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British Journal of Cancer logoLink to British Journal of Cancer
. 2022 Dec 19;128(5):844–856. doi: 10.1038/s41416-022-02106-9

Hyperglycaemia induces metabolic reprogramming into a glycolytic phenotype and promotes epithelial-mesenchymal transitions via YAP/TAZ-Hedgehog signalling axis in pancreatic cancer

Zhao Liu 1, Hiromitsu Hayashi 1, Kazuki Matsumura 1, Yoko Ogata 1, Hiroki Sato 1, Yuta Shiraishi 1, Norio Uemura 1, Tatsunori Miyata 1, Takaaki Higashi 1, Shigeki Nakagawa 1, Kosuke Mima 1, Katsunori Imai 1, Hideo Baba 1,✉
PMCID: PMC9977781  PMID: 36536047

Abstract

Background

Hyperglycaemia is a well-known initial symptom in patients with pancreatic ductal adenocarcinoma (PDAC). Metabolic reprogramming in cancer, described as the Warburg effect, can induce epithelial-mesenchymal transition (EMT).

Methods

The biological impact of hyperglycaemia on malignant behaviour in PDAC was examined by in vitro and in vivo experiments.

Results

Hyperglycaemia promoted EMT by inducing metabolic reprogramming into a glycolytic phenotype via yes-associated protein (YAP)/PDZ-binding motif (TAZ) overexpression, accompanied by GLUT1 overexpression and enhanced phosphorylation Akt in PDAC. In addition, hyperglycaemia enhanced chemoresistance by upregulating ABCB1 expression and triggered PDAC switch into pure basal-like subtype with activated Hedgehog pathway (GLI1 high, GATA6 low expression) through YAP/TAZ overexpression. PDAC is characterised by abundant stroma that harbours tumour-promoting properties and chemoresistance. Hyperglycaemia promotes the production of collagen fibre-related proteins (fibronectin, fibroblast activation protein, COL1A1 and COL11A1) by stimulating YAP/TAZ expression in cancer-associated fibroblasts (CAFs). Knockdown of YAP and/or TAZ or treatment with YAP/TAZ inhibitor (K975) abolished EMT, chemoresistance and a favourable tumour microenvironment even under hyperglycemic conditions in vitro and in vivo.

Conclusion

Hyperglycaemia induces metabolic reprogramming into glycolytic phenotype and promotes EMT via YAP/TAZ-Hedgehog signalling axis, and YAP/TAZ could be a novel therapeutic target in PDAC.

Subject terms: Oncology, Pancreatic cancer

Introduction

Pancreatic ductal adenocarcinoma (PDAC) displays aggressive malignant behaviour and remains the most lethal cancer type [1]. Prognostic outcomes of patients with PDACs considerably worsen when the tumour size exceeds 2 cm [2]. All primary tumours possess metastatic potential at diagnosis [3]. The probability of metastasis varies as a function of tumour size, with 28% at 1 cm and 94% at 3 cm [4]. However, why PDAC has such a high malignant behaviour even in small tumours compared to other solid gastrointestinal cancers is unclear. Hyperglycaemia or type 2 diabetes (T2D) is associated with the development of several cancers, including liver and bladder cancers [5]. In PDAC, hyperglycaemia is well known as an initial symptom, especially in patients with the occult disease. These clinical backgrounds led us to hypothesise that hyperglycaemia, as an initial symptom, plays a key role in the malignant behaviour of PDAC. Cancer cells produce ATP under aerobic conditions through glycolysis rather than by oxidative phosphorylation. Metabolic reprogramming, or “Warburg effect”, can induce epithelial-mesenchymal transition (EMT) in cancer cells [6]. Disruption of cell-cell contacts during EMT allows the cells to detach and migration away from their neighbours, and the trigger remains poorly understood.

Previous studies have linked the Hippo pathway to cell proliferation regulation and apoptosis [7, 8]. Dysregulation of the Hippo signalling pathway can occur through yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) overexpression [9]. YAP/TAZ, downstream effectors of the Hippo pathway, have a pivotal role in oncogenic effects in human cancers, including PDAC. However, the biological roles of hyperglycaemia and its biological impact on YAP/TAZ during PDAC progression are yet unidentified.

We explored the hypothesis that hyperglycaemia promotes metabolic reprogramming into a glycolytic phenotype and accelerates malignant behaviour via YAP/TAZ signalling activation in PDAC.

Methods

Cell culture

Human pancreatic cancer (PC) cell lines (AsPC-1, PK8, MIA PaCa-2, PANC-1, PK-59, KLM-1, S2-013 and S2-VP10) were purchased from RIKEN Bioresource Center cell Bank (Tsukuba, Japan) and the Japanese Collection of Research Bioresource Cell Bank (Ibaraki, Japan). These cells were routinely cultured in RPMI-1640 (Wako, Osaka, Japan), replenished with 10% foetal bovine serum (FBS; Mediatech, Osaka, Japan) and maintained at 37 °C in a sterile humidified atmosphere of 5% CO2.

Establishment of cancer-associated fibroblasts (CAFs)

CAFs were established as described previously [10]. Briefly, patient-sourced material was acquired from pancreatic resection of primary cancers. CAFs were extracted from PC tissues, then transferred to a culture medium and put on ice. Each sample was cut into small slices ~2–4 mm with scalpels, and the tissue slices were transferred into a gentleMACS C tube containing an enzyme mix by adding (4.7 mL RPMI 1640, 200 mL Enzyme H, 100 mL Enzyme R and 25 mL Enzyme A). Tissue was dispersed and disrupted using a gentle MACS dissociator. The sample was then centrifuged for a short time at room temperature (22–25 °C) at 300 × g for 30 s to collect the sample materials at the bottom of the tube. Pellet was washed with 20 mL of RPMI 1640 to collect any remaining cells that stuck to the cell strainer. The cell suspension was then centrifuged at 300 × g at 4 °C for 7 min, and the supernatant was removed. Cells were cultured in 10 mL medium in a 10 cm collagen-coated dish at 37 °C in a sterile humidified atmosphere of 5% CO2 and expanded in a 15 cm dish until 90% fusion was achieved.

RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

RNA extraction, cDNA synthesis and qRT-PCR were performed as previously described [11, 12]. Total RNA was isolated from PDAC cell lines using RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Five hundred nanograms of purified total RNA were used for cDNA synthesis with SuperScript III reverse transcriptase (Invitrogen) following the manufacturer’s instructions. All qRT-PCR reactions were run on a LightCycler 480 II instrument (Roche). mRNA levels were quantified by SYBR Green qRT-PCR using a LightCycler 480 SYBR Green I Master (Roche Diagnostics, Mannheim, Germany) and normalised to β-actin. Fold-change was measured using 2 − ΔΔCt method (LightCycler® 480 Software) to determine the differences in gene expression levels between samples. Independent experiments were performed in triplicate and repeated at least three times. To perform qRT-PCR, primers were designed using the Universal Probe Library (Roche), according to the manufacturer’s proposals. See primers in Supplementary Table S1.

Extracellular metabolic flux assays

The extracellular acidification rate (ECAR; mpH/min) was measured as previously described [13]. Briefly, ECAR was measured using a Seahorse XF24 analyser (Seahorse Bioscience, North Billerica, MA, USA), according to the manufacturer’s instructions. Cells were grown in complete RPMI1640 (Wako, Tokyo, Japan) to approximately 80% confluency, trypsinized and inoculated at 6.0 × 104 cells/well into XF24 cell culture microtiter plates. Cells were then incubated with 5% CO2 at 37 °C overnight. The initial medium was replaced and the plate incubated at 37 °C for 1 h prior to the assay. Then, the plates were transferred into an XF24 analyser to calculate ECAR values before and after successive additions of 2-deoxy-D-glucose (2-DG). All values were recorded at set intervals. All compounds and materials were obtained from Agilent (Santa Clara, CA, USA).

Protein sample preparation and western blot analysis

Protein extraction from cultured PDAC cells and subsequent western blot analysis were performed as previously described [11, 12]. Briefly, whole-cell lysates from cell lines were prepared in RIPA buffer [25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)] containing protease and phosphatase inhibitors. Equal amounts of protein from cell lysates were electrophoretically separated on 5–15% SDS gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% non-fat dry milk in tris-buffered saline (TBS)/Tween-20 (0.1%) for 1 h at room temperature, then incubated overnight at 4 °C with primary antibody. After washing the membrane with TBS/Tween-20, membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody diluted 1:5000 in TBS/Tween-20 for 1 h at room temperature. Membranes were washed with TBS/Tween-20, incubated with an enhanced chemiluminescence detection system [ECL (GE Healthcare Corp) or ECL-Prime (GE Healthcare)] and visualised using a ChemiDocTM Touch Gel Imaging System (Bio-Rad, Hercules, CA, USA).

Immunohistochemistry, immunocytofluorescence and sirius red staining

Sample processing and immunohistochemical procedures were performed as previously described [12, 14]. Briefly, immunohistochemical staining (IHS) was performed on formalin-fixed, paraffin-embedded 4 µm thick sections. Pre-treated sections were autoclaved in Histofine antigen retrieval solution (pH 9) (Nichirei, Tokyo, Japan). Endogenous peroxidase activity with 3% hydrogen peroxide (H2O2) was blocked, and sections incubated with diluted antibodies overnight at 4 °C. Subsequent reactions were performed using a biotin-free HRP enzyme-labelled polymer from Envision Plus detection system (Dako Co. Tokyo, Japan). Positive reactions were shown with diaminobenzidine (DAB) solution, followed by counterstaining with Mayer’s hematoxylin. Negative controls were prepared for immunostaining by omitting primary antibodies. IHS results were evaluated independently by three researchers who were unaware of the clinical data. The method was scored as previously described [15]. Briefly, YAP/TAZ immunoreactivity was considered positive when intense staining was uniformly observed in nuclei of cancer cells. Counts were performed in six random high-magnification fields, then the positive cancer cells were calculated. Researchers analysed and scored the means of the six fields on a 4-tier scale as follows: 0% (negative), 1–10% (weak), 11–50% (moderate), 51–100% (strong); and classified low expression as negative and weak; high expression as moderate and strong. For immunocytofluorescence, the cells were fixed for 10 min with ice-cold 4% paraformaldehyde (PFA). After blocking with 3% bovine serum albumin (BSA) (Sigma) in phosphate buffer saline (PBS), cells were permeabilized with 0.2% Triton X-100 and incubated with the primary antibody in 1% BSA in PBS overnight, followed by secondary antibody labelling with Alexa Fluor 488 (1:200). After washing twice with PBS, the nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Negative control staining was performed by omitting the primary antibody. For Sirius Red staining, hydrated paraffin sections were stained in Picro-Sirius for 1 h, then washed in two changes of acidified water, and blotted with damp filter paper to remove most of the water. Finally, the samples were dehydrated in three portions of 100% ethanol, cleared in xylene and mounted in a resinous medium. Then three fields of view were randomly captured under high magnification and quantified using ImageJ (NIH) to analyse the stained areas.

Antibodies, cytokines and reagents

See this study’s primary antibodies in Supplementary Table S2. Gemcitabine was purchased from Wako (PTF0153, 10 mg; Osaka, Japan). Oxaliplatin and K975 were obtained from MedChemExpress (NJ, USA). Streptozotocin (STZ) was purchased from Sigma–Aldrich (MO, USA). 30% H2O2 was obtained from Wako.

YAP and TAZ suppression with siRNA duplex oligoribonucleotides

YAP or TAZ suppression was induced with siRNA and Stealth RNAi siRNA duplex oligonucleotides (Invitrogen) as previously described [12]. Briefly, PDAC cells were seeded in six-well plates and transfected with duplex siRNA using Lipofectamine 3000 transfection reagent (Invitrogen), following the manufacturer’s instructions. After 48 h transfection, whole-cell lysates were generated. We designed and purchased three different siRNAs for each target gene (Stealth RNAi siRNA, Invitrogen), and two validated siRNAs were used in subsequent research. The most validated sequences were: siYAP: 5’-GGAAGGAGAUGGAAUGAACAUAGAA-3’, siTAZ (WWTR1), 5’-CCCAGACAUGAGAUCCAUCACUAAU-3’, and each oligo’s complementary sequence. The Stealth RNAi Negative Control Duplexes (Medium GC Duplex) (Invitrogen) was used as a negative control.

YAP and TAZ overexpression using plasmid vectors

The cDNA corresponding to human YAP/TAZ was inserted into pLenti-C-Myc-DDK-IRES-Puro lentiviral gene expression vector (#PS100069; Origene) as previously described [12]. Human YAP/TAZ cDNA was amplified with forward primers (5’-ATGGATCCCGGGCAGCA-3’)/(5’-ATGAATCCGGCCTCGGC-3’) and reverse primers (5’-CTATAACCATGTAAGAAAGCTTTC-3’) and (5’-TTACAGCCAGGTTAGAAAGG-3’) using cDNA from isolated SK-Hep-1 cells as a template, and the product was ligated into Target CloneTM –Plus- (TOYOBO). Verification of the complete sequence of the YAP/TAZ cDNA was done by Sanger sequencing. The open reading frame (ORFs) of YAP/TAZ were PCR amplified with forward primers using the AsiSI site (5’-ATAGCGATCGCCACCATGGATCCCGGGCAGCA-3’)/(5’-ATAGCGATCGCCACCATGAATCCGGCCTCGGC-3’) and reverse primers using MluI site (5’-TATACGCGTCTATAACCATGTAAGAAAGCTTTC-3’)/(5’-TATACGCGTTTACAGCCAGGTTAGAAAGG-3’) to attach cloning sites. ORF was cloned into target vector pLenti-C-Myc-DDK-IRES-Puro Tagged Cloning Vector (Origene, #PS100069). PCR product and pLenti-C-Myc-DDK-IRES-Puro labelled cloning vector were digested using AsiSI and MluI, purified and ligated using a ligation mix. The transformation was performed in NEB stable competent Escherichia coli (NEW ENGLAND BioLabs, #C3040I) following the manufacturer’s instructions. The final vector sequences were validated by Sanger sequencing. Lentivirus particles were produced using a lentiviral high-titre packaging mix (TaKaRa, #6194) according to the manufacturer’s instructions. Briefly, target vectors were transfected with lentiviral high-titre packaging mix into the cells from Lenti-X 293 T cell line (TaKaRa, #Z2180N) using Lipofectamine 3000 (Thermo Fisher Scientific). After 48 h of incubation, the medium containing lentivirus particles was collected and passed through a 0.45 mm filter. To establish PDAC cells overexpressing YAP/TAZ, PDAC cells were cultured for 24 h in a medium containing viral particles with 8 µg/mL polybrene.

Cell proliferation assay

Cell proliferation assays were performed in 96-well plates, and live cells were counted at each time point in the WST-8 assay using a cell counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan), as previously described. Absorbance was measured at 450 nm. Various doses of gemcitabine (Wako, PTF0153), oxaliplatin (MedChemExpress, HY-17371) and K975 (MedChemExpress, HY-138565) were appended to the cells 48 h after seeding, and the medium was changed daily. The final concentration of dimethyl sulfoxide was controlled within 0.1% (v/v) in each experiment.

Transwell cell invasion assays

Cell invasion assays were performed as follows: 3 × 104 cells were suspended in BioCoat Matrigel invasion chambers (24-well plate, 8 μm pores; BD Biosciences, San Jose, CA, USA) and inserted into Transwell device (Corning Life Sciences). The lower chamber was chemically primed using a medium containing 20% FBS. Cells were placed in an incubator at 37 °C for 24 h to enable transfection. They were fixed on the lower surface using 4% paraformaldehyde and stained with Wright Giemsa dye (Sangon). Cells were counted under a microscope (×20, ×40 magnification) in six predetermined fields, per replicate.

Animal studies

To assess whether high glucose affects PDAC tumour growth and invasiveness in vivo, we established a xenograft nude mouse model of STZ-induced diabetes and continuously measured blood glucose values 1–3 days before injecting cancer cells. Twelve 4–6-weeks-old male athymic nude mice (BALB/c nu/nu) (CLEA Japan, Inc.) were randomly divided into two groups (six mice per group). The control group fed an ordinary diet and given insulin injections to maintain blood glucose at levels <130 mg/dL, was considered euglycemic. The treatment group, injected with STZ (180 mg/kg body weight), then fed a high-glucose diet to preserve blood glucose at levels ≥450 mg/dL, were considered hyperglycemic. Next, 1 × 106 AsPC-1 cells were implanted in mice on Day 0. Tumour volumes were gauged using the following formula: volume [mm3] = (length [mm]) × (width [mm])2 × 0.52. The mice were sacrificed on Day 21, and tumours were dissected.

Orthotopic model of PC

Twelve 4–6-weeks-old male athymic nude mice (BALB/c nu/nu) (CLEA Japan, Inc.) were randomly divided into three groups (four mice per group), and luciferase labelled AsPC-1 cells were transplanted.

Hyperglycemic and euglycemic mice were developed, as mentioned above. The treatment group was administrated K975 p.o. twice daily (130 mg/kg weight) starting 3 days after surgery for 14 days. For surgery, mice were anaesthetised intraperitoneally and placed in a supine position, as described previously [16]. The abdomen was disinfected with 70% alcohol, and a 0.5–1 cm midline incision was made to expose the mice pancreas. AsPC-1 cells (1 × 106) were injected into the tail of the pancreas, and the incision was sutured. Three groups of orthotopically transplanted mice were anaesthetised by isoflurane inhalation, and tumour metastasis was assessed using an in vivo imaging system (Caliper IVIS Kinetic In Vivo Optical Imaging System) after a luciferin injection (180 mg/kg) (Promega, P1043) on the 21st day. Then, mice were sacrificed by CO2 asphyxiation. The pancreas was removed, and tumours were weighed.

All animal studies were conducted in accordance with the guidelines of the Animal Care and Use Committee of Kumamoto University (approval number A2021-090).

Patients and tissue samples

PDAC paraffin-embedded sections were obtained from patients who underwent pancreatic resection at the Department of Gastroenterological Surgery, Kumamoto University Hospital, from March 2011 to July 2020. The study was approved by the Medical Ethics Committee of Kumamoto University (Project No. 1291), and written informed consent was obtained from all human subjects.

Statistical analysis

All experiments were performed in triplicate, and the data shown are representative of consistently observed results. Data are expressed as the mean ± standard deviation (SD). Mann–Whitney U test was used to compare continuous variables between two groups. Kaplan–Meier curves and generalised Wilcoxon test were used to evaluating the statistical significance of the differences. Data analysis was performed with JMP (version 9, SAS Institute, Japan). Statistical significance was set at P < 0.05.

Results

Hyperglycaemia induces YAP/TAZ expressions, and their overexpression triggers glycolytic phenotype in metabolic pathways in PDAC cells

To investigate the biological impact of hyperglycaemia on YAP/TAZ expression in PDAC cells, cells were cultured with a dose-dependent glucose solution (100, 300 and 500 mg/dL in glucose concentration). PDAC cells cultured at high-glucose concentrations upregulated YAP/TAZ expression and downregulated YAPSer127 expression phosphorylation in a dose-dependent manner (Fig. 1a, b). To validate YAP/TAZ signal activation due to hyperglycaemia, we demonstrated increased expression of cysteine-rich angiogenic inducer 61 (CYR61) and connective tissue growth factor (CTGF) protein, which are both well-known downstream targets of YAP/TAZ [7] (Fig. 1c). To investigate the impact of hyperglycaemia on metabolic pathways, we analysed metabolic reprogramming using an extracellular flux analyser in PDAC cells. Hyperglycaemia enhanced glycolysis, compared to normal conditions, was indicated by increased ECAR (Fig. 1d). Furthermore, under normal glucose concentration, YAP/TAZ overexpression also induced glycolysis, indicated by increased ECAR, compared to the control (Fig. 1e). Controversially, YAP or TAZ knockdown diminished glycolysis, as indicated by decreased ECAR compared to control under hyperglycaemia (Fig. 1f). These results suggest that hyperglycaemia enhances glycolysis through YAP/TAZ overexpression in cancer cells even in the presence of oxygen and fully functioning mitochondria, thereby increasing glucose uptake. Mammalian cells obtain energy and carbon primarily through glucose, and glucose transporters (GLUTs) assist in glucose uptake and then metabolise to pyruvate in the cytoplasmic matrix during glycolysis. In this study, hyperglycaemia upregulated GLUT1 expression in PDAC cells (Fig. 1g). YAP or TAZ overexpression also upregulated GLUT1 expression under normal glucose concentrations (Fig. 1h). In contrast, YAP and/or TAZ knockdown attenuated GLUT1 expression under hyperglycaemia (Fig. 1i). As for intracellular signals, hyperglycaemia upregulated phosphorylated Akt expression in PDAC cells (Fig. 1j). Collectively, hyperglycaemia promotes metabolic reprogramming into a glycolytic phenotype via YAP/TAZ overexpression, accompanied by GLUT1 overexpression and enhanced Akt phosphorylation.

Fig. 1. Hyperglycaemia induces YAP / TAZ overexpression and trigger the glycolytic phenotype in the metabolic pathway in PDAC cells.

Fig. 1

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a Real-time PCR of YAP/TAZ mRNA expressions in human PDAC cell lines (AsPC-1 and PK8) treated at different glucose concentrations. (*P < 0.05 and **P < 0.01; n = 3). b Western blot analysis of YAP/TAZ, phosphorylation YAPSer127 proteins in human PDAC cell lines (AsPC-1 and PK8) treated at different glucose concentrations. β-actin protein expression served as a loading control. Representative blots are shown. Top and bottom arrows indicate 70 kDa (YAP), and 50 kDa (TAZ). c Western blot analysis of Cyr61 and CTGF proteins expression in human PDAC cell lines (AsPC-1 and PK8) treated at different glucose concentrations. β-actin protein expression served as a loading control. Representative blots are shown. d Flux assay analysis of ECAR in AsPC-1 cell line treated at different glucose concentration. High-glucose concentrations shifted the metabolic reprogramming into glycolysis as indicated by an increased ECAR. (*P < 0.05 and **P < 0.01; n = 3). e Flux assay analysis of ECAR in AsPC-1 cell lines (Mock, YAP overexpressed and TAZ overexpressed) treated at the normal glucose concentration (100 mg/dL). YAP/TAZ overexpressed shifted the metabolic reprogramming into glycolysis as indicated by an increased ECAR. (*P < 0.05; n = 3). f Flux assay analysis of ECAR in AsPC-1 cell line with knockdown of YAP (left panel) or TAZ (right panel) under the high-glucose concentration (500 mg/dL). The ECAR was decreased by YAP or TAZ downregulation. (*P < 0.05 and **P < 0.01; n = 3). g Real-time PCR and western blot analysis of GLUT1 expression in AsPC-1 cell line treated at different glucose concentrations. β-actin protein expression served as the loading control. Representative blots are shown. (**P < 0.01; n = 3). h Real-time PCR and western blot analysis of GLUT1 expression in AsPC-1 cell lines (Mock, YAP overexpressed and TAZ overexpressed) treated at the normal glucose concentration (100 mg/dL). β-actin protein expression served as the loading control. Representative blots are shown. (**P < 0.01; n = 3). i Real-time PCR analysis and western blot analysis of GLUT1 expression in AsPC-1 cell line with knockdown of YAP and/or TAZ under a high-glucose concentration (500 mg/dL). Upper) knockdown of YAP; middle) knockdown of TAZ; and lower) knockdown of YAP/TAZ. β-actin protein expression served as the loading control. Representative blots are shown. (**P < 0.01; n = 3). j Western blot analysis of intracellular signals in human PDAC cell lines (AsPC-1 and PK8) treated at different glucose concentrations. β-actin protein expression served as the loading control. Representative blots are shown. PCR polymerase chain reaction, PDAC pancreatic ductal adenocarcinoma, ECAR extracellular acidification rate.

Hyperglycaemia promotes EMT by YAP/TAZ overexpression in PDAC cell

EMT is well known as an important process occurring prior to tumour metastasis. During EMT, tumour cells undergo morphological changes, transforming from an epithelial-like morphology to mesenchymal cell morphology, with metabolism elevated and intercellular adhesion diminished, the latter promoting cell migration. The most important landmark change associated with EMT is decreased or loss of E-cadherin, a type I cadherin. In this study, hyperglycaemia decreased E-cadherin expression, but increased vimentin and snail expression levels were in the culture with hyperglycaemia (Fig. 2a). Hyperglycaemia accelerated the PDAC cells invasiveness (Fig. 2b). PDAC cells overexpressing YAP or TAZ also showed increased invasiveness under normal glucose concentration (Fig. 2c). In contrast, YAP or TAZ knockdown upregulated E-cadherin (CDH1) expression and downregulated vimentin and snail expression under hyperglycaemia (Fig. 2d). YAP or TAZ knockdown resulted in less invasiveness compared to controls in PDAC cells (Fig. 2e). The YAP/TAZ inhibitor (K975) had similar effects on E-cadherin (CDH1), vimentin and snail expression, and successfully suppressed PDAC cells invasiveness (Fig. 2f, g). Metformin did not inhibit YAP/TAZ expression in PDAC cells under hyperglycaemia in vitro but may indirectly regulate YAP/TAZ expression by downregulating blood glucose in vivo (Supplementary Fig. S1 and S4b). Above all, these results show that hyperglycaemia-induced EMT by YAP/TAZ overexpression in PDAC cells, and YAP/TAZ inhibition could be a therapeutic target. Interestingly, hyperglycaemia had no impact on PDAC cell growth ability (Supplementary Fig. S2a). However, hyperglycaemia inhibited Cleaved Caspase3 and Cleaved PARP expression in PDAC cells, which may demonstrate that hyperglycaemia enhanced the anti-apoptotic ability of PDAC cells (Supplementary Fig. S2b).

Fig. 2. Hyperglycaemia promotes EMT by YAP/TAZ overexpression in PDAC cells.

Fig. 2

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a Western blot analysis of EMT markers (E-cadherin, vimentin and Snail) expression in human PDAC cell line (AsPC-1) treated at different glucose concentrations (100, 300, 500 mg/dL), β-actin protein expression served as a loading control. Representative blots are shown. b Cell invasion assay in human PDAC cell line (AsPC-1) treated at different glucose concentrations (100, 300, 500 mg/dL). Representative pictures are shown. Scale bars, 50μm. (*P < 0.05; n = 3). c Cell invasion assay in human PDAC cell line (AsPC-1) (Mock, YAP overexpressed and TAZ overexpressed) treated at the normal glucose concentration (100 mg/dL). Representative pictures are shown. Scale bars, 50 μm. (**P < 0.01; n = 3). d Western blot analysis of EMT markers (E-cadherin, vimentin and Snail) expression in human PDAC cell line (AsPC-1) with YAP (left) or TAZ (right) knockdown under a high-glucose concentration (500 mg/dL). β-actin protein expression served as a loading control. Representative blots are shown. e Cell invasion assay in human PDAC cell line (AsPC-1) with YAP or TAZ knockdown under a high-glucose concentration (500 mg/dL). Scale bars, 50 μm. (*P < 0.05 and **P < 0.01; n = 3). f Western blot analysis of EMT markers (E-cadherin, vimentin and Snail) expression in human PDAC cell line (AsPC-1) treated with K975 under a high-glucose concentration (500 mg/dL), β-actin protein expression served as a loading control. Representative blots are shown. g Cell invasion assay in human PDAC cell line (AsPC-1) treated with K975 (1, 5 μM) and control (PBS). Representative pictures are shown. Scale bars, 100 μm. (**P < 0.01; n = 3). PDAC pancreatic ductal adenocarcinoma, EMT epithelial-mesenchymal transition.

Hyperglycaemia enhances chemoresistance by upregulating ABCB1 expression via YAP/TAZ overexpression in PDAC cell

In most cancers, drug resistance is the main reason for chemotherapy failure. Glycolysis and Akt signal activation are associated with drug resistance [17, 18]. ATP-binding cassette subfamily B member 1 (ABCB1) is an ATP-dependent drug efflux pump for xenobiotic compounds with broad substrate specificity. It is responsible for reduced drug accumulation in multidrug-resistant cells and often mediates the development of anti-cancer drug resistance. Thus, ABCB1 plays an important role in the chemoresistance of cancer cells and is a cancer stem cell transcription factor [19]. In our study, hyperglycaemia-induced ABCB1 expression in a dose-dependent manner (Fig. 3a), and was also upregulated by YAP/TAZ overexpression under normal glucose concentrations in PDAC cells (Fig. 3b, c). Conversely, YAP or TAZ knockdown attenuated ABCB1 expression compared to that of controls, even in a high-glucose environment (Fig. 3d). Indeed, PDAC cells cultured with hyperglycaemia displayed an accelerated chemoresistance response to gemcitabine and oxaliplatin (Fig. 3e), which are key anti-cancer drugs for PDAC in the present clinical setting. Furthermore, YAP/TAZ inhibitor (K975) successfully overcame the chemoresistance induced by hyperglycaemia in PDAC cells (Fig. 3f). These findings indicate that hyperglycaemia promotes chemoresistance by upregulating ABCB1 expression via YAP/TAZ overexpression in PDAC cells.

Fig. 3. Hyperglycaemia enhances chemoresistance by upregulating ABCB1 expression via YAP/TAZ overexpression in PDAC cells.

Fig. 3

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a Real-time PCR and western blot analysis of ABCB1 expressions in human PDAC cell line (AsPC-1) treated at different glucose concentrations (100, 300, 500 mg/dL), β-actin protein expression served as a loading control. Representative blots are shown. (**P < 0.01; n = 3). b Western blot analysis of ABCB1 expressions in human PDAC cell line (AsPC-1) (Mock, YAP overexpressed and TAZ overexpressed) treated under a normal glucose concentration (100 mg/dL), β-actin protein expression served as a loading control. Representative blots are shown. c Immunofluorescence staining of ABCB1 (green)/phalloidin (red)/4’,6-diamidino-2-phenylindole (DAPI, blue) in human PDAC cell line (AsPC-1) (Mock, YAP overexpressed and TAZ overexpressed) under a normal glucose concentration (100 mg/dL). Representative pictures are shown. Scale bars, 50 μm. d Western blot analysis of ABCB1 expressions in human PDAC cell line (AsPC-1) with YAP (left panel) and TAZ (right panel) knockdown, under a normal glucose concentration (100 mg/dL), β-actin protein expression served as a loading control. Representative blots are shown. e Chemoresistance assay was performed in human PDAC cell line (AsPC-1) treated with gemcitabine (5 μM) (left panel) or oxaliplatin (5 μM) (right panel) and control (PBS) under normal (100 mg/dL) and high (500 mg/dL) glucose concentration. f Chemoresistance assay was performed in human PDAC cell line (AsPC-1) treated with K975, gemcitabine (left panel) or oxaliplatin (right panel), K975 + gemcitabine (left panel) or K975 + oxaliplatin (right panel) and control (PBS) under a high-glucose concentration (500 mg/dL). PCR polymerase chain reaction, PDAC pancreatic ductal adenocarcinoma, ABCB1 ATP-binding cassette subfamily B member 1.

Hyperglycaemia triggers an alteration of molecular subtype in cancer cells via the YAP/TAZ- Hedgehog signalling axis

Recent comprehensive genomic analysis in PDAC revealed that the cancer cell subtype has an impact on malignant behaviour, including prognostic outcomes [20]. High GLI1 and low GATA6 expressions are well-known subtype markers of the pure basal-like subtype in PDAC [21]. GLI1 is downstream, while GATA6 is an inhibitor of the hedgehog signalling pathway, which, as reported, serves an essential role as an oncogenic signalling pathway in PDAC [22]. The Hippo pathway exhibits widespread interlocution with other signalling pathways, such as transforming growth factor-beta [23, 24], Wnt [25, 26], Sonic hedgehog [27, 28] and Notch [29, 30]. Hyperglycaemia upregulated hedgehog signalling pathway-related markers (Shh and GLI1) expression and downregulated GATA6 expression (inhibiting Hedgehog signalling pathway) (Fig. 4a). YAP or TAZ overexpression enhanced the nuclear localisation of GLI1 protein compared to that of controls, under normal glucose concentration in PDAC cells (Fig. 4b). In contrast, YAP and/or TAZ knockdown downregulated hedgehog signalling pathway-related markers (Shh and GLI1) expression and upregulated GATA6 expression (Fig. 4c). YAP/TAZ inhibitor (K975) displayed similar effects on hedgehog signalling pathway-related markers, including GATA6, under hyperglycaemia (Fig. 4d). Some investigators indicated that SFK can drive PDAC cells into a hyper-metastatic state [31], EMT and reduced dependence on KRAS signalling by promoting YAP expression, which is a distinctive feature of the aggressive basal-like/squamous subtype of PDAC. Interestingly, when we blocked SFK expression, YAP expression was also suppressed, and YAP phosphorylation was promoted in the cytoplasm (Supplementary Fig. S6). Combining the above data, hyperglycaemia triggers an alteration of molecular subtype to the pure basal-like subtype PDAC via YAP/TAZ overexpression, which is characterised by worse prognosis and higher malignant potential.

Fig. 4. Hyperglycaemia triggers an alteration of molecular subtype in cancer cells via YAP/TAZ- Hedgehog signalling axis.

Fig. 4

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a Real-time PCR and western blot analysis of hedgehog signalling pathway markers (Shh, GLI1) expression and GATA6 in human PDAC cell line (AsPC-1) treated at different glucose concentrations (100, 300, 500 mg/dL), β-actin protein expression served as a loading control. Representative blots are shown. (**P < 0.01; n = 3). b Immunofluorescence staining of GLI1 (green)/phalloidin (red)/4’,6-diamidino-2-phenylindole (DAPI, blue) in human PDAC cell line (AsPC-1) (Mock, YAP overexpressed and TAZ overexpressed) under the normal glucose concentration (100 mg/dL). Representative pictures are shown. Scale bars, 50 μm. c Real-time PCR analysis of hedgehog signalling pathway markers (Shh, GLI1) expression in human PDAC cell line (AsPC-1) with upper) YAP; middle) TAZ; and lower) YAP/TAZ knockdown under a high-glucose concentration (500 mg/dL). Western blot analysis of Hedgehog signalling pathway markers (Shh, GLI1) expression in human PDAC cell line (AsPC-1) with upper) YAP or middle) TAZ knockdown under a high-glucose concentration (500 mg/dL). β-actin protein expression served as a loading control. Representative blots are shown. (*P < 0.05 and **P < 0.01; n = 3). d Western blot analysis of Hedgehog signalling pathway markers (Shh, GLI1) expression in human PDAC cell line (AsPC-1) after add YAP/TAZ inhibitor (K975) under a high-glucose concentration (500 mg/dL). β-actin protein expression served as a loading control. Representative blots are shown. PCR polymerase chain reaction, PDAC pancreatic ductal adenocarcinoma.

High YAP /TAZ expression is associated with impaired glucose tolerance and worse prognostic outcomes in PDAC patients

Next, we examined YAP and TAZ expression patterns by IHS of 147 PDAC human tissues. YAP was predominantly expressed in cancer cells, and high YAP (moderate and strong) expression was detected in 73 cases (49.7%) (Supplementary Fig. S3a). Furthermore, high YAP expression was significantly associated with impaired glucose tolerance (HbA1c ≥ 7.0) compared with low YAP expression (Supplementary Fig. S3b) and was significantly associated with worse prognostic outcomes (recurrence-free survival and overall survival) compared with low YAP expression (Supplementary Fig. S3c). Furthermore, TAZ was also predominantly expressed in cancer cells, and high TAZ (moderate and strong) expression was detected in 81 cases (55%) (Supplementary Fig. S3d). High TAZ expression was significantly associated with impaired glucose tolerance (HbA1c ≥ 7.0) compared with low TAZ expression (Supplementary Fig. S3e) and was significantly associated with worse prognostic outcomes (recurrence-free survival and overall survival) compared with low TAZ expression (Supplementary Fig. S3f). In univariate analysis, both YAP and TAZ were a significant prognostic factor. Moreover, in multivariate analysis, TAZ overexpression rather than YAP overexpression was an independent worse prognostic factor for patients with resected PDAC, suggesting that IHC of TAZ is useful for predicting the prognostic outcomes in PDAC. Tumour size and male were also independent of worse prognostic factors in patients with resected PDAC. Contrariwise, DM was not a significant prognostic factor in patients with resected PDAC (Supplementary Table S3). These results suggest that YAP/TAZ overexpression is associated with impaired glucose tolerance and is a poor prognostic factor in patients with resected PDAC.

Hyperglycaemia promoted malignant behaviour of PDAC via YAP/TAZ in vivo

To confirm the biological impact of hyperglycaemia on the malignant behaviour of PDAC cells in vivo, an STZ-induced diabetic model was established in nude mice (Fig. 5a). Human PDAC cells were subcutaneously or orthotopically transplanted (xenograft model). Subcutaneously transplanted tumours under conditions of hyperglycemic conditions (blood glucose levels ≥ 450 mg/dL) were significantly enlarged both in terms of weight and volume compared with euglycemia mice (blood glucose levels < 130 mg/dL) (Fig. 5b–d). Histological analysis of subcutaneously transplanted tumours in hyperglycemic mice showed elevated YAP, TAZ and vimentin expression compared with those in euglycemia mice (Fig. 5e). In contrast, E-cadherin expression was suppressed in hyperglycemic mice (Fig. 5e). The orthotopically transplanted tumours under hyperglycaemia condition displayed enhanced tumour metastatic capacity and increased tumour weight compared with euglycemia, and the phenotype was diminished by oral administration of YAP/TAZ inhibitor (K975) in hyperglycemic mice (Fig. 5f and Supplementary Fig. S4c). Thus, hyperglycaemia promoted the malignant behaviour of PDAC via YAP/TAZ in vivo.

Fig. 5. Hyperglycaemia promoted malignant behaviour of PDAC via YAP/TAZ in vivo.

Fig. 5

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a Schema of streptozotocin (STZ)-induced nude mouse models of hyperglycaemia were established. Human PDAC cell line (AsPC-1) was injected subcutaneously in nude mice. Mice were sacrificed at 21 days. b Tumour volume in the two groups were measured twice weekly for 3 weeks (n = 6 in each group). The volume of tumour was calculated by the formula: Volume [mm3] = (length [mm]) × (width [mm])2 × 0.52. (*P < 0.05; n = 6). c Comparing the gross morphologies of the tumour between the two groups at 21 days post-implantation. d Tumour weight was measured after extraction at 21 days. (**P < 0.01; n = 6). e Representative image of microscopic findings (hematoxylin-eosin, YAP, TAZ, vimentin and E-cadherin) at the invasive front in the extracted tumours with euglycemic and hyperglycemic tumours. Scale bars, 50 μm. f In vivo imaging system images of the euglycemic, hyperglycemic and hyperglycemic K975-supplemented orthotopically transplanted pancreatic tumours (left panel). The overall morphology (middle panel) and tumour weight (right panel) (red dotted line scope) of the extracted euglycemic, hyperglycemic and hyperglycemic K975-supplemented orthotopically transplanted pancreatic tumours were compared. Representative pictures are shown. (**P < 0.01; n = 4). PDAC pancreatic ductal adenocarcinoma.

Hyperglycaemia promotes collagen deposition by activating CAFs via YAP/TAZ overexpression

Although from a genetic perspective, primary and metastatic tumours are very similar, their cell proliferation capacity differs greatly because it is greatly influenced by the tumour microenvironment (TME) [3]. Because of its abundant stroma characteristics, PDAC has significantly enhanced chemoresistance and harbours tumour-promoting features. Recent research shows that CAFs support tumorigenesis by enhancing the deposition of collagen, which is the main component of the extracellular matrix (ECM). In our study, hyperglycaemia upregulated YAP/TAZ expression in CAFs and further induced protein interacting with never in mitosis A1 (Pin1), whose overexpression in PDAC correlates with the desmoplastic and immunosuppressive TME and poor patient survival [32, 33]. Collagen fibre-related marker proteins (fibronectin, FAP, COL1A1 and COL11A1) were also induced in CAFs with hyperglycaemia (Fig. 6a). YAP/TAZ inhibitor (K975) successfully attenuated Pin1 expression and collagen-related proteins (Fig. 6b). The orthotopically transplanted tumours under hyperglycemic condition, compared with euglycemia, displayed much collagen deposition and activated fibroblasts indicated as Sirius red and alfa-SMA positive (Fig. 6c, d). The phenotypes were diminished by oral administration of YAP/TAZ inhibitor (K975) in hyperglycemic mice (Fig. 6c, d). Thus, hyperglycaemia provides a favourable TME for cancer cells by increasing pro-fibrogenic CAFs via YAP/TAZ overexpression.

Fig. 6. Hyperglycaemia promotes collagen deposition by activating cancer-associated fibroblasts via YAP/TAZ overexpression.

Fig. 6

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a Western blot analysis of YAP/TAZ, Pin1 and fibro-related proteins marker (fibronectin, FAP, COL1A1, COL11A1) expression in human PDAC CAFs cell line (CAF15) treated at different glucose concentrations (100, 300, 500 mg/dL). Top and bottom arrows indicate 70 kDa (YAP), and 50 kDa (TAZ), β-actin protein expression served as a loading control. Representative blots are shown. b Western blot analysis of YAP/TAZ, Pin1 and fibro-related proteins marker (fibronectin, FAP, COL1A1, COL11A1) expression in human PDAC CAFs cell line (CAF15) after adding YAP/TAZ inhibitor (K975) under high-glucose concentration (500 mg/dL). β-actin protein expression served as a loading control. Representative blots are shown. c Sirius red staining of collagen deposition of euglycemic, hyperglycemic and hyperglycemic K975-supplemented orthotopically transplanted pancreatic tumours after the hyperglycemic mice were treated with K975 for 2 weeks. Representative pictures are shown. Scale bars, 200 μm. (**P < 0.01; n = 4). d Immunofluorescence staining of αSMA (red)/4’,6-diamidino-2-phenylindole (DAPI, blue) in euglycemic, hyperglycemic and hyperglycemic K975-supplemented orthotopically transplanted pancreatic tumours after the hyperglycemic mice were treated with K975 for 2 weeks. Representative pictures are shown. Scale bars, 50 μm. (**P < 0.01; n = 4). PDAC pancreatic ductal adenocarcinoma, CAF cancer-associated fibroblast.

Discussion

This is the first study to provide compelling evidence that hyperglycaemia triggers metabolic reprogramming into a glycolytic phenotype and promotes EMT via YAP/TAZ overexpression in PC. Our study supports a cohort study finding that patients with PDAC and impaired glucose tolerance display worse prognostic outcomes [34]. In the present study, hyperglycaemia, a well-known initial symptom, may contribute to the clinical course of PC. Therefore, early detection of PDAC and treatment strategy for YAP/TAZ overexpression induced by hyperglycaemia would be important to improve the prognosis of this deadly disease.

The Hippo pathway has an indispensable regulatory role in organ regeneration and tumorigenesis, and YAP/TAZ is the most important downstream effector of this pathway [9]. Dysregulation of the Hippo pathway induces YAP/TAZ overexpression, resulting in enlarged organs and tumour development, such as liver cancer [9]. In previous research, YAP/TAZ plays a pivotal role as a pro-oncogenic factor of PDAC in genetically engineered mouse models (GEMMs) [35, 36]. In the present study, hyperglycaemia-induced YAP/TAZ overexpression triggered metabolic reprogramming of the glycolytic phenotype.

Cancer cells obtain energy mainly through glycolysis, accompanied by the accumulation of lactate and a small amount of ATP under a hypoxic tumour environment, and the metabolic reprogramming in cancer cells is referred to as the “Warburg effect” [37]. Since glucose cannot freely pass through the membrane, it requires the assistance of GLUTs for glycolysis. Here, hyperglycaemia upregulated GLUT1 expression through YAP/TAZ overexpression. In our previous study, GLUT1 was reported as an unfavourable prognostic factor for PDAC [15]. In the present study, YAP/TAZ overexpression in tumour tissues was significantly associated with impaired glucose tolerance (HbA1c ≥ 7.0) and worse prognosis outcomes in patients with PDAC. Thus, hyperglycaemia supports the Warburg effect in cancer cells by activating the YAP/YAZ-GLUT1 axis. The glycolytic phenotype induced by the Warburg effect can enhance EMT in cancer cells [6, 38], and provides sufficient energy for cancer cells to maintain the undifferentiated state during EMT. In the present study, hyperglycaemia may provide cancer cells with sufficient energy to carry out EMT by activating the YAP/YAZ-GLUT1 axis.

Gemcitabine and oxaliplatin have been widely used in patients with advanced PC and have improved patient’s prognoses, but the development of chemoresistance can still lead to poor outcomes [39]. The drug efflux pump, ABCB1, is a key driver of chemoresistance and is a cancer stem cell marker in PDAC [40]. In the present study, hyperglycaemia promoted chemoresistance with ABCB1 overexpression, and chemoresistance was overcome by YAP/TAZ inhibition.

Metformin, a commonly used drug for controlling blood glucose, has been shown to have an anti-cancer effect, including against PC [41]. Interestingly, in this study’s in vitro, metformin did not induce any changes in YAP/TAZ expression under hyperglycaemia, yet in vivo, YAP/TAZ expression was significantly suppressed in tumour specimens from the metformin-treated group; perhaps the indirect regulation of YAP/TAZ by some unknown pathway after hyperglycaemia was reversed. However, this phenomenon will be further investigated in future.

Recent comprehensive gene expression studies have identified the PDAC subtypes with biological and prognostic relevance [42]. Among several subtypes, as reported, a pure basal-like subtype is closely related to poorly differentiated tumours of highly metastatic potential, with the activation of the Hedgehog signalling pathway (high GLI1 and low GATA6 expressions) [20, 43]. As reported, the Hedgehog pathway has a key regulatory role in the development of PC [22]. The present study shows that hyperglycaemia activates the hedgehog pathway through YAP/TAZ and promotes the transformation of PDAC cells into a pure basal-like subtype (high GLI1 and low GATA6 expression).

PDAC is characterised by abundant stroma and has tumour-promoting and chemoresistance features. A previous study reported that ECM stiffness also stimulates YAP/TAZ overexpression in cancer cells [44]. A recent study has found that targeting Pin1, which is a peptidyl-prolyl isomerase, disrupts collagen deposition by acting on CAFs [32]. There are also reports indicating that diabetes-related EMT contributes to CAF formation in tumours, enabling epithelial and endothelial extravasation of tumour cells [45]. In parallel, the Hippo pathway is activated by stromal stiffness in solid tumour tissue, and there is increasing evidence that the transcription factor, YAP, is activated in CAF [46, 47]. In the present study, hyperglycaemia promoted collagen production by upregulating YAP/TAZ expression in CAFs, and YAP/TAZ inhibition attenuated the collagen deposition induced by hyperglycaemia. Pin1 was also downregulated by YAP/TAZ inhibition. Hyperglycaemia may accelerate the malignant behaviour of PDAC not only by promoting glycolysis and EMT in cancer cells but also by enhancing ECM stiffness through YAP/TAZ overexpression in CAFs.

Our study had some limitations. The molecular mechanism between hyperglycaemia and YAP/TAZ lacks direct mechanism validation. Another limitation is that immuno-oncology also plays an important role in the TME, but the related exploration of hyperglycaemia and immunology was lacking in this study.

In conclusion, hyperglycaemia can induce metabolic reprogramming into the glycolytic phenotype and promote EMT via the YAP/TAZ-Hedgehog signalling axis in PDAC. Hyperglycaemia as an initial symptom may be the cause of highly invasive and metastatic potential by inducing YAP/TAZ overexpression, while YAP/TAZ could be a novel therapeutic target in PDAC patients.

Supplementary information

Supplementary informations (26.3MB, pdf)

Acknowledgements

We thank Dr. Hiromitsu Hayashi, Dr. Norio Uemura and Dr. Kazuki Matsumura for helping with the study; Ms. Ogata, Ms. Yasuda and Ms. Taniguchi for their excellent technical assistance; Dr. Feng Wei, Dr. Chuan Lan and Dr. Xiyu Wu for giving me good advices; Shigeki Nakagawa, Kosuke Mima, Katsunori Imai, Yo-ichi Yamashita and Prof. Hideo Baba for assistance with revision of the paper. Data sharing requests will be considered by the management group upon written request to the corresponding author.

Author contributions

Conceived and designed the experiments: ZL and HH. Performed the experiments: ZL, NU, KM and YO. Analysed the data: ZL and HH. Wrote the manuscript: HH and ZL. Collected clinical samples: ZL, NU, KM, HS, YS, TM and TH. Organised the paper and approved the final version to be published: HH, SN, KM, KI and HB.

Funding information

This work was supported by a Grant-in-Aid for Scientists (C); the Ministry of Education, Culture, Sports, Science, and Technology of Japan, No. 19K09177 (to HH); the Takeda Science Foundation, Japan (to HH); Japanese Foundation for Multidisciplinary Treatment of Cancer, Japan (to HH) and JST SPRING, Grant Number JPMJSP2127 (to ZL).

Data availability

All presented data are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

The study was approved by the Medical Ethics Committee of Kumamoto University (Project No. 1291), and written informed consent was obtained from all human subjects.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Consent for publication Not applicable

Supplementary information

The online version contains supplementary material available at 10.1038/s41416-022-02106-9.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary informations (26.3MB, pdf)

Data Availability Statement

All presented data are available from the corresponding author upon reasonable request.

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

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