Molecular and Cellular Pathobiology Prolyl Hydroxylase 3 Attenuates MCL-1–Mediated ATP Production to Suppress the Metastatic Potential of Colorectal Cancer Cells Praveenkumar Radhakrishnan 1 , Nadine Ruh 1 , Jonathan M. Harnoss 1 , Judit Kiss 1 , Martin Mollenhauer 1 , Anna-Lena Scherr 2 , Lisa K. Platzer 1 , Thomas Schmidt 1 , Klaus Podar 2 , Joseph T. Opferman 3 , Juergen Weitz 1,4 , Henning Schulze-Bergkamen 2 , Bruno C. Koehler 2 , Alexis Ulrich 1 , and Martin Schneider 1 Abstract Hypoxia is a common feature of solid tumors. Prolyl hydrox- ylase enzymes (PHD1–3) are molecular oxygen sensors that reg- ulate hypoxia-inducible factor activity, but their functions in met- astatic disease remain unclear. Here, we assessed the significance of PHD enzymes during the metastatic spread of colorectal cancer. PHD expression analysis in 124 colorectal cancer patients revealed that reduced tumoral expression of PHD3 correlated with increased frequency of distant metastases and poor outcome. Tumorigenicity and metastatic potential of colorectal tumor cells over and under- expressing PHD3 were investigated in orthotopic and heterotopic tumor models. PHD3 overexpression in a syngeneic tumor model resulted in fewer liver metastases, whereas PHD3 knockdown induced tumor spread. The migration of PHD3-overexpressing tumor cells was also attenuated in vitro. Conversely, migratory potential and colony formation were enhanced in PHD3-deficient cells, and this phenotype was associated with enhanced mito- chondrial ATP production. Furthermore, the effects of PHD3 deficiency were accompanied by increased mitochondrial expres- sion of the BCL-2 family member, member myeloid cell leukemia sequence 1 (MCL-1), and could be reversed by simultaneous inhibition of MCL-1. MCL-1 protein expression was likewise enhanced in human colorectal tumors expressing low levels of PHD3. Therefore, we demonstrate that downregulation of PHD3 augments metastatic spread in human colorectal cancer and iden- tify MCL-1 as a novel downstream effector of oxygen sensing. Importantly, these findings offer new insight into the possible, context-specific deleterious effects of pharmacologic PHD inhibi- tion. Cancer Res; 76(8); 2219–30. Ó2016 AACR. Introduction Metastatic colorectal cancer represents a leading cause for cancer-related deaths worldwide (1). When tumor growth exceeds the formation of nourishing blood vessels, hypoxia occurs and leads to the stabilization of hypoxia-inducible transcription fac- tors (HIF; ref. 2). In hypoxia, transcriptionally active HIF com- plexes bind to the promoter region of downstream target genes, which collectively mount an adaptive response aiming at securing cellular survival and restoring oxygen supply (3). Three HIF prolyl hydroxylases (PHD1, PHD2, and PHD3) regulate the stability of HIFs in an oxygen-dependent manner (4, 5). Their capacity to abrogate the hypoxic response depending on the availability of oxygen predestines these PHD enzymes as molecular oxygen sensors and makes them interesting targets for pharmacologic intervention (6). HIFs are frequently overexpressed in human tumors (7), and several studies have revealed that the PHD enzymes are implicated in cancer growth. Albeit conflicting evidence has likewise been reported (8, 9), a majority of these studies suggest that PHD enzymes exert tumor-suppressive effects. For instance, forced overexpression of PHD1 in tumor cells suppresses HIF-1a acti- vation and inhibits tumor growth in mice (10). Loss of PHD2 increases the growth of tumors derived from human colorectal and pancreatic cancer cells (11, 12), and silencing of PHD3 expression enhances the growth of heterotopically implanted colorectal tumors in mice (13). However, although PHD3 has been assigned a tumor-suppressive role in colorectal cancer, its function in metastatic tumor spread has not been studied. Here, we assessed the significance of PHD enzymes in meta- static colorectal cancer. We report that underexpression of PHD3 in human colorectal tumors is associated with the occurrence of distant metastases and impaired patient survival. Furthermore, we identify the BCL-2 family member myeloid cell leukemia sequence 1 (MCL-1) as a downstream effector, causing improved mitochondrial efficacy and enhanced metastatic spread of PHD3- deficient tumor cells. These studies confirm the significance of PHD3 as a tumor suppressor in human metastatic colorectal cancer and identify MCL-1 as a novel link between the hypox- ia-sensing pathway and energy homeostasis in tumor cells. 1 Department of General,Visceral and Transplantation Surgery, Heidel- berg University Hospital, Heidelberg, Germany. 2 Department of Med- ical Oncology, Internal Medicine VI, National Center for Tumor Dis- eases, Heidelberg University Hospital, Heidelberg, Germany. 3 St. Jude Children's Research Hospital, Cell & Molecular Biology, Memphis, Tennessee. 4 Department of Visceral, Thoracic and Vascular Surgery, Dresden University Hospital, Dresden, Germany. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). P. Radhakrishnan and N. Ruh contributed equally to this article. Corresponding Author: Martin Schneider, Heidelberg University Hospital, Im Neuenheimer Feld 110, Heidelberg 69120, Germany. Phone: 4962-2156-37876; Fax: 4962-2156-5264; E-mail: m.schneider@uni-heidelberg.de doi: 10.1158/0008-5472.CAN-15-1474 Ó2016 American Association for Cancer Research. Cancer Research www.aacrjournals.org 2219 on May 25, 2020. © 2016 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 26, 2016; DOI: 10.1158/0008-5472.CAN-15-1474