Inflammatory Conditions Induce IRES-Dependent Translation of cyp24a1

Abstract

Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR)-kinase, alternative modes of translation, such as internal ribosome entry site (IRES)-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase) to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM). Using bicistronic reporter assays, we identified and validated an IRES within the 5′ untranslated region (5′UTR) of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K) inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option.

Function of the vitamin D endocrine system in mammary gland and breast cancer

“…The presence of CYP24A1 splice variants that may alter enzyme activity have been reported in breast cancer cell lines and tissues (64; 65), and characterization of these variants may help clarify the discrepancies noted above with respect to CYP24A1 expression in breast cancer. The regulation of CYP24A1 by factors other than 1,25D in breast cancer has not been particularly well studied, although inflammation was shown to increase, and miR125b shown to decrease, CYP24A1 in vitro (66; 67). Epigenetic regulation of CYP24A1 by promoter methylation has been reported in lung and prostate cancer (68; 69), but studies in breast cancer are lacking.…”

Exploring Internal Ribosome Entry Sites as Therapeutic Targets

“…Another interesting recent report has identified an IRES element in the cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase) mRNA, which encodes a protein that inactivates vitamin D3-mediated signaling ( 100 ). This IRES was induced by vitamin D3 and inflammation and involved the PI3K-AKT1 signaling pathway ( 100 ).…”

“…Another interesting recent report has identified an IRES element in the cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase) mRNA, which encodes a protein that inactivates vitamin D3-mediated signaling ( 100 ). This IRES was induced by vitamin D3 and inflammation and involved the PI3K-AKT1 signaling pathway ( 100 ). Cyp24a1 was also found to be induced in breast and colon cancer cells and its enhanced expression may therefore explain the development of tumor resistance to chemotherapies in which D3 is used as an adjuvant agent ( 100 ).…”

“…This IRES was induced by vitamin D3 and inflammation and involved the PI3K-AKT1 signaling pathway ( 100 ). Cyp24a1 was also found to be induced in breast and colon cancer cells and its enhanced expression may therefore explain the development of tumor resistance to chemotherapies in which D3 is used as an adjuvant agent ( 100 ). Understanding how inflammation causes translational reprogramming in different cancers will be an important step toward targeting IRES-mediated translation for treatment of these diseases.…”

Hydrogen peroxide formation by Nox4 limits malignant transformation

“…Polysome analysis was performed as described previously (21). Briefly, after incubation with 100 µg/ml cycloheximide for 10 min at 37°C cells were scraped and centrifuged.…”