The Role of Egln2/Proline Hydroxylase 1 in Regulation of Cyclin D1 in Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma characterized by a translocation that juxtaposes the CCND1 gene (which encodes for cyclin D1) on chromosome 11q13 and an immunoglobulin heavy chain gene promoter on chromosome 14q32. Despite a high rate of complete remission (response 20-80%) by many chemotherapy regimens, MCL typically relapses after treatment, with a median survival time of approximately 3-4 years. Novel therapeutic approaches target several cellular pathways of MCL (CDK, proteasome, mTOR, or NFκB inhibitors and others) but so far have shown only modest effects on overall survival.

Iron is essential for cell proliferation. Although iron chelators were developed for treatment of iron overload diseases, depletion by chelators leads to cancer cell cycle arrest and apoptosis. Iron chelation directly or indirectly affects multiple proteins controlling DNA replication, DNA repair, cell cycle progression and multiple metabolic processes; one known target of iron chelation is cyclin D1. Besides iron chelation-induced suppression of CCND1 gene transcription, Tjendraputra, et al (Blood, 2007), postulated that iron chelation causes posttranslational degradation of cyclin D1 via von Hippel-Lindau protein-independent ubiquitination and subsequent proteasomal degradation.

We hypothesized that iron chelation would be particularly effective in MCL with upregulated cyclin D1. We treated human MCL cell lines (Jeko-1, Mino and HBL-2) with 250 µM deferoxamine mesylate (DFO), which caused decreased cell growth, increased apoptosis, and decreased cyclin D1 mRNA and protein levels. These cell lines were more susceptible to treatment with DFO than B-cell lymphoma derived cell lines without constitutively active cyclin D1 (SUDHL-6, DG-75). A possible cytotoxic effect due to a high concentration of DFO was ruled out by abrogating the DFO effect by concomitant administration of ferric ammonium citrate.

We further investigated the molecular mechanism underlying decreased cyclin D1 mRNA and protein levels in MCL cell lines after DFO treatment. We tested whether the DFO effect may be due to inhibition of one of the iron-dependent hypoxia-inducible factor (HIF) hydroxylases. Expression analysis of several hypoxia related genes in MCL cell lines treated with DFO revealed down-regulation of PHD1 encoded by the EglN2 gene, a member of the PHD family. All members of the PHD protein family contribute to regulation of HIF, but only EglN1 gene encoding PHD2 has a significant role in oxygen sensing under physiological conditions. EglN2 /PHD1 and EglN3 /PHD3 also have HIF-independent functions in control of cell proliferation and apoptosis. In breast cancer, a loss of EglN2 /PHD1 leads to down-regulation of cyclin D1 and decreases cell proliferation in a HIF-independent manner (Zhang et al, Cell, 2009). To further confirm that EglN2 /PHD1 inhibition is linked to suppression of cyclin D1, we treated MCL cell lines with known PHD inhibitors, 1 mM DMOG and 50 µM FG-4497. Proliferation was markedly reduced and down-regulation of cyclin D1 was confirmed in mRNA and protein levels. It has been proposed that the EglN 2/PHD1 substrate linked to repression of cyclin D1 is the transcription factor FOXO3a. An inability of EglN2 /PHD1 to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses cyclin D1 expression by a yet unknown mechanism (Zheng X et al, Genes & Development, 2014). We measured FOXO3a expression levels in MCL cell lines after treatment with DFO and DMOG and found it significantly upregulated. However, knock-down of FOXO3a by CRISPR/Cas9 technology in MCL line Mino did not affect cyclin D1 levels after DFO treatment. These data suggest that in MCL cell lines treated with DFO, accumulation of FOXO3a is present, but not required for cyclin D1 repression and that cyclin D1 down-regulation mediated by inhibition of PHD1 is a result of another, as yet unknown mechanism. Thus, our preliminary data unveiled new insights into regulation of cyclin D1 in MCL and opens new possibilities for MCL targeted therapy with chelation therapy and/or PHD inhibitors.

This study was supported by research funding from Czech Science Foundation, project GACR 15-18046Y (OB, LL), by Ministry of Health Czech Republic, grant AZV 16-31689A (VD) and from the Ministry of Education, Youth and Sports, Czech Republic, Program KONTAKT II (LH15223) (LL, KK, VD).