HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer

Han, Shuyan; Tian, Zhihua; Tian, Huifang; Han, Haibo; Zhao, Jun; Jiao, Yanna; Wang, Chunli; Hao, Huifeng; Wang, Shan; Fu, Jialei; Xue, Dong; Sun, Hong; Li, ****

doi:10.1038/s41420-023-01476-0

HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer

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Published: 10 June 2023

Volume 9, article number 181, (2023)
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Cell Death Discovery

HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer

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Shuyan Han ORCID: orcid.org/0000-0001-6544-8090¹^na1,
Zhihua Tian²^na1,
Huifang Tian²^na1,
Haibo Han³,
Jun Zhao⁴,
Yanna Jiao¹,
Chunli Wang⁵,
Huifeng Hao ORCID: orcid.org/0000-0003-3396-1759¹,
Shan Wang¹,
Jialei Fu¹,
Dong Xue¹,
Hong Sun ORCID: orcid.org/0000-0002-0445-8954¹ &
…
**** Li ORCID: orcid.org/0009-0006-0955-3164¹

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Abstract

Hepatoma-derived growth factor (HDGF) expression is associated with poor prognosis in non-small cell lung cancer (NSCLC); however, whether HDGF affects gefitinib resistance in NSCLC remains unknown. This study aimed to explore the role of HDGF in gefitinib resistance in NSCLC and to discover the underlying mechanisms. Stable HDGF knockout or overexpression cell lines were generated to perform experiments in vitro and in vivo. HDGF concentrations were determined using an ELISA kit. HDGF overexpression exacerbated the malignant phenotype of NSCLC cells, while HDGF knockdown exerted the opposite effects. Furthermore, PC-9 cells, which were initially gefitinib-sensitive, became resistant to gefitinib treatment after HDGF overexpression, whereas HDGF knockdown enhanced gefitinib sensitivity in H1975 cells, which were initially gefitinib-resistant. Higher levels of HDGF in plasma or tumor tissue also indicated gefitinib resistance. The effects of HDGF on promoting the gefitinib resistance were largely attenuated by MK2206 (Akt inhibitor) or U0126 (ERK inhibitor). Mechanistically, gefitinib treatment provoked HDGF expression and activated the Akt and ERK pathways, which were independent of EGFR phosphorylation. In summary, HDGF contributes to gefitinib resistance by activating the Akt and ERK signaling pathways. The higher HDGF levels may predict poor efficacy for TKI treatment, thus it has the potential to serve as a new target for overcoming tyrosine kinase inhibitor resistance in combating NSCLC.

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Introduction

Lung cancer is the most common cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of all lung carcinoma cases. Patients with epidermal growth factor receptor (EGFR) mutations respond well to tyrosine kinase inhibitors (TKIs), but all responding patients eventually develop acquired resistance to TKIs [1]. The underlying mechanisms of TKI resistance include secondary resistance mutations of targeted genes, activation of bypass signaling pathways, dysregulation of downstream effectors, and phenotypic transformation [2]. T790M mutation in exon 20 of EGFR accounts for approximately 50% of resistant cases and is the predominant resistance mechanism of TKIs. However, approximately 19% of resistance mechanisms remain unknown [3].

Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor that was first identified from the conditioned medium of the Huh-7 hepatoma cell line [4], indicating that it can be secreted. HDGF has been reported to be involved in cancer cell growth, angiogenesis, anti-apoptosis, and tumor metastasis [4, 5]. In a range of tumor types, including NSCLC, ovarian cancer, oral cancer, gastric cancer and melanoma, HDGF is overexpressed, and its higher expression is strongly correlated with poor prognosis [6,7,8,9,10,11]. HDGF enhances vascular endothelial growth factor (VEGF)-dependent angiogenesis in NSCLC [12], and high serum HDGF levels predict bone metastasis and unfavorable prognosis in NSCLC [13]. In surgically resected stage I NSCLC, HDGF is regarded as a biomarker for molecular staging and prognosis and provides prediction for postoperative adjuvant chemotherapy [14]. Inhibition of HDGF suppressed tumor cell growth both in vitro and in vivo [15, 16]. Antibodies targeting HDGF could prevent NSCLC relapse after chemotherapy by suppressing cancer stem cells [17, 18]. Some microRNAs (miRNAs) inhibit NSCLC proliferation and invasion by targeting HDGF [19,20,Full size image

We then asked whether HDGF and EGFR have complementary roles in responding to gefitinib in NSCLC cells with HDGF loss and gain. As shown in Fig. 6G, I, HDGF knockdown caused a decrease in HDGF expression, and p-EGFR upregulation was reversed by gefitinib treatment in H1975 cells. In H292 and PC-9 cells, HDGF overexpression resulted in HDGF elevation and p-EGFR suppression, and gefitinib treatment further inhibited p-EGFR but without an obvious influence on HDGF expression. As shown in Fig. 6H, J, HDGF was increased by gefitinib administration in PC-9 xenograft tumors with HDGF overexpression (P < 0.05), and other changes in HDGF and p-EGFR in tumor tissues were consistent with the cell experiments. Therefore, the above data indicate crosstalk between HDGF and EGFR in NSCLC, and HDGF may serve as a bypass signaling molecule of EGFR to activate the downstream molecules Akt and ERK. Furthermore, HDGF and EGFR have complementary roles in maintaining the survival of tumor cells exposed to gefitinib. The function of HDGF in gefitinib resistance is illustrated by a schematic diagram in Fig. 7.

Discussion

In the present study, we found that higher HDGF levels are not only associated with the malignant phenotype of NSCLC but can also induce gefitinib resistance. TKI resistance-related signaling, such as the PI3K/Akt and MEK/ERK pathways, could be bypass-activated by HDGF in an EGFR-independent manner. Furthermore, suppression of HDGF increased gefitinib efficacy in resistant NSCLC cells both in vitro and in vivo, suggesting that HDGF is a potential target to overcome gefitinib resistance.

First, our study demonstrated that HDGF promoted NSCLC tumorigenesis and facilitated metastasis. In H292 and PC-9 cells, which have relatively low levels of HDGF expression, forced HDGF expression increased the cells’ proliferation, colony formation, and migration and invasion abilities. Meanwhile, exogenous rhHDGF expression enhanced H292 and PC-9 cell proliferation, confirming the role of HDGF as a growth stimulator. The tumorigenesis function of HDGF was also confirmed in HDGF-overexpressing PC-9 xenograft mice. In contrast, silencing HDGF by CRISPR inhibited the malignant phenotype of H1975 cells and retarded tumor development in vivo. Studies have shown that targeting HDGF by miRNAs or antibodies is effective in inhibiting the growth of NSCLC [16, 19,20,21] or lung squamous cell carcinoma [30]. However, until now, most studies on HDGF have mainly focused on its tumorigenic role, while only limited attention has been given to determining its function in anticancer therapy resistance. As reported, HDGF participated in the development of chemotherapy drug resistance in colorectal cancer [25] and tongue squamous cell carcinoma [26] or promoted radioresistance in breast cancer [27]. Inhibiting HDGF with the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) could increase the NSCLC response to chemotherapy [31].

Gefitinib is an extensively used first-generation EGFR-TKI for NSCLC patients with EGFR-sensitive mutations, but inevitable drug resistance hampers its clinical benefits. The major mechanisms accounting for TKI resistance include alterations in drug targets, such as T790M, which accounts for half of all cases of acquired resistance, while bypass signaling activation comprises 20 to 25% of all cases [32]. However, there are still approximately 15–20% of NSCLC patients in whom no mechanisms for acquired TKI resistance have been identified [29]. Our results revealed that HDGF knockdown could increase gefitinib sensitivity, while HDGF augmentation abrogated gefitinib efficacy to some extent in NSCLC. In detail, knockdown of HDGF inhibited tumor growth in H1975 cells in vitro and in vivo, while overexpression of HDGF retarded the gefitinib response in PC-9 cells. Immunohistochemical staining of Ki-67 and HDGF in mouse tumor tissue sections was consistent with the above results. Therefore, our data indicated that HDGF expression was correlated with gefitinib efficacy in NSCLC. These results were partly supported by a recent study that provides some clues for HDGF-S165 and TKI resistance via site-specific peptide reporters and targeted mass spectrometry at the nanogram level in cells [28].

High serum HDGF levels predicted bone metastasis and unfavorable prognosis in NSCLC [13]. First, we found a parallel correlation between plasma HDGF levels and the antitumor effect of gefitinib in NSCLC xenografts. In a small number of clinical samples, the HDGF levels were also elevated in the peripheral blood of NSCLC patients who took gefitinib and AZD9291 after drug resistance occurred. Meanwhile, HDFG overexpression was associated with gefitinib resistance in clinical samples. Thus, HDGF is involved in gefitinib resistance, and HDGF levels in plasma or tissue samples may predict TKI efficacy to some extent. However, only limited clinical samples were tested in the present study; thus, more clinical specimens are needed to verify these results.

PI3K/Akt and MAPK/ERK signaling are major EGFR downstream pathways leading to cell proliferation. TKIs bind to the tyrosine kinase domain of EGFR and block EGFR-driven signaling downstream to exert their tumor-inhibitory effects. The EGFR T790M mutation interferes with the interaction by enhancing its affinity to ATP to confer gefitinib resistance [33]. In EGFR-independent conditions, dysregulation of other receptor tyrosine kinases or abnormal activation of downstream signals has a compensatory function to abrogate EGFR inhibition and results in TKI resistance [3]. The bypass signaling pathway shares the same downstream pathways with EGFR and converges to trigger the PI3K/Akt and MAPK/ERK signaling axes [30, 34]. A great number of studies have demonstrated aberrant activation of p-Akt and p-ERK when EGFR-TKI resistance occurs [29], but restraining p-Akt and p-ERK expression could recover gefitinib sensitivity in NSCLC [35, 36]. In the present study, HDGF knockdown inhibited Akt and ERK phosphorylation, while HDGF overexpression exerted the opposite effect, suggesting that it regulates p-Akt and p-ERK activation. In gefitinib-sensitive NSCLC cells, gefitinib-restrained p-Akt and p-ERK levels were abrogated by HDGF overexpression; however, in resistant H1975 cells, the activation of Akt and ERK was strongly suppressed by HDGF depletion. Inhibitors of Akt or ERK were used to validate whether HDGF inducing cell growth and gefitinib resistance by these two pathways. These data indicated that HDGF could enhance EGFR downstream p-Akt and p-ERK signaling, which is related to gefitinib resistance.

As both EGFR and HDGF regulate the activation of Akt and ERK, we were not surprised to find that the majority of known pathways of HDGF overlapped with EGFR, including Akt and ERK signaling, by searching GeneCards (https://www.genecards.org). This is consistent with a study in ovarian cancer cells in which extracellular HDGF stimulated the phosphorylation of ERK [37] and promoted tumor development and progression by regulating the PI3K-Akt pathway in bladder cancer cells [38]. Tumor cells usually orchestrate a network of signaling pathways to sustain their survival when facing death caused by anticancer therapy. When the dominant pathway, such as EGFR, is inhibited by a TKI, tumor cells tend to turn on the critical downstream effector through bypass signaling pathways, maintaining continued cell survival and growth [39]. In the present study, we found that HDGF and p-EGFR expression levels were complementary to each other in NSCLC cells with or without HDGF loss and gain. Gefitinib treatment could reduce EGFR phosphorylation but increase HDGF expression, aberrantly activating the PI3K/Akt and MEK/ERK pathways. However, knockdown of HDGF in H1975 cells could reactivate EGFR and decrease HDGF when exposed to gefitinib, suggesting both a delicate balance and complementarity among HDGF and p-EGFR in EGFR-dependent NSCLC cells when exposed to gefitinib. Considering all the relevant information, the increase of HDGF levels may serve as a bypass survival signal to trigger resistance to gefitinib, and drug resistance can be overcome by silencing HDGF.

In summary, the present research found that HDGF not only promoted the NSCLC cell malignant phenotype but also contributed to gefitinib resistance. As a bypass and compensatory signaling pathway, HDGF activates the EGFR downstream PI3K/Akt and MEK/ERK pathways, which are associated with gefitinib resistance. Targeting HDGF could restore gefitinib sensitivity through a delicate balance between HDGF expression and EGFR reactivation as well as their shared downstream molecules. Therefore, HDGF can be regarded as a potential underlying mechanism of gefitinib resistance and is a promising target both against NSCLC and to increase TKI efficacy in NSCLC.

Materials and methods

Cell lines and culture

Human NSCLC cell lines H1975, H157, H460, H292 and PC-9 were purchased from American Type Culture Collection (Manassas, Virginia, USA), while A549 and H1650 cells were obtained from National Infrastructure of Cell Line Resource (Bei**g, China). All cells were maintained in RPMI-1640 with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO₂.

Drugs and reagents

Gefitinib was obtained from AstraZeneca (Cheshire, UK). Antibodies against Akt (9272), ERK1/2 (9102) and p-ERK1/2 (T202/Y204) (9101) were purchased from Cell Signaling Technology (Beverly, MA). HDGF and p-Akt (Ser473) were obtained from Abcam (Cambridge, UK). EGFR and p-EGFR were purchased from ABclonal (Wuhan, China). GAPDH, β-actin, β-tubulin and Ki-67 were purchased from TDYbio (Bei**g, China). Recombinant human HDGF (rhHDGF) was obtained from ProSpec-Tany TechnoGene Ltd. (Israel).

Establishment of stably transfected NSCLC cells

The recombinant HDGF plasmid in the pLenti6/TR lentiviral vector (Invitrogen) and the pcDNA3.1-EGFR T790M mutation plasmid were obtained by molecular cloning. The single-guide RNA (sgRNA) for HDGF was ligated into the lentiCRISPRv2 plasmid. NSCLC cells were transfected with plasmid and selected to generate a stable cell line for further study. The sequences for HDGF sgRNA1, sgRNA2, control sgRNA (sgRNA-Con) and other primers are listed in Tables S1 and S2.

Quantitative RT‒PCR (qRT‒PCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). qRT‒PCR was performed as previously described [40]. The primer sequences of HDGF and GAPDH are presented in Table S2.

Cell proliferation assay

The influence of HDGF knockdown or overexpression, as well as the induction of rhHDGF on NSLCL cell growth, was determined by counting cell numbers. The MTT assay was used to assess cell growth inhibition following gefitinib treatment. Briefly, 1 × 10⁴ cells in 96-well plates were treated with gefitinib (0.001 ~ 50 μM) for 72 h. The optical density at 570 nm was measured, and the IC50 values were calculated by GraphPad Prism 6.0 software (San Diego, CA).

Colony formation assay

Cells were seeded in 6 cm culture dishes at 200 cells per dish. After 14 days of incubation, cells were fixed in 4% paraformaldehyde and stained with crystal violet, and colonies (diameter > 40 μm) were counted and compared.

Transwell assay

Experiments were performed in a 24-well Boyden chamber as previously described [40]. Cells were used at a density of 3 × 10⁴ for migration and invasion assays without or with Matrigel. Photographs of four randomly selected fields were taken, and cell numbers were counted under a microscope.

Western blot

Immunoblotting was carried out as described previously [22]. Protein bands were visualized using an enhanced chemiluminescence kit, and their intensities were measured by ImageJ software.

In vivo study of the correlation between HDGF and gefitinib efficacy

All animals were obtained from Bei**g HFK Bioscience Co. Ltd., (China), and were randomly divided into experimental groups using the random number generated by Excel. NSCLC xenografts were established by subcutaneously inoculating cells into 6–8 weeks male BALB/c nude mic (SCXK-2016-006). When the tumor volumes reached approximately 50–100 mm³, the mice were randomly divided and treated as described below. At the end of the test, the mice were sacrificed, and plasma and tumors were collected for further analysis.

Mice were divided into three groups and implanted with H1975 cells (5 × 10⁵) with sgRNA control, sgRNA1 or sgRNA2. Other mice were inoculated with PC-9 cells (8 × 10⁵) overexpressing HDGF or its vector control. The effect of HDGF knockdown on gefitinib efficacy in H1975 cells was evaluated in four groups of mice: sgRNA-control or sgRNA2 treated with gefitinib (50 mg/kg) or vehicle. The effect of HDGF overexpression on gefitinib efficacy in PC-9 cells was assessed in four groups: vector control or HDGF overexpression treated with gefitinib (10 mg/kg) or vehicle. The data of tumor volume was collected in a blind manner. The student who measured the tumor volume had no idea of the group information.

ELISA

HDGF concentrations in mouse plasma were evaluated by a kit from Enzyme-linked Biotechnology Co., Ltd. (Shanghai, China). HDGF levels in the plasma of NSCLC patients were determined by an ELISA kit from Anrc (Tian**, China).

Immunohistochemistry

The immunohistochemistry (IHC) procedure was performed as described elsewhere. The tissue sections were incubated with antibodies against HDGF or Ki-67. A peroxidase-conjugated goat anti-rabbit secondary antibody from Jackson (West Grove, PA) was diluted in PBS. Specific binding was visualized with the peroxidase substrate diaminobenzidine (DAB) from Pierce (Rockford, IL).

Statistical analysis

The data are presented as the means ± SDs. For the in vitro studies, experiments were performed at least in triplicates. For the in vivo studies, 5 mice were included in each group. The data which were out of the ranges of the means ± 3 × SDs were excluded following the pre-established criteria. The IC50 values were determined by fitting the data in GraphPad Prism 6.0. The Mann‒Whitney U test was performed to compare gene expression between different groups, and other comparisons were made using Student’s t-test. P-values < 0.05 were considered statistically significant. Data variances were compared using F tests. Mann Whitney test was used to compare the data when p-value of F tests is smaller than 0.05. All statistical analyses were performed with SPSS 18.0 (SPSS, Chicago, IL).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Funding

This study was supported by the National Natural Science Foundation of China (grant numbers 81673730 and 82072583) and the Bei**g Municipal Natural Science Foundation (grant number 7152034).

Author information

These authors contributed equally: Shuyan Han, Zhihua Tian, Huifang Tian.

Authors and Affiliations

Department of Integration of Chinese and Western Medicine, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Bei**g, 100142, China
Shuyan Han, Yanna Jiao, Huifeng Hao, Shan Wang, Jialei Fu, Dong Xue, Hong Sun & **** Li
Central Laboratory, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Bei**g, 100142, China
Zhihua Tian & Huifang Tian
The Tissue Bank, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Bei**g, 100142, China
Haibo Han
Department of Thoracic Medical Oncology, Key laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Bei**g, 100142, China
Jun Zhao
Department of Oncology, Infectious Disease Hospital of Heilongjiang Province, Harbin, 150030, China
Chunli Wang

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Shuyan Han
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Zhihua Tian
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Haibo Han
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Hong Sun
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**** Li
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Contributions

SH, HS, and PL conceived and designed the work. ZT, HT, HBH, YJ, HFH, SW, and JF performed the experiments and data analyses. JZ and CW provided clinical samples, information and conducted the clinical data analyses. SH, JZ, and HFH provided technical support. SH, ZT, HT, HS, DX, and HBH drafted and revised the manuscript. All authors read and approved the final paper.

Corresponding authors

Correspondence to Shuyan Han, Hong Sun or **** Li.

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The authors declare no competing interests.

Ethical approval

These studies were approved by the Peking University Cancer Hospital & Institute Animal Ethics Committee (No. 2015KT10), the Ethics Committee of Infectious Disease Hospital of Heilongjiang Province (No. 2022LS008). All patients enrolled in this study provided written informed consent for sample collection and data analyses. The experimental methods complied with the Declaration of Helsinki.

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Han, S., Tian, Z., Tian, H. et al. HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer. Cell Death Discov. 9, 181 (2023). https://doi.org/10.1038/s41420-023-01476-0

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Received: 21 January 2023
Revised: 29 April 2023
Accepted: 30 May 2023
Published: 10 June 2023
DOI: https://doi.org/10.1038/s41420-023-01476-0
Springer Nature Limited

HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer

Abstract

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Antitumor activity of afatinib in EGFR T790M-negative human oral cancer therapeutically targets mTOR/Mcl-1 signaling axis

Targeting the PI3K/AKT/mTOR and RAF/MEK/ERK pathways for cancer therapy

Targeting KRASG12C in Non-Small-Cell Lung Cancer: Current Standards and Developments

Introduction

Discussion

Materials and methods

Cell lines and culture

Drugs and reagents

Establishment of stably transfected NSCLC cells

Quantitative RT‒PCR (qRT‒PCR)

Cell proliferation assay

Colony formation assay

Transwell assay

Western blot

In vivo study of the correlation between HDGF and gefitinib efficacy

ELISA

Immunohistochemistry

Statistical analysis

Data availability

References

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Ethical approval

Additional information

Supplementary information

Combined supplementary materials in one flie

Original Western Blots

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HDGF promotes gefitinib resistance by activating the PI3K/AKT and MEK/ERK signaling pathways in non-small cell lung cancer

Abstract

Similar content being viewed by others

Antitumor activity of afatinib in EGFR T790M-negative human oral cancer therapeutically targets mTOR/Mcl-1 signaling axis

Targeting the PI3K/AKT/mTOR and RAF/MEK/ERK pathways for cancer therapy

Targeting KRASG12C in Non-Small-Cell Lung Cancer: Current Standards and Developments

Introduction

Discussion

Materials and methods

Cell lines and culture

Drugs and reagents

Establishment of stably transfected NSCLC cells

Quantitative RT‒PCR (qRT‒PCR)

Cell proliferation assay

Colony formation assay

Transwell assay

Western blot

In vivo study of the correlation between HDGF and gefitinib efficacy

ELISA

Immunohistochemistry

Statistical analysis

Data availability

References

Funding

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Ethical approval

Additional information

Supplementary information

Combined supplementary materials in one flie

Original Western Blots

Rights and permissions

About this article

Cite this article

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