Background
Non-small cell lung cancer (NSCLC) is one of the most lethal cancers. Epidermal growth factor receptor (EGFR) activating mutations are considered as a driving force for tumorigenesis of some NSCLC. Over 90% of EGFR activating mutations which occur in both Asian and Western NSCLC patient present as an exon 19 deletion (60%) or exon 21 point mutation (30%) [
1‐
3]. Targeting therapy with the tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, has become the first-line treatment for these patients with EGFR activating mutations. However, most patients who initially respond to TKIs eventually develop acquired resistance. Beyond c-Met amplification, previous studies reveal that over 60% of acquired resistant cases associated with the emergence of a secondary mutation of EGFR, T790M. The threonine to methionine mutation, which occurs in the EGFR tyrosine kinase domain, promotes ATP binding affinity and attenuates the interaction between EGFR tyrosine kinase domain and the first-generation reversible EGFR-TKIs [
4,
5]. Osimertinib represents the third-generation EGFR-TKIs, which irreversibly inhibit EGFR activating mutations, overcomes EGFR T790M secondary mutation conferred acquired resistance to first- and second-generation TKIs. Although osimertinib significantly improved the progression-free survival (PFS) of NSCLC patients with EGFR T790M mutation, the development of acquired resistance to the third-generation EGFR-TKIs has already been described and increased in the clinic [
6‐
8]. However, the precise mechanisms mediating resistance to osimertinib remain largely unknown, and the strategies to overcome osimertinib resistance are still limited.
Myeloid cell leukemia sequence 1 (Mcl-1) is a member of the pro-survival Bcl-2 family that negatively regulates the mitochondrial apoptotic pathway. Overexpression or amplification of Mcl-1 is frequently observed in human cancers and associated with poor prognosis. Inhibition of Mcl-1 sensitizes chemo/radiotherapy induced apoptosis in multiple cancer models [
9‐
11]. Recent studies showed that Mcl-1 is upregulated by EGFR signaling. For example, EGF stimulation enhances Mcl-1 transcription in a transcription factor Elk-1 dependent manner [
12]. In EGFR mutant NSCLC cells, hyperactivation of mTORC1 increased Mcl-1 mRNA level and conferred EGFR TKI resistance [
13]. The mechanisms regarding EGFR activation and Mcl-1 transcription were well studied previously. However, the mechanisms underlying how EGFR signaling regulates Mcl-1 protein stability, as well as ubiquitination, remains elusive.
Previous studies have demonstrated that the natural compound, formononetin (C
16H
12O
4), exhibits significant anti-tumor potentials against human cancers [
14,
15]. The evidence from in vitro and in vivo studies reveal that Formo acts as a novel anti-tumorigenic agent to induce cell cycle arrest, apoptosis, anti-angiogenesis, and metastasis in a panel of solid tumors, including lung cancer [
16], colorectal cancer [
17], breast cancer [
18], and gastric cancer [
19]. The mechanism studies indicate that deactivation of protein kinases and signal transduction, or dysfunction of oncogenetic-related transcription factors, are involved in Formo-induced anti-tumor activities [
14,
15]. However, the inhibitory efficacy of Formo on EGFR signaling, and the anti-tumor effect of Formo on both osimertinib sensitive and resistant NSCLC cells, is not clear.
In the present study, by the screening of a natural products library, we identified Formo, a flavonoid derivative [
18], as a potential anti-tumor agent for use in NSCLC therapy. We investigated the therapeutic effect using NSCLC cell lines and determined the underlying mechanism of action.
Materials and methods
Reagents and antibodies
The screened compound library (L1400) was a product of Selleck Chemicals (Houston, TX). The inhibitors, including Necrostatin-1, GSK’872, z-VAD-fmk, MG132, SB216763, PD98059, and LY294002, were purchased from Selleck Chemicals. Cell culture medium, Fetal Bovine Serum (FBS), and supplements were purchased from Invitrogen (Grand Island, NY). Primary antibodies against EGFR (#4267, 1:1000), ERK1/2 (#9102, 1:1000), Akt (#4691, 1:1000), p-EGFR-Tyr1068 (#3777, 1:1000), p-ERK1/2-Thr202/Tyr204 (#4370, 1:1000), p-Akt-Ser473 (#4060, 1:1000), PARP (#9532, 1:1000), cleaved-caspase 3 (#9664, 1:1000), Bax (#14796, 1:1000), VDAC1 (#4661, 1:2000), cytochrome C (#11940, 1:1000), Mcl-1 (#39224, 1:1000), ubiquitin (#3936, 1:1000), ubiquitin (#43124, 1:1000), α-Tubulin (#2125, 1:5000), GSK3β (#12456, 1:1000), and β-actin (#3700, 1:10000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Flag-tag (F3165, 1:5000) antibody was obtained from Sigma Aldrich (St. Louis, MO). Antibodies against FBW7 (ab109617, 1:1000) and FBW7 (ab187815, 1:1000) were obtained from Abcam (Cambridge, UK). Antibodies for immunohistochemistry staining (IHC), including anti-ki67 (ab15580, 1:300) and anti-p-Akt (ab81283, 1:100) were obtained from Abcam. Anti-p-EGFR (#3777, 1:200), and anti-Mcl-1 (#39224, 1:100) were purchased from Cell Signaling Technology, Inc.
Cell culture
Human NSCLC cells, including HCC827 (EGFR Del E746-A750), H3255 (EGFR L858R), H1975 (EGFR L858R/T790M), A549 (EGFR WT), and H1299 (EGFR WT) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were maintained at 37 °C in a humidified incubator with 5% CO2 according to ATCC protocols. The osimertinib acquired resistance cell line HCC827 OR was established in our laboratory by exposing HCC827 cells to gradually increasing concentration of osimertinib for approximately 5 months (starting at 5 nM and ending with 500 nM). All NSCLC cells were subjected to mycoplasma analysis and cytogenetically tested before being frozen. The immortalized epithelial cells NL20 and HBE were purchased from ATCC and cultured following the standard protocols. Ba/F3 cell was obtained from Cell Engineering Division/RIKEN BioResource Center (Tsukuba, Ibaraki, Japan) and maintained in RPMI1640 + 10% FBS + 10% WEHI-3 cell conditioned medium according to instructions provided. Lipofectamine® 2000 (Thermo Fisher Scientific) transfection reagent was used for plasmid transfection following the manufacturer’s instructions.
Immunoblotting
Immunoblotting (IB) was performed as described previously [
20]. Whole-cell lysate was prepared with RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS. 140 mM NaCl) supplied with phosphatase and protease inhibitors, BCA kit (#23225, Thermo Fisher Scientific) was used for protein concentration. The whole-cell lysate was boiled with loading buffer and subjected to SDS-PAGE gel electrophoresis and antibody hybridization. The target proteins were visualized by chemiluminescence (Amersham Biosciences, Piscataway, NJ).
Isolation of subcellular fractions
The Mitochondria Isolation Kit (#89874, Thermo Fisher Scientific) was used for subcellular fractions preparation according to the standard instructions.
MTS assay
NSCLC cells were seeded (2.5 × 103 /well/100 μL) in 96-well plates and treated with Formo or osimertinib, as indicated. Cell viability was examined using MTS assay (Promega, Madison, WI) following the instructions provided.
Anchorage-independent cell growth
The anchorage-independent cell growth assay was performed as described previously [
21]. Briefly, the Eagle’s basal medium containing 0.6% agar, 10% FBS, and different concentration of Formo or osimertinib was loading to a six-well plate as an agar base. Cells were then counted at the concentration of 8000 cells/ml with the Eagle’s basal medium containing 10% FBS, 0.3% agar, Formo, or osimertinib, followed by overlaid into the six-well plate containing a 0.6% agar base. The cultures were maintained in a 37 °C, 5% CO
2 incubator for 2 weeks and counted using a microscope.
Stable lines generation
EGFR cDNA clones, including WT EGFR, L858R EGFR, L858R/T790M EGFR, and Del E746-A750 EGFR, were subcloned into the lentivirus vector by SgfI and MluI. The Lentiviral Packaging Kit (TR30037, Origene) was used for virus package in 293 T cells. The Ba/F3 cells were infected with lentivirus together with 8 μg/mL polybrene for 24 h. Two days later, 2 μg/mL puromycin was added to the cell culture medium and maintained for another 7 days for stable cell selection.
In vitro EGFR kinase assay
The recombinant active WT EGFR, L858R EGFR, L858R/T790M EGFR, and Del E746-A750 EGFR were purchased from Millipore. The in vitro EGFR kinase assay was performed as described previously [
22]. Briefly, active EGFR (100 ng) was mixed with various doses of Formo or 100 nM osimertinib. The reaction was incubated with 500 μM angiotensin II for 5 min at room temperature, followed by incubation with 10 μL of ATP mixture (25 mM MgAc and 0.25 μM ATP containing 10 μCi [γ-32P] ATP) for 15 min at 30 °C and then 25 μL of the reaction mixture was transferred onto P81 papers. The papers were washed with 0.75% phosphoric acid twice and then with acetone once. The radioactive incorporation was determined using a scintillation counter.
In vitro pulldown and ATP competition assays
The in vitro pulldown and ATP competition assays were performed as described previously [
23]. Formo-Sepharose 4B beads were prepared following the protocol provided by GE Healthcare Biosciences. NSCLC cell lysate (500 μg) or an active kinase with different concentrations of ATP was incubated with Formo-Sepharose 4B beads or Sepharose 4B beads only in reaction buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM DTT, 0.01% Nonidet P-40, 0.02 mM phenylmethylsulfonyl fluoride, 1 × protease inhibitor mixture and 2 μg/mL bovine serum albumin) at 4 °C with gentle rocking overnight, followed by washing with washing buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM DTT, and 0.01% Nonidet P-40, and 0.02 mM phenylmethylsulfonyl fluoride) 5 times. Protein binding was analyzed by IB.
Molecular Modeling
Homology modeling
The three-dimensional structure of EGFR with exon 19 deletion mutant (residues E746–A750) was modeled based on the wild type (WT) crystal structure of EGFR using Modeller [
24]. Through extensive analysis of the deposited structures in Protein Data Bank (PDB) [
25], the crystal structure of EGFR (PDB: 4JR3) was used as the template for homology modeling. Ten models were generated and evaluated with the Discrete Optimized Protein Energy (DOPE) score implemented in Modeller. Finally, the best model was adapted for the subsequent docking studies.
Molecular docking
The structures of WT EGFR (PDB: 4JR3), L858R EGFR (PDB: 2ITV), L858R/T790M EGFR (PDB: 3W2P), and exon 19 deletion EGFR were prepared, including filling in missing side chains, adding hydrogens and minimizing heavy atoms with default parameters using Protein Preparation Wizard in Schrödinger Suite 2013. Then the structure file of the ligand, Formo, was well pretreated in LigPrep, and docking was performed based on the standard precision mode of Glide with default settings. Docking poses for each receptor-ligand complex were then analyzed for binding modes, and final figures were generated using PyMOL. Hydrogen bond lengths were defined as the distance between the donor and acceptor atom centers.
Refine and MM-GBSA
Prime was employed to further refine the binding pose and calculate binding free energy (i.e. ΔG) by the MM-GBSA method using the Prime MM-GBSA module in Schrödinger Suite 2013. Residues with distances from the ligand within 12.0 Å were set as flexible. Other settings were kept in default.
Ubiquitination assay
The ubiquitination assay was performed as described previously [
26]. Cells were lysed with modified RIPA buffer containing 1% SDS (20 mM NAP, pH 7.4, 150 mM NaCl, 1% Triton, and 0.5% Sodium-deoxycholate), protease inhibitors, and 10 mM N-Ethylmaleimide (NEM). The lysates were sonicated for 30 s and boiled at 95 °C for 15 min, then diluted with 0.1% SDS containing RIPA buffer and centrifuged at 16000×g for 15 min. The supernatant was transferred to a new tube and incubated with Mcl-1 antibody plus protein A-Sepharose beads overnight at 4 °C. Beads were washed and subjected to IB analysis.
In vivo tumor growth
All mice experiments were performed according to strict guidelines established by the Medical Research Animal Ethics Committee, Central South University, China. NSCLC cells, including HCC827 cells (2 × 106), H1975 (1 × 106), A549 (2 × 106) and H3255 (2 × 106) were counted and suspended in 100 μL RPMI-1640 medium and inoculated s.c. into the right flank of 6-week-old female athymic nude mice (6 mice per group). Formo (10 mg/kg) or vehicle control was administrated daily by i.p. Injection when the tumor volume reached 100 mm3, whereas osimertinib (2 mg/kg) was initiated and repeated daily by oral gavage. Mouse body weight was recorded, and tumor volume was determined by caliper. Tumor volume was calculated following the formula of A × B2 × 0.5, wherein A is the longest diameter of the tumor, B is the shortest diameter, and B2 is B squared.
Immunohistochemical (IHC) staining
IHC staining was performed as described previously [
27]. Briefly, tissue sections from xenograft tumor tissues were deparaffinized and rehydrated. The slide was unmasked by submersion into boiling sodium citrate buffer (10 mM, pH 6.0) for 10 min, followed by treating with 3% H
2O
2 for 10 min at room temperature. The tissue slide was blocked with 50% goat serum albumin in a humidified chamber at room temperature for 1 h, then incubated with the primary antibody at 4 °C overnight. After hybridized with the second antibody for 45 min at room temperature, the slide was incubated with DAB substrate for target protein visualization. Hematoxylin was used for counterstaining.
Blood analysis
Mouse whole blood (200 μL) was collected in EDTA-coated tubes via cardiac puncture. The count for red blood cells (RBC) and white blood cells (WBC), hemoglobin (Hb), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) were analyzed at the Laboratory of the Third Xiangya Hospital (Changsha, China).
Statistical analysis
Statistical analyses were performed using SPSS (version 16.0 for Windows, SPSS Inc., Chicago, IL, USA) and GraphPad Prism 5 (GraphPad 5.0, San Diego, CA, USA). The quantitative data were expressed as means ± SD. Significant differences were determined by the Student t-test or ANOVA. A probability value of less than 0.05 was used as the criterion for statistical significance.
Discussion
EGFR activating mutations serve as driver mutations and play a crucial role in the tumorigenesis of NSCLC. Therapeutic strategies using TKI to inhibit EGFR signaling have become an effective approach for these patients [
1,
29]. However, most patients relapsed on TKIs due to acquired resistance. It is generally believed that hyperactivation of Akt and ERK1/2 signaling are the most common mechanisms of resistance to multiple targeted therapeutics [
30]. Thus, identification of novel small molecule inhibitors to overcome primary or acquired TKI resistance is still an urgent need in NSCLC treatment. In this study, with natural compound screening, we identified Formo acted as a potential EGFR inhibitor to suppress NSCLC cells. Our data indicated that Formo docked into the EGFR ATP-binding pocket and decreased EGFR kinase activity. The in vitro and in vivo data further confirmed this inhibitory effect. Moreover, Formo reduced Mcl-1 protein level in an Akt kinase-dependent manner and promoted mitochondrial apoptosis. Our data extend the understanding of the Formo-mediated anti-tumor mechanism and suggest that suppression of both EGFR signaling and downstream oncoprotein is an alternative strategy to enhance the efficacy of the anti-tumor agent.
As the unique pro-survival Bcl-2 family member, Mcl-1 is required for maintaining cell growth and survival of NSCLC, prostate, colorectal, liver, and gastric cancer cells [
10,
31]. Mcl-1 is a short half-life protein, and the expression of Mcl-1 is regulated at multiple levels, including transcription, translation, and post-translation [
32]. Mcl-1 inhibits mitochondrial outer membrane permeabilization (MOMP) and suppresses cytochrome c release from mitochondria to antagonist apoptosis. Thus, overexpression of Mcl-1 was frequently observed in therapeutic resistant tumors. NF-κB dependent upregulation of Mcl-1 confers chemo/radiotherapy resistance in NSCLC and esophageal carcinoma [
33]. The reduction of Mcl-1 protein by GSK3β- β-TrCP axis-mediated Mcl-1 ubiquitination and degradation enhances tumor-killing efficacy of chemotherapy agents [
34]. Furthermore, degradation of Mcl-1 in colon cancer is required for targeted therapeutics induced tumor suppression [
35]. Recent studies revealed that the decrease of Mcl-1 plays a crucial role in TKI or other targets therapy-mediated tumor suppression. For example, TKI promotes Mcl-1 degradation and, in combination with Bcl-XL/Bcl2 inhibitors, induced apoptosis in cancer cells [
36]. Inhibition of EGFR by TKI, erlotinib, disrupts the interaction between Bim and Mcl-1 and sensitizes tumor cells to ABT-737 treatment [
37]. Degradation of Mcl-1 overcome acquired resistance to osimertinib in EGFR-mutant lung cancer [
38,
39]. These reports indicate that target Mcl-1 is a promising anti-tumor strategy for clinical treatment. However, there is no Mcl-1 inhibitors have been approved currently, especially the small compound, which can directly reduce the Mcl-1 protein level by ubiquitination-mediated degradation [
40]. Our results revealed that Formo is a well tolerate compound that decreased Mcl-1 expression through ubiquitination-dependent degradation. Formo enhances the interaction between Mcl-1 and E3 ligase, therefore shortens Mcl-1 half-life and promotes protein destruction. These findings offer an alternative strategy to degrade the non-enzymatic activity oncoproteins through ubiquitination.
Currently, four different E3 ligases have been identified as Mcl-1 negative regulators to attenuate Mcl-1 stability, including β-Trcp [
34], Trim17 [
41], Mule [
42], and FBW7 [
43]. In contrast, multiple deubiquitinase, such as DUB3 [
44], USP9X [
45], and USP13 [
46], were required for reversing Mcl-1 ubiquitination. In this study, we found that Formo-induced Mcl-1 degradation in an FBW7-dependent manner, depletion of FBW7 compromised Formo-promoted Mcl-1 ubiquitination. Importantly, we demonstrated that Formo suppresses EGFR-Akt signaling, which in turn activates GSK3β and eventually enhanced GSK3β-mediated Mcl-1 phosphorylation and FBW7-mediated degradation. Indeed, depletion of FBW7 causes the upregulation of Mcl-1 and confers TKI resistance in PC9 cells [
47]. Our data are consistent with the previous reports and indicate that the decrease of Mcl-1 is a promising strategy to overcome TKI resistance in NSCLC treatment.
Formo is a flavonoid which originates mainly from red clovers and the Chinese herb
Astragalus membranaceus. Accumulating evidence suggests that Formo was a multiple kinase inhibitor. A panel of kinases was identified as the protein targets of Formo, including MAPK (ERK1/2, P38), PI3K/Akt, VEGFR, FGFR, and mTOR [
14,
15]. Our data showed that Formo suppressed EGFR signaling and decreased the protein level of Mcl-1. However, we cannot exclude the possibility that other oncogenic signalings are involved in Formo-mediated anti-tumor activity. Currently, numerous preclinical investigations have demonstrated that Formo exhibits chemopreventive and therapeutic potentials in multiple cancer models. The combination with Formo, significantly enhanced the anti-tumor effect of chemotherapeutic agents in a wide range of human cancers [
14,
15]. Our data showed that Formo reduced the in vivo tumor growth in both EGFR WT and mutant xenograft tumors. Importantly, Formo is well-tolerated in vivo and exhibited no significant toxicity to vital organs, indicating that Formo is a potential anti-tumor candidate compound for EGFR WT and activating mutation NSCLC treatment.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.