Many tumor suppressor genes are known to be inactivated by epigenetic modifications including DNA methylation and histone deacetylation. RECK is a membrane-anchored glycoprotein that may negatively regulate matrix metalloproteinase (MMP) activity and inhibit tumor metastasis. Our previous study demonstrated that oncogenic ras inhibited RECK expression via Sp1 binding site in the RECK promoter by histone deacetylation mechanism. In this study, we tried to characterize the molecular pathway that mediates the inhibitory effect of ras on RECK. Methylation specific PCR (MSP) indicated that RECK gene promoter is hypermethylated in ras-activated 2-12 cells. We also tested whether ras activation induced the binding of DNA methyltransferases (DNMTs) to Sp1 to repress RECK expression. Our data showed Sp1-associated DNMT3b in cells was increased after ras induction. By using DNA affinity precipitation assay (DAPA) and chromatin immunoprecipitation (ChIP) , we found that induction of oncogenic ras enhanced the binding of DNMT3b to the Sp1 site in the RECK promoter in vitro and in vivo. Additionally, treatment of DNMT inhibitor 5'-azacytidine led to the re-expression of RECK in ras-activated 2-12 cells. The signaling pathway by which ras suppresses RECK was also addressed. Chemical inhibitor of ERK signaling pathway U0126 reversed the methylation of RECK promoter and up-regulated RECK expression in ras-activated 2-12 cells. More importantly, 5'-azacytidine and DNMT3b siRNA may suppress the invasive ability of 2-12 cells. Taken together, our results suggest that oncogenic ras inhibit the metastasis suppressor gene RECK via a DNA methylation mechanism and provide valuable insights for the understanding of the mechanisms by which ras promotes tumor invasion and metastasis

The major cause of therapy failure and death of cancer patients is metastasis. RECK is a newly identified gene which was isolated by screening for human fibroblast cDNA clones giving rise to flat colonies when transfected into v-Ki-ras-transformed NIH3T3 cells. It can inhibit the release and activation of MMP-2 and MMP-9, and prevent cell invasion in vitro. In addition, RECK can repress tumor metastasis in experimental animal. Thus, RECK is a metastasis suppressor gene. However, RECK gene is a common target that is negatively regulated by oncogenic signals. Overexpression of c-myc protooncogene is frequently found in several types of human cancer and contributes to multiple steps of tumorigenesis. Ecto-expression of c-Myc in NIH3T3 cells inhibited RECK expression. Promoter activity assay suggested c-Myc repressed RECK at transcriptional level. By using DNA affinity precipitation assay and chromatin immunoprecipitation assay, we found that oncogenic c-Myc bound to the SP1 sites of RECK promoter in vitro and in vivo. It is possible that c-Myc could repress RECK expression via SP1. Our data suggest that c-Myc may inhibit the metastatic/angiogenic suppressor RECK to enhance cell invasiveness and restoration of RECK may be a novel strategy to inhibit c-Myc-mediated invasion.

Orientador: Hernandes Faustino de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T10:11:12Z (GMT). No. of bitstreams: 1 Peters_Helene_M.pdf: 3147205 bytes, checksum: 29d84ddab25f17a17efc857545126663 (MD5) Previous issue date: 2005 / Resumo: A próstata tem merecido crescente atenção devido à maior incidência de câncer prostático e outras afecções do órgão, que resultam do aumento na longevidade dos indivíduos do sexo masculino em todo o mundo. Além disto, o desenvolvimento e crescimento prostático normal apresenta regulação androgênica e está sujeito a uma série de disruptores endócrinos que afetam o seu crescimento e função, assim como predispõem ao desenvolvimento tumoral. Nosso interesse reside principalmente na remodelação prostática seguida à castração e nas interações epitélio estroma que ocorrem neste órgão. Neste trabalho, investigamos a expressão do inibidor de metaloproteinases (MMPs) RECK, em nível de RNAm, procurando correlacioná-Io com o desenvolvimento pós-natal e com a regressão prostática seguida à castração. Para isto, foram utilizadas técnicas de RT-PCR semiquantitativo, Real time RT-PCR e de hibridação in situ,pareados sempre que possível com a expressão do RNAm e com a atividade de algumas MMPs. Os resultados demonstram que o gene RECK é expresso na próstata ventral de ratos, que existe uma significativa redução na sua expressão ao longo do desenvolvimento pós-natal, que há mecanismos diferenciados controlando a expressão dos pares RECKlMMP-2 e MMP-7/MMP-14. Foi observado também um crescente aCÚInulo da forma ativa da MMP-9, conforme o animal se aproxima da idade adulta. Utilizando RT-PCR semiquantitativo, pudemos determinar que o conteúdo relativo do RNAm para o RECK após a castração não muda, embora haja uma inversão no balanço entre a expressão epitelial (células epiteliais) e estromal (células musculares lisas e fibroblastos), nesta situação. No conjunto, os resultados sugerem que o RECK é expresso por diferentes tipos celulares da próstata ventral de ratos, com mecanismos de regulação complexos provavelmente oriundos da existência de diferentes compartimentos no órgão, ao contrário do que se observa para células isoladas / Abstract: The prostate has deserved increasingly attention due to the growing incidence of prostatic cancer and other prostatic diseases, which can be related to the longevity increase of men around the world. Besides, the normal prostatic development is under androgen regulation and as so is subject to a series of endocrine disruptors which affect its growth and function and predisposes to prostate cancer. Our interest resides on the prostatic remodelling following castration and on the epithelial-stromal relationships known to occur in the organ. In this work, we have investigated the expression of the matrix metalloproteinase inhibitor RECK, at the rnRNA leveI, trying to correlate its expression with the post natal prostatic development and regression after castration, using semiquantitative RT-PCR, Real time RT-PCR and in situ hybridization, paralleled with the determination of some MMPs expression and activity. Tbe results demonstrate that RECK is expressed in the rat ventral prostate, that there is a significative reduction in its expression during the post natal development, which is paralleled by the expression of some MMPs and that the mechanisms controling the pairs RECKJMMP-2 and MMP-7/MMP-14 are different. It was also observed an increased proportion of the active form of MMP-9, as the animal approaches adulthood. Using semiquantitative RT-PCR, we could determine that the relative content ofRECK rnRNA remains unchanged by castration, spite detecting an inversion in the balance between the epithelial (epithelial cells) and stromal (smooth muscle cells and fibroblasts) in this situation. Taken together, the results indicate that RECK is expressed by different cell types of the rat ventral prostate, with regulatory mechanisms appearing more complex, likely resulting ftom the existence of different compartments in the organ opposing what was seen for isolated cells / Mestrado / Biologia Celular / Mestre em Biologia Celular e Estrutural

Lung cancer is the common cause of cancer death. STAT3 (signal transducer and activator of transcription 3) has been reported to be an oncogenic transcription factor and high expression of STAT3 is associated with lung cancer progression. RECK (reversion-inducing cysteine-rich protein with Kazal motifs) is a tumor suppressor gene and a membrane-anchored glycoprotein that reduces the matrix metalloproteinases (MMPs)-induced destruction of extra-cellular matrix (ECM) and tumor metastasis. RECK also inhibits tumor angiogenesis. We have previously elucidated the transcriptional regulation of RECK gene. Recently, microRNAs (miRs) are shown to be key players in gene regulation and cancer progression. In this study, we try to elucidate whether ovexpression of STAT3 can affect microRNA expression to regulate RECK via post-transcriptional modulation. miR-17-92a cluster is a well-known oncomir which is highly expressed in lung cancer tissue. We find that miR-92a, a member of miR-17-92a cluster can target RECK 3¡¦UTR. In addition, our data suggest that STAT3 regulates the expression of miR-92a and inhibition of STAT3 can decrease miR-92a expression. Furthermore, overexpression of miR-92a can decrease RECK protein level. While knockdown of miR-92a expression in STAT3-overexpressing cell lines can restore RECK protein level, and reduce invasion and migration. Results of this study suggest that STAT3 up-regulates miR-92a to inhibit RECK expression and promote lung cancer metastasis.

RECK stands for ¡¥reversion-inducing cysteine-rich protein with Kazal motifs¡¦. This gene was initially discovered by screening a human fibroblast cDNA library for genes giving rise to reversion-inducing clones when transfected into v-Ki-ras transformed NIH3T3 cells. The key action of RECK is to inhibit matrix metalloproteinases (MMPs), and it has a significant effect on limiting tumor invasion. Located within the middle part of RECK protein are three serine protease inhibitor-like (SPI) domains (635-654,716-735 and 754-772 amino acids, respectively) which are similar to Kazal motif. Kazal motif is a peptidase inhibitor motif containing disulfide bonds with small alpha and beta folds. The first of these SPI is identical to the Kazal motif (named as K1) and the other two SPIs are highly similar to the Kazal motif (named as K2 & K3). Given RECK is a MMP inhibitor, these SPI-like domains are likely to have a significant role in MMP inhibition. Our previous data indicated that K23 motifs of RECK protein can inhibit MMP-9 secretion and activity and attenuate metastasis of lung cancer cells. To go a step further, we constructed secretory mammalian expression vectors which could produce K1, K2 and K3 to investigate their effect on MMP activity and cell invasion. We found that K2 also exhibited inhibitory activity on MMP activity and cell invasion. Thus, these finding indicate that the K2 domain of RECK function may be developed as a peptide inhibitor of tumor invasion.

RECK(reversion-inducing cysteine-rich protein with Kazal motifs) encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeat, four N-glycosylation sites and three Kazal-like domains. RECK functions as a tumor suppressor gene which may inhibit the release and activation of MMP-2 and MMP-9. Previous studies indicated that RECK-mediated suppression of tumor cell invasion is regulated by glycosylation of RECK in human tumor cell lines. However, the anti-cancer action of other functional domains of RECK have not been studied. In the study, We investigated the effects of different functional domains of RECK protein on the invasion of tumor cell lines and on the activation of matrix metalloproteinase. We constructed bacterial expression vector and secretory mammalian expression vector which could produce full-length, Kazal-like motifs 1~3, Kazal-like motifs 2~3 and CKM5 polypeptides. Recombinant proteins were purified and used for treatment of human lung cancer cell lines. We found that treatment of K23 and RECK recombinant proteins resulted in suppression of invasive ability and MMP activity. Moreover,K23 and RECK proteins were found to inhibit the secretion of matrix metallo- protease-9 (MMP-9). K23 also formed a complex with MMP-9 and inhibited its proteolytic activity noncompetitively. Experimental metastasis assay revealed that there were fewer tumor nodule formation in the lungs injected with A549 cells stably expressing K23 than control vector. Thus, these findings indicate that the K23 domain of RECK functions as an inhibitor of tumor invasion and metastasis.

Reversion-inducing cysteine-rich with Kazal motif (RECK) is a cell surface anchoring protein, which known for the ability to inhibit matrix metalloproteinases (MMPs) and participate in angiogenesis regulation. The inhibition of membrane type-1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-7 and, MMP-9 by RECK has been demonstrated. Our previous studies show that RECK expression is suppressed by Ras and Her-2/neu oncogene. In addition, oncogenic Ras activates downstream ERK signaling pathway to increase Sp1/HDAC promoter binding affinity which results in reduction of RECK gene transcription and increase of tumor progression and metastasis. From the clinical investigation, RECK expression is down-regulated in a number of cancer types. In breast cancer, RECK expression is associated with the prognosis of the patients. Recently, single nucleotide polymorphisms (SNPs) of RECK promoter have been suggested to be linked with survival rate and prognosis of breast cancer patients. Whether SNP of the RECK promoter has any effect on RECK expression and its clinical significance is still unclear. . In this study, we investigate -402 SNP at RECK promoter and find this SNP directly affects RECK expression through progesterone receptor binding. Additionally, we also address the -402 SNP in the sample collected from patients and analyze its association with clinicopathological parameters to clarify its clinical significance. Our results suggest that RECK SNP may be an valuable prognosis factor for breast cancer.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18513号 / 医博第3933号 / 新制||医||1006(附属図書館) / 31399 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松田 道行, 教授 羽賀 博典, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Os tumores da cérvice-uterina, que representam uma das principais doenças ginecológicas em mulheres na idade reprodutiva em todo o mundo, estão etiologicamente associados com a infecção pelo papilomavírus humano (HPV). A progressão de uma lesão intraepitelial escamosa de baixo-grau (LSIL) a um carcinoma invasivo de cérvix uterina está acompanhada da degradação da matriz extracelular (MEC) devido à ação progressiva das metaloproteinases de matriz (MMP-2, MMP-9 e MMP-14) no processo de invasão e metástase. Entretanto, o balanço entre as MMPs e seus reguladores como RECK e TIMPs é necessário para controlar esta invasão. O objetivo deste projeto consiste em avaliar a atividade e a expressão das metaloproteinases 2, 9, e 14, e caracterizar a expressão do gene supressor de metástase RECK e do inibidor tecidual de metaloproteinases (TIMP-2), em modelo de queratinócitos humanos infectados com retrovírus recombinantes que expressam os oncogenes E6 e/ou E7 de HPV 16, em culturas cultivadas em monocamada e organotípicas. Para isso, utilizamos ensaios de real-time PCR, zimografia, western blot, imunocitoquímica, ensaio de ELISA e imunohistoquímica. Em culturas em monocamada observamos que as células que expressam as oncoproteínas E6E7 de HPV16 apresentaram menores níveis protéicos de RECK e TIMP-2 em relação ao controle pXLSN. Quando analisamos as culturas organotípicas, também observamos esta diminuição dos níveis de RNAm e protéicos de RECK em rafts que expressam E6E7, acompanhado pelo aumento da atividade de MMP-9, em relação ao controle. Também observamos que o tratamento das culturas com a citocina TNF aumenta a expressão gênica, protéica e atividade de MMP-9 em todas as linhagens analisadas. Além disso, os oncogenes E6 e/ou E7 não afetam a expressão e/ou atividade de MMP-2, MT1-MMP. Nossos dados demonstraram que a expressão das oncoproteínas E6E7 de HPV16 estão relacionadas com o desequilíbrio entre MMPS e seus inibidores, sugerindo que em uma fase pré-invasiva do carcinoma cervical, não somente as MMPs, mas, principalmente seus inibidores são críticos para início da progressão tumoral. / Cervical cancer is etiologically associated with to high-risk human papillomavirus (HPV) infection. It has been observed that matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation during cervical carcinoma progression. Moreover, a counterbalancing among MMPs and their regulators, such as TIMPs and RECK, is necessary to modulate invasion. In order to study the effect of HPV oncogenes on MMPs expression, primary human keratinocytes (PHKs) were infected with recombinant retroviruses expressing wild-type HPV16 E6 and/or E7 oncogenes and were used to seed monolayers and organotypic cultures. Quantitative real-time PCR (Q-PCR), western blot, zimography, immunocitochemistry, ELISA assay and immunohistochemistry were used to determine the expression level and activity of MMP-2, MMP-9, MT1-MMP and their inhibitors RECK and TIMP-2. We observed that cultures expressing E6E7 presented lower RECK and TIMP-2 protein levels than control keratinocytes. In addition, rafts cultures presented the same lower RECK levels additionally presenting higher MMP-9 activity than control. Furthermore, we observed that expression of E6 and/or E7 proteins do not affect MMP-2 and MT1-MMP protein levels and/or activity. We also observed that TNF treatment enhance the MMP-9 gene and protein expression and activity in all studied cell lines. Taken together, our results demonstrate that HPV16E6E7 expression is related with the unbalance between MMPs and their inhibitors, suggesting that in the initial steps of HPV-related cervical disease, not only MMPs but also RECK and TIMP-2 are critical for tumor progression.

Os gliomas são o tipo de tumor primário cerebral mais comum em adultos. A sobrevida média dos pacientes é de cerca de um ano para pacientes de glioblastoma multiforme, o maior grau de malignidade. As terapias disponíveis atualmente, que incluem cirurgia, radioterapia e quimioterapia, não têm sido eficientes devido a vários fatores, em particular a capacidade invasiva das células tumorais. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) é um importante gene supressor de tumor cuja atividade anti-tumoral têm sido associada à sua atividade inibitória sobre algumas MMPs. A perda da função de RECK compromete a integridade tecidual, em parte causada pela atividade aumentada das MMPs. A linhagem celular T98G, derivada de glioblastoma multiforme (GBM), apresenta grande capacidade invasiva e níveis elevados de MMP-2 e -9. A fim de avaliar o efeito de RECK na contenção da invasão no modelo de glioma humano, foi feita a superexpressão de RECK nesta linhagem. A expressão de RECK, MMP-2, MMP-9 a MT1-MMP foram avaliadas por qPCR e por western blotting nas células T98G/RECK+ (células T98G superexpressando RECK após transfecção estável utilizando o vetor pCXN2). O potencial invasivo e migratório das células T98G/RECK+foi inibido, verificado por ensaio transwell. Foram observadas nas células T98G/RECK+ alterações importantes no arranjo do citoesqueleto, mas não nas células controle. Arranjos de actina na forma de \"stress fibers\" presentes no clone positivo podem ser responsáveis pela alteração observada na migração. A distribuição de FAK foi avaliada por imunocitoquímica e sua expressão por Western blot, mostrando que RECK só altera a distribuição desta proteína no citoesqueleto celular. Quanto ao potencial de inibição de MMPs, observou-se uma diminuição significativa dos níveis gênicos de MMP-9, mas não em termos protéicos ou de atividade de MMPs. Assim, este trabalho contribui para a discussão do papel de RECK na migração de células do modelo de glioma humano, uma das características responsáveis pela ausência de terapia efetiva deste tipo de tumor. / Malignant gliomas are the most common type of primary brain tumors in adults. Patient survival is less than one year in average for glioblastoma, the most malignant glioma. Therapies available today, which include surgery, radiotherapy and chemotherapy, have not been successful due to several factors, specially the invasiveness of the tumor. RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) is an important tumor suppressor gene whose anti-tumoral activity is associated to its anti-MMPs activity. Lack of functional RECK compromises tissue integrity, in part due to elevated MMPs activity. T98G cells, derived from a human multiform glioblastoma (GBM), were described as a highly invasive glioma cell line, which displays high levels of MMPs 2 and 9. In order to evaluate the effect of RECK in restraining glioma invasion, we overexpressed RECK in these invasive cell line derived from GBM. The expression of RECK, MMP-2, MMP-9 and MT1-MMP was evaluated by qPCR and by western blotting in T98G/RECK+ (T98G cells overexpressing RECK cells, where RECK gene was cloned into the pCXN2 vector generating a stable transfection). The invasion and migration capacity of T98G/RECK+ cells was inhibited in transwell assay. Important cytoskeleton modifications were also observed in T98G/RECK+ cells but not on the control cells. Actin arrangements representing stress fibers on the positive clones may be responsible for motility alteration patterns observed. FAK distribution was assessed through imunocytochemical staining, and its expression evaluated by western blot analyses, showing that RECK forced expression changed the distribution pattern but not FAK expression. Concerning MMPs inhibition, a significant inhibition of MMP-9 gene expression was observed in T98G/RECK+, but neither protein levels nor protein activity were affected. Thus, the present study improves the discussion about RECK role in the migration of glioma cells, an important feature for the failure of current therapies for this kind of tumor.