RESUMO
The zinc finger homeobox 3 (ZFHX3, also named ATBF1 for AT motif binding factor 1) is a transcription factor that suppresses prostatic carcinogenesis and induces neuronal differentiation. It also interacts with estrogen receptor α to inhibit cell proliferation and regulate pubertal mammary gland development in mice. In the present study, we examined whether and how Zfhx3 regulates lactogenic differentiation in mouse mammary glands. At different stages of mammary gland development, Zfhx3 protein was expressed at varying levels, with the highest level at lactation. In the HC11 mouse mammary epithelial cell line, an in vitro model of lactogenesis, knockdown of Zfhx3 attenuated prolactin-induced ß-casein expression and morphological changes, indicators of lactogenic differentiation. In mouse mammary tissue, knock-out of Zfhx3 interrupted lactogenesis, resulting in underdeveloped glands with much smaller and fewer alveoli, reduced ß-casein expression, accumulation of large cytoplasmic lipid droplets in luminal cells after parturition, and failure in lactation. Mechanistically, Zfhx3 maintained the expression of Prlr (prolactin receptor) and Prlr-Jak2-Stat5 signaling activity, whereas knockdown and knock-out of Zfhx3 in HC11 cells and mammary tissues, respectively, decreased Prlr expression, Stat5 phosphorylation, and the expression of Prlr-Jak2-Stat5 target genes. These findings indicate that Zfhx3 plays an essential role in proper lactogenic development in mammary glands, at least in part by maintaining Prlr expression and Prlr-Jak2-Stat5 signaling activity.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Glândulas Mamárias Animais/metabolismo , Prolactina/metabolismo , Transdução de Sinais , Animais , Western Blotting , Caseínas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Imuno-Histoquímica , Janus Quinase 2/metabolismo , Lactação/genética , Lactação/metabolismo , Células MCF-7 , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prolactina/farmacologia , Receptores da Prolactina/genética , Receptores da Prolactina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT5/metabolismoRESUMO
α-Fetoprotein (AFP) is known to be highly produced in fetal liver despite its barely detectable level in normal adult liver. On the other hand, hepatocellular carcinoma often shows high expression of AFP. Thus, AFP seems to be an oncogenic marker. In our present study, we investigated how TGF-ß signaling cooperates with AT motif-binding factor-1 (ATBF1) to inhibit AFP transcription. Indeed, the expression of AFP mRNA in HuH-7 cells was negatively regulated by TGF-ß signaling. To further understand how TGF-ß suppresses the transcription of the AFP gene, we analyzed the activity of the AFP promoter in the presence of TGF-ß. We found that the TGF-ß signaling and ATBF1 suppressed AFP transcription through two ATBF1 binding elements (AT-motifs). Using a heterologous reporter system, both AT-motifs were required for transcriptional repression upon TGF-ß stimulation. Furthermore, Smads were found to interact with ATBF1 at both its N-terminal and C-terminal regions. Since the N-terminal (ATBF1N) and C-terminal regions of ATBF1 (ATBF1C) lack the ability of DNA binding, both truncated mutants rescued the cooperative inhibitory action by the TGF-ß signaling and ATBF1 in a dose-dependent manner. Taken together, these findings indicate that TGF-ß signaling can act in concert with ATBF1 to suppress the activity of the AFP promoter through direct interaction of ATBF1 with Smads.
RESUMO
BACKGROUND: The AT-motif binding factor 1 (ATBF1) gene is frequently altered at the genetic level in several types of cancer, but its protein expression and subcellular localization have not been well studied in human cancers, including head and neck squamous cell carcinomas (HNSCCs). METHODS: ATBF1 expression and localization were examined in 5 cell lines and 197 clinical specimens of HNSCC, and correlated with pathologic and clinical characteristics. RESULTS: ATBF1 was predominantly localized in the nucleus of hyperplastic squamous epithelium. Whereas nuclear ATBF1 dramatically decreased in invasive tumors (p = .0012), cytoplasmic ATBF1 levels progressively increased from dysplasia to invasive tumors (p < .0001), and the increase correlated with poor survival. Reduced nuclear ATBF1 level was also detected in HNSCC cell lines. CONCLUSIONS: Nuclear localization of ATBF1 is frequently interrupted in HNSCC, and the interruption is significantly associated with the progression of HNSCC. The cytoplasmic ATBF1 level could be useful for predicting patient survival.
Assuntos
Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Núcleo Celular/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias de Cabeça e Pescoço/diagnóstico , Neoplasias de Cabeça e Pescoço/genética , Proteínas de Homeodomínio/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Imunofluorescência , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Carcinoma de Células Escamosas de Cabeça e Pescoço , Adulto JovemRESUMO
We reported previously that the tumour suppressor ATBF1 (AT motif-binding factor 1) formed an autoregulatory feedback loop with oestrogen-ERα (oestrogen receptor α) signalling to regulate oestrogen-dependent cell proliferation in breast cancer cells. In this loop ATBF1 inhibits the function of oestrogen-ERα signalling, whereas ATBF1 protein levels are fine-tuned by oestrogen-induced transcriptional up-regulation as well as UPP (ubiquitin-proteasome pathway)-mediated protein degradation. In the present study we show that EFP (oestrogen-responsive finger protein) is an E3 ubiquitin ligase mediating oestrogen-induced ATBF1 protein degradation. Knockdown of EFP increases ATBF1 protein levels, whereas overexpression of EFP decreases ATBF1 protein levels. EFP interacts with and ubiquitinates ATBF1 protein. Furthermore, we show that EFP is an important factor in oestrogen-induced ATBF1 protein degradation in which some other factors are also involved. In human primary breast tumours the levels of ATBF1 protein are positively correlated with the levels of EFP protein, as both are directly up-regulated ERα target gene products. However, the ratio of ATBF1 protein to EFP protein is negatively correlated with EFP protein levels. Functionally, ATBF1 antagonizes EFP-mediated cell proliferation. These findings not only establish EFP as the E3 ubiquitin ligase for oestrogen-induced ATBF1 protein degradation, but further support the autoregulatory feedback loop between ATBF1 and oestrogen-ERα signalling and thus implicate ATBF1 in oestrogen-dependent breast development and carcinogenesis.
Assuntos
Estrogênios/fisiologia , Proteínas de Homeodomínio/metabolismo , Proteólise , Fatores de Transcrição/biossíntese , Ubiquitina-Proteína Ligases/biossíntese , Linhagem Celular Tumoral , Feminino , Proteínas de Homeodomínio/biossíntese , Humanos , Proteínas com Motivo TripartidoRESUMO
Whereas estrogen-estrogen receptor α (ER) signaling plays an important role in breast cancer growth, it is also necessary for the differentiation of normal breast epithelial cells. How this functional conversion occurs, however, remains unknown. Based on a genome-wide sequencing study that identified mutations in several breast cancer genes, we examined some of the genes for mutations, expression levels, and functional effects on cell proliferation and tumorigenesis. We present the data for C1orf64 or ER-related factor (ERRF) from 31 cell lines and 367 primary breast cancer tumors. Whereas mutation of ERRF was infrequent (1 of 79 or 1.3%), its expression was up-regulated in breast cancer, and the up-regulation was more common in lower-stage tumors. In addition, increased ERRF expression was significantly associated with ER and/or progesterone receptor (PR) positivity, which was still valid in human epidermal growth factor receptor 2 (HER2)-negative tumors. In ER-positive tumors, ERRF expression was inversely correlated with HER2 status. Furthermore, higher ERRF protein expression was significantly associated with better disease-free survival and overall survival, particularly in ER- and/or PR-positive and HER2-negative tumors (luminal A subtype). Functionally, knockdown of ERRF in two ER-positive breast cancer cell lines, T-47D and MDA-MB-361, suppressed cell growth in vitro and tumorigenesis in xenograft models. These results suggest that ERRF plays a role in estrogen-ER-mediated growth of breast cancer cells and could, thus, be a potential therapeutic target.
Assuntos
Neoplasias da Mama/genética , Receptor alfa de Estrogênio/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Genes erbB-2/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Proteínas Nucleares/genética , RNA Interferente Pequeno/farmacologia , Transplante Heterólogo , Regulação para CimaRESUMO
The proper level of estrogen-estrogen receptor (ER) signaling is important for the maintenance of epithelial homeostasis in the breast. In a previous study we demonstrated that ATBF1, which has been suggested as a tumor suppressor in breast cancer, inhibited estrogen-mediated cell proliferation by selectively competing with AIB1 for binding to the ER. However, the expression of ATBF1 mRNA was shown to positively correlate with ER in breast cancer specimens. We, therefore, examined whether estrogen regulates ATBF1. We demonstrated that estrogen up-regulated the transcription of ATBF1, which was mediated by the direct binding of the ER onto the ATBF1 promoter, and that a half-estrogen-responsive element in the ATBF1 promoter was essential for ER direct binding. Furthermore, we found that estrogen at lower levels increased, but at higher levels decreased the expression of ATBF1 protein, which involved the degradation of ATBF1 protein by the estrogen-responsive proteasome system. ATBF1 protein levels fluctuate with estrogen levels. Although lower levels of estrogen increased ATBF1 protein expression, ATBF1 still inhibited cell proliferation caused by lower levels of estrogen. These findings not only reveal an autoregulatory feedback loop between ATBF1 and estrogen-ER signaling but also suggest that ATBF1 plays a role in both the maintenance of breast epithelial homeostasis and breast tumorigenesis caused by elevated estrogen levels.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/biossíntese , Estrogênios/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/biossíntese , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cicloeximida/farmacologia , Feminino , Perfilação da Expressão Gênica , Humanos , Oligonucleotídeos/farmacologia , Regiões Promotoras Genéticas , Interferência de RNA , Transdução de Sinais , Regulação para CimaRESUMO
Loss of the q22 band of chromosome 16 is a frequent genetic event in breast cancer, and the candidate tumor suppressor gene, ATBF1, has been implicated in breast cancer by genomic deletion, transcriptional down-regulation, and association with better prognostic parameters. In addition, estrogen receptor (ER)-positive breast cancer expresses a higher level of ATBF1, suggesting a role of ATBF1 in ER-positive breast cancer. In this study, we examined whether and how ATBF1 affects the ER function in breast cancer cells. We found that ATBF1 inhibited ER-mediated gene transcription, cell growth, and proliferation in ER-positive breast cancer cells. In vitro and in vivo immunoprecipitation experiments revealed that ATBF1 interacted physically with the ER and that multiple domains in both ATBF1 and ER proteins mediated the interaction. Furthermore, we demonstrated that ATBF1 inhibited ER function by selectively competing with the steroid receptor coactivator AIB1 but not GRIP1 or SRC1 for binding to the ER. These findings not only support the concept that ATBF1 plays a tumor-suppressive role in breast cancer, they also provide a mechanism for how ATBF1 functions as a tumor suppressor in breast cancer.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptores de Estrogênio/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Feminino , Deleção de Genes , Proteínas de Homeodomínio/genética , Humanos , Masculino , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Coativador 3 de Receptor Nuclear/genética , Coativador 3 de Receptor Nuclear/metabolismo , Ligação Proteica , Receptores de Estrogênio/genética , Transcrição Gênica/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Kruppel-like factor 5 (KLF5) is implicated in human breast cancer by frequent genomic deletion and expressional deregulation, but the molecular mechanisms by which KLF5 affects breast tumorigenesis are still unknown. This study was conducted to examine whether and how KLF5 affects the function of estrogen receptor (ER) in breast cancer cells. Using different cell lines, we found that restored expression of KLF5 inhibited estrogen-promoted cell proliferation in ER-positive MCF-7 and T-47D cell lines but had no effect on ER-negative SK-BR-3 cells. Transcriptional activity of ER was also suppressed by KLF5, as detected by using estrogen-stimulated ER responsive element-mediated reporter assay and expression analysis of ER target genes including c-MYC and Cathepsin D (CSTD). Chromatin immunoprecipitation assays showed that KLF5 inhibited ERalpha binding to the promoter of c-myc and CSTD. Furthermore, estrogen induced an interaction between KLF5 and ERalpha. These results suggest that KLF5 inhibits the function of ERalpha in gene regulation and cell proliferation through protein interaction that interrupts the binding of ERalpha to target gene promoters to prevent target gene induction.
Assuntos
Neoplasias da Mama/metabolismo , Receptor alfa de Estrogênio/fisiologia , Estrogênios/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Sequência de Bases , Neoplasias da Mama/patologia , Catepsina D/genética , Linhagem Celular Tumoral , Proliferação de Células , Imunoprecipitação da Cromatina , Primers do DNA , Receptor alfa de Estrogênio/metabolismo , Estrogênios/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Genes myc , Humanos , Fatores de Transcrição Kruppel-Like/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , RNA Interferente Pequeno , Transcrição Gênica/fisiologiaRESUMO
Deletion of chromosome 6q is frequent in breast cancer, and the deletion often involves a region in 6q14-q16. At present, however, the underlying tumor suppressor gene has not been established. Based on a recent study identifying snoRNA U50 as a candidate for the 6q14-16 tumor suppressor gene in prostate cancer, we investigated whether U50 is also involved in breast cancer. PCR-based approaches showed that U50 underwent frequent genomic deletion and transcriptional downregulation in cell lines derived from breast cancer. Mutation screening identified the same 2-bp deletion of U50 as in prostate cancer in both cell lines and primary tumors from breast cancer, and the deletion was both somatic and in germline. Genotyping of a cohort of breast cancer cases and controls for the mutation demonstrated that, while homozygous genotype of the mutation was rare, its heterozygous genotype occurred more frequently in women with breast cancer. Functionally, re-expression of U50 resulted in the inhibition of colony formation in breast cancer cell lines. These results suggest that noncoding snoRNA U50 plays a role in the development and/or progression of breast cancer.
Assuntos
Neoplasias da Mama/genética , RNA Nucleolar Pequeno/genética , Alelos , Sequência de Bases , Células Cultivadas , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Humanos , Mutação , Fatores de Risco , Transcrição GênicaRESUMO
The proto-oncogene MYC plays a critical role in cell proliferation and tumorigenesis, and its down-regulation by transforming growth factor beta (TGFbeta) signaling is necessary for TGFbeta to inhibit cell proliferation. KLF5, on the other hand, is a pro-proliferative basic transcription factor that reverses function to become an anti-proliferative TGFbeta cofactor upon TGFbeta stimulation in epithelial homeostasis. In this study we investigated whether KLF5 directly regulates MYC transcription in epithelial cells in the context of TGFbeta. Knockdown of KLF5 significantly reduced MYC expression in the HaCaT epidermal epithelial cells. When TGFbeta was applied, however, whereas MYC expression was significantly inhibited, knockdown of KLF5 increased MYC expression. Furthermore, re-expression of KLF5 restored the inhibitory effect of TGFbeta on MYC expression in two cancer cell lines. Chromatin immunoprecipitation and oligo pulldown experiments demonstrated that whereas binding of KLF5 to both KLF5 binding element (KBE) and TGFbeta inhibitory element (TIE) DNA elements was necessary for MYC transcription, binding to KBE was decreased by TGFbeta, and binding to TIE was increased by TGFbeta. These results suggest that KLF5 is not only essential for MYC transcription in proliferating epithelial cells but also mediates the inhibitory effect of TGFbeta on MYC transcription. Furthermore, different binding sites mediate different effects of KLF5 in the context of TGFbeta.
Assuntos
Proliferação de Células , Células Epiteliais/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular , Células Epiteliais/citologia , Humanos , Fatores de Transcrição Kruppel-Like/genética , Mutação , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-myc/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/genéticaRESUMO
KLF5 plays important roles in a variety of cellular processes including proliferation and differentiation. Recently KLF5 was shown to reverse its function in proliferative and p15 regulation upon transforming growth factor-beta (TGFbeta)-mediated acetylation. To understand how KLF5 acetylation functions in TGFbeta-induced p15 transcription, we characterized the interactions of KLF5 with other transcription factors and promoter DNA elements in the context of TGFbeta. KLF5 interacted with Smad2-4 and Miz-1 in a TGFbeta-independent manner, but interacted with Myc only when TGFbeta was activated, and at least some of the interactions had an additive effect on TGFbeta-induced p15 transcription. Oligo pulldown assays showed that binding of Myc to the Inr element was KLF5-dependent, and TGFbeta could enhance the binding when more KLF5 was available. Furthermore, TGFbeta induced an interaction between KLF5 and the p300 acetylase, and acetylation of KLF5 was necessary for Smad4 to associate with p300. Failure in KLF5 acetylation not only prevented p300-assembled Smad4-KLF5 complex formation on p15 promoter but also affected the binding of Smad4 and FOXO3 on the p15 promoter in vivo. These findings suggest that without TGFbeta, some KLF5 associates with Smads in the nucleus and other KLF5 associates with Miz-1 on the p15 promoter to repress its transcription. Activation of TGFbeta recruits p300 to the KLF5-Smad complex to acetylate KLF5, and the complex with acetylated KLF5 binds to the Smad binding element and alters the binding of other factors to p15 promoter to induce its transcription.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Acetilação , Animais , Células COS , Chlorocebus aethiops , Células Epiteliais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/citologia , Luciferases/genética , Regiões Promotoras Genéticas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Proteína Smad4/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
During epithelial homeostasis, stem cells divide to produce progenitor cells, which not only proliferate to generate the cell mass but also respond to cellular signaling to transition from a proliferative state to a differentiation state. Such a transition involves functional alterations of transcriptional factors, yet the underlying molecular mechanisms are poorly understood. Recent studies have implicated Kruppel-like factors (KLFs) including KLF5 in the renewal and maintenance of stem/progenitor cells. Here we demonstrate that the pro-proliferative factor KLF5 becomes anti-proliferative upon TGFbeta-mediated acetylation in an in vitro model of epithelial homeostasis. In the HaCaT epidermal cell line treated with or without TGFbeta, we found that KLF5 was not only essential for cell proliferation, it was also indispensable for TGFbeta-induced anti-proliferation in these cells. KLF5 inhibited the expression of p15 (CDKN2B), a cell cycle inhibitor, without TGFbeta, but became a coactivator in TGFbeta-induced p15 expression in the same cells. Mechanistically, TGFbeta recruited acetylase p300 to acetylate KLF5, and acetylation in turn altered the binding of KLF5 to p15 promoter, resulting in the reversal of KLF5 function. These studies not only demonstrate that a basic transcription factor can be both pro-proliferation and anti-proliferation in epithelial homeostasis, they also present a unique mechanism for how transcriptional regulation changes during the transition from proliferation to inhibition of proliferation. Furthermore, they establish KLF5 as an essential cofactor for TGFbeta signaling.
Assuntos
Proliferação de Células , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Fatores de Transcrição Kruppel-Like/metabolismo , Transdução de Sinais/fisiologia , Acetilação , Animais , Células COS , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor de Quinase Dependente de Ciclina p15/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Modelos Biológicos , Regiões Promotoras Genéticas/fisiologia , Ligação Proteica/fisiologia , Células-Tronco/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
FEZ1/LZTS1 is a tumor suppressor gene located in chromosomal band 8p22, and methylation has been identified as a mechanism for its loss of function in tumors. Chromosomal deletion at 8p22 is also frequent in breast cancer. We therefore examined whether LZTS1 plays a role in breast cancer. We analyzed expression of LZTS1 at both the RNA and protein levels, and promoter methylation in a number of primary tumors and cell lines from breast cancer. We also examined the association between LZTS1 expression and different clinicopathological parameters of breast cancer. We found that the expression of LZTS1 mRNA was reduced in 25 of 50 (50%) primary tumors and 29 of 30 (97%) breast cancer cell lines. Immunohistochemical staining showed that LZTS1 protein was absent or down-regulated in 72 (72%) of 100 primary breast carcinomas. Reduced expression of LZTS1 at either the RNA or protein level was significantly correlated with lymph node metastases (P < 0.05). DNA methylation analysis revealed that the LZTS1 gene was frequently methylated in both cell lines and primary tumors from breast cancer, and the extent of DNA methylation was correlated with reduced expression of the gene. These findings suggest that LZTS1 plays a role in the development and progression of breast cancer at least through promoter methylation-mediated transcriptional downregulation.
Assuntos
Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Papilar/genética , Metilação de DNA , Neoplasias Ductais, Lobulares e Medulares/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Intraductal não Infiltrante/metabolismo , Carcinoma Intraductal não Infiltrante/secundário , Carcinoma Papilar/metabolismo , Carcinoma Papilar/secundário , Estudos de Casos e Controles , Cromossomos Humanos Par 8/genética , Ilhas de CpG , Primers do DNA/química , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Ductais, Lobulares e Medulares/metabolismo , Neoplasias Ductais, Lobulares e Medulares/secundário , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Células Tumorais CultivadasRESUMO
The presence of somatic beta-catenin mutations in some prostate cancers implies that aberrant WNT signaling is involved in the cancer development. Although beta-catenin stability is regulated by a multicomponent destruction complex, mutational alterations of beta-catenin or other components of the destruction complexes are rare in prostate tumors. Therefore, beta-catenin may be regulated by another protein in the prostate. In fact, recent linkage and somatic deletion analyses in prostate cancers reveal a 1.4-Mb candidate tumor suppressor locus on 8p23.1, which includes the Sox7 gene. Here we show that Sox7 protein expression was indeed down-regulated in 47% (15 of 32) of prostate adenocarcinomas. In addition, Sox7 mRNA was down-regulated in 60% of snap-frozen tumors. This down-regulation was found to be due to tumor-specific promoter hypermethylation, which was present in 48% (10 of 21) of primary prostate tumors and 44% (11 of 25) of prostate cancer cell lines/xenografts. We discovered that Sox7 protein physically interacts with beta-catenin and suppresses beta-catenin-mediated transcription by depleting active beta-catenin. Furthermore, in HCT116 colorectal cancer cell lines with Sox7 inactivation, ectopic Sox7 expression suppressed cell proliferation and inhibited transcription that was activated by an endogenous mutant beta-catenin. Although nearly all colorectal cancers contain mutations in beta-catenin or adenomatous polyposis coli/axin, epigenetic silencing of Sox7 was still observed. These data suggest that Sox7 is a tumor suppressor that functions as an independent checkpoint for beta-catenin transcriptional activity. Inactivation of Sox7 could promote the development of a majority of colorectal tumors and approximately half of prostate tumors.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Neoplasias da Próstata/genética , Fatores de Transcrição/genética , beta Catenina/fisiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/secundário , Desequilíbrio Alélico , Sequência de Aminoácidos , Animais , Proliferação de Células , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Metilação de DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Immunoblotting , Técnicas Imunoenzimáticas , Imunoprecipitação , Luciferases/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXF , Homologia de Sequência de Aminoácidos , Fatores de Transcrição TCF/genética , Fatores de Transcrição TCF/metabolismo , Análise Serial de Tecidos , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transplante HeterólogoRESUMO
BACKGROUND & AIMS: The present study was undertaken to determine the expression of a newly identified tumor antigen cancer-placenta 1 (CP1) in colorectal carcinoma (CRC) and explore the CP1-specific immune response in CRC patients and its correlation with patient survival. METHODS: CP1 expression was determined by reverse-transcription polymerase chain reaction, immunohistochemistry, and Western blot analysis. Serum antibodies against CP1 were detected by enzyme-linked immunosorbent assay, and T-cell response was measured by interferon-gamma/granzyme-B release enzyme-linked immunospot assays. The HLA-A2-restricted epitopes in CP1 were predicted by bioinformatics and then experimentally validated by enzyme-linked immunospot assay. RESULTS: CP1 expression was detected in a significant number of CRC tissues, reaching 47.6% at the messenger RNA (mRNA) level and 28.6% at the protein level. Of patients with CP1 mRNA(+) tumors, more than 50% had CP1-responsive CD4(+) and CD8(+) T cells and 30% spontaneous-occurring antibodies against CP1. Further studies revealed 2 dominant HLA-A2-restricted epitopes in the CP1 antigen: p31-39 and p58-66. In a follow-up study up to 33 months after surgery, 9 of the 10 patients with CP1-specific CD8 T-cell response survived, whereas 6 of the 8 nonresponders died. Kaplan-Meier analysis indicated a significant correlation between T-cell response and patient survival. CONCLUSIONS: CP1 represents a new class of tumor-specific shared antigen. Its high expression in CRC tissues, prevalence of CP1-specific immune responses in CP1 mRNA(+) CRC patients, and positive correlation with survival suggest that the antigen may be a useful target for cancer immunotherapy.
Assuntos
Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Neoplasias do Colo/imunologia , Regulação Neoplásica da Expressão Gênica , RNA Neoplásico/genética , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Relação CD4-CD8 , China/epidemiologia , Neoplasias do Colo/genética , Neoplasias do Colo/mortalidade , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Taxa de Sobrevida , Dedos de ZincoRESUMO
Immunoselection and tumor evasion constitutes one of the major obstacles in cancer immunotherapy. A potential solution to this problem is the development of polyvalent vaccines, and the identification of more tumor-specific antigens is a prerequisite for the development of cancer vaccines. To identify novel tumor-specific antigens, suppression subtractive hybridization (SSH) was performed to isolate genes differentially expressed in human hepatocellular cancer (HCC) tissues. PLAC1 (PLACenta-specific 1) was one of the genes identified highly expressed in HCC tissues but not in paired noncancerous tissues. Further analyses revealed its expression in several other types of cancer tissues as well as tumor cell lines, but not in normal tissues except for placenta. Among HCC samples tested, 32% (22/69) showed PLAC1 mRNA expression while the protein was detected in 23.3% (7/30). A serological survey revealed that 3.8% (4/101) of HCC patients had anti-PLAC1 antibody response, suggesting the immunogenicity of PLAC1 in HCC patients. PLAC1 represents a new class of tumor associated antigen with restricted expression in placenta and cancer tissues, that may serve as a target for cancer vaccination.
Assuntos
Anticorpos Antineoplásicos/sangue , Carcinoma Hepatocelular/imunologia , Neoplasias Hepáticas/imunologia , Proteínas da Gravidez/imunologia , Formação de Anticorpos , Northern Blotting , Western Blotting , Vacinas Anticâncer/imunologia , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização de Ácido Nucleico/métodos , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Técnica de SubtraçãoRESUMO
Deletion of chromosome 6q14-q22 is common in multiple human cancers including prostate cancer, and chromosome 6 transferred into cancer cells induces senescence and reduces cell growth, tumorigenicity and metastasis, indicating the existence of one or more tumor-suppressor genes in 6q. To identify the 6q tumor-suppressor gene, we first narrowed the common region of deletion to a 2.5 Mb interval at 6q14-15. Of the 11 genes located in this minimal deletion region and expressed in normal prostates, only snoRNA U50 was mutated, demonstrated transcriptional downregulation and inhibited colony formation in prostate cancer cells. The mutation, a homozygous 2 bp (TT) deletion, was found in two of 30 prostate cancer cell lines/xenografts and nine of 89 localized prostate cancers (eleven of 119 or 9% cancers). Two of 89 (2%) patients with prostate cancer also showed the same mutation in their germline DNA, but none of 104 cancer-free control men did. The homozygous deletion abolished U50 function in a colony formation assay. Analysis of 1371 prostate cancer cases and 1371 matched control men from a case-control study nested in a prospective cohort showed that, although a germline heterozygous genotype of the deletion was detected in both patients and controls at similar frequencies, the homozygosity of the deletion was significantly associated with clinically significant prostate cancer (odds ratio 2.9; 95% confidence interval 1.17-7.21). These findings establish snoRNA U50 as a reasonable candidate for the 6q tumor-suppressor gene in prostate cancer and likely in other types of cancers.
Assuntos
Cromossomos Humanos Par 6 , Genes Supressores de Tumor , Mutação , Neoplasias da Próstata/genética , RNA Nucleolar Pequeno/genética , Sequência de Bases , Estudos de Casos e Controles , Linhagem Celular Tumoral , Mapeamento Cromossômico , Estudos de Coortes , Regulação Neoplásica da Expressão Gênica , Genes Recessivos , Mutação em Linhagem Germinativa , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Estudos Prospectivos , RNA Nucleolar Pequeno/metabolismo , Deleção de SequênciaRESUMO
Chromosomal deletion is frequent at the region between BRCA2 and RB1 in the q14 band of chromosome 13 (13q14) in human cancers, including prostate cancer, suggesting the presence of a tumor suppressor gene. However, no reasonable candidate has been identified thus far. In this study, we did genetic and functional analyses to identify and evaluate the 13q14 tumor suppressor gene. Hemizygous and homozygous deletions in cell lines/xenografts of prostate cancer mapped the deletion locus to 919 kb, which harbors only one known gene, the FOXO1A transcription factor. Deletion at FOXO1A was detected in 31% to 34% in 6 cell lines, 27 xenografts, and 72 clinical specimens of prostate cancer, and was significantly more frequent than deletions at surrounding loci. In addition, FOXO1A was transcriptionally down-regulated in some prostate cancers. Functionally, ectopic expression of FOXO1A inhibited, and its knockdown promoted, cell proliferation or survival. Furthermore, FOXO1A inhibited androgen- and androgen receptor-mediated gene regulation and cell proliferation. Consistent with the understanding of FOXO1A biology, our findings suggest that FOXO1A is the 13q14 tumor suppressor gene, at least in prostate cancer. As a well-established negative effector in the phosphatidylinositol 3-kinase/AKT signaling pathway, FOXO1A inactivation in cancer would impair the therapeutic effect of phosphatidylinositol 3-kinase/AKT inhibitors in cancer treatment.
Assuntos
Antagonistas de Receptores de Andrógenos , Cromossomos Humanos Par 13/genética , Fatores de Transcrição Forkhead/genética , Genes Supressores de Tumor , Neoplasias da Próstata/genética , Metilação de DNA , Proteína Forkhead Box O1 , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Neoplasias da Próstata/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Transdução de Sinais/genéticaRESUMO
OBJECTIVE: To investigate PLAC1/CP1 as a potential candidate gene in gastric cancer therapy. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of PLAC1/CP1 mRNA in gastric cancer tissues and paired adjacent non-cancerous tissues resected from 28 patients with gastric cancer. Specific antibodies against PLAC1/CP1 were also detected by ELISA method in gastric cancer patients. RESULTS: Fourteen (50%,14/28) out of the 28 gastric cancer samples were PLAC1/CP1 mRNA positive. Of the 28 serum samples tested,eight (29%,8/28) displayed positive seroreactivity against PLAC1/CP1 antigen, accounting for 57% (8/14) of PLAC1/CP1 mRNA positive samples. CONCLUSION: PLAC1/CP1 mRNA was expressed in high frequency and induced spontaneous antibody responses in gastric cancer patients. PLAC1/CP1 may be a valuable candidate antigen in gastric cancer immunotherapy.
Assuntos
Imunidade Humoral , Proteínas da Gravidez/metabolismo , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/metabolismo , Anticorpos Antineoplásicos/imunologia , Feminino , Expressão Gênica , Humanos , Masculino , Proteínas da Gravidez/genética , Neoplasias Gástricas/genéticaRESUMO
KLF5 is a transcription factor that plays important roles in multiple physical and pathological processes, including cell growth, cell cycle regulation, and angiogenesis. To better characterize KLF5 function in bladder carcinogenesis, we established stable TSU-Pr1 cell clones expressing different levels of KLF5. These clones were then characterized for cell growth, cell cycle progression, tumorigenesis, and alteration in gene expression. Overexpression of KLF5 promoted tumorigenesis of the TSU-Pr1 cancer cells in mice. Consistently, KLF5 increased G1 to S phase transition, which was accompanied by the upregulation of cyclin D1, phosphorylation of MAPK and Akt, and reduced protein levels for CDK inhibitors p27 and p15. Microarray analysis combined with expression verification in different cell systems identified a number of additional genes that are potentially regulated by KLF5, including HBP17, ITGA6, and RAIG1. These findings suggest that the KLF5 transcription factor plays an oncogenic role in the TSU-Pr1 bladder cancer cell line through the regulation of a subset of genes.