RESUMO
We have previously observed a paradoxical relationship of the psoriasin/S100A7 gene with estrogen response in-vitro in ERalpha positive cells but its association with ERalpha negative status in-vivo raising the possibility that S100A7 might be regulated by ERbeta in breast cancer. Using doxycycline-inducible ERbeta and ERalpha expressing MCF-7 cells the hypothesis that psoriasin/S100A7 is ERbeta regulated was investigated To explore the relationship between psoriasin/S100A7 and ERbeta expression in-vivo, we also assessed a cohort of 233 ERalpha negative breast tumors using tissue microarrays and immunohistochemistry. Psoriasin/S100A7 was increased by 17beta-estradiol (E2) following ERbeta induction, in several clones of ERbeta over-expressing but not in the original MCF-7 cells, nor clones over-expressing ERalpha. The effect of E2 on psoriasin/S100A7 was inhibited by 4-hydroxytamoxifen and ICI 182780 but not with a selective ERalpha antagonist. An ERbeta selective-agonist but not an ERalpha selective-agonist, induced psoriasin/S100A7. This induction still occurred after stable down-regulation of ERalpha using siRNA in ERbeta inducible cells. E2 increased psoriasin/S100A7 mRNA but cycloheximide treatment inhibited this effect. A relationship between ERbeta and psoriasin/S100A7 was observed in the p53 immunohistochemically negative subset of invasive breast tumors in-vivo (r = 0.225, p = 0.046, n = 79). In conclusion we demonstrate that E2 induction of psoriasin/S100A7 can be specifically regulated through ERbeta in-vitro and associated with ERbeta in-vivo. These data support the hypothesis that psoriasin/S100A7 is specifically regulated by ERbeta activity and could be useful to guide future therapies targeting ERbeta in certain phenotypic subsets of breast cancers in-vivo.
Assuntos
Neoplasias da Mama/genética , Proteínas de Ligação ao Cálcio/genética , Receptor beta de Estrogênio/genética , Neoplasias Hormônio-Dependentes/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Primers do DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Hormônio-Dependentes/patologia , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A7 Ligante de Cálcio S100 , Proteínas S100RESUMO
Upregulation of the p16 tumor suppressor is a hallmark of senescence in human fibroblasts. In this study, we investigated potential protein modification of p16 in senescent human fibroblasts using 2D SDS-PAGE analysis. Three distinct p16 variants with isoelectric points of 5.2, 5.4, and 5.6, were consistently detected in normal human IMR90 fibroblasts that had undergone senescence due to forced expression of oncogenic H-ras or culture passage. Moreover, in contrast to short-term serum starvation, which induces quiescence, IMR90 fibroblasts cultured in low serum for a prolonged period exhibited senescent phenotypes and expression of the three p16 variants. All three p16 variants are unlikely phosphoproteins since they failed to react with antibodies against phospho-serine, and were resistant to the treatment with phosphatases. Functionally, co-immunoprecipitation assays using antibodies against cdk4 and/or cdk6 revealed that only the two most acidic p16 variants associated with cdk4/6. Moreover, senescence induced by the forced expression of p16 in early passage IMR90 fibroblasts or osteosarcoma U2OS cells was accompanied by expression of the two most acidic p16 variants, which also associated with cdk4/6. In summary, we report that prolonged serum starvation-induced senescence may provide an additional model for studying biochemical changes in senescence, including p16 regulation. Furthermore, induction of endogenous p16 in senescent human fibroblasts correlates with the expression of three distinct p16 variants independent of protein phosphorylation. Lastly, expression of the two cdk-bound variants is sufficient to induce senescence in human cells.
