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ERK Regulates Hif1α-Mediated Platinum Resistance by Directly Targeting PHD2 in Ovarian Cancer

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ERK Regulates Hif1α-Mediated Platinum Resistance by Directly Targeting PHD2 in Ovarian Cancer Published OnlineFirst July 8, 2019; DOI: 10.1158/1078-0432.CCR-18-4145 Translational Cancer Mechanisms and Therapy Clinical Cancer Research ERK Regulates HIF1a-Mediated Platinum Resistance by Directly Targeting PHD2 in Ovarian Cancer Zhuqing Li1,2, Wei Zhou1,2,3, Yi Zhang1,2, Wei Sun4, Mingo M.H. Yung5, Jing Sun1,2, Jing Li1,2, Chi-Wei Chen1,2, Zongzhu Li1,2, Yunxiao Meng1,2, Jie Chai1,2, Yuan Zhou1,2, Stephanie S. Liu6, Annie N.Y. Cheung6, Hextan Y.S. Ngan5, David W. Chan5, Wei Zheng4, and Wenge Zhu1,2 Abstract Purpose: Up to 80% of patients with ovarian cancer Results: YC-1 and selumetinib resensitized PROC cells to develop platinum resistance over time to platinum-based cisplatin. Next, the prolyl hydroxylase domain-containing chemotherapy. Increased HIF1a level is an important mech- protein 2 (PHD2) was shown to be a direct substrate of anism governing platinum resistance in platinum-resistant ERK. Phosphorylation of PHD2 by ERK prevents its binding ovarian cancer (PROC). However, the mechanism regulat- to HIF1a, thus inhibiting HIF1a hydroxylation and degra- ing HIF1a stability in PROC remains largely unknown. dation—increasing HIF1a stability. Significantly, ERK/ Here, we elucidate the mechanism of HIF1a stability reg- PHD2 signaling in PROC cells is dependent on TGFb1, ulation in PROC and explore therapeutic approaches to promoting platinum resistance by stabilizing HIF1a.Inhi- overcome cisplatin resistance in ovarian cancer. bition of TGFb1 by SB431542, ERK by selumetinib, or Experimental Design: We first used a quantitative high- HIF1a by YC-1 efficiently overcame platinum resistance throughput combinational screen (qHTCS) to identify novel both in vitro and in vivo. The results from clinical samples drugs that could resensitize PROC cells to cisplatin. Next, we confirm activation of the ERK/PHD2/HIF1a axis in patients evaluated the combination efficacy of inhibitors of HIF1a with PROC, correlating highly with poor prognoses for (YC-1), ERK (selumetinib), and TGFb1 (SB431542) with patients. platinum drugs by in vitro and in vivo experiments. Moreover, Conclusions: HIF1a stabilization is regulated by TGFb1/ anovelTGFb1/ERK/PHD2-mediated pathway regulating ERK/PHD2 axis in PROC. Hence, inhibiting TGFb1, ERK, or HIF1a stability in PROC was discovered. HIF1a is potential strategy for treating patients with PROC. Introduction platinum drugs is a big hurdle for the treatment of ovarian cancer. Thus, treatments for platinum-resistant ovarian cancer (PROC) Most patients with ovarian cancer initially respond well to remain an unmet need; research into treatments will improve platinum drug-based chemotherapy, but up to 80% patients survival and quality of life for these patients with PROC (1–4). relapse when becoming resistant to platinum. Resistance to Hypoxia-inducible factor-1a (HIF1a) is a subunit of HIF1, a heterodimeric transcription factor that is considered the master 1Department of Biochemistry and Molecular Medicine, The George Washington transcriptional regulator of multiple cellular pathways including University School of Medicine and Health Sciences, Washington, District of cell proliferation, tumor migration, and angiogenesis (5, 6). Ele- Columbia. 2GW Cancer Center, The George Washington University, Washington, vated levels of HIF1a are associated with tumor metastasis and District of Columbia. 3Department of Colorectal Surgery, Sir Run Shaw Hospital, poor patient prognosis as well as chemoresistance in several School of Medicine, Zhejiang University, Hangzhou, China. 4National Center for 5 tumors including breast cancer, oropharyngeal cancer, and ovar- Advancing Translational Sciences, NIH, Bethesda, Maryland. Department of ian cancer (7, 8). HIF1a contains an oxygen-dependent degra- Obstetrics and Gynecology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China. 6Department of Pathology, LKS Faculty of dation domain (ODDD, residues 410-603), which is responsible Medicine, The University of Hong Kong, Hong Kong SAR, China. for its rapid degradation under normoxic conditions (9). In the presence of oxygen, HIF1a protein is hydroxylated by prolyl Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). hydroxylase (PHD) domain enzymes on two key proline residues (P402/P564) located within the ODDD, resulting in HIF1a deg- Z. Li, W. Zhou, and Y. Zhang are co-first authors. radation by an ubiquitin-mediated pathway. Hydroxylation of Corresponding Authors: Wenge Zhu, George Washington University, Washing- HIF1a by PHDs creates a binding site for the von Hippel-Lindau ton, DC 20052. Phone: 202-994-3125; Fax: 2029943125; E-mail: (VHL) E3 ligase, which promotes subsequent ubiquitylation and [email protected]; David W. Chan, [email protected]; and Wei Zheng, [email protected] proteasomal degradation of HIF1a. Therefore, PHDs are key factors regulating HIF1a levels in cells; inhibition of PHDs' Clin Cancer Res 2019;XX:XX–XX activity limits HIF1a degradation (10). Nonetheless, the regula- doi: 10.1158/1078-0432.CCR-18-4145 tory pathway governing hydroxylation of HIF1a by PHDs Ó2019 American Association for Cancer Research. remains largely unknown. www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst July 8, 2019; DOI: 10.1158/1078-0432.CCR-18-4145 Li et al. and anti-phospho-PHD2 (Ser 125) antibody (MABC1612, Translational Relevance 1:1,000) were from Sigma-Aldrich. p-ERK (CST, 4370S, Identification of mechanisms regulating platinum resis- 1:1000), ERK (4695S, 1:1000), hydroxy-HIF1a (3434S, tance and new approaches for treatment of platinum- 1:1000), phospho-MAPK/CDK substrates (2325S, 1:1000), TGFb resistant ovarian cancer (PROC) will benefit patients with (3711S, 1:1000), and PHD2 (4835, 1:1000) were from Cell cancer. The amount of HIF1a plays an important role in Signaling Technology. Normal mouse IgG (sc-2025), mouse platinum resistance in ovarian cancer, but the mechanism anti-goat IgG-HRP (sc-2354), and mouse anti-rabbit IgG-HRP regulating HIF1a stability, in this context, remained largely (sc-2357) were from Santa Cruz Biotechnologies. Cis-diamine- unknown. This study demonstrates that PHD2 is a direct platinum(II) dichloride (Cisplatin; Sigma-Aldrich, 479306), substrate of ERK and phosphorylation of PHD2 by increased HIF1a inhibitor YC-1 (Selleck Chemicals, S7958), MEK inhibitor, ERK activity in PROC cells prevents PHD2 binding to HIF1a, selumetinib (Selleck Chemicals, S1008), and TGFb receptor resulting in the inhibition of HIFa hydroxylation and degra- inhibitor, SB431542 (Selleck Chemicals, S1067), were dissolved dation. Further studies indicate that this ERK/PHD2/HIF1a in DMSO and sterile 0.9% saline respectively for the in vitro and in pathway is potentiated in PROC cells or tumors by TGFb1. vivo assay. Lambda Protein Phosphatase (Lambda PP; P0753S) Inhibition of HIF1a by YC-1, ERK by selumetinib, or TGFb1by were purchased from New England Biolabs (NEB). SB431542 overcomes platinum resistance in vitro or in vivo. These findings reveal an unexpected TGFb1/ERK/PHD2/ qHTCS HIF1a pathway regulating platinum resistance in ovarian Compound screen experiments were performed in two rounds cancer. Additionally, this study points to three potential drugs as described previously (16). Cisplatin-resistant IGROV1 CR cells to treat patients with PROC. were used against 6,016 compounds from multiple compound libraries including NPC (NCGC Pharmaceutical Collection), MIPE (Mechanism Interrogation Plate), and LOPAC (The Library of Pharmacologically Active Compounds). Briefly, IGROV CR Extracellular regulated kinase (ERK) is a member of the MAPK cells (1,500 cells/well) were plated in 1,536-well polystyrene family and has been implicated in diverse cellular pathways plates (Greiner Bio-One) with 5-mL DMEM medium (10% FBS) including proliferation, survival, differentiation, and motili- and incubated for 16 hours. In the first screen, drugs were tested at ty (11). Although ERK was shown to control HIF1a transcrip- 11 different concentrations from 0.8 nmol/L to 46 mmol/L by tional activation (12, 13), it is unknown if ERK directly regulates serial dilution (1:3). After incubation for 72 hours, a total of 112 HIF1a protein stability and how ERK participates in platinum compounds that efficiently inhibited the proliferation of IGROV1 resistance in PROC. The TGFb, which can activate the ERK CR cells were identified. To further identify compounds that have pathway, belongs to the TGF superfamily comprising a large synergy with cisplatin, these 112 compounds were screened at number of cytokines secreted by many cell types (14). A growing 11 different concentrations in combination with vehicle, or body of evidence indicates that TGFb signaling is altered in 8.5 mmol/L cisplatin (IC50 of cisplatin). Then 4-mL/well ATP cancer. A dual role for TGFb in tumors, depending on tumor content cell viability assay reagent (Promega, G7570) was added stage, cellular context, and tumor microenvironment has long into each well and incubated for 30 minutes followed by detec- been noted. TGFb exerts tumor-suppressive effects; paradoxical- tion of cell viability. Through the second screen, compounds that ly, TGFb can also act as a tumor activator because of its activity exhibited the increased cytotoxicity of cisplatin in IGROV1 CR in suppressing immune and inflammatory responses (15). cells were identified (Supplementary Table S1). The primary We have established
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