Knockdown of prolyl‐4‐hydroxylase domain 2 inhibits tumor growth of human breast cancer MDA‐MB‐231 cells by affecting TGF‐β1 processing

Abstract

The prolyl-4-hydroxylase domain 1-3 (PHD1-3) enzymes are regulating the protein stability of the a-subunit of the hypoxiainducible factor-1 (HIF-1), which mediates oxygen-dependent gene expression. PHD2 is the main isoform regulating HIF-1a hydroxylation and thus stability in normoxia. In human cancers, HIF-1a is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA-MB-231 breast cancer cells and analyzed their tumorforming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA-MB-231 cells, which could be partially rescued by re-establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA-MB-231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)-b1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro-protein convertase furin. Thus, our data demonstrate that in MDA-MB-231 cells PHD2 might affect tumor-relevant TGF-b1 target gene expression by altering the TGF-b1 processing capacity.

Hypoxia inducible factor prolyl hydroxylase 2 (PHD2) is a direct regulator of epidermal growth factor receptor (EGFR) signaling in breast cancer

“…efficiency of EGFR mRNA [16] or via the attenuation of rabaptin-5 transcription, which leads to longer halflives of activated EGFR due to delayed late endosomal EGFR sorting [33]. Therefore, we initially expected that the knockdown of PHD2 and, as shown previously [28], the stabilization of HIF-1α and HIF-2α in our cell model (Supplementary Figure 2) would favor the upregulation of EGFR. However, the experimental results were quite opposite.…”

The Cancer Cell Oxygen Sensor PHD2 Promotes Metastasis via Activation of Cancer-Associated Fibroblasts

“…Silencing of PHD2 in BC cells reduces TGF-b1 secretion (Wottawa et al, 2013). In agreement, PHD2 haplodeficient PyMT cancer cells released less TGF-b1.…”

“…Selective PHD2 haplodeficiency in ECs reduces metastasis without affecting tumor growth by normalizing tumor vessels (Leite de Oliveira et al, 2012;Mazzone et al, 2009), while deficiency of PHD2 in immune cells decreases tumor growth (Mamlouk et al, 2014). However, others reported that PHD2 silencing in cancer cells increases or decreases tumor growth via different underlying mechanisms (Klotzsche-von Ameln et al, 2011;Bordoli et al, 2011;Chan et al, 2009;Su et al, 2012;Wottawa et al, 2013).…”

“…Given that silencing of PHD2 in BC cells reduced TGF-b1 secretion (Wottawa et al, 2013), we investigated if reduced activation of CAFs was due to a reduction in TGF-b1 secretion by PyMT +/À cancer cells. ELISA showed that TGF-b1 levels were reduced in the supernatant of PyMT +/À cancer cells ( Figure 5A).…”

Prolyl hydroxylase-2 inhibits liver tumor cell proliferation and cyclin D1 expression in a hydroxylase-dependent manner

“…Unlike HIFα, the role of PHDs in tumorigenesis remains poorly understood. PHDs can function as tumor suppressors as well as tumor oncogenes depending on the cancer context (Bordoli et al, 2011;Erez et al, 2003;Heindryckx et al, 2012;Jokilehto and Jaakkola, 2010;Kato et al, 2006;Klotzschevon Ameln et al, 2012;Klotzsche-von Ameln et al, 2011;Lee et al, 2008;Lee et al, 2005;Mamlouk et al, 2014;Su et al, 2012;Wottawa et al, 2013;Xie et al, 2014).…”