RESUMEN
To identify potential effectors of transforming growth factor (TGF)-beta-mediated suppression of colon cancer, we used GeneChip expression microarrays to identify TGF-beta-induced genes in VACO 330, a nontransformed TGF-beta-sensitive cell line derived from a human adenomatous colon polyp. PMEPA1 was identified as a gene highly up-regulated by TGF-beta treatment of VACO 330. Northern blot analysis confirmed TGF-beta induction of PMEPA1 in VACO 330, as well as a panel of three other TGF-beta-sensitive colon cell lines. PMEPA1 induction could be detected as early as 2 h after TGF-beta treatment and was not inhibited by pretreatment of cells with cycloheximide, suggesting that PMEPA1 is a direct target of TGF-beta signaling. Wild-type PMEPA1 and an alternative splice variant lacking the putative transmembrane domain were encoded by the PMEPA1 locus and were shown by epitope tagging to encode proteins with differing subcellular localization. Both variants were found to be expressed in normal colonic epithelium, and both were shown to be induced by TGF-beta. Consistent with TGF-beta playing a role in terminal differentiation of colonocytes, in situ hybridization of normal colonic epithelium localized PMEPA1 expression to nonproliferating, terminally differentiated epithelium located at the top of colonic crypts. Intriguingly, in situ hybridization and Northern blot analysis showed that the expression of PMEPA1 was well maintained both in colon cancer primary tumors and in colon cancer liver metastases. PMEPA1 is thus a novel TGF-beta-induced marker of a differentiated crypt cell population. Moreover, as PMEPA1 expression is maintained, presumptively in a TGF-beta-independent manner after malignant transformation and metastasis, it demonstrates that even late colon cancers retain a strong capacity to execute many steps of the normal colonic differentiation program.
Asunto(s)
Neoplasias del Colon/metabolismo , Proteínas de la Membrana/biosíntesis , Factor de Crecimiento Transformador beta/fisiología , Empalme Alternativo , Secuencia de Aminoácidos , Secuencia de Bases , Diferenciación Celular/genética , División Celular/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Colon/citología , Colon/metabolismo , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas , Transducción de Señal/fisiología , Fracciones Subcelulares/metabolismo , Transfección , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia ArribaRESUMEN
Transforming growth factor (TGF) beta mediates a tumor suppressor pathway in human colon epithelial cells. We were interested in identifying and characterizing novel genes regulated by the TGF beta pathway in the colon. We employed expression microarrays to identify transcripts induced by TGF beta in Vaco 330, a colon adenoma cell line. We then used expression microarrays to determine which of these TGF beta induced transcripts are down-regulated in metastatic colon cancer. Northern analysis and real-time reverse transcription PCR confirmed and quantified our findings from the microarrays. These analyses highlighted C8orf4 as induced by TGF beta in colon cells. Moreover, C8orf4 is expressed in most normal colon mucosa samples, and is not expressed in most colon cancer metastases or colon cancer cell lines. Colon cancer primary tumors showed reduced expression of C8orf4 relative to normal mucosa, possibly reflecting contributions of C8orf4 expression in stromal cells. C8orf4 is a gene regulated by TGF beta signaling and loss in advanced colon cancer suggests C8orf4 may play a role in colon cell differentiation or growth regulation.