Up to 80% of patients with ovarian cancer develop platinum resistance over time to platinum-based chemotherapy. Increased HIF1α level is an important mechanism governing platinum resistance in platinum-resistant ovarian cancer (PROC). However, the mechanism regulating HIF1α stability in PROC remains largely unknown. Here, we elucidate the mechanism of HIF1α stability regulation in PROC and explore therapeutic approaches to overcome cisplatin resistance in ovarian cancer.
We first used a quantitative high-throughput combinational screen (qHTCS) to identify novel drugs that could resensitize PROC cells to cisplatin. Next, we evaluated the combination efficacy of inhibitors of HIF1α (YC-1), ERK (selumetinib), and TGFβ1 (SB431542) with platinum drugs by in vitro and in vivo experiments. Moreover, a novel TGFβ1/ERK/PHD2-mediated pathway regulating HIF1α stability in PROC was discovered.
YC-1 and selumetinib resensitized PROC cells to cisplatin. Next, the prolyl hydroxylase domain-containing protein 2 (PHD2) was shown to be a direct substrate of ERK. Phosphorylation of PHD2 by ERK prevents its binding to HIF1α, thus inhibiting HIF1α hydroxylation and degradation—increasing HIF1α stability. Significantly, ERK/PHD2 signaling in PROC cells is dependent on TGFβ1, promoting platinum resistance by stabilizing HIF1α. Inhibition of TGFβ1 by SB431542, ERK by selumetinib, or HIF1α by YC-1 efficiently overcame platinum resistance both in vitro and in vivo. The results from clinical samples confirm activation of the ERK/PHD2/HIF1α axis in patients with PROC, correlating highly with poor prognoses for patients.
HIF1α stabilization is regulated by TGFβ1/ERK/PHD2 axis in PROC. Hence, inhibiting TGFβ1, ERK, or HIF1α is potential strategy for treating patients with PROC.
Identification of mechanisms regulating platinum resistance and new approaches for treatment of platinum-resistant ovarian cancer (PROC) will benefit patients with cancer. The amount of HIF1α plays an important role in platinum resistance in ovarian cancer, but the mechanism regulating HIF1α stability, in this context, remained largely unknown. This study demonstrates that PHD2 is a direct substrate of ERK and phosphorylation of PHD2 by increased ERK activity in PROC cells prevents PHD2 binding to HIF1α, resulting in the inhibition of HIFα hydroxylation and degradation. Further studies indicate that this ERK/PHD2/HIF1α pathway is potentiated in PROC cells or tumors by TGFβ1. Inhibition of HIF1α by YC-1, ERK by selumetinib, or TGFβ1 by SB431542 overcomes platinum resistance in vitro or in vivo. These findings reveal an unexpected TGFβ1/ERK/PHD2/HIF1α pathway regulating platinum resistance in ovarian cancer. Additionally, this study points to three potential drugs to treat patients with PROC.
Most patients with ovarian cancer initially respond well to platinum drug-based chemotherapy, but up to 80% patients relapse when becoming resistant to platinum. Resistance to platinum drugs is a big hurdle for the treatment of ovarian cancer. Thus, treatments for platinum-resistant ovarian cancer (PROC) remain an unmet need; research into treatments will improve survival and quality of life for these patients with PROC (1–4).
Hypoxia-inducible factor-1α (HIF1α) is a subunit of HIF1, a heterodimeric transcription factor that is considered the master transcriptional regulator of multiple cellular pathways including cell proliferation, tumor migration, and angiogenesis (5, 6). Elevated levels of HIF1α are associated with tumor metastasis and poor patient prognosis as well as chemoresistance in several tumors including breast cancer, oropharyngeal cancer, and ovarian cancer (7, 8). HIF1α contains an oxygen-dependent degradation domain (ODDD, residues 410-603), which is responsible for its rapid degradation under normoxic conditions (9). In the presence of oxygen, HIF1α protein is hydroxylated by prolyl hydroxylase (PHD) domain enzymes on two key proline residues (P402/P564) located within the ODDD, resulting in HIF1α degradation by an ubiquitin-mediated pathway. Hydroxylation of HIF1α by PHDs creates a binding site for the von Hippel-Lindau (VHL) E3 ligase, which promotes subsequent ubiquitylation and proteasomal degradation of HIF1α. Therefore, PHDs are key factors regulating HIF1α levels in cells; inhibition of PHDs' activity limits HIF1α degradation (10). Nonetheless, the regulatory pathway governing hydroxylation of HIF1α by PHDs remains largely unknown.
Extracellular regulated kinase (ERK) is a member of the MAPK family and has been implicated in diverse cellular pathways including proliferation, survival, differentiation, and motility (11). Although ERK was shown to control HIF1α transcriptional activation (12, 13), it is unknown if ERK directly regulates HIF1α protein stability and how ERK participates in platinum resistance in PROC. The TGFβ, which can activate the ERK pathway, belongs to the TGF superfamily comprising a large number of cytokines secreted by many cell types (14). A growing body of evidence indicates that TGFβ signaling is altered in cancer. A dual role for TGFβ in tumors, depending on tumor stage, cellular context, and tumor microenvironment has long been noted. TGFβ exerts tumor-suppressive effects; paradoxically, TGFβ can also act as a tumor activator because of its activity in suppressing immune and inflammatory responses (15).
We have established a quantitative high-throughput combinational screen (qHTCS) to study platinum resistance in PROC cells (16). Using this qHTCS, we identified the HIF1α inhibitor (YC-1) and ERK inhibitor (selumetinib) that could overcome platinum resistance in PROC cells. Further mechanistic studies revealed a novel ERK/PHD2/HIF1α-mediated pathway regulating platinum resistance in ovarian cancer cells. Significantly, we found that ERK directly interacts with and phosphorylates PHD2; the phosphorylated PHD2 compromises the interaction of PHD2 with HIF1α, resulting in the reduction of HIF1α hydroxylation, which stabilizes the HIF1α protein in PROC cells. Importantly, both in vitro and in vivo studies indicated that this ERK/PHD2/HIF1α pathway is activated in PROC cells and is a major driving force to promote platinum resistance. Furthermore, our clinical studies confirmed the activation of ERK and HIF1α in tumors from patients with PROC.
Materials and Methods
Antibodies and reagents
Antibodies used in immunoblotting: HIF1α (BD Biosciences, 610958, 1:1,000) were purchased from BD Biosciences. BCL-XL (ab32370, 1:1,000) were from Abcam. β-Actin (A5441, 1:1,000) and anti-phospho-PHD2 (Ser 125) antibody (MABC1612, 1:1,000) were from Sigma-Aldrich. p-ERK (CST, 4370S, 1:1000), ERK (4695S, 1:1000), hydroxy-HIF1α (3434S, 1:1000), phospho-MAPK/CDK substrates (2325S, 1:1000), TGFβ (3711S, 1:1000), and PHD2 (4835, 1:1000) were from Cell Signaling Technology. Normal mouse IgG (sc-2025), mouse anti-goat IgG-HRP (sc-2354), and mouse anti-rabbit IgG-HRP (sc-2357) were from Santa Cruz Biotechnologies. Cis-diamineplatinum(II) dichloride (Cisplatin; Sigma-Aldrich, 479306), HIF1α inhibitor YC-1 (Selleck Chemicals, S7958), MEK inhibitor, selumetinib (Selleck Chemicals, S1008), and TGFβ receptor inhibitor, SB431542 (Selleck Chemicals, S1067), were dissolved in DMSO and sterile 0.9% saline respectively for the in vitro and in vivo assay. Lambda Protein Phosphatase (Lambda PP; P0753S) were purchased from New England Biolabs (NEB).
Compound screen experiments were performed in two rounds as described previously (16). Cisplatin-resistant IGROV1 CR cells 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 cells (1,500 cells/well) were plated in 1,536-well polystyrene plates (Greiner Bio-One) with 5-μL DMEM medium (10% FBS) and incubated for 16 hours. In the first screen, drugs were tested at 11 different concentrations from 0.8 nmol/L to 46 μmol/L by serial dilution (1:3). After incubation for 72 hours, a total of 112 compounds that efficiently inhibited the proliferation of IGROV1 CR cells were identified. To further identify compounds that have synergy with cisplatin, these 112 compounds were screened at 11 different concentrations in combination with vehicle, or 8.5 μmol/L cisplatin (IC50 of cisplatin). Then 4-μL/well ATP content cell viability assay reagent (Promega, G7570) was added into each well and incubated for 30 minutes followed by detection of cell viability. Through the second screen, compounds that exhibited the increased cytotoxicity of cisplatin in IGROV1 CR cells were identified (Supplementary Table S1). The primary screen data and curve fitting were analyzed using software developed at the NIH Chemical Genomics Center (NCGC). The half-maximal inhibitory concentration (IC50 values) of the two-drug combinations was compared with that of a single drug.
Cell culture and establishment of resistance cell lines
Human ovarian cancer cell line SKOV3 (ATCC) and IGROV1 (gift from Wei Zheng's lab) were cultured in DMEM containing 10% FBS. PEO1/4 cell lines (Sigma) were cultured in RPMI1640 containing 10% FBS. All the cells were cultured at 37°C in a cell incubator containing 5% CO2. For hydroxylated HIF1α protein analysis, all the cell samples were treated with 10 μmol/L MG-132 (Selleck, S2619) for 8 hours before harvest (17). The cisplatin-resistant cell lines (SKOV3 CR and IGROV1 CR) were generated by 6 cycles of cisplatin treatment in our lab as described in published papers (16). The early passages (less than 10 passages) of the resistant cell lines were used in our present study.
Cells were harvested and total RNA was isolated using an RNeasy Mini Kit (Qiagen, 74106) according to the manufacturer's instructions. Using an iScript cDNA Synthesis Kit (Bio-Rad, 1708891), cDNAs were prepared and analyzed for the expression of genes by real-time PCR (qPCR) using SsoAdvanced Universal SYBR Green Supermi (Bio-Rad, 1725271). The expression of each gene was normalized to the expression of β-actin. The sequences of the HIF1α primers were: forward, 5′-CGGCGAGAACGAGAAGAA-3′ and reverse, 5′-GCAACTGATGAGCAAGCTCATA-3′ (18).
Fifty nmol/L HIF1α siRNA (Thermo Fisher Scientific, 106498) was transferred into cells using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150) according to the manufacturer's instructions. HIF1α was stably knocked down using short hairpin RNA (shRNA; OriGene, TG320380) using Lipofectamine 2000 according to the manufacturer's instructions. Cells were then treated with indicated agents for 48 hours after siRNA transfection or shRNA infection (19, 20).
Co-immunoprecipitation and Western blotting
For co-immunoprecipitation (Co-IP), cells were harvested and lysed in Co-IP buffer (25 mmol/L Hepes, 150 mmol/L KAc, 5 mmol/L MgCl2, 1 mmol/L Na2EGTA, 10% Glycerol, 0.1% NP-40, and 1% complete protease inhibitor cocktail; Roche). Cell lysates were centrifuged and the supernatant was incubated with indicated antibodies or nonspecific IgG as a negative control at 4°C overnight. Then the protein beads (protein A/G plus-Agarose sc-2003; Santa Cruz) were added into the tubes and incubated with the samples at 4°C for 1 hour. After that, the beads were washed at least three times using Co-IP buffer, and the precipitated proteins were used for further analysis after being mixed with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, NP0007) and heated at 75°C for 10 minutes. For Western blotting, cells were harvested and lysed with RIPA cell lysis buffer (Thermo Fisher Scientific, 89900). Protein samples mixed with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific, NP0007) and NuPAGE Sample Reducing Agent (Thermo Fisher Scientific, NP0004) were then separated by NuPAGE 4-12% Bis-Tris Protein Gels (Thermo Fisher Scientific, NP0335BOX) and were transferred to a nitrocellulose membrane (Bio-Rad, 1620115) using transfer buffer (192 mmol/L glycine, 25 mmol/L Tris-HCl, 20% methanol) at 4°C for 2 hours at 100 V. The membrane was then blocked with blocking solution (3% BSA in 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.1% Tween 20, TBST) at room temperature for 30 minutes. Finally, the membrane was incubated with the indicated primary antibodies at 4°C overnight, followed by HRP-conjugated secondary antibodies at room temperature for 2 hours. Western blot analysis was performed using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, 34580).
Formalin-fixed paraffin-embedded tissue sections were deparaffinized in xylene and hydrated with ethanol solutions. After antigen-retrieval treatment (121°C for 10 minutes in 10 mmol/L sodium citrate buffer), slides were treated with a 3% hydrogen peroxide methanol solution for 10 minutes to quench endogenous peroxidase activity. Ten percent normal goat serum was used to block nonspecific binding. Slides were then incubated with a primary antibody at 4°C overnight and the next day incubation of sections with IHC detection reagent (CST; mouse 8125, rabbit 8124) was carried out for 30 minutes at room temperature. An ImmPACT DAB peroxidase (HRP) Substrate Kit (Vector, SK-4105) was used to visualize the staining according to the manufacturer's instructions. The slides were lightly counterstained with hematoxylin. Staining was quantified for at least five fields at 200× magnification. IHC score = summation (1 + i)pi, where i is the intensity score and pi is the percent of the cells with that intensity. In our experiment, we set i from 0 to 5 (no stain = 0; ≤1/100 cells stained = 1; ≤1/10 cells stained = 2; ≤1/3 cells stained = 3, ≤2/3 cells stained = 4; all cells stained = 5). The quantification of IHC staining was scored blindly by three independent observers (21).
Cell viability and clonogenic assay
Sulforhodamine B (SRB) assay was used to detect cell viability as in published papers (22, 23). For the clonogenic assay, cells were seeded in a 6-well dishes with a density of 500 to 800 cells per dish, then treated with 0.3 μmol/L cisplatin, 0.15 μmol/L YC-1, or both for 14 days to allow colony formation. Colonies were stained with crystal violet. Colonies with >50 cells were counted.
Cell apoptosis assay
In vitro cell apoptosis was analyzed by flow cytometry using a FITC Annexin-V Apoptosis Detection Kit (BD Biosciences, 556547), whereas in vivo apoptotic tumor cells were detected using an In Situ Cell Death Detection Kit (Roche, 11684817910) according to the manufacturer's instructions.
TGFβ1 content in the cell culture supernatant was detected using a Human TGFβ1 Quantikine ELISA Kit (R&D, DB100B) according to the manufacturer's instructions.
Recombinant protein purification and GST pull-down assay
His-tagged ERK1/ERK2 and GST-tagged PHD2 were expressed in E. coli BL21 treated with 0.1 mmol/L IPTG at 16°C for 12 hours to induce protein expression. Bacteria were then harvested and resuspended in PBS containing 0.5% Triton X-100 and 1 mmol/L PMSF, followed by ultrasonication for 20 minutes. The recombinant His-tagged and GST-tagged proteins were purified using Ni-NTA Agarose (25214; Thermo Fisher Scientific) and Glutathione Agarose (16100; Thermo Fisher Scientific), respectively. For the ERK1/2 and PHD2 GST pull-down assay, GST-tagged PHD2 was incubated with purified ERK1 or ERK2 at 4°C for 2 hours. The elution was analyzed by Western blot analysis with indicated antibodies (24).
In vitro kinase assay
For the ERK in vitro kinase assay, the bead-bound ERK1/2 (His-tagged ERK1/ERK2 expressed from E. coli BL21) were washed at least three times with PBS. The proteins bound to histidine-tagged protein purification resin were separated by SDS-PAGE and visualized by Coomassie Blue staining. Immunoprecipitated IgG or His-ERK1/2 were incubated with recombinant human PHD2 Protein (H00054583; Novus Biologicals) and ATP in a kinase buffer. The supernatants containing phosphorylated protein were subjected to Western blot assay (25).
Five- to 6-week-old female BALB/c athymic nude mice of 20 to 25 g were purchased from the Jackson Laboratory. The mouse housing conditions and all the animal experiment procedures were performed in accordance with the Institutional Animal Care and Use Committees (IACUC) of George Washington University. After 10 days of recovery from the shipping process, mice were subcutaneously inoculated with IGROV1 or IGROV1 CR cells (suspended in 100-μL PBS, 5 × 106 cells/mouse) into the dorsal flank to establish the subcutaneous cisplatin sensitive and resistant xenografts. Mice were randomized into subsequent experiment groups when the average tumor volume reached 100 to 150 mm3. To investigate the synergized effect of cisplatin and YC-1, four groups of mice (eight mice per group) with IGROV1 CR xenograft tumors were intraperitoneally treated with vehicle, cisplatin (4 mg/kg), YC-1 (30 mg/kg), or a combination of cisplatin plus YC-1 twice a week for 3 weeks. To investigate the synergized effect of cisplatin and selumetinib, four groups of IGROV1 CR xenograft tumors-bearing mice (eight mice per group) were intraperitoneally treated with vehicle, cisplatin (2 mg/kg), selumetinib (50 mg/kg), or combination of cisplatin plus selumetinib for 20 days, once every 2 days (n = 8). For single cisplatin treatment, mice bearing IGROV1 xenograft tumors were intraperitoneally treated with vehicle or cisplatin [0.5, 1, 2 mg/kg i.p. for 2 weeks, twice a week (n = 8)]. Tumor size and body weight were measured every 2 days. The relative tumor volume was calculated using the formula: a × (b2)/2, for which a and b represent the longest and shortest diameters, respectively. All the tissue and tumor samples were obtained 3 days after the final drug administration for subsequent evaluation.
Patients with ovarian cancer
The tumor samples of all the chemosensitive and matched chemoresistant patients with ovarian cancer were kept as formalin-fixed paraffin-embedded sections in the Department of Pathology at the University of Hong Kong. The study was performed in accordance with the Declaration of Helsinki statement on ethical biomedical research and studies using human tissues were approved by the local institutional ethics committee (institutional review board reference No. UW 05-143 T/806 and UW 11-298). Written informed consent was received from each patient prior to their inclusion in the study. The histological types, disease stages, and cancer cell contents in each FFPE section were examined by experienced pathologists. Tumors from patients who had a total response to platinum-based therapy and no recurrence within 6 months were defined as platinum-sensitive, and tumors from patients who had the recurrence occur within 6 months following the completion of platinum-based therapy were defined as platinum resistant.
Statistical analysis was performed using GraphPad Prism 7.0 Software. Data are represented as the mean ± the SD; P values were calculated with Student t tests (unless otherwise indicated) in all the data comparing control to treatment. *P < 0.05, **P < 0.01, and ***P < 0.001. Progression-free survival (PFS) rates of patients were analyzed via Kaplan–Meier analysis by online databases (http://kmplot.com) and a log-rank (Mantel–Cox) test was used in comparison of each arm (26).
qHTCS to identify HIF1α inhibitor YC-1 overcoming cisplatin resistance
To explore new effective therapeutic strategies for treatment of PROC, we compared three paired PROC cell lines with their platinum-sensitive controls (Supplementary Fig. S1). Among them, two paired cells (IGROV1 vs. IGROV1 CR and SKOV3 vs. SKOV3 CR) have been described previously (16). Another pair of platinum-sensitive and -resistant ovarian cancer cells (PEO1 and PEO4) was derived from the same patient before (PEO1) and after (PEO4) development of chemoresistant to platinum-based drug treatment (16).
We carried out a two-round drug screen in the qHTCS format, using IGROV1 CR cells, against 6,016 approved drugs and bioactive compounds as described previously (Data shown in Supplementary Table S1; ref. 16). HIF1α inhibitor (YC-1) and MEK inhibitor (PD-0325901) were found to exert a very strong growth inhibition on IGROV CR cells when combined with cisplatin (Fig. 1A). The results suggest that ERK and HIF1α may regulate platinum resistance in ovarian cancer.
To validate the inhibitory efficacy of YC-1 (the validation for MEK inhibitor was included in Fig. 3), three PROC cell lines were treated with a combination of YC-1 and cisplatin for 72 hours and cell viability was measured by a SRB viability assay. Significantly, the combined treatment of YC-1 with cisplatin showed an excellent synergy to inhibit the growth of PROC cells (Fig. 1B and C). The addition of 0.2 and 0.4 μmol/L YC-1 reduced the IC50 of cisplatin in IGROV CR cells from 9.8 to 2.4 μmol/L and 0.8 μmol/L, respectively (Supplementary Fig. S2A). Consistently, the clonogenic assay indicated that combinatorial treatment with YC-1 and cisplatin significantly reduced the survival of IGROV1 CR cells compared with cisplatin or YC-1 treatment alone (Fig. 1C). Together, these results suggest that HIF1α may regulate platinum resistance in ovarian cancer cells.
Elevated HIF1α levels in PROC were reduced by YC-1 treatment both in vitro and in vivo
To study how YC-1 regulates cisplatin-resistance in ovarian cancer, we examined the expression of HIF1α and its target gene BCL-XL in three paired sensitive and resistant cell lines. Strikingly, HIF1α and BCL-XL protein expression levels were increased in all three PROC cells compared with their sensitive counterparts (Fig. 2A). However, the HIF1α mRNA levels in all three paired parental and resistant cell lines were not changed (Supplementary Fig. S2B), suggesting that increased HIF1α protein levels are due to an alteration of the protein posttranscription pathway(s) in PROC cells.
We next depleted HIF1α by using two independent short-hairpin RNAs (shRNAs) to explore possible HIF1α regulation of cisplatin resistance in ovarian cancer. Downregulation of HIF1α by shRNA redcued the BCL-XL level and resensitized IGROV1 CR cells to cisplatin (Supplementary Fig. S2C and S2D). HIF1α knockdown by siRNA in IGROV1 CR cells also reduced BCL-XL levels and resensitized IGROV1 CR cells to cisplatin (Supplementary Fig. S2E and S2F). To further confirm HIF1α's important role in cispaltin-resistance, we treated sensitive IGROV1 cell with CoCl2, a chemical inducer of HIF1α, followed by cisplatin treatment (Supplementary Fig. S2G; ref. 27). The cells with higher levels of HIF1α induced by CoCl2 exhibited stronger resistance to cisplatin compared with cells treated with vehicle (Supplementary Fig. S2H). Together, these observations suggest that HIF1α is crucial for the regulation of platinum resistance in ovarian cancer.
In addition, YC-1 treatment dramatically reduced both HIF1α and BCL-XL protein levels in a dose-dependent manner (Fig. 2B). Consistent with the synergistic effect of YC-1 and cisplatin on growth inhibition of resistant cells, the combination of cisplatin and YC-1 significantly increased the apoptotic population (Supplementary Fig. S2I), and promoted the cleavage of PARP-1 (Supplementary Fig. S2J), a hallmark of apoptosis (28).
To test the hypothesis of YC-1 inhibiting the growth of PROC cells in vivo, we applied a tumor graft model by subcutaneously implanting IGROV1 CR cells into nude mice to form tumors. Mice were randomized for treatment with vehicle, cisplatin (4 mg/kg), YC-1 (30 mg/kg), or a combination of cisplatin and YC-1. Three weeks after the treatment, cisplatin or YC-1 alone had little effects on IGROV1 CR xenograft tumors, but the combined treatment of YC-1 and cisplatin significantly reduced the tumor size (Fig. 2C). IHC in these treated tumors indicated that treatment of YC-1, either in monotherapy or combination with cisplatin, significantly reduced the HIF1α and BCL-XL levels (Fig. 2D and E); furthermore, the combined treatment with YC-1 and cisplatin increased the apoptotic population as indicated by TUNEL staining (Fig. 2F). During the treatment course, there was no change in body weight or obvious histologic change in heart, liver, kidney, or lung among the various treatment groups (Supplementary Fig. S2K and S2L).
ERK activation upregulates HIF1α levels in PROC
MAPK has been shown to regulate HIF1α expression (29, 30). Given the identification of a MEK inhibitor, PD-0325901, that overcame cisplatin resistance in IGROV CR cells from qHTCS (Fig. 1A) and the fact that PD-0325901 suppresses phosphorylation of ERK1/2 (31), we speculated that the ERK pathway may regulate HIF1α as well as cisplatin-resistance in ovarian cancer cells. To test this hypothesis, we examined the activation of ERK in all three PROC cell lines. Significantly increased phosphorylated-ERK (p-ERK) as well as HIF1α were seen in all three resistant cell lines compared with their sensitive counterparts (Fig. 3A), indicating the activation of the ERK pathway in PROC cell lines.
To test the ability of ERK1/2 to regulate HIF1α protein levels, we used a selective MEK1/2 inhibitor, selumetinib, which has been tested in phase II clinical trials and proved to significantly inhibit phosphorylation of ERK1/2 (32). As expected, selumetinib was able to reduce the p-ERK and HIF1α levels in a dose-dependent manner (Supplementary Fig. S3A). Moreover, selumetinib efficiently inhibited the ERK/HIF1α pathway and reduced cell viability when combined with cisplatin and the combination treatment exhibited an excellent synergistic effect in all three resistant cell lines (Fig. 3B and C; Supplementary Fig. S3B–S3D). These results demonstrate that ERK is critical to maintaining HIF1α levels in PROC cell lines.
We next established the IGROV1 CR xenograft tumor-bearing mouse system to further examine the effect of cisplatin and selumetinib in combination on tumors in vivo. Although cisplatin alone had no effects on tumor growth due to resistance, selumetinib alone significantly inhibited tumor growth and combined drug treatments exhibited further inhibition on tumor growth (Fig. 3D). Immunoblot and IHC analysis of tumor samples indicated selumetinib could inactivate ERK/HIF1α signaling in the xenograft tumors after the treatment (Fig. 3E–G). Importantly, combined treatment of selumetinib and cisplatin had no effects on body weight with no histologic changes in heart, liver, kidney, or lung among the various treatment groups (Supplementary Fig. S3E and S3F).
Because ERK can regulate protein expression either transcriptionally or posttranscriptionally (33), we therefore examined mRNA levels of HIF1α by reverse transcription-quantitative real-time PCR (qRT-PCR) in IGROV1 CR cells after treatment with cisplatin, selumetinib, or both. The results indicated that selumetinib or selumetinib plus cisplatin did not change HIF1α mRNA level in IGROV1 CR cells (Supplementary Fig. S3G), indicating that ERK regulates HIF1α expression through a posttranscriptional regulatory pathway.
ERK increases HIF1α stability by directly interacting and phosphorylating PHD2
HIF1α protein stability is regulated by a ubiquitin-mediated pathway after hydroxylation by PHDs (9). Among all three PHDs, PHD2 is the most abundant form, we explored the possibility that ERK regulates HIF1α expression by directly targeting PHD2 (34, 35).
We first examined the hydroxylation levels of HIF1α in PROC cell lines and found that hydroxylated HIF1α (OH-HIF1α) was decreased in all three resistant cell lines, whereas higher levels of HIF1α were detected (Fig. 4A). Given that the total PHD2 levels were unchanged in paired resistant cells (Supplementary Fig. S4A), we hypothesized that ERK might directly target PHD2 and regulate its activity. To test this hypothesis, we examined the phosphorylation levels of PHD2 at serine residues using a specific phosphorylated-MAPK substrate antibody against serine residues in PROC cells. Significantly, in all three PROC cell lines, phosphorylation of PHD2 was increased compared with their sensitive counterparts (Fig. 4B; Supplementary Fig. S4B). Strikingly, the interaction between HIF1α and PHD2 in all resistant cells was decreased (Fig. 4B; Supplementary Fig. S4B), suggesting that the phosphorylation of PHD2 by ERK may interfere the interaction between HIF1α and PHD2.
We, therefore, tested ERK interactions with PHD2 by a Co-IP assay. From endogenous Co-IP of PHD2 in both IGROV1 and PEO1 cells, we could detect ERK proteins and vice versa (Fig. 4C; Supplementary Fig. S4C). Consistently, the in vitro GST pull-down assay indicated that both His-tagged ERK1 and ERK 2 interacted with GST-fused PHD2 (Fig. 4D), suggesting a direct interaction between PHD2 and ERK1/2.
We assumed that in resistant cells, enhanced phosphorylation of PHD2 by ERK may be due to the increased direct interaction between PHD2 and ERK. Indeed, the result showed that the interaction between PHD2 and ERK was significantlty increased in all resistant cells compared with their sensitive counterparts (Supplementary Fig. S4D). We next observed that inhibition of ERK by selumetinib reduced phosphorylated-PHD2 on serine residues and increased the interaction between PHD2 and HIF1α (Fig. 4E; Supplementary Fig. S4E). Also, selumetinib treatment further increased HIF1α hydroxylation as well as its degradation (Fig. 4F; Supplementary Fig. S4F).
Because both ERK1 and ERK2 directly interact with PHD2, we hypothesized that PHD2 might be a phosphorylated substrate of ERK. To test this hypothesis, we screened the potential phosphorylation sites of PHD2 using an in vitro kinase assay, and found that PHD2 was phosphorylated at Ser125 by ERK1/2, as reconized by an anti p-PHD2 (Ser125) antibody (Fig. 4G). Consistently, inhibition of ERK by MEK inhibitor selumetinib significantly reduced phosphorylation of PHD2 at Ser 125, increased HIF1α hydroxylation as well as its degradation (Fig. 4H). Furthermore, S125A mutation of PHD2 significantly increased the interaction between PHD2 and HIF1α compared with wild-type PHD2 (Fig. 4I). Together, the results suggest that ERK directly phosphorylate PHD2 at Ser125 site and phosphorylation of PHD2 by ERK prevents its binding to HIF1α, thus preventing HIF1α hydroxylation and subsequently preventing HIF1α ubiquitination and degradation.
Autocrine TGFβ1 activates ERK/PHD2/HIF1α pathway to cause platinum resistance
We next sought to determine how ERK is activated in PROC cell lines. Because cytokines including TGFβ1 and growth factors are known activators of ERK (36, 37), we hypothesized that ERK may be activated by TGFβ1, resulting in platinum resistance in PROC cell lines. We first collected conditioned medium from PROC cells (IGROV1 CR, PEO4, SKOV3 CR) and used the medium to culture platinum-sensitive cells (IGROV1, PEO1, SKOV3). Interestingly, we found that cells cultured with the conditioned medium from PROC cells exhibited a better survival when treated with cisplatin (Supplementary Fig. S5A), indicating that secreted factors from PROC cells may contribute to the acquired resistance in platinum-sensitive cells. We also found that TGFβ1 levels in the cell culture medium were increased in all three resistant cell lines (Fig. 5A). We next treated platinum-sensitive cells with recombinant human TGFβ1 (R&D, 7754-BH-005), which induced cisplatin resistance in sensitive cells (Fig. 5B; Supplementary Fig. S5B). The TGFβ1 levels were also increased inside all resistant cell lines (Fig. 5C). Moreover, recombinant TGFβ1 increased p-ERK and HIF1α levels in both sensitive IGROV1 and PEO1 cells and this activation of the ERK/HIF1α pathway was reversed by selumetinib treatment (Fig. 5D; Supplementary Fig. S5C).
To further test the notion that the TGFβ1/ERK/PHD2/HIF1α pathway contributes to platinum resistance in ovarian cancer cells, we treated all these resistant cells with a TGFβ1 inhibitor, SB431542, and found that inhibition of TGFβ1 decreased p-ERK and HIF1α levels but increased hydroxylated HIF1α (Fig. 5E; Supplementary Fig. S5D). Consistently, inhibition of TGFβ1 by SB431542 decreased PHD2 phosphorylation and increased the interaction between PHD2 and HI1α (Fig. 5F; Supplementary Fig. S5E). Similar to YC-1 and selumetinib, SB431542 also exhibited a synergistic effect with cisplatin on growth inhibition in PROC cell lines (Fig. 5G; Supplementary Fig. S5F).
Collectively, these findings demonstrate that secreted TGFβ1 acts as an autocrine factor to stimulate the activation of the ERK/PHD2/HIF1α pathway, thereby inducing platinum resistance in PROC cells.
Cisplatin induces the activation of TGFβ1/ERK/HIF1α signaling in vitro and in vivo
Given that TGFβ1/ERK/HIF1α signaling is upregulated in cisplatin-resistant cells, we then assumed that cisplatin treatment may cause an activation of the TGFβ1/ERK/PHD2/HIF1α pathway in sensitive ovarian cancer cells. To test this possibility, three sensitive cell lines were treated with multiple dosages of cisplatin for 4 days. The TGFβ1/ERK/HIF1α signaling was dramatically activated in all the three cisplatin sensitive cell lines (IGROV1, SKOV3, and PEO1) after cisplatin treatment (Supplementary Fig. S6A). The TGFβ1/ERK/PHD2/HIF1α activation was also observed in a time-dependent manner after 1 μmol/L cisplatin treatment (Supplementary Fig. S6B). Consistent with these results, the cisplatin treatment increased phosphorylated-PHD2 levels and decreased the interaction of PHD2 with HIF1α (Supplementary Fig. S6C).
We then established IGROV1 xenograft tumors that were treated with cisplatin for 2 weeks to determine the in vivo effect of cisplatin on the ERK/HIF1α pathway. IHC analysis indicated a significant cisplatin-doses-dependent increase of p-ERK, HIF1α, and BCL-XL protein levels in IGROV1 tumors (Supplementary Fig. S6D and S6E). Thus, the results indicate that cisplatin treatment induces the activation of the ERK/HIF1α/BCL-XL pathway in tumor cells in vivo which contributes to the development of cisplatin resistance.
Clinical evidence that TGFβ1/ERK/HIF1α is activated in tumors from platinum- sensitive or -resistant patient tumors
To verify the TGFβ1/ERK/HIF1α pathway is activated in platinum-resistant tumors from patients with ovarian cancer, we used a Kaplan–Meier plotter to analyze the correlations of TGFβ1, ERK, or HIF1α levels with the survival of patients with ovarian cancer who received platinum-based chemotherapy. The results showed that the higher TGFβ1/HIF1α levels and the higher the ERK signature genes expression levels, the worse the 5-year PFS (Fig. 6A).
We also evaluated these correlations in the samples from the same patients before platinum drug treatment and after development of resistance to platinum drugs. To this end, IHC staining was performed to detect the p-ERK and HIF1α levels in tumors (Fig. 6B). The HIF1α level was significantly increased in all seven resistant tumors, whereas the p-ERK level was elevated in six of seven resistant tumors (Fig. 6C). The Pearson correlation coefficient (r = 0.729, P < 0.01) indicates a strong correlation between elevated levels of p-ERK and HIF1α (Fig. 6D).
Therefore, our results indicate that the activation of the TGFβ1/ERK/HIF1α pathway is highly correlated with platinum resistance and poor prognoses for patients with ovarian cancer.
HIF1α has been reported to promote cisplatin resistance in ovarian cancer (38–40); however, the mechanism regulating HIF1α in ovarian cancer remains largely unknown. In this study, a qHTCS assay was used to identify inhibitors of HIF1α and MEK, which resensitized PROC cells to cisplatin. The follow-up mechanistic study not only revealed a novel TGFβ1/ERK/PHD2/HIF1α pathway, the activation of which causes cisplatin resistance in PROC cells, but also points to three potential drugs to treat patients with PROC. More importantly, for the first time, we provide clinical evidence confirming the link between the activation of ERK and HIF1α in tumors from patients with PROC.
We have demonstrated that in PROC cells activated ERK direclty phoshorylates PHD2; this phosphorylation reduces the interaction of PHD2 with HIF1α, preventing HIF1α hydroxylation and degradation (Fig. 6E). Importantly, this study shows targeting of TGFβ1 by SB431542, ERK by selumetinib, or HIF1α by YC-1 overcame cisplatin resistance in vitro and in vivo. Previously YC-1 was found to degrade HIF1α, thus inhibiting tumor growth (38). Selumetinib is currently under phase II clinical trials for cancer treatments of advanced hepatocellular carcinoma and multiple myeloma (41, 42). A phase I trial has shown low toxicity of selumetinib (43, 44). Thus, our study provides a solid mechanistic basis and preliminary clinic evidence, supporting the clinical application of combining selumetinib and platinum drugs for treatment of PROC.
Under normoxic conditions, HIF1α is hydroxylated on two key proline residues P402 and P564 located within the ODDD domain by PHDs in the presence of oxygen, α-ketoglutarate, and iron. Hydroxylation of HIF1α creates a binding site for the VHL tumor suppressor E3 ligase complex, resulting in polyubiquitylation and proteasomal degradation of HIF1α (45, 46). PHD1/2 can be regulated by the posttranscriptional level, which affects their interaction with HIF1α. For example, PHD1 phosphorylation at Ser130 in CDK-dependent manner lowers its activity towards HIF1α (47). JNK2-mediated phosphorylation of PHD1 at Ser74 and Ser162 increases the degradation of HIF1α (48). PHD2 phosphorylated on Ser125 by rapamycin (mTOR), a downstream kinase of P70S6K increases its ability to degrade HIF1α (49). Our study elucidates an ERK1/2-mediated pathway to regulate HIF1α levels by phosphorylation of PHD2 on Ser125 in platinum drug-resistant ovarian cancer cells in a way different from P70S6K. The different effects of P70S6K- and ERK1/2-mediated phosphorylation of PHD2 on HIF1α levels may be because the different cell lines with different stress are used in two studies, or ERK1/2 and P70S6K have additional phosphorylation sites on PHD2 that affects PHD2-HIF1α interaction in coordination with PHD2 phosphorylation at Ser125. Another possibility is that other targets by ERK1/2 and P70S6K may also participate in controlling PHD2–HIF1α interaction. Further studies using different cancer cells with the same stress are expected to elucidate the interplay between PHD2 phosphorylation at S125 by ERK1/2 and P70S6K. Overall, our study suggest that phosphorylation of PHD2 by ERK stabilizes HIF1α, thus leading to resistance to cisplatin in PROC cells. It is not known whether this mechanism exists in other physiologic or pathologic conditions. Our study sheds the light for further study to investigate whether this novel mechanism regulates tumorigenesis and chemoresponse in the future.
ERK/MAPK signaling is activated in many cancers in response to growth factors. TGFβ signaling has been reported to activate the MEK/ERK pathway; inhibition of TGFβ signaling restores drug responsiveness in MED12KD cells (36). Silencing TGFβ1 expression in lung cancer cells may deactivate the ERK, JNK, and p38 MAPK pathways (37). Consistant with these findings, the results of this study revealed the autocine signaling in TGFβ1 in PROC cell lines caused the activation of the ERK/PHD2/HIF1α pathway, resulting in resistance to platinum treatments. The clinic evidence showed TGFβ1 levels in tumor samples strongly correlate with poor patient prognosis. Thus, determination of TGFβ1 levels may be used as a biomarker to predict platinum resistance in patients with ovarian cancer. Of course, studies using a large amount of clinical samples need be conducted to confirm it in the future.
Platimun drugs have been used to treat several other cancers, including small cell lung cancer and pancreatic adenocarcinoma (50); HIF1α has been shown to regulate cancer cell survival in many types of cancer (38–40). These two facts, along with our discoveries detailed in this article, offer insights and hope for treating PROC. Future work will illuminate the role of the ERK/PHD2/HIF1α pathway in other drug-resistant cancers. These studies are expected not only to elucidate the mechanistic basis of cellular response to platinum treatments, but also provide potential new therapeutic approaches for the treatment of drug-resistant cancers.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: Z. Li, W. Zhou, Y. Zhang, J. Sun, C.-W. Chen, Z. Li, W. Zheng, W. Zhu
Development of methodology: Z. Li, W. Zhou, W. Sun, Y. Meng, W. Zheng, W. Zhu
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): W. Zhou, Y. Zhang, W. Sun, M.M.H. Yung, W. Sun, J. Li, Y. Meng, S.S. Liu, A.N.Y. Cheung, H.Y.S. Ngan, D.W. Chan, W. Zheng
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Z. Li, W. Zhou, Y. Zhang, W. Sun, W. Sun, J. Li, Y. Meng, Y. Zhou, W. Zhu
Writing, review, and/or revision of the manuscript: Z. Li, W. Zhou, W. Sun, J. Li, Y. Meng, D.W. Chan, W. Zheng, W. Zhu
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Z. Li, W. Zhou, Z. Li, J. Chai, W. Zhu
Study supervision: J. Chai, W. Zheng, W. Zhu
This work was partially supported by funding from the NIH (CA177898 and CA184717 to W. Zheng), the McCormick Genomic and Proteomic Center. W. Zhu was supported by a Research Scholar Grant, RSG-13-214-01-DMC from the American Cancer Society. The authors thank Dr. DeeAnn Visk for her assistance in editing the manuscript.