Purpose
We explored the mechanisms leading to the distinct overexpression of EPOR as well as the effects of EPO signaling on ETV6/RUNX1-positive acute lymphoblastic leukemias.
Experimental Design
ETV6/RUNX1-expressing model cell lines and leukemic cells were used for real-time PCR of EPOR expression. Proliferation, viability, and apoptosis were analyzed on cells exposed to EPO, prednisone, or inhibitors of EPOR pathways by [3H]thymidine incorporation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and Annexin V/propidium iodide staining. Western blot analysis was done to detect activation of signaling proteins. Serum EPO levels and sequences of the EPOR (n = 53) as well as hemoglobin levels were taken from children with acute lymphoblastic leukemia enrolled in Austrian protocols.
Results
We show here that ectopic expression of ETV6/RUNX1 induced EPOR up-regulation. Anemia, however, did not appear to influence EPOR expression on leukemic cells, although children with ETV6/RUNX1-positive leukemias had a lower median hemoglobin than controls. Exposure to EPO increased proliferation and survival of ETV6/RUNX1-positive leukemias in vitro, whereas blocking its binding site did not alter cell survival. The latter was not caused by activating mutations in the EPOR but might be triggered by constitutive activation of phosphatidylinositol 3-kinase/Akt, the major signaling pathway of EPOR in these cells. Moreover, prednisone-induced apoptosis was attenuated in the presence of EPO in this genetic subgroup.
Conclusions
Our data suggest that ETV6/RUNX1 leads to EPOR up-regulation and that activation by EPO might be of relevance to the biology of this leukemia subtype. Further studies are, however, needed to assess the clinical implications of its apoptosis-modulating properties.
Metastasis is associated with poor prognosis for melanoma responsible for about 90% of skin cancer-related mortality. To metastasize, melanoma cells must escape keratinocyte control, invade across the basement membrane and survive in the dermis by resisting apoptosis before they can intravasate into the circulation. a-Catulin (CTNNAL1) is a cytoplasmic molecule that integrates the crosstalk between nuclear factor-kappa B and Rho signaling pathways, binds to b-catenin and increases the level of both a-catenin and b-catenin and therefore has potential effects on inflammation, apoptosis and cytoskeletal reorganization. Here, we show that a-catulin is highly expressed in melanoma cells. Expression of a-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of expression of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. Knockdown of a-catulin promoted adhesion to and inhibited migration away from keratinocytes in an E-cadherin-dependent manner and decreased the transmigration through a keratinocyte monolayer, as well as in Transwell assays using collagens, laminin and fibronectin coating. Moreover, knockdown promoted homotypic spheroid formation and concomitantly increased E-cadherin expression along with downregulation of transcription factors implicated in its repression (Snail/Slug, Twist and ZEB). Consistent with the molecular changes, a-catulin provoked invasion of melanoma cells in a three-dimensional culture assay by the upregulation of matrix metalloproteinases 2 and 9 and the activation of ROCK/Rho. As such, a-catulin may represent a key driver of the metastatic process, implicating potential for therapeutic interference.
We describe here the identification and initial characterization of a novel human gene termed IKIP (I kappa B kinase interacting protein) that is located on chromosome 12 in close proximity to APAF1 (apoptotic protease-activating factor-1). IKIP and APAF1 share a common 488 bp promoter from which the two genes are transcribed in opposite directions. Three IKIP transcripts are generated by differential splicing and alternative exon usage that do not show significant homology to other genes in the databases. Similar to APAF1, expression of IKIP is enhanced by X-irradiation, and both genes are dependent on p53. Moreover, IKIP promotes apoptosis when transfected into endothelial cells. We conclude that IKIP is a novel p53 target gene with proapoptotic function.
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