Interleukin-8 expression is regulated by histone deacetylases through the nuclear factor-kappaB pathway in breast cancer.

We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ERalpha)-negative breast cancer cells compared with ERalpha-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ERalpha-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-kappaB (NF-kappaB) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-kappaB activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-kappaB pathway by adenoviral delivery of a dominant-negative IkappaBorIkappaB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IkappaBalpha, which accounts for NF-kappaB activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-kappaB pathway repression by HDAC is responsible for the low expression of IL-8 in ERalpha-positive breast cancer cells.

Mechanisms underlying differential expression of interleukin-8 in breast cancer cells.

We have recently reported that interleukin-8 (IL-8) expression was inversely correlated to estrogen receptor (ER) status and was overexpressed in invasive breast cancer cells. In the present study, we show that IL-8 overexpression in breast cancer cells involves a higher transcriptional activity of IL-8 gene promoter. Cloning of IL-8 promoter from MDA-MB-231 and MCF-7 cells expressing high and low levels of IL-8, respectively, shows the integrity of the promoter in both cell lines. Deletion and site-directed mutagenesis of the promoter demonstrate that NF-kappaB and AP-1 and to a lesser extent C/EBP binding sites play a crucial role in the control of IL-8 promoter activity in MDA-MB-231 cells. Knockdown of NF-kappaB and AP-1 activities by adenovirus-mediated expression of an NF-kappaB super-repressor and RNA interference, respectively, decreased IL-8 expression in MDA-MB-231 cells. On the contrary, restoration of Fra-1, Fra-2, c-Jun, p50, p65, C/EBPalpha and C/EBPbeta expression levels in MCF-7 cells led to a promoter activity comparable to that observed in MDA-MB-231 cells. Our data constitute the first extensive study of IL-8 gene overexpression in breast cancer cells and suggest that the high expression of IL-8 in invasive cancer cells requires a complex cooperation between NF-kappaB, AP-1 and C/EBP transcription factors.

IL-8 expression and its possible relationship with estrogen-receptor-negative status of breast cancer cells.

Estrogen-receptor (ER) status is an important parameter in breast cancer management as ER-positive breast cancers have a better prognosis than ER-negative tumors. This difference comes essentially from the lower aggressiveness and invasiveness of ER-positive tumors. Here, we demonstrate, that interleukin-8 (IL-8) was clearly overexpressed in most ER-negative breast, ovary cell lines and breast tumor samples tested, whereas no significant IL-8 level could be detected in ER-positive breast or ovarian cell lines. We have also cloned human IL-8 from ER-negative MDA-MB-231 cells, and we show that IL-8 produced by breast cancer cells is identical to monocyte-derived IL-8. Interestingly, the invasion potential of ER-negative breast cancer cells is associated at least in part with expression of IL-8, but not with IL-8 receptor levels. Moreover, IL-8 increases the invasiveness of ER-positive breast cancer cells by two fold, thus confirming the invasion-promoting role of IL-8. On the other hand, exogenous expression of estrogen receptors in ER-negative cells led to a decrease of IL-8 levels. In summary, our data show that IL-8 expression is negatively linked to ER status of breast and ovarian cancer cells. We also support the idea that IL-8 expression is associated with a higher invasiveness potential of cancer cells in vitro, which suggests that IL-8 could be a novel marker of tumor aggressiveness.