No abstract

Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37-47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

Telomerase activation is crucial for cells that tend to be immortalized. Increased telomerase activity is correlated with upregulation of telomerase reverse transcriptase (TERT) expression. In most human somatic cells, hTERT expression is suppressed by multiple factors. We have previously shown that human chromosome 5 carries a possible suppressor of mouse tert mtert expression in a mouse melanoma cell line, B16-F10 cells. However, the function of the transcriptional regulator of TERT on this chromosome remains unclear. To examine the functional role of a putative hTERT regulator(s) on this chromosome, we transferred human chromosome 5 in a human melanoma cell line, A2058 cells by microcell-mediated chromosome transfer (MMCT). Microcell hybrid clones with an introduced chromosome 5, but not chromosome 10, showed a remarkable decrease in the growth rate with an obvious cellular morphological alteration and eventually cellular senescence. Moreover, this phenomenon was accompanied by a reduction of hTERT expression and telomerase activity. Most importantly, we found that transcriptional suppression of hTERT by the introduction of chromosome 5 is largely mediated by regulating hTERT promoter activity. Furthermore, the hTERT promoter region between -1623 and -1047 was responsible for this function. These results provide evidence that transcriptional regulator(s) of the hTERT is carried on human chromosome 5 as an endogenous mechanism of hTERT suppression.

Germline RB1 mutations strongly predispose humans to cone-precursor-derived retinoblastomas and strongly predispose mice to pituitary tumors, yet shared cell type-specific circuitry that sensitizes these different cell types to the loss of RB1 has not been defined. Here we show that the cell type-restricted thyroid hormone receptor isoform TRβ2 sensitizes to RB1 loss in both settings by antagonizing the widely expressed and tumor suppressive TRβ1. TRβ2 promoted expression of the E3 ubiquitin ligase SKP2, a critical factor for RB1-mutant tumors, by enabling EMI1/FBXO5-dependent inhibition of SKP2 degradation. In RB1-wild type neuroblastoma cells, endogenous Rb or ectopic TRβ2 was required to sustain SKP2 expression as well as cell viability and proliferation. These results suggest that in certain contexts, Rb loss enables TRβ1-dependent suppression of SKP2 as a safeguard against RB1-deficient tumorigenesis. TRβ2 counteracts TRβ1, thus disrupting this safeguard and promoting development of RB1-deficient malignancies.

MYC family oncoproteins MYC, MYCN, and MYCL are deregulated in diverse cancers and via diverse mechanisms. Recent studies established a novel form of MYCN regulation in MYCN-overexpressing retinoblastoma and neuroblastoma cells in which the MDM2 oncoprotein promotes MYCN translation and MYCN-dependent proliferation via a p53-independent mechanism. However, it is unclear if MDM2 also promotes expression of other MYC family members and has similar effects in other cancers. Conversely, MYCN has been shown to induce MDM2 expression in neuroblastoma cells, yet it is unclear if MYC shares this ability, if MYC family proteins upregulate MDM2 in other malignancies, and if this regulation occurs during tumorigenesis as well as in cancer cell lines. Here, we report that intrinsically high MDM2 expression is required for high-level expression of MYCN, but not for expression of MYC, in retinoblastoma, neuroblastoma, small cell lung cancer, and medulloblastoma cells. Conversely, ectopic overexpression of MYC as well as MYCN induced high-level MDM2 expression and gave rise to rapidly proliferating and MDM2-dependent cone-precursor-derived masses in a cultured retinoblastoma genesis model. These findings reveal a highly specific collaboration between the MDM2 and MYCN oncoproteins and demonstrate the origin of their oncogenic positive feedback circuit within a normal neuronal tissue.