Reactive oxygen species induce phosphorylation of serine 118 and 167 on estrogen receptor alpha.

Estrogen receptor alpha (ERalpha) is a well-known target for signaling pathways originating from growth factor receptors. Reactive oxygen species (ROS) can induce activation of extracellular response kinase 1/2 (Erk1/2) and protein kinase B (Akt). Both kinases have been implicated in the phosphorylation of serine 118 and serine 167 on ERalpha, respectively. This activity may lead either to ligand-independent activation of ERalpha or down-regulation of ERalpha and may contribute to development of the resistance to endocrine therapy. Treatment of MCF-7 human breast cancer cells with glucose oxidase (GO, 0.1 un/ml) induced transient phosphorylation of serine 118 and serine 167. The increase in expression of p-ser118-ERalpha was 355 +/- 98% (mean +/- SD) and of p-ser167-ERalpha was 632 +/- 355%. These effects were enhanced in Her2 over-expressing MCF7 cells. ERalpha expression declined to 63 +/- 20% within the first 90 min of treatment and was below 10% 24 h later. ROS induced phosphorylation of ERalpha resulted in decreased expression of pS2 and progesterone receptor. Activation of Erk1/2 and Akt was transient with highest levels of Erk1/2 being 595 +/- 143% and p-Akt 311 +/- 125%. Inhibition of Erk1/2 by U0126 (10 microM) decreased p-ser118-ERalpha by 51.7 +/- 8.5% and decreased p-ser167-ERalpha by 41.9 +/- 16.9% whereas inhibition of Akt by LY294002 (20 microM) and wortmannin (500 nM) or by siRNA knock-down, had no effect on p-ser167-ERalpha expression. Our data show for the first time that ROS can induce post-translational modifications of ERalpha at serine 118 and serine 167, and may lead to ERalpha down-regulation in human breast cancer cells. Both the phosphorylation and consequent down-regulation of ERalpha may be a mechanism associated with development of endocrine therapy resistance.

Estrogen receptor-beta regulates psoriasin (S100A7) in human breast cancer.

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.

Expression of p16INK4A variants in senescent human fibroblasts independent of protein phosphorylation.

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.