RESUMEN
MicroRNA-101, a tumor suppressor microRNA (miR), is often downregulated in cancer and is known to target multiple oncogenes. Some of the genes that are negatively regulated by miR-101 expression include histone methyltransferase EZH2 (enhancer of zeste homolog 2), COX2 (cyclooxygenase-2), POMP (proteasome maturation protein), CERS6, STMN1, MCL-1 and ROCK2, among others. In the present study, we show that miR-101 targets transcriptional coactivator SUB1 homolog (Saccharomyces cerevisiae)/PC4 (positive cofactor 4) and regulates its expression. SUB1 is known to have diverse role in vital cell processes such as DNA replication, repair and heterochromatinization. SUB1 is known to modulate transcription and acts as a mediator between the upstream activators and general transcription machinery. Expression profiling in several cancers revealed SUB1 overexpression, suggesting a potential role in tumorigenesis. However, detailed regulation and function of SUB1 has not been elucidated. In this study, we show elevated expression of SUB1 in aggressive prostate cancer. Knockdown of SUB1 in prostate cancer cells resulted in reduced cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Gene expression analyses coupled with chromatin immunoprecipitation revealed that SUB1 binds to the promoter regions of several oncogenes such as PLK1 (Polo-like kinase 1), C-MYC, serine-threonine kinase BUB1B and regulates their expression. Additionally, we observed SUB1 downregulated CDKN1B expression. PLK1 knockdown or use of PLK1 inhibitor can mitigate oncogenic function of SUB1 in benign prostate cancer cells. Thus, our study suggests that miR-101 loss results in increased SUB1 expression and subsequent activation of known oncogenes driving prostate cancer progression and metastasis. This study therefore demonstrates functional role of SUB1 in prostate cancer, and identifies its regulation and potential downstream therapeutic targets of SUB1 in prostate cancer.
Asunto(s)
Proteínas de Unión al ADN/genética , MicroARNs/genética , Neoplasias de la Próstata/genética , Factores de Transcripción/genética , Animales , Proliferación Celular/genética , Proteínas de Unión al ADN/biosíntesis , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Xenoinjertos , Humanos , Masculino , Ratones , MicroARNs/biosíntesis , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Factores de Transcripción/biosíntesisRESUMEN
A comprehensive comparison of Sonic (Shh), Indian (Ihh), and Desert (Dhh) hedgehog biological activities has not previously been undertaken. To test whether the three higher vertebrate Hh proteins have distinct biological properties, we compared recombinant forms of the N-terminal domains of human Shh, Ihh, and Dhh in a variety of cell-based and tissue explant assays in which their activities could be assessed at a range of concentrations. While we observed that the proteins were similar in their affinities for the Hh-binding proteins; Patched (Ptc) and Hedgehog-interacting protein (Hip), and were equipotent in their ability to induce Islet-1 in chick neural plate explant; there were dramatic differences in their potencies in several other assays. Most dramatic were the Hh-dependent responses of C3H10T1/2 cells, where relative potencies ranged from 80nM for Shh, to 500nM for Ihh, to >5microM for Dhh. Similar trends in potency were seen in the ability of the three Hh proteins to induce differentiation of chondrocytes in embryonic mouse limbs, and to induce the expression of nodal in the lateral plate mesoderm of early chick embryos. However, in a chick embryo digit duplication assay used to measure polarizing activity, Ihh was the least active, and Dhh was almost as potent as Shh. These findings suggest that a mechanism for fine-tuning the biological actions of Shh, Ihh, and Dhh, exists beyond the simple temporal and spatial control of their expression domains within the developing and adult organism.
Asunto(s)
Tipificación del Cuerpo , Diferenciación Celular , Inducción Embrionaria , Osteoblastos/citología , Transactivadores/farmacología , Transactivadores/fisiología , Fosfatasa Alcalina/biosíntesis , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , División Celular , Línea Celular , Embrión de Pollo , Condrocitos/citología , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Técnicas de Cultivo de Órganos , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular , Proteínas Recombinantes/farmacología , Transducción de Señal , Transactivadores/química , Alas de Animales/embriologíaRESUMEN
Bone morphogenetic proteins (BMPs) have been implicated in regulating multiple stages of bone development. Recently it has been shown that constitutive activation of the BMP receptor-IA blocks chondrocyte differentiation in a similar manner as misexpression of Indian hedgehog. In this paper we analyze the role of BMPs as possible mediators of Indian hedgehog signaling and use Noggin misexpression to gain insight into additional roles of BMPs during cartilage differentiation. We show by comparative analysis of BMP and Ihh expression domains that the borders of Indian hedgehog expression in the chondrocytes are reflected in changes of the expression level of several BMP genes in the adjacent perichondrium. We further demonstrate that misexpression of Indian hedgehog appears to directly upregulate BMP2 and BMP4 expression, independent of the differentiation state of the flanking chondrocytes. In contrast, changes in BMP5 and BMP7 expression in the perichondrium correspond to altered differentiation states of the flanking chondrocytes. In addition, Noggin and Chordin, which are both expressed in the developing cartilage elements, also change their expression pattern after Ihh misexpression. Finally, we use retroviral misexpression of Noggin, a potent antagonist of BMP signaling, to gain insight into additional roles of BMP signaling during cartilage differentiation. We find that BMP signaling is necessary for the growth and differentiation of the cartilage elements. In addition, this analysis revealed that the members of the BMP/Noggin signaling pathway are linked in a complex autoregulatory network.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Cartílago/crecimiento & desarrollo , Condrogénesis/fisiología , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Proteínas/fisiología , Transactivadores , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Portadoras , Diferenciación Celular , Embrión de Pollo , Condrogénesis/genética , Retroalimentación , Glicoproteínas/genética , Glicoproteínas/fisiología , Proteínas Hedgehog , Hibridación in Situ , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Alas de Animales/embriologíaRESUMEN
A number of proteins have recently been identified which play roles in regulating bone development. One important example is Indian hedgehog (Ihh) which is secreted by the prehyprtrophic chondrocytes. Ihh acts as an activator of a second secreted factor, parathyroid hormone-related protein (PTHrP), which, in turn, negatively regulates the rate of chondrocyte differentiation. Here we examine the expression of these genes and their molecular targets during different stages of bone development. In addition to regulating PTHrP expression in the perichondrium, we find evidence that Ihh may also act on the chondrocytes themselves at particular stages. As bone growth continues postnatally in mammals and the developmental process is reactivated during fracture repair, understanding the molecular basis regulating bone development is of medical relevance. We find that the same molecules that regulate embryonic endochondral ossification are also expressed during postnatal bone growth and fracture healing, suggesting that these processes are controlled by similar mechanisms.