Activation of EGFR-DNA-PKcs pathway by IGFBP2 protects esophageal adenocarcinoma cells from acidic bile salts-induced DNA damage
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The incidence of esophageal adenocarcinoma (EAC) is rising rapidly in the US and Western countries. The development of Barrett’s esophagus (BE) and its progression to EAC have been linked to chronic gastroesophageal reflux disease (GERD). Exposure of BE and EAC cells to acidic bile salts (ABS) in GERD conditions induces high levels of oxidative stress and DNA damage. In this study, we investigated the role of insulin-like growth factor binding protein 2 (IGFBP2) in regulating ABS-induced DNA double-strand breaks.
Real-time RT-PCR, western blot, immunohistochemistry, immunofluorescence, co-immunoprecipitation, flow cytometry, and cycloheximide (CHX) chase assays were used in this study. To mimic GERD conditions, a cocktail of acidic bile salts (pH 4) was used in 2D and 3D organotypic culture models. Overexpression and knockdown of IGFBP2 in EAC cells were established to examine the functional and mechanistic roles of IGFBP2 in ABS-induced DNA damage.
Our results demonstrated high levels of IGFBP2 mRNA and protein in EAC cell lines as compared to precancerous Barrett’s cell lines, and IGFBP2 is frequently overexpressed in EACs (31/57). Treatment of EAC cells with ABS, to mimic GERD conditions, induced high levels of IGFBP2 expression. Knocking down endogenous IGFBP2 in FLO1 cells (with constitutive high levels of IGFBP2) led to a significant increase in DNA double-strand breaks and apoptosis, following transient exposure to ABS. On the other hand, overexpression of exogenous IGFBP2 in OE33 cells (with low endogenous levels of IGFBP2) had a protective effect against ABS-induced double-strand breaks and apoptosis. We found that IGFBP2 is required for ABS-induced nuclear accumulation and phosphorylation of EGFR and DNA-PKcs, which are necessary for DNA damage repair activity. Using co-immunoprecipitation assay, we detected co-localization of IGFBP2 with EGFR and DNA-PKcs, following acidic bile salts treatment. We further demonstrated, using cycloheximide chase assay, that IGFBP2 promotes EGFR protein stability in response to ABS exposure.
IGFBP2 protects EAC cells against ABS-induced DNA damage and apoptosis through stabilization and activation of EGFR - DNA-PKcs signaling axis.
KeywordsIGFBP2 EGFR DNA-PKcs DNA damage Acidic bile salts Esophageal adenocarcinoma
acidic bile salts
analysis of variance
DNA-dependent protein kinase, catalytic subunit
epidermal growth factor receptor
gastroesophageal reflux disease
insulin-like growth factor binding protein 2
non-homologous end joining
quantitative real time reverse transcription-polymerase chain reaction
reactive oxygen species
Over the past few decades, the incidence of esophageal adenocarcinoma (EAC) has increased rapidly in the United States and Western countries [1, 2]. Abnormal exposure of esophageal cells to a mixture of acid and bile salts in patients with chronic gastroesophageal reflux disease (GERD) is a major risk factor for the development of pre-malignant Barrett’s esophagus (BE) and its progression to EAC [3, 4]. Previous studies have shown that exposure to acidic bile salts (ABS) induces DNA damage in BE and EAC cells [5, 6, 7]. Accumulation of unrepaired DNA damage in cells can lead to massive genomic instability that can mediate cell death . To maintain DNA damage at tolerable sublethal levels, cancer cells must acquire adaptive pro-survival protective mechanisms.
DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is an enzyme encoded by PRKDC in humans . It contributes to the repair of DNA double-strand breaks (DSBs) by accessing broken ends of DNA in combination with the other two DNA-binding factors, Ku70 and Ku80 . This complex serves as a molecular scaffold for recruiting DNA repair factors to DNA strand breaks, such as XRCC4 and DNA ligase IV . The kinase activity of DNA-PKcs is required for the non-homologous end joining (NHEJ) pathway of DNA repair, which rejoin double-strand breaks [12, 13, 14]. Phosphorylation at Thr2609 of DNA-PKcs plays a key role in NHEJ [15, 16]. Earlier reports have shown that epidermal growth factor receptor (EGFR) plays an important role in the regulation of DNA-PKcs activity in response to radiation or anti-cancer drugs that induce DNA damage [17, 18]. In addition, EGFR nuclear localization is required for modulation of the repair of cisplatin and ionizing radiation-induced DNA damage [17, 18, 19].
Insulin-like growth factor binding protein 2 (IGFBP2) is a member of the IGFBP’s family which shares cysteine-rich amino- and carboxyterminal domains for the IGF-binding site . High levels of IGFBP2 have been detected in patients’ sera of some cancers with poor prognostic outcome [21, 22]. In addition to its functions as a secretory protein, IGFBP2 intracellular oncogenic functions promote cancer cell proliferation, invasion, metastasis and drug resistance [23, 24, 25]. Interestingly, IGFBP2 protein has a nuclear localization signal sequence, which is required for its nuclear translocation to regulate cell functions or signaling pathways [26, 27]. IGFBP2 overexpression in EAC is associated with drug resistance . However, its signaling and functional role in response to bile salts-induced DNA damage in esophageal tumorigenesis have not been studied.
In the present study, we investigated the expression, signaling and functional roles of IGFPB2 in response to bile salts-induced DNA damage in esophageal tumorigenesis. We detected nuclear localization and associations of IGFPB2 with EGFR-DNA-PKcs in response to acidic bile salts, a mechanism that promotes DNA damage repair and esophageal cell survival in response to the toxic effects of acidic bile salts.
Methods and materials
Three immortalized cell lines originated from Barrett’s esophagus, BAR10T (kindly provided by Dr. Rhonda Souza), CPA and CPB (ATCC, Manassas, VA, USA), were cultured with DMEM/F12 (GIBCO, New York, NY, USA), supplemented with 5% fetal bovine serum (FBS, GIBCO), 1% penicillin/streptomycin (GIBCO), 0.4 μg/ml hydrocortisone (Sigma-Aldrich, Saint Louis, MO, USA), 20 mg/L adenine hydrochloride hydrate (Sigma-Aldrich), 140 μg/ml bovine pituitary extract (Thermo Fisher Scientific, Waltham, MA, USA), Insulin-transferrin-sodium selenite media supplement (Sigma-Aldrich) and 20 ng/ml recombinant epidermal growth factor (Sigma-Aldrich). EAC cell lines, FLO1 (ATCC), OE19 (Sigma-Aldrich), SK-GT4 (kindly provided by Dr. Xiaochun Xu at MD Anderson), ESO26 (Sigma-Aldrich) and OE33 (kindly provided by Dr. David Beer) were cultured in DMEM or RPMI 1640 medium (GIBCO), supplemented with 10% FBS and 1% penicillin/streptomycin. All cell lines were grown at 37 °C in 5% CO2. Cell lines were authenticated by Genetica DNA Laboratories using short tandem repeat profiling (Genetica DNA Laboratories, Burlington, NC, USA). Cells used were from the stocks immediately after authentication and cultured less than 6 months. Mycoplasma was tested periodically using the qRT-PCR method (Southernbiotech, Birmingham, AL, USA).
A bile salts cocktail, consisting of an equal molar mixture of sodium salts of glycocholic acid, taurocholic acid, glycodeoxycholic acid, deoxycholic acid (CALBIOCHEM, La Jolla, CA, USA), and glycochenodeoxycholic acid (Sigma-Aldrich), was prepared to reflect the mixture of bile acids in the distal esophagus during GERD, as previously reported [29, 30]. In all experiments, 200 μM final concentration of the bile salts cocktail (40 μM of each of the above bile acids) in pH 4 medium was used.
Organotypic 3D culture
Organotypic 3D reconstruction cultures were performed as previously described [31, 32]. Briefly, human esophageal fibroblasts (ScienCell, Carlsbad, CA, USA) were seeded into a 3D matrix (75,000 cells/well) containing collagen I (Corning, Tewksbury, MA, USA) and Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) and were incubated for 7 days at 37 °C. Following incubation, FLO1 and OE33 cells were seeded (500,000 cells/well) on top of esophageal fibroblast matrix. Cultures were incubated for an additional 14 days. Cells were then treated with ABS (pH 4, 200 μM) for 30 min then recovered for 1 h. Cultures were harvested, fixed in 4% paraformaldehyde for 1 h, and then transferred to 70% ethanol until pathological process. Sectioning of paraffin-embedded blocks and H&E staining were processed in the Pathology Research Resource at the University of Miami.
Plasmids and siRNA transfection
The IGFBP2 expression plasmid was designed and obtained from Vectorbuilder (https://en.vectorbuilder.com/). IGFBP2 siRNAs were purchased from Santa Cruz Biotechnology (sc-37,195; Dallas, TX, USA) and Dharmacon (SMARTpool; Lafayette, CO, USA). The siRNA target sequences are provided in Additional file 1: Table S1. FLO1 and OE33 cells were seeded at 2 × 105 cells/well in 6-well plates 24 h before transfection. 40 nM siRNA or 1.5 μg plasmid/well were transfected using the Lipofectamine 3000 Transfection kit (ThermoFisher).
Quantitative real time RT-PCR
Total RNA was isolated using the Direct-zol RNA MiniPrep Plus kit (ZYMO Research, Irvine, CA, USA). Single-stranded complementary DNA was synthesized using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA). Quantitative real time RT-PCR (qRT-PCR) was performed using the CFX Connect real-time system (Bio-Rad) with the threshold cycle number determined by CFX manager 3.0 software. The primer sequences for IGFBP2 are GCGAGGGCACTTGTGAGA (forward) and CCAACATGTTCATGGTGCTG (reverse). The primer sequences for EGFR are CCAGTATTGATCGGGAGAGC (forward) and TGCCTTGGCAAACTTTCTTT (reverse). Reactions were performed in triplicate. The relative mRNA expression fold was normalized to the average value of the reference gene, HPRT1, of the same sample as previously described [33, 34].
Western blotting analysis
Whole cell lysates were extracted using RIPA Lysis Buffer with proteinase and phosphatase inhibitors (Santa Cruz Biotechnology). Extraction of cytoplasmic and nuclear proteins was performed using the Cell Fractionation Kit (Cell Signaling, Danvers, MA, USA). Protein concentrations of lysates were measured by the Pierce™ BCA Protein Assay (ThermoFisher Scientific) using a FLUO Star OPTIMA microplate reader (BMG, Cary, NC). Equal amounts of proteins were loaded onto gradient 8–10% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). Western blotting was performed following standard procedure. The information of antibodies is provided in Additional file 1: Table S2. The ChemiDoc™ XRS+ Imaging System (Bio-Rad) was used to capture western blot signals. Bands’ intensity was measured using Image lab software (Bio-Rad). The quantification of the proteins was normalized to the intensity of β-actin of the same sample.
Approximately 1 × 104 cells were seeded in 8-chamber Slides (Corning) 24 h after siRNA or plasmid transfection. Cells were treated with ABS (pH 4, 200 μM) for 20 min and recovered for designated time points. After the treatment, cells were fixed with fresh-made 4% formaldehyde for 45 min. Then immunofluorescence staining was performed as previously described . Antibodies for IGFBP2 (Cell signaling), EGFR (Invitrogen) and Phospho-DNA-PKcs (Thr2609) (Invitrogen) were incubated at 4 °C for overnight. Alexa fluor-568 conjugated anti-rabbit and Alexa fluor-488 conjugated anti-mouse secondary antibody (Invitrogen) were incubated for 1 h at room temperature. Cells were mounted with DAPI solution. Images were captured using the All-in-one Fluorescence Microscope (BZ-X700) (Keyence, Itasca, IL, USA).
Flow cytometry analysis of Annexin V
To quantitate ABS-induced apoptosis, flow cytometry analysis of Annexin V were performed using a FITC Annexin V apoptosis detection kit (BD Biosciences, San Jose, CA). EAC Cells were transfected with 40 nM IGFBP2 SMARTpool siRNA or control siRNA (Dharmacon, Lafayette, CO) using LipoJet transfection reagent (SignaGen Laboratories, Rockville, MD). 48 h post transfection, cells were treated with ABS for 30 min then recovered in regular medium for 3 h. Cells then were collected for FITC Annexin V and PI staining according to the manufacturer’s instruction and subjected to flow cytometry analysis at Flow Cytometry Shared Resource at Sylvester Comprehensive Cancer Center.
Protein G magnetic beads (Millipore, Billerica, MA, USA) were used to capture the primary antibodies for IGFBP2, EGFR, and DNA-PKcs according to the supplier’s instruction. IgG from rabbit or mouse sources (Santa Cruz Biotechnology) were used as the negative control. The same amount of protein from each group was incubated with antibody-bound beads at 4 °C overnight with continuous mixing. The beads were washed and re-suspended, on the second day, using NuPAGE™ LDS Sample Buffer (ThermoFisher) suitable for electrophoresis. Denature was performed at 90 °C for 10 min. Western blotting analysis was performed following standard protocols.
Cycloheximide (CHX) chase assay
Cycloheximide (CHX) is a protein synthesis inhibitor in eukaryotes, which is used in cell biology to determine the half-life of a given protein [36, 37]. FLO1 cells with IGFBP2 knockdown and control cells were treated with or without ABS for 20 min and then recovered in full medium with 50 μg/ml CHX. Whole cell lysates were collected and analyzed using anti-EGFR antibody by Western blotting. The bands’ intensities of EGFR and β-actin were measured with Bio-Rad Image lab software. The ratio of EGFR/β-actin was obtained for each sample at different time points.
Tissue microarrays containing 60 de-identified archival cases of EACs as well as the normal esophagus, and 3 non-dysplastic BE was obtained from Tissue Pathology Core at Vanderbilt University Medical Center, Nashville, TN. Immunohistochemical staining was performed using The IHC Select® Immunoperoxidase Secondary Detection system (MilliporeSigma, Burlington, MA, USA) following manufactory’s instruction. The sections were incubated with the IGFBP2 primary antibody (Cell Signaling) overnight. Immunohistochemical results were evaluated for intensity and frequency of the staining and an index score was applied as previously described .
Data are expressed as the mean ± SD for parametric data. One-way ANOVA and Newman-Keuls test were used to compare multiple groups. Unpaired Student t-test was performed for two independent variables. All statistical analyses were done using GraphPad Prism 5 software. For all analyses, P < 0.05 is considered statistically significant.
IGFBP2 is overexpressed and induced by acidic bile salts in EACs
To mimic the physiological and pathological conditions of GERD, we cultured EAC cells together with esophageal fibroblasts in a 3D organotypic culture model. We exposed 3D cultured EAC cells to ABS, and examined IGFBP2 protein expression levels using immunofluorescence assay. Data shows that IGFBP2 levels were clearly enhanced by ABS in both FLO1 (Fig. 2e) and OE33 cells (Fig. 2f) cells. The HE staining images of the organotypic cultures for FLO1 and OE33 cells are provided in Additional file 2: Figure S4.
IGFBP2 protects EAC cells from acidic bile salts-induced DNA double-strand breaks and apoptosis
IGFBP2 is required for acidic bile salts-induced activation of EGFR-DNA-PKcs pathway in EAC cells
IGFBP2 is required for nuclear accumulation and activation of EGFR and DNA-PKcs in EAC cells in response to ABS
Earlier studies have suggested that EGFR phosphorylation at Tyr1068 is required for its nuclear translocation in response to cisplatin-induced DNA damage [19, 43]. We, therefore, used tyrosine kinase inhibitor, gefitinib, to examine whether phosphorylation of EGFR is required for its nuclear translocation in response to ABS. As shown in Fig. 5d, treatment with gefitinib attenuated EGFR nuclear accumulation in response to ABS. As expected, less phospho-DNA-PKcs in the nucleus was also observed in gefitinib-treated cells. To confirm that the DNA damage repair function of IGFBP2 is mediated by EGFR, we performed rescue experiments by overexpressing IGFPB2 in OE33 cells, followed by treatment with ABS and gefitinib (Fig. 5e). Overexpression of IGFBP2 enhanced p-EGFR and inhibited ABS-induced cleaved PARP and p-H2AX. However, administration of gefitinib abrogated the protective function of IGFBP2 and impaired ABS-induced phosphorylation of EGFR with a notable increase in the ABS-induced cleaved PARP and p-H2AX levels.
IGFBP2 forms a complex with EGFR and DNA-PKcs in EAC cells in response to ABS
IGFBP2 is required for stabilization of EGFR after ABS treatment in EAC cells
Chronic gastroesophageal reflux disease, where acidic bile salts abnormally reflux into the lower esophagus, is a major risk factor for the development of Barrett’s esophagus and EAC [4, 44]. Acidic bile salts induce high levels of oxidative stress that damage cellular components including proteins, lipids, and DNA . This cellular stress environment persists along the stages of BE and its progression to EAC [5, 6, 46]. Therefore, EAC cells must develop adaptation mechanisms to overcome this toxic environment, promote cell survival, and maintain DNA damage below lethal levels. In this study, we discovered that IGFBP2 is expressed at high levels in EAC tissues and cell lines. We also found that IGFPB2 is further induced in both mRNA and protein levels in EAC cells following exposure to ABS. Our findings demonstrate that IGFBP2, under ABS-induced stress condition, promotes stability and nuclear translocation of EGFR and co-exists with EFGR-DNA-PKcs protein complex, essential for DNA repair under stress condition.
IGFBP2 was originally discovered in serum for its binding to IGF1/2, ligands for IGF1R, to maintain activation of the IGF1R signaling pathway [47, 48]. Although IGFBP2 is a secretory protein, IGFBP2 has a nuclear translocation sequence, necessary for its import into the nucleus . Overexpression of intracellular IGFBP2 has been described in several human cancers where it is associated with cellular proliferation, progression and drug-resistance [25, 26, 27]. In this study, we detected high levels of IGFPB2 in EAC tissues and in vitro cell models, suggesting a possible role in Barrett’s esophageal tumorigenesis. We focused our studies on the mechanistic relationship between IGFBP2 and ABS, a major etiological risk factor that underlies the biology of EAC. Because acidic bile salts induce high levels of ROS and DNA damage [6, 7], EAC cells must evolve to endure this harsh environment for survival. Our data indicate that IGFBP2 protects against ABS-induced double strand breaks and may thus favor tumor cell survival under reflux conditions.
In response to DNA damage caused by anti-cancer drugs or radiation, DNA-PKcs is phosphorylated at Thr2609, an essential step for its DNA repair capacity [15, 16]. DNA-PKcs recruits other DNA damage repair proteins such as Ku70 and Ku80, XLF, XRCC4 and LigIV to repair damaged DNA . We found that phosphorylation of DNA-PKcs at Thr2609, following ABS, was regulated by levels of IGFBP2. Earlier studies suggested that EGFR binds to DNA-PKcs and induces its phosphorylation [19, 42]. In agreement with this note, our data suggest that IGFBP2 is required for stabilization and nuclear translocation of EGFR, and is required for formation of the EGFR-DNA-PKcs nuclear complex. Of note, we found that the interaction between IGFPB2 and the EGFR-DNA-PKcs complex is markedly enhanced, in response to ABS-induced stress. However, the precise mechanism by which IGFBP2 stabilizes EGFR under ABS stress conditions warrants further investigations.
We postulated that induction of IGFBP2 in response to ABS could be due to regulation of miRNAs. An earlier study has shown that MiR-126 can target IGFBP2 for degradation . Our studies demonstrated that MiR-126 is significantly downregulated in response to ABS, inversely correlating with IGFBP2 levels. This finding provides a plausible mechanism for regulation of IGFBP2 in our system. It is also possible that a similar mechanism may be involved in regulation of EGFR in response to ABS, such as MiR-141 . Of note, downregulation of MiR-141 has been reported in Barrett’s esophagus . However, further studies are needed to validate this mechanism.
IGFBP2 is a secretory protein with high levels in the serum of patients with several types of cancer [21, 22, 51]. High levels of IGFBP2 in patients’ sera predicted poor prognostic outcome [21, 22]. Therefore, the high levels of IGFBP2 in EAC may also have a promising application in diagnosis and / or prognosis in patients with EAC. A comprehensive large size clinical study is needed to clarify this note. In addition, targeting the IGFBP2-EGFR-DNA-PKcs signaling axis may be beneficial in patients with EAC. EGFR inhibitors are used in the treatment of selected cancer patients; especially those with non-small cell lung cancer [52, 53]. A number of experimental DNA-PK inhibitors, such as NU7026, are also under investigation for various cancers [54, 55]. Considering the role of IGFBP2 in forming the complex with EGFR and DNA-PKcs in DNA damage or drug resistance in EAC, the use of EGFR, DNA-PKcs inhibitors or the development of specific IGFBP2 inhibitors will be a promising therapeutic strategy as single agents or in combination with DNA damaging anti-cancer agents.
We have discovered that IGFBP2 is overexpressed in EAC and ABS induces IGFBP2 probably through degradation of MiRNAs targeting IGFBP2. IGFBP2 protects EAC cells from ABS-induced DNA damage and apoptosis through activating nuclear EGFR-DNA-PKcs pathway. IGFBP2 promotes stabilization of EGFR protein levels and interacts with the EGFR-DNA-PKcs complex to promote DNA repair and cancer cell survival (a graphic summary is shown in Fig. 7c).
The authors gratefully acknowledge financial support from the China Scholarship Council. The content of this work is solely the responsibility of the authors and do not necessarily represent the official views of the Department of Veterans Affairs, National Institutes of Health, or the University of Miami.
This study was supported by development funds from Sylvester Comprehensive Cancer Center, and grants from the U.S. National Institutes of Health (R01CA206563 and R01CA224366) and the U.S. Department of Veterans affairs (1IK6BX003787 and I01BX001179).
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
ZZ designed methodology, performed research, analyzed data and wrote the manuscript; HL, SZ, ZC, AG and JY contributed new reagents or analytic tools and results interpretation; KW performed histology and tissue microarray analysis; WER, CD and DP designed research, interpretated results, supervised research and manuscript writing.
Ethics approval and consent to participate
All de-identified tissue samples were obtained from the archives of pathology at Vanderbilt University (Nashville, Tennessee, USA). The use of specimens from the tissue repository was approved by the Vanderbilt institutional review board.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 21.Russell MR, Graham C, D'Amato A, Gentry-Maharaj A, Ryan A, Kalsi JK, et al. A combined biomarker panel shows improved sensitivity for the early detection of ovarian cancer allowing the identification of the most aggressive type II tumours. Br J Cancer. 2017;117(5):666–74.PubMedPubMedCentralCrossRefGoogle Scholar
- 36.Kao SH, Wang WL, Chen CY, Chang YL, Wu YY, Wang YT, et al. Analysis of protein stability by the Cycloheximide chase assay. Bio Protoc. 2015;5(1).Google Scholar
- 42.Javvadi P, Makino H, Das AK, Lin YF, Chen DJ, Chen BP, et al. Threonine 2609 phosphorylation of the DNA-dependent protein kinase is a critical prerequisite for epidermal growth factor receptor-mediated radiation resistance. Mol Cancer Res. 2012;10(10):1359–68.PubMedPubMedCentralCrossRefGoogle Scholar
- 52.Rosell R, Carcereny E, Gervais R, Vergnenegre A, Massuti B, Felip E, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13(3):239–46.PubMedCrossRefGoogle Scholar
- 53.Zhou C, Wu YL, Chen G, Feng J, Liu XQ, Wang C, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol. 2011;12(8):735–42.PubMedCrossRefGoogle Scholar
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