongly correlated (r(s)â=â0.65, p<0.001) and both were associated with pT3 disease staging. Furthermore, immunohistochemistry results showed CRISP3 protein overexpression in 63% of the carcinomas and chromatin immunoprecipitation with an anti-ERG antibody showed that CRISP3 is a direct target of the transcription factor ERG. We conclude that ERG rearrangement is associated with significant expression alterations in genes involved in critical cellular pathways that define a subset of locally advanced PCa. In particular, we show that CRISP3 is a direct target of ERG that is strongly overexpressed in PCa with the TMPRSS2-ERG fusion gene.
Asunto(s)
Biomarcadores de Tumor/genética , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/genética , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , Proteínas y Péptidos Salivales/genética , Proteínas de Plasma Seminal/genética , Anciano , Biomarcadores de Tumor/metabolismo , Western Blotting , Estudios de Casos y Controles , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Genoma Humano , Humanos , Técnicas para Inmunoenzimas , Hibridación Fluorescente in Situ , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Fusión Oncogénica/metabolismo , Próstata/metabolismo , Próstata/patología , Hiperplasia Prostática/metabolismo , Hiperplasia Prostática/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas y Péptidos Salivales/metabolismo , Proteínas de Plasma Seminal/metabolismo , Regulación hacia ArribaRESUMEN
Screening tools have greatly improved prostate cancer (PCa) detection, but the disease course is heterogeneous, and standard clinicopathological parameters do not fully discriminate aggressive from indolent tumors. To evaluate the prognostic value of the TMPRSS2-ERG fusion gene combined with chromosome arm 8q relative gain in diagnostic biopsies of PCa, we studied a consecutive series of 200 diagnostic needle biopsies from patients with 10-year disease-specific survival data. TMPRSS2-ERG fusion gene status and relative 8q gain were assessed by fluorescent in situ hybridization in whole formalin fixed paraffin-embedded biopsies. The TMPRSS2-ERG fusion gene was detected in 43.5% of PCa and was associated with lower Gleason score (P = 0.045), whereas relative 8q gain was present in 48% of PCa and was associated in high-Gleason score (P < 0.001). ERG rearrangement alone was not associated with clinical outcome, whereas relative 8q gain predicted worse disease-specific survival in PCa patients both with and without the TMPRSS2-ERG fusion gene (P < 0.001), independently of Gleason score, clinical stage, and treatment modality. We conclude that relative 8q gain in diagnostic needle biopsies is a poor prognostic factor independent of the TMPRSS2-ERG fusion gene status and of standard clinicopathological parameters.
Asunto(s)
Cromosomas Humanos Par 8 , Proteínas de Fusión Oncogénica , Neoplasias de la Próstata/genética , Serina Endopeptidasas/genética , Transactivadores/genética , Anciano , Biopsia/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Estimación de Kaplan-Meier , Masculino , Pronóstico , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/patología , Regulador Transcripcional ERGRESUMEN
The association of a genetic analysis that could improve the diagnostic accuracy of renal cell tumors in biopsy samples would allow better-informed therapeutic decisions. We performed comparative genomic hybridization (CGH) on an ex vivo fine-needle aspiration (FNA) biopsy and a tumor fragment obtained from 75 patients consecutively diagnosed with renal tumors and subjected to radical nephrectomy. The pattern of genomic changes by CGH was used blindly to classify the renal tumors and the genetic findings were subsequently compared with the histopathologic diagnosis. In particular cases, including in two carcinomas with morphologically distinct tumor areas, we performed FISH with several locus-specific probes, and looked for VHL point mutations, exonic rearrangements, or promoter methylation. CGH was successful in 82.7% FNA biopsies and in 96% tumor fragments, with the former allowing genetic diagnosis in 75% of renal cell tumors. The genetic and the initial histological classification differed in two renal neoplasias, but the genetic diagnosis was confirmed after review. The genetic pattern correctly diagnosed 93.5% of clear cell renal cell carcinomas (RCC), 61.5% of chromophobe RCC, 100% of papillary RCC, and 14.3% of oncocytomas, with the negative predictive value being 93.9, 90.7, 100, and 90.2%, respectively. The positive predictive value and specificity of copy number profiles was 100%. We demonstrate that genetic diagnosis by CGH on FNA biopsies can improve differential diagnosis in patients with kidney tumors.