RESUMO
Long non-coding RNAs are increasingly being recognized as cancer biomarkers in various malignancies, acting as either tumor suppressors or oncogenes. The long non-coding MALINC1 intergenic RNA was identified as significantly upregulated in breast ductal carcinoma in situ. The aim of this study was to characterize MALINC1 expression, localization, and phenotypic and molecular effects in non-invasive and invasive breast cancer cells. We determined that MALINC1 is an estrogen-estrogen receptor-modulated lncRNA enriched in the cytoplasmic fraction of luminal A/B breast cancer cells that is associated with worse overall survival in patients with primary invasive breast carcinomas. Transcriptomic studies in normal and DCIS cells identified the main signaling pathways modulated by MALINC1, which mainly involve bioprocesses related to innate and adaptive immune responses, extracellular matrix remodeling, cell adhesion, and activation of AP-1 signaling pathway. We determined that MALINC1 induces premalignant phenotypic changes by increasing cell migration in normal breast cells. Moreover, high MALINC1 expression in invasive carcinomas was associated with a pro-tumorigenic immune environment and a favorable predicted response to immunotherapy both in luminal and basal-like subtypes compared with low-MALINC1-expression tumors. We conclude that MALINC1 behaves as an oncogenic and immune-related lncRNA involved with early-stage breast cancer progression.
RESUMO
The long-non-coding HOX transcript antisense intergenic RNA (HOTAIR) was identified as significantly upregulated in breast ductal carcinoma in situ (DCIS). The aim of this study was to characterize the phenotypic effects and signaling pathways modulated by HOTAIR in early-stage breast cancer progression. We determined that HOTAIR induces premalignant phenotypic changes by increasing cell proliferation, migration, invasion and in vivo growth in normal and DCIS breast cell lines. Transcriptomic studies (RNA-seq) identified the main signaling pathways modulated by HOTAIR which include bioprocesses related to epithelial to mesenchymal transition, cell migration, extracellular matrix remodeling and activation of several signaling pathways (HIF1A, AP1 and FGFR). Similar pathways were identified as activated in primary invasive breast carcinomas with HOTAIR over-expression. We conclude that HOTAIR over-expression behaves as a positive regulator of cell growth and migration both in normal and DCIS breast cells involved with early-stage breast cancer progression.
RESUMO
Long intergenic non-protein coding RNA 885 (LINC00885) was identified as significantly upregulated in breast ductal carcinoma in situ (DCIS). The aim of this study was to characterize the phenotypic effects and signaling pathways modulated by LINC00885 in non-invasive and invasive breast cancer models. We determined that LINC00885 induces premalignant phenotypic changes by increasing cell proliferation, motility, migration and altering 3D growth in normal and DCIS breast cell lines. Transcriptomic studies (RNA-seq) identified the main signaling pathways modulated by LINC00885, which include bioprocesses related to TP53 signaling pathway and proliferative signatures such as activation of EREG, EGFR and FOXM1 pathways. LINC00885 silencing in breast cancer lines overexpressing this lncRNA leads to downregulation of proliferation related transcripts such as EREG, CMYC, CCND1 and to significant decrease in cell migration and motility. TCGA-BRCA data analyses show an association between high LINC00885 expression and worse overall survival in patients with primary invasive breast carcinomas (p = 0.024), suggesting that the pro-tumorigenic effects of LINC00885 overexpression persist post-invasion. We conclude that LINC00885 behaves as a positive regulator of cell growth both in normal and DCIS breast cells possibly operating as a ceRNA and representing a novel oncogenic lncRNA associated with early stage breast cancer progression.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/metabolismo , Progressão da Doença , Oncogenes , RNA Longo não Codificante/genética , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinoma Intraductal não Infiltrante/patologia , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Células MCF-7 , Invasividade Neoplásica/genética , Estadiamento de Neoplasias , Interferência de RNA , RNA Longo não Codificante/metabolismo , Transcriptoma , Regulação para Cima/genéticaRESUMO
Controversy always existed on the utility of chemically induced mouse mammary carcinogenesis models as valid equivalents for the study of human breast cancer. Here, we performed whole exome and RNA sequencing on long latency mammary tumors (218 ± 27 days) induced by the carcinogen 7,12-Dimethylbenzathracene (DMBA) and short latency tumors (65 ± 11 days) induced by the progestin Medroxyprogesterone Acetate (MPA) plus DMBA in CD2F1 mice. Long latency tumors displayed a high frequency of Pi3kca and/or Pten mutations detected in 11 of 13 (85%) long latency cases (14/22, 64% overall). Eighty-two percent (9/11) of tumors carried the Pik3ca H1047L/R hot-spot mutation, as frequently found in human breast cancer. These tumors were luminal-like and mostly ER/PR+, as in humans. Transcriptome profiling indicated a significant activation of the PI3K-Akt pathway (p=3.82e-6). On the other hand MPA+DMBA induced short latency tumors displayed mutations in cancer drivers not commonly found mutated in human breast cancer (e.g. Hras and Apc). These tumors were mostly basal-like and MPA exposure led to Rankl overexpression (60 fold induction) and immunosuppressive gene expression signatures. In summary, long latency DMBA induced mouse mammary tumors reproduce the molecular profile of human luminal breast carcinomas representing an excellent preclinical model for the testing of PIK3CA/Akt/mTOR pathway inhibitory therapies and a good platform for the developing of additional preclinical tools such as syngeneic transplants in immunocompetent hosts.
Assuntos
9,10-Dimetil-1,2-benzantraceno , Transformação Celular Neoplásica/genética , Neoplasias Mamárias Experimentais/genética , Mutação , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Animais , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Classe I de Fosfatidilinositol 3-Quinases , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Mamárias Experimentais/induzido quimicamente , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Acetato de Medroxiprogesterona , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ligante RANK/genética , Ligante RANK/metabolismo , Análise de Sequência de RNA , Fatores de Tempo , Transcriptoma , Sequenciamento do ExomaRESUMO
Ductal carcinoma in situ (DCIS) is a noninvasive precursor lesion to invasive breast carcinoma. We still have no understanding on why only some DCIS lesions evolve to invasive cancer whereas others appear not to do so during the life span of the patient. Here, we performed full exome (tumor vs. matching normal), transcriptome, and methylome analysis of 30 pure high-grade DCIS (HG-DCIS) and 10 normal breast epithelial samples. Sixty-two percent of HG-DCIS cases displayed mutations affecting cancer driver genes or potential drivers. Mutations were observed affecting PIK3CA (21% of cases), TP53 (17%), GATA3 (7%), MLL3 (7%) and single cases of mutations affecting CDH1, MAP2K4, TBX3, NF1, ATM, and ARID1A. Significantly, 83% of lesions displayed numerous large chromosomal copy number alterations, suggesting they might precede selection of cancer driver mutations. Integrated pathway-based modeling analysis of RNA-seq data allowed us to identify two DCIS subgroups (DCIS-C1 and DCIS-C2) based on their tumor-intrinsic subtypes, proliferative, immune scores, and in the activity of specific signaling pathways. The more aggressive DCIS-C1 (highly proliferative, basal-like, or ERBB2(+)) displayed signatures characteristic of activated Treg cells (CD4(+)/CD25(+)/FOXP3(+)) and CTLA4(+)/CD86(+) complexes indicative of a tumor-associated immunosuppressive phenotype. Strikingly, all lesions showed evidence of TP53 pathway inactivation. Similarly, ncRNA and methylation profiles reproduce changes observed postinvasion. Among the most significant findings, we observed upregulation of lncRNA HOTAIR in DCIS-C1 lesions and hypermethylation of HOXA5 and SOX genes. We conclude that most HG-DCIS lesions, in spite of representing a preinvasive stage of tumor progression, displayed molecular profiles indistinguishable from invasive breast cancer.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/química , Metilação de DNA , DNA de Neoplasias/genética , Perfilação da Expressão Gênica , Proteínas de Neoplasias/genética , Transcriptoma , Antígenos de Diferenciação de Linfócitos T/análise , Mama/química , Neoplasias da Mama/química , Neoplasias da Mama/imunologia , Antígeno CTLA-4/análise , Carcinoma Intraductal não Infiltrante/classificação , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Intraductal não Infiltrante/imunologia , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Humanos , Linfócitos do Interstício Tumoral/química , Linfócitos do Interstício Tumoral/imunologia , Mutação , Invasividade Neoplásica/genética , Proteínas de Neoplasias/análise , RNA Mensageiro/genética , RNA Neoplásico/genética , RNA não Traduzido/genética , Linfócitos T Reguladores/imunologiaRESUMO
Oral leukoplakia is the most prevalent and potentially malignant disorder of the oral mucosa. Previous studies have demonstrated that molecular changes of the WWOX gene (WW-domain containing oxidoreductase), a candidate tumor suppressor gene located at 16q23.3-24.1 that spans FRA16D, the second most common fragile site, are present in several malignant neoplasias, including oral squamous cell carcinoma. In this report, the role of the WWOX gene was investigated in 23 cases of oral leukoplakias. Using nested RT-PCR and immunohistochemistry, altered mRNA transcription and/or reduced Wwox protein expression was observed in 35% of the lesions when compared with normal mucosa. The majority of lesions (4/6) with altered transcripts had a reduction in the expression of Wwox protein. Although normal WWOX expression was found in some lesions with dysplasia, all lesions with WWOX mRNA and/or protein expression showed histological evidence of dysplasia and none of the cases without dysplasia presented this alteration. These results show that the WWOX gene alteration is an early genetic alteration and may contribute to oral carcinogenesis.
Assuntos
Genes Supressores de Tumor , Leucoplasia Oral/genética , Oxirredutases/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Estudos de Casos e Controles , Sítios Frágeis do Cromossomo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Leucoplasia Oral/metabolismo , Leucoplasia Oral/patologia , Masculino , Pessoa de Meia-Idade , Oxirredutases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tabagismo/genética , Tabagismo/patologia , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WWRESUMO
Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm of the oral cavity, representing 90% of all oral carcinomas and accounting for 3-5% of all malignancies. The WWOX gene (WW-domain containing oxidoreductase) is a candidate tumor suppressor gene located at 16q23.3-24.1, spanning the second most common fragile site, FRA16D. In this report, the role of the WWOX gene was investigated in 20 tumors and 10 normal oral mucosas, and we demonstrated an altered WWOX gene in 50% (10/20) of OSCCs. Using nested RT-PCR, mRNA transcription was altered in 35% of the tumors, with the complete absence of transcripts in 2 samples as well as absence of exons 6-8 (2 tumors), exon 7 (1 tumor), exon 7 and exon 6-8 (1 tumor) and partial loss of exons 8 and 9 (1 tumor). To determine if the aberrant transcripts were translated, Western blots were performed in all samples; however, only the normal protein was detected. By immunohistochemistry, a reduction in Wwox protein expression was observed, affecting 40% of the tumors when compared with normal mucosa. In addition, a novel somatic mutation (S329F) was found. The presence of alterations in mRNA transcription correlated with the reduced expression of Wwox protein in the tumors. These results show that the WWOX gene is frequently altered in OSCC and may contribute to the carcinogenesis processes in oral cancer.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Oxirredutases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Sequência de Bases , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , DNA Complementar/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Oxirredutases/genética , RNA Mensageiro/genética , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WWRESUMO
We studied the karyotypes of four different mammary carcinoma cell lines derived from a medroxy-progesterone acetate (MPA)-induced mouse mammary carcinoma using G-banding and fluorescence in situ hybridization. All the cell lines showed the same four marker chromosomes (M1-M4) as the parental tumor and also acquired new markers. M1 and M2 are Robertsonian translocations between chromosomes 1 and 10 and 2 and 17. M3 is an acrocentric marker derived from chromosomes 4, 5, and 12; M4 is derived from chromosomes 6 and 8. The parental tumor disclosed a modal number of 39, with a trisomy of chromosomes 3, 4, 10, and 11 and monosomies of 9, 13, and 16. MC4-L1 and MC4-L3 lines had a chromosome number similar to that of the parental tumor in early passages, which increased to the triploid range in late passages. MC4-L5 showed a near-diploid modal number in both early and late passages. MC4-L2 cells had a high chromosome number even in early passages. To our knowledge, this is the first study in which a complete characterization of the cytogenetics of murine mammary carcinoma cell lines and of their parental tumor is described. No associations between changes in ploidy, invasiveness, or hormone dependence were found. Conversely, the presence of one exclusive marker chromosome, a translocation between chromosomes 1 and 18 (M5), in the most aggressive and in vivo hormone-independent line suggests that this rearrangement may be associated with these biologic features. The constant presence of common marker chromosomes in both the parental tumor and the derived cell lines suggests that they are involved in the maintenance of this tumor phenotype.