Abstract
This study aimed to determine if the (pro)renin receptor (ATP6AP2) changes the cellular profile of choriocarcinomas from cytotrophoblast cells to terminally syncytialised cells and ascertain whether this impacts the invasive potential of choriocarcinoma cells. Additionally, we aimed to confirm that FURIN and/or site 1 protease (MBTPS1) cleave soluble ATP6AP2 (sATP6AP2) in BeWo choriocarcinoma cells and determine whether sATP6AP2 levels reflect the cellular profile of choriocarcinomas. BeWo choriocarcinoma cells were treated with ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK (to inhibit FURIN activity), or PF 429242 (to inhibit MBTPS1 activity). Cells were also treated with forskolin, to induce syncytialisation, or vehicle and incubated for 48 h before collection of cells and supernatants. Syncytialisation was assessed by measuring hCG secretion (by ELISA) and E-cadherin protein levels (by immunoblot and immunocytochemistry). Cellular invasion was measured using the xCELLigence real-time cell analysis system and secreted sATP6AP2 levels measured by ELISA. Forskolin successfully induced syncytialisation and significantly increased both BeWo choriocarcinoma cell invasion (P < 0.0001) and sATP6AP2 levels (P = 0.02). Treatment with ATP6AP2 siRNA significantly inhibited syncytialisation (decreased hCG secretion (P = 0.005), the percent of nuclei in syncytia (P = 0.05)), forskolin-induced invasion (P = 0.046), and sATP6AP2 levels (P < 0.0001). FURIN siRNA and DEC-RVKR-CMK significantly decreased sATP6AP2 levels (both P < 0.0001). In conclusion, ATP6AP2 is important for syncytialisation of choriocarcinoma cells and thereby limits choriocarcinoma cell invasion. We postulate that sATP6AP2 could be used as a biomarker measuring the invasive potential of choriocarcinomas. Additionally, we confirmed that FURIN, not MBTPS1, cleaves sATP6AP2 in BeWo cells, but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.
Introduction
The (pro)renin receptor ((P)RR/ATP6AP2) is a multi-functioning receptor that stimulates numerous cell signalling pathways, including proliferation, invasion, and angiogenesis, that enhance cancer progression. It does this by activating tissue renin-angiotensin systems (RASs) (Sanchez-Lopez et al. 2005, Herr et al. 2008), wingless/integrated (Wnt)/β-catenin, and extracellular signal-regulated kinases 1/2 (ERK1/2)/mitogen-activated protein kinase (MAPK) signalling pathways (Pollheimer et al. 2006, Olsen et al. 2017, Wang et al. 2019, Guo et al. 2020) and via its role as part of the vacuolar-ATPase (Kinouchi et al. 2010, Capecci & Forgac 2013, Kulshrestha et al. 2015). ATP6AP2 is over expressed in pancreatic ductal adenocarcinoma (PDAC) (Shibayama et al. 2015), endometrial (Delforce et al. 2017) and colorectal cancer (Wang et al. 2019) and, through its essential role in Wnt/β-catenin signalling, has been shown to directly contribute to cancer progression (Shibayama et al. 2015, Wang et al. 2019). ATP6AP2 may also play a role in other epithelial cancers, such as choriocarcinoma. Choriocarcinomas account for 5% of gestational trophoblastic tumours and are highly malignant (Markman & Kavanagh 2011). The prevalence of choriocarcinoma is highest in Asian populations (Bishop & Edemekong 2019) but its pathophysiology is not well understood.
While exploring the role of ATP6AP2 in placentation, we have shown that although ATP6AP2 is abundant in the placenta (Pringle et al. 2011, Morosin et al. 2020a), it is not involved in trophoblast syncytialisation (Morosin et al. 2020a). Because of the intimate links between ATP6AP2 and cancer and the fact that Wnt/β-catenin signalling is elevated in various cancers (Pai et al. 2017) and is important for BeWo choriocarcinoma cell syncytialisation (Matsuura et al. 2011), we wanted to find out if ATP6AP2 affected the syncytialisation of choriocarcinoma-derived trophoblasts. Additionally, the invasive characteristics of the syncytiotrophoblast and cytotrophoblast cells that comprise choriocarcinoma have, to our knowledge, not been investigated. Therefore, this study also aimed to determine whether the syncytiotrophoblast in choriocarcinoma more closely resembles the early invasive syncytiotrophoblast of the blastocyst at implantation (Huppertz 2019) or the late non-invasive syncytiotrophoblast of the term placenta (James et al. 2005, Wang & Zhao 2010). This will also aid in better ascertaining whether the ATP6AP2 could be involved in choriocarcinoma cell invasion.
The extracellular domain of ATP6AP2 can be cleaved and secreted as a soluble protein (sATP6AP2). Soluble ATP6AP2 is detected in both plasma and urine, making it an ideal biomarker candidate (Cousin et al. 2009, Lu et al. 2016). Soluble ATP6AP2 is increased in chronic kidney disease, heart failure, PDAC, preeclampsia, and gestational diabetes (Ichihara & Yatabe 2019). Recently, Endo et al. found that sATP6AP2 is a marker of autophagy in lung cancer (Endo et al. 2020). Therefore, sATP6AP2 may serve both as a novel biomarker in choriocarcinoma and have important carcinogenic functions.
Soluble ATP6AP2 is secreted by both JAR and JEG choriocarcinoma cells (Suda et al. 2020) as well as by primary human trophoblast cells (Morosin et al. 2020b). We aimed to find out if sATP6AP2 is also secreted by BeWo cells and determine if sATP6AP2 could potentially be used as a biomarker of tissue ATP6AP2 expression, and of the ratio of syncytiotrophoblast to cytotrophoblast so that it becomes a potential indicator of the invasiveness of the choriocarcinoma. Additionally, if sATP6AP2 is a biomarker of invasiveness of choriocarcinoma, it is important to determine which proteases cleave it from membrane-bound ATP6AP2. Therefore, we also aimed to find out which proteases (FURIN or site 1 protease (MBTPS1)) cleave sATP6AP2 in BeWo cells.
Materials and methods
BeWo choriocarcinoma cell culture
BeWo choriocarcinoma cells (American Type Culture Collection, CCL-98, USA; passages 7–15; University of Newcastle Human Research Ethics H-2020-0398) were cultured in 1× high-glucose Dulbecco’s Modified Eagles Medium (DMEM-HG; Hyclone, UT, USA) supplemented with 10% non-heat inactivated foetal bovine serum (FBS; Bovogen Biologicals, VIC, Australia), 2 mM l-glutamine (Gibco) and 1% antibiotic-antimycotic (Gibco) in 5% CO2 in room air at 37°C. For all experiments (apart from xCELLigence invasion experiments described below), cells were plated at 200,000 cells/well in six-well plates. For immunocytochemistry (ICC) experiments, cells were plated on 0.1% gelatin-coated coverslips.
siRNA transfection
Transfection was performed as described previously by Morosin et al. (2020a). At 24 h after plating, Lipofectamine 2000 (Invitrogen) and Opti-MEM (Gibco) were combined with either 125 nM ATP6AP2 siRNA (HSS115474, Life Technologies), 125 nM FURIN siRNA (HSS107545, Life Technologies) or negative control siRNA (medium GC; Invitrogen) and added to the culture medium (without antibiotic-antimycotic). Cells were also treated with media containing lipofectamine and Opti-MEM alone as non-transfected controls. To induce syncytialisation, 24 h after transfection, cells were treated with either 100 μM forskolin or vehicle (DMSO; UNIVAR, USA) and incubated for a further 48 h. At 48 h, cells and supernatants were collected, snap-frozen, and stored at –80°C. For ICC experiments, cells were fixed in 4% paraformaldehyde (PFA) in PBS for 20 min and stored in 0.02% sodium azide in PBS at 4°C until required.
Protease inhibitor treatment
Treatment with protease inhibitors was commenced 24 h after plating. BeWo cells were treated with either:
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50 μM of broad protease inhibitor (DEC-RVKR-CMK; Merck);
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10 μM of site 1 protease (MBTPS1) inhibitor (PF-429242; Adoo-Q Bioscience, USA); or
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0.5% DMSO (Sigma–Aldrich) as the vehicle control.
To induce syncytialisation, all cells were simultaneously treated with 100 μM forskolin (Sigma–Aldrich) or vehicle (DMSO, 0.5%). BeWo cells were then incubated for 48 h before cells were collected in TRIzol reagent (Invitrogen) directly from the culture dish, supernatants were also collected, and both were snap-frozen and stored at –80°C. ICC experiments were collected as described previously.
RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)
Briefly, RNA was extracted from BeWo cell lysates using an RNeasy-mini Kit (Qiagen) according to the manufacturer’s instructions. RNA was then incubated with DNase I (Qiagen) to purify the sample of contaminating DNA. Total RNA was quantified using a nanodrop ND-1000 spectrophotometer, A260/A280 and A260/A230 ratios were examined for purity. Agarose gel electrophoresis was performed to assess the integrity of RNA samples.
Purified RNA samples were spiked with Alien RNA (107 copies per µg of total RNA; Stratagene) to ensure efficient RT across all samples. RT was then performed using a Superscript III RT kit with random hexamers (Invitrogen). Samples were prepared for PCR by combining 5 μL of SYBR Green PCR master mix (Applied Biosystems), primers for ATP6AP2 (Morosin et al. 2020a), Alien (Stratagene), β-actin (ACTB) (Morosin et al. 2020a), or tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) (Morosin et al. 2020a), cDNA (10 ng), and water to 10 μL. An Applied Biosystems 7500 Real-Time PCR System was used. The 2−ΔΔCT method was used for calculating the mRNA relative abundance for each gene, and data were expressed relative to the mean of three housekeepers (Alien, ACTB, and YWHAZ) and a calibrator sample (term human placenta).
Protein extraction
For siRNA experiments, the protein was extracted from cell lysates using a radio immuno-precipitation assay (RIPA) method. Briefly 100 μL of RIPA buffer (50 mM Tris–HCl (Invitrogen), 150 mM NaCl (VWR international), 1 mM EDTA, 1% Nonidet P-40, 0.5% C24H39NaO4, 1 nM Na3VO4, complete mini protease inhibitor cocktail tablets (Sigma–Aldrich)) and 1 μL of 100 nM phenyl methyl sulfonyl fluoride (PMSF)) were added to each sample. Samples were incubated on ice for 30 min, centrifuged at 16,200 g at 4°C for 10 min, and supernatants were collected. For inhibitor experiments, the protein was extracted from cell lysates using TRIzol according to the manufacturer’s instructions as previously described (Morosin et al. 2020a).
Bicinchoninic acid (BCA) assay (ThermoFisher Scientific) was used to measure protein concentrations. A SPECTROstarnano microplate reader (BMG LABTECH) was used to measure optical densities at 570 nm.
Immunoblotting
E-cadherin and ATP6AP2 protein levels were determined via immunoblot. Refer to Supplementary Table 1 (see section on supplementary materials given at the end of this article) for optimised protein concentrations and primary and secondary antibody details and concentrations; Refer to Supplementary Figure 1 for representative full-length immunoblots and primary antibody specificity information. Briefly, isolated BeWo cell protein was loaded onto 4–12% Bis-Tris precast NuPAGE Novex gels (Life Technologies) and separated by electrophoresis, before transferring onto PVDF membranes (GE Healthcare). Protein isolated from term human placenta (for E-cadherin immunoblots) or term human amnion (for ATP6AP2 immunoblots) was also included on every immunoblot and used as an internal control. Membranes were dried and reactivated in methanol before blocking overnight with 5% skim milk/5% BSA in tris-buffered saline with 20% Tween 20 (TBST), at 4°C on a rocker. Membranes were then incubated with the primary antibody (in 5% skim milk/TBST) for 2 h at room temperature on a rocker (as per Supplementary Table 1) and washed in TBST before incubation in the secondary antibody (in 1 or 3% skim milk/TBST) for 1 h at room temperature on a rocker (as per Supplementary Table 1). Membranes were again washed in TBST before signal detection using an Amersham Imager 600 (GE Healthcare) and Amersham ECL Western blotting detection reagent kit (GE Healthcare). Membranes were then stripped in 0.2 M sodium hydroxide and re-probed for β-actin (as per Supplementary Table 1), which was used as a loading control for all immunoblots.
Densitometry was analysed using the Amersham Imager 600 software, using the rubber band method of background subtraction (GElifesciences 2014). Data are presented as a ratio of the band/s of interest divided by β-actin.
Enzyme linked immunosorbent assay (ELISA)
Soluble ATP6AP2 (IBL, USA) and hCG (Thermofisher Scientific) ELISA kits were used to measure the amounts of these secreted proteins in culture media, according to the manufacturer’s instructions. Optical densities were measured using a SPECTROstarnano microplate reader. Each sample was normalised to its media blank to correct for background interference. Intra-assay coefficients of variability (CV) for sATP6AP2 and hCG were 3 and 2.1%, respectively. Inter-assay CV for sATP6AP2 and hCG were 12.8 and 3.7%, respectively.
Immunocytochemistry
Immunocytochemistry was performed as previously described (Morosin et al. 2020a). Briefly, fixed cells were permeabilised in 0.1% Triton X-100 in PBS (Bio-Rad) before blocking in 1% BSA. Cells were then incubated with E-cadherin primary antibody (Abcam ab1416; 0.4 µg/mL) before being washed with PBS and incubated with Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody (Thermofisher, A-11029; 1.3 µg/mL). Negative controls were included where cells were incubated in the absence of either the primary antibody or both the primary and secondary antibodies. Cells were mounted onto microscope slides using a DAPI mounting medium (prolong diamond anti-fade mountant with DAPI; Thermofisher Scientific, P36962). Slides were stored at 4°C and three images were taken per slide (imaged areas were selected at random) using a Nikon C2 confocal microscope at 40× magnification.
ImageJ cell counting software was used to determine the degree of syncytialisation as previously described (Morosin et al. 2020a). Briefly, to determine the percent of nuclei that were in a syncytium, the total number of nuclei in a syncytium was divided by the total number of nuclei present in each image and converted to percentage by multiplying by 100. When multiple nuclei (blue) were within one green E-cadherin boundary, these were counted as ‘nuclei in a syncytium’.
xCELLigence real-time cell analysis-invasion
Invasion was measured using an xCELLigence real-time cell analyser (RTCA) dual-purpose (DP) instrument (Agilent) and its accompanying cell invasion and migration (CIM) plate. The xCELLigence RTCA is placed inside the cell incubator at standard culture conditions (5% CO2 and 37°C). BeWo choriocarcinoma cells (passage 18–30) were transfected as mentioned previously (10 mM ATP6AP2 siRNA or 25 mM negative control siRNA), 48 h after plating. 24 h after transfection, and prior to cell seeding, the CIM plate was set up as follows:
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Each well of the CIM plate’s upper chamber was coated with 20 μL of 800 μg/mL matrigel growth factor-reduced (Cat#354230, Corning) in serum-free media and allowed to set at room temperature for 2 h.
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In total, 160 µL of complete media (as described previously) was added to the lower chamber of the CIM plate. The upper chamber was then attached and 30 µL of media (without FBS) was added to each well.
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The plate was then placed in the incubator for 1 h to calibrate at 5% CO2 and 37°C before taking a baseline reading using the xCELLigence RTCA.
Transfected cells were then digested with trypsin (Gibco), counted, and seeded at a density of 5000 cells/well (in FBS and antibiotic-antimycotic-free media) into the upper chamber of the prepared CIM plate. Concurrently, cells were treated with either 100 µM forskolin to induce syncytialisation or vehicle (DMSO) in a final volume of 150 μL in the upper chamber. The plate was then incubated at room temperature for a further 30 min before being returned to the xCELLigence RTCA. Cell index (as a measure of electrical impedance) was measured at 30 min intervals over 40 h (when the slope of the invasion curve reached its peak). The interval slope (1/h) was calculated using the RTCA software to evaluate the rate of cell invasion which was then exported for analysis.
Statistical analysis
BeWo cells (apart from the ICC experiments) were plated in triplicate (at a minimum), and experiments were repeated three to four times to ensure reproducibility. ICC experiments were plated in singlicate, and three separate areas were imaged per well. Significance was set at P < 0.05, and GraphPad Prism version 8 was used for all statistical analyses.
Effects of forskolin treatment on hCG, E-cadherin, and the percent of nuclei in syncytia were determined using Mann–Whitney t-tests. Effects of ATP6AP2 siRNA, FURIN siRNA, DEC-RVKR-CMK, or PF 429242 on ATP6AP2 gene and protein expression, markers of syncytialisation (hCG and E-cadherin), and/or sATP6AP2 secretion were determined using two-way ANOVA tests with Sidak’s multiple comparisons. As were the effects forskolin and ATP6AP2 siRNA on invasion.
Results
Forskolin successfully induced syncytialisation of BeWo choriocarcinoma cells
Forskolin-induced syncytialisation was determined by measuring hCG and E-cadherin levels, well-known indicators of syncytialisation (Coutifaris et al. 1991, Costa 2016). Treatment with forskolin significantly increased hCG secretion (Fig. 1A; P < 0.0001), decreased E-cadherin protein levels (Fig. 1B; P = 0.004), and increased the percent of nuclei in syncytia (as measured by ICC for E-cadherin; Fig. 1C, D, E and F ; P < 0.0001). Thus, syncytialisation was successfully induced.
Forskolin-induced syncytialisation of BeWo choriocarcinoma cells. BeWo cells were treated with forskolin or vehicle and left for 48 h. Treatment with forskolin significantly increased (A) hCG secretion, decreased (B) intracellular E-cadherin protein levels, and increased (C, D and E) the percent of nuclei in syncytia (as determined by E-cadherin immunostaining), compared with the vehicle control. β-actin was used as a loading control in the representative immunoblot image in (B). Images in (D, E and F) were stained with E-cadherin (green) and DAPI (blue). (D) Representative image of vehicle-treated cells, there are 97 total nuclei and no nuclei in a syncytium. (E) Representative image of forskolin-treated cells, there are 64 total nuclei and 28 (44%) of these nuclei are in a syncytium (these are depicted by the white dot). (F) Represents a no primary antibody control, controlling for non-specific secondary antibody binding. ANOVA values are reported in the text above each graph and represented by #, where # denotes a significant difference from the vehicle control. Data are presented as median and interquartile ranges. The effect of forskolin (PForsk) is reported. n =3 experiments in triplicate. For immunocytochemistry only: n = 3 in singlicate (three images/well).
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
Forskolin-induced syncytialisation of BeWo choriocarcinoma cells. BeWo cells were treated with forskolin or vehicle and left for 48 h. Treatment with forskolin significantly increased (A) hCG secretion, decreased (B) intracellular E-cadherin protein levels, and increased (C, D and E) the percent of nuclei in syncytia (as determined by E-cadherin immunostaining), compared with the vehicle control. β-actin was used as a loading control in the representative immunoblot image in (B). Images in (D, E and F) were stained with E-cadherin (green) and DAPI (blue). (D) Representative image of vehicle-treated cells, there are 97 total nuclei and no nuclei in a syncytium. (E) Representative image of forskolin-treated cells, there are 64 total nuclei and 28 (44%) of these nuclei are in a syncytium (these are depicted by the white dot). (F) Represents a no primary antibody control, controlling for non-specific secondary antibody binding. ANOVA values are reported in the text above each graph and represented by #, where # denotes a significant difference from the vehicle control. Data are presented as median and interquartile ranges. The effect of forskolin (PForsk) is reported. n =3 experiments in triplicate. For immunocytochemistry only: n = 3 in singlicate (three images/well).
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
Forskolin-induced syncytialisation of BeWo choriocarcinoma cells. BeWo cells were treated with forskolin or vehicle and left for 48 h. Treatment with forskolin significantly increased (A) hCG secretion, decreased (B) intracellular E-cadherin protein levels, and increased (C, D and E) the percent of nuclei in syncytia (as determined by E-cadherin immunostaining), compared with the vehicle control. β-actin was used as a loading control in the representative immunoblot image in (B). Images in (D, E and F) were stained with E-cadherin (green) and DAPI (blue). (D) Representative image of vehicle-treated cells, there are 97 total nuclei and no nuclei in a syncytium. (E) Representative image of forskolin-treated cells, there are 64 total nuclei and 28 (44%) of these nuclei are in a syncytium (these are depicted by the white dot). (F) Represents a no primary antibody control, controlling for non-specific secondary antibody binding. ANOVA values are reported in the text above each graph and represented by #, where # denotes a significant difference from the vehicle control. Data are presented as median and interquartile ranges. The effect of forskolin (PForsk) is reported. n =3 experiments in triplicate. For immunocytochemistry only: n = 3 in singlicate (three images/well).
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
ATP6AP2 is important for syncytialisation in BeWo choriocarcincoma cells
BeWo cells were treated with ATP6AP2 siRNA before being induced to syncytialise with forskolin. Treatment with forskolin had no effect on ATP6AP2 mRNA expression or protein levels (Fig. 2A and B). ATP6AP2 siRNA successfully decreased ATP6AP2 mRNA expression by 57 and 70% in the vehicle- and forskolin-treated groups, respectively (Fig. 2A; both P < 0.0001) and protein levels by >50%, in both the vehicle- and forskolin-treated groups (Fig. 2B; P = 0.002 and P = 0.009, respectively).
The (pro)renin receptor (ATP6AP2) is important for BeWo choriocarcinoma cell syncytialisation. BeWo cells were treated with ATP6AP2 siRNA to knockdown ATP6AP2 expression before cells were treated with either forskolin, to induce syncytialisation, or vehicle and left for 48 h. Forskolin-induced syncytialisation had no effect on either (A) ATP6AP2 mRNA or (B) ATP6AP2 protein levels. ATP6AP2 siRNA significantly decreased (A) ATP6AP2 mRNA expression (both P < 0.0001 in the vehicle and forskolin groups) and (B) ATP6AP2 protein levels (P = 0.002 and P = 0.009 in the vehicle and forskolin groups, respectively) compared with the negative control siRNA. ATP6AP2 siRNA treatment significantly (C) decreased hCG secretion in forskolin-treated cells (P = 0.0005). While ATP6AP2 siRNA had no effect on (D) E-cadherin protein levels, it decreased (E, F and G) the percent of nuclei in syncytia in forskolin-treated cells, (measured by E-cadherin immunostaining; P = 0.05), compared with the negative control siRNA. β-actin was used as a loading control in the representative immunoblot images in (B and D). Representative images in (F, G and H) were stained with E-cadherin (green) and DAPI (blue). Nuclei within a syncytium are depicted by a white dot. (F) Representative image of cells treated with both negative control siRNA and forskolin, there are 31 total nuclei and 9 nuclei (29%) within a syncytium. (G) Representative image- of cells treated with ATP6AP2 siRNA and forskolin, there are 34 total nuclei and 4 nuclei (12%) in a syncytia. (H) A representative image of a no primary antibody control, controlling for non-specific secondary antibody binding. ANOVA values are reported in the text above each graph and multiple comparisons are represented by # and *, where # denotes a significant difference from the vehicle control and * a significant difference from the negative control siRNA in its respective vehicle- or forskolin-treated group. ^ A near-significant difference (P = 0.05) from the negative control siRNA. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA (PsiRNA), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 3 experiments in triplicate. For immunocytochemistry only: n = 3 in singlicate (three images/well).
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
The (pro)renin receptor (ATP6AP2) is important for BeWo choriocarcinoma cell syncytialisation. BeWo cells were treated with ATP6AP2 siRNA to knockdown ATP6AP2 expression before cells were treated with either forskolin, to induce syncytialisation, or vehicle and left for 48 h. Forskolin-induced syncytialisation had no effect on either (A) ATP6AP2 mRNA or (B) ATP6AP2 protein levels. ATP6AP2 siRNA significantly decreased (A) ATP6AP2 mRNA expression (both P < 0.0001 in the vehicle and forskolin groups) and (B) ATP6AP2 protein levels (P = 0.002 and P = 0.009 in the vehicle and forskolin groups, respectively) compared with the negative control siRNA. ATP6AP2 siRNA treatment significantly (C) decreased hCG secretion in forskolin-treated cells (P = 0.0005). While ATP6AP2 siRNA had no effect on (D) E-cadherin protein levels, it decreased (E, F and G) the percent of nuclei in syncytia in forskolin-treated cells, (measured by E-cadherin immunostaining; P = 0.05), compared with the negative control siRNA. β-actin was used as a loading control in the representative immunoblot images in (B and D). Representative images in (F, G and H) were stained with E-cadherin (green) and DAPI (blue). Nuclei within a syncytium are depicted by a white dot. (F) Representative image of cells treated with both negative control siRNA and forskolin, there are 31 total nuclei and 9 nuclei (29%) within a syncytium. (G) Representative image- of cells treated with ATP6AP2 siRNA and forskolin, there are 34 total nuclei and 4 nuclei (12%) in a syncytia. (H) A representative image of a no primary antibody control, controlling for non-specific secondary antibody binding. ANOVA values are reported in the text above each graph and multiple comparisons are represented by # and *, where # denotes a significant difference from the vehicle control and * a significant difference from the negative control siRNA in its respective vehicle- or forskolin-treated group. ^ A near-significant difference (P = 0.05) from the negative control siRNA. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA (PsiRNA), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 3 experiments in triplicate. For immunocytochemistry only: n = 3 in singlicate (three images/well).
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
The (pro)renin receptor (ATP6AP2) is important for BeWo choriocarcinoma cell syncytialisation. BeWo cells were treated with ATP6AP2 siRNA to knockdown ATP6AP2 expression before cells were treated with either forskolin, to induce syncytialisation, or vehicle and left for 48 h. Forskolin-induced syncytialisation had no effect on either (A) ATP6AP2 mRNA or (B) ATP6AP2 protein levels. ATP6AP2 siRNA significantly decreased (A) ATP6AP2 mRNA expression (both P < 0.0001 in the vehicle and forskolin groups) and (B) ATP6AP2 protein levels (P = 0.002 and P = 0.009 in the vehicle and forskolin groups, respectively) compared with the negative control siRNA. ATP6AP2 siRNA treatment significantly (C) decreased hCG secretion in forskolin-treated cells (P = 0.0005). While ATP6AP2 siRNA had no effect on (D) E-cadherin protein levels, it decreased (E, F and G) the percent of nuclei in syncytia in forskolin-treated cells, (measured by E-cadherin immunostaining; P = 0.05), compared with the negative control siRNA. β-actin was used as a loading control in the representative immunoblot images in (B and D). Representative images in (F, G and H) were stained with E-cadherin (green) and DAPI (blue). Nuclei within a syncytium are depicted by a white dot. (F) Representative image of cells treated with both negative control siRNA and forskolin, there are 31 total nuclei and 9 nuclei (29%) within a syncytium. (G) Representative image- of cells treated with ATP6AP2 siRNA and forskolin, there are 34 total nuclei and 4 nuclei (12%) in a syncytia. (H) A representative image of a no primary antibody control, controlling for non-specific secondary antibody binding. ANOVA values are reported in the text above each graph and multiple comparisons are represented by # and *, where # denotes a significant difference from the vehicle control and * a significant difference from the negative control siRNA in its respective vehicle- or forskolin-treated group. ^ A near-significant difference (P = 0.05) from the negative control siRNA. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA (PsiRNA), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 3 experiments in triplicate. For immunocytochemistry only: n = 3 in singlicate (three images/well).
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
Treatment of BeWo choriocarcinoma cells with ATP6AP2 siRNA significantly inhibited forskolin-induced hCG secretion by 21% (Fig. 2C; P = 0.0005). Additionally, and while this did not quite reach statistical significance, the percent of nuclei in syncytia was decreased by 15% (Fig. 2E; P = 0.05). ATP6AP2 siRNA treatment had no effect on E-cadherin protein levels (Fig. 2D).
Forskolin increases BeWo choriocarcinoma cell invasion and this is reduced by treatment with ATP6AP2 siRNA
BeWo cells were treated with ATP6AP2 siRNA before being induced to syncytialise with forskolin and invasion was measured. Treatment with forskolin significantly increased BeWo choriocarcinoma cell invasion by >400% in both the negative control siRNA (P < 0.0001; Fig. 3) and ATP6AP2 siRNA (P = 0.001; Fig. 3) groups. ATP6AP2 siRNA significantly decreased cellular invasion by 26% compared with the negative control siRNA group, when cells were treated with forskolin (P = 0.01; Fig. 3).
Forskolin increases BeWo choriocarcinoma cell invasion, and (pro)renin receptor (ATP6AP2) knockdown reduces this. BeWo cells were treated with ATP6AP2 siRNA to knockdown ATP6AP2 expression before treatment with forskolin, cells were then left to invade for 40 h. Forskolin significantly increased BeWo choriocarcinoma cell invasion compared with the DMSO control, regardless of siRNA treatment (P < 0.0001 for the negative control siRNA group; P = 0.001 for the ATP6AP2 siRNA treatment group). Treatment with ATP6AP2 siRNA significantly decreased invasion compared with the negative control siRNA in the forskolin-treated group alone (P = 0.01). ANOVA values are reported in the text above each graph and multiple comparisons are represented by the # and *, where # denotes a significant difference from the vehicle control, and * a significant difference from the negative control siRNA treated with forskolin. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA (PsiRNA), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 4 experiments in triplicate/quadruplicate.
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
Forskolin increases BeWo choriocarcinoma cell invasion, and (pro)renin receptor (ATP6AP2) knockdown reduces this. BeWo cells were treated with ATP6AP2 siRNA to knockdown ATP6AP2 expression before treatment with forskolin, cells were then left to invade for 40 h. Forskolin significantly increased BeWo choriocarcinoma cell invasion compared with the DMSO control, regardless of siRNA treatment (P < 0.0001 for the negative control siRNA group; P = 0.001 for the ATP6AP2 siRNA treatment group). Treatment with ATP6AP2 siRNA significantly decreased invasion compared with the negative control siRNA in the forskolin-treated group alone (P = 0.01). ANOVA values are reported in the text above each graph and multiple comparisons are represented by the # and *, where # denotes a significant difference from the vehicle control, and * a significant difference from the negative control siRNA treated with forskolin. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA (PsiRNA), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 4 experiments in triplicate/quadruplicate.
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
Forskolin increases BeWo choriocarcinoma cell invasion, and (pro)renin receptor (ATP6AP2) knockdown reduces this. BeWo cells were treated with ATP6AP2 siRNA to knockdown ATP6AP2 expression before treatment with forskolin, cells were then left to invade for 40 h. Forskolin significantly increased BeWo choriocarcinoma cell invasion compared with the DMSO control, regardless of siRNA treatment (P < 0.0001 for the negative control siRNA group; P = 0.001 for the ATP6AP2 siRNA treatment group). Treatment with ATP6AP2 siRNA significantly decreased invasion compared with the negative control siRNA in the forskolin-treated group alone (P = 0.01). ANOVA values are reported in the text above each graph and multiple comparisons are represented by the # and *, where # denotes a significant difference from the vehicle control, and * a significant difference from the negative control siRNA treated with forskolin. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA (PsiRNA), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 4 experiments in triplicate/quadruplicate.
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
sATP6AP2 secretion reflects ATP6AP2 levels in BeWo choriocarcinoma cells
BeWo choriocarcinoma cells secrete sATP6AP2 and levels in the supernatant were increased following treatment with forskolin in the negative control siRNA treatment group (P = 0.003; Fig. 4A). Treatment with ATP6AP2 siRNA significantly decreased sATP6AP2 secretion by BeWo cells by 65 and 74%, in both vehicle- and forskolin-treated groups, respectively (Fig. 4A; both P < 0.0001).
BeWo choriocarcinoma cells secrete soluble (pro)renin receptor (sATP6AP2) and its levels decrease with both ATP6AP2 and FURIN inhibition. BeWo cells were treated with either ATP6AP2 siRNA, FURIN siRNA or DEC-RVKR-CMK to inhibit FURIN activity or PF 429242 to inhibit site 1 protease (MBTPS1) activity. Forskolin-induced syncytialisation significantly increased sATP6AP2 levels compared with the vehicle control in all experiments. (A) ATP6AP2 siRNA significantly decreased sATP6AP2 levels compared with the negative control siRNA (both P < 0.0001 in vehicle and forskolin groups). Treatment with (B) FURIN siRNA significantly decreased sATP6AP2 levels compared with the negative control siRNA (P = 0.007 and P = 0.0002 in vehicle and forskolin groups, respectively). (C) Treatment with DEC-RVKR-CMK also significantly decreased sATP6AP2 levels compared with the inhibitor vehicle control (both P < 0.0001 in vehicle and forskolin groups). (D) PF 429242 had no effect on sATP6AP2 levels. ANOVA values are reported in the text above each graph and multiple comparisons are represented by # and *, where # denotes a significant difference from the vehicle control, and * a significant difference from the negative control siRNA or inhibitor vehicle control in its respective vehicle- or forskolin-treated group. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA and FURIN siRNA (PsiRNA), DEC-RVKR-CMK (PDEC), PF 429242 (PPF), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 3 experiments in triplicate.
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
BeWo choriocarcinoma cells secrete soluble (pro)renin receptor (sATP6AP2) and its levels decrease with both ATP6AP2 and FURIN inhibition. BeWo cells were treated with either ATP6AP2 siRNA, FURIN siRNA or DEC-RVKR-CMK to inhibit FURIN activity or PF 429242 to inhibit site 1 protease (MBTPS1) activity. Forskolin-induced syncytialisation significantly increased sATP6AP2 levels compared with the vehicle control in all experiments. (A) ATP6AP2 siRNA significantly decreased sATP6AP2 levels compared with the negative control siRNA (both P < 0.0001 in vehicle and forskolin groups). Treatment with (B) FURIN siRNA significantly decreased sATP6AP2 levels compared with the negative control siRNA (P = 0.007 and P = 0.0002 in vehicle and forskolin groups, respectively). (C) Treatment with DEC-RVKR-CMK also significantly decreased sATP6AP2 levels compared with the inhibitor vehicle control (both P < 0.0001 in vehicle and forskolin groups). (D) PF 429242 had no effect on sATP6AP2 levels. ANOVA values are reported in the text above each graph and multiple comparisons are represented by # and *, where # denotes a significant difference from the vehicle control, and * a significant difference from the negative control siRNA or inhibitor vehicle control in its respective vehicle- or forskolin-treated group. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA and FURIN siRNA (PsiRNA), DEC-RVKR-CMK (PDEC), PF 429242 (PPF), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 3 experiments in triplicate.
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
BeWo choriocarcinoma cells secrete soluble (pro)renin receptor (sATP6AP2) and its levels decrease with both ATP6AP2 and FURIN inhibition. BeWo cells were treated with either ATP6AP2 siRNA, FURIN siRNA or DEC-RVKR-CMK to inhibit FURIN activity or PF 429242 to inhibit site 1 protease (MBTPS1) activity. Forskolin-induced syncytialisation significantly increased sATP6AP2 levels compared with the vehicle control in all experiments. (A) ATP6AP2 siRNA significantly decreased sATP6AP2 levels compared with the negative control siRNA (both P < 0.0001 in vehicle and forskolin groups). Treatment with (B) FURIN siRNA significantly decreased sATP6AP2 levels compared with the negative control siRNA (P = 0.007 and P = 0.0002 in vehicle and forskolin groups, respectively). (C) Treatment with DEC-RVKR-CMK also significantly decreased sATP6AP2 levels compared with the inhibitor vehicle control (both P < 0.0001 in vehicle and forskolin groups). (D) PF 429242 had no effect on sATP6AP2 levels. ANOVA values are reported in the text above each graph and multiple comparisons are represented by # and *, where # denotes a significant difference from the vehicle control, and * a significant difference from the negative control siRNA or inhibitor vehicle control in its respective vehicle- or forskolin-treated group. Data are presented as median and interquartile ranges. The effect of ATP6AP2 siRNA and FURIN siRNA (PsiRNA), DEC-RVKR-CMK (PDEC), PF 429242 (PPF), forskolin (PForsk), and the interaction between these parameters (PInt) are reported. n = 3 experiments in triplicate.
Citation: Reproduction 162, 5; 10.1530/REP-20-0650
FURIN cleaves sATP6AP2 in BeWo choriocarcinoma cells
FURIN siRNA knockdown was successful (Morosin et al. 2021). FURIN siRNA significantly decreased levels of sATP6AP2 in the supernatant by 23 and 28%, in both the vehicle and forskolin groups, respectively (Fig. 4B; P = 0.007 and P = 0.0002, respectively).
In a separate experiment, BeWo cells were simultaneously treated with forskolin and the broad protease inhibitor, DEC-RVKR-CMK, which inhibits FURIN (Morosin et al. 2021) as well as other proteases. DEC-RVKR-CMK significantly decreased sATP6AP2 levels in the supernatant by 75 and 69% in both the vehicle and forskolin groups, respectively (Fig. 4C; both P < 0.0001).
MBTPS1 does not cleave the sATP6AP2 in BeWo choriocarcinoma cells
BeWo cells were treated with forskolin and the MBTPS1 inhibitor PF 429242, simultaneously. PF 429242 decreased MBTPS1 enzyme activity (Supplementary Fig. 2) but had no effect on sATP6AP2 secretion by BeWo cells (Fig. 4D).
Discussion
Our study is the first to investigate the potential role of ATP6AP2 in choriocarcinoma cells. ATP6AP2 is involved in BeWo choriocarcinoma cell syncytialisation and, therefore, can change its cellular profile. We have shown that forskolin treatment increases BeWo choriocarcinoma cell invasion and that this is inhibited by treatment with ATP6AP2 siRNA. Therefore, ATP6AP2 could be involved in the pathogenesis of choriocarcinoma. Furthermore, sATP6AP2 is secreted by BeWo cells, and sATP6AP2 levels reflect ATP6AP2 expression. Thus, our results indicate that levels of sATP6AP2 could be a measure of the degree of syncytialisation and the invasive potential of choriocarcinomas, which is regulated by ATP6AP2. In choriocarcinoma cells, sATP6AP2 is cleaved by FURIN but not by MBTPS1.
We have shown for the first time that ATP6AP2 is important for syncytialisation of BeWo choriocarcinoma cells. Successful knockdown of ATP6AP2 was associated with a significant reduction in hCG production and a decrease in the percent of nuclei in syncytia (Fig. 2); both are measures of syncytialisation. While the decrease in the percent of nuclei in syncytia did not quite reach significance (at P = 0.05), this decrease in conjunction with the significant decrease in hCG secretion suggests that ATP6AP2 siRNA inhibited syncytiotrophoblast formation, increasing the presence of mononuclear cytotrophoblasts. Interestingly, while it is unlikely that the ATP6AP2 impacts syncytialisation via the tissue RAS (due to BeWo cells having little to no REN expression (Wang et al. 2013)), it is possible that ATP6AP2 could be acting via the Wnt/β-catenin pathway (Matsuura et al. 2011, Müller et al. 2012).
It is clear that while ATP6AP2 does play a role, it is not essential for BeWo choriocarcinoma cell syncytialisation. Syncytialisation is a complex process that is regulated by multiple pathways (Gupta et al. 2016), therefore it would be unreasonable to expect ATP6AP2 to be the sole regulator. Importantly, forskolin is a potent inducer of cyclic-AMP and thereby could mask some effects of ATP6AP2 knockdown on syncytialisation. Finally, because this is a knockdown model, there is still some ATP6AP2 present. Therefore, a knock-out model may show a greater effect of ATP6AP2 in syncytialisation. This may also explain why ATP6AP2 siRNA did not affect E-cadherin protein levels despite it having decreased the percent of nuclei in a syncytium (which was measured using E-cadherin to mark cell boundaries; Fig. 2). As the change in the percent of nuclei is modest with ATP6AP2 knockdown, it may only be impacting the distribution of E-cadherin protein, at this point, rather than its total protein levels. Therefore, while we have shown that ATP6AP2 does play a role in BeWo choriocarcinoma cell syncytialisation, further experimentation using a ATP6AP2 knock-out model is required to determine the full extent to which this occurs.
Our study has also demonstrated that by changing the cellular profile in choriocarcinomas, ATP6AP2 may also be involved in regulating their invasive potential. Alterations in the activity of RAS independent pathways that are stimulated by ATP6AP2 (such as Wnt/β-catenin and ERK1/2/MAPK pathways) have been linked to the progression and pathogenesis of other cancers (Dhillon et al. 2007, Shibayama et al. 2015, Pai et al. 2017, Wang et al. 2019). Specifically, ATP6AP2 has been implicated in the pathogenesis of PDAC and colorectal cancer by regulating cell proliferation and apoptosis, because it activates the Wnt/β-catenin pathway (Shibayama et al. 2015, Wang et al. 2019). However, neither study looked at the role of ATP6AP2 in the invasion. Our data show that the syncytiotrophoblast in choriocarcinoma is invasive and that knockdown of ATP6AP2 inhibits this invasion (Fig. 3). In this way, ATP6AP2 may also be involved in the pathogenesis of choriocarcinoma. However, it is important to note that forskolin (a potent cAMP inducer used to induce syncytialisation in BeWo cells) could also be having effects on invasion via activation of other signalling pathways and not solely through influencing syncytialisation.
To our knowledge, there is no available literature that shows whether the syncytiotrophoblast in choriocarcinomas resembles the early or late syncytiotrophoblast in terms of invasive potential. Specifically, while the syncytiotrophoblast in late pregnancy is not considered invasive (James et al. 2005, Wang & Zhao 2010), there have been reports of initial trophoblast invasion by the syncytiotrophoblast into the endometrium and uterine glands during embryo implantation (Huppertz 2019). We have demonstrated that BeWo choriocarcinoma syncytiotrophoblasts are more representative of the early syncytiotrophoblasts in terms of invasive potential. This is critically important considering most studies of syncytialisation use forskolin-induced syncytialisation of BeWo cells to model physiological placental syncytialisation. This highlights that BeWo cells should only be used to model early trophoblast syncytialisaton and invasion and not syncytialisation at term. Additionally, being a choriocarcinoma cell line, the BeWo model may only reflect pathological responses. This would also explain why primary trophoblasts and BeWo cells respond very differently when induced to syncytialise, with BeWo cells but not primary trophoblast cells (Morosin et al. 2020a) acting via the ATP6AP2 to syncytialise. This is again important, as the two models are often used interchangeably in studies involving syncytialisation.
We showed that sATP6AP2 was secreted by BeWo choriocarcinoma cells (Fig. 4), confirming findings by Suda et al. in JEG and JAR choriocarcinoma cells (Suda et al. 2020). Additionally, we have shown that sATP6AP2 levels in the supernatant decreased correspondingly when ATP6AP2 is knocked down with an siRNA (Fig. 4), indicating that sATP6AP2 levels reflect ATP6AP2 levels in choriocarcinoma. This also supports previous data by Suda et al. in other choriocarcinoma cell lines (Suda et al. 2020) and indicates that sATP6AP2 could be used as a biomarker for ATP6AP2 expression in choriocarcinoma.
In BeWo cells, sATP6AP2 levels increased with syncytialisation. Therefore, as well as providing an index of levels of ATP6AP2 expression, sATP6AP2 levels also reflect the amount of syncytiotrophoblast formed by choriocarcinoma cells. Hence, high levels of sATP6AP2 in choriocarcinoma could reflect syncytialisation and hence indicate increased invasion. To our knowledge, circulating levels of sATP6AP2 in choriocarcinoma patients are unknown. sATP6AP2 levels are, however, increased in other cancers of epithelial origin, including in PDAC (Shibayama et al. 2015). Additionally, these findings are opposite to what we have previously shown in spontaneously syncytialisating primary human trophoblast cells (Morosin et al. 2020b), indicating that sATP6AP2 levels are dysregulated in choriocarcinoma.
Because sATP6AP2 levels could indicate the degree of choriocarcinoma malignancy, we need to know how it is released from these cancer cells. We showed that FURIN, but not MBTPS1, produced sATP6AP2 in BeWo choriocarcinoma cells (Fig. 4), confirming findings by Suda et al. who showed that sATP6AP2 is cleaved by FURIN in JAR choriocarcinoma cells. However, Suda et al. found that MBTPS1 was also involved in sATP6AP2 cleavage, this was not confirmed in our study. These conflicting findings could be the result of hypoxic conditions (1%) used by Suda et al. which could have increased the enzyme activity of MBTPS1 (and FURIN) in JAR cells (Hughes et al. 2005, McMahon et al. 2005).
We used both FURIN siRNA and the broad protease inhibitor, DEC-RVKR-CMK, to determine if FURIN was involved in the formation of sATP6AP2 in choriocarcinoma. While both these treatments decreased sATP6AP2 levels in the supernatant, DEC-RVKR-CMK treatment appeared to have a greater effect than FURIN siRNA. DEC-RVKR-CMK inhibits the activity of pro-protein convertases 1 through to 7 (pro-protein convertase 3 is FURIN) (Couture et al. 2011). Thus, while both treatments inhibited FURIN enzyme activity, DEC-RVKR-CMK may have also inhibited other proteases responsible for sATP6AP2 cleavage.
In other studies in our laboratory using the term primary trophoblast cells, we have shown that another protease inhibited by DEC-RVKR-CMK was responsible for sATP6AP2 cleavage and FURIN was not involved (Morosin et al. 2020b). FURIN expression is increased in multiple cancers, including lung adenocarcinoma (Mbikay et al. 1997). Thus, it is reasonable to postulate that because choriocarcinoma is also epithelial in origin, FURIN expression is increased. Further research is needed to determine whether or not this explains why FURIN cleaves sATP6AP2 in choriocarcinoma but not healthy primary trophoblasts.
In conclusion, we have shown that ATP6AP2 plays some role in syncytialisation of BeWo choriocarcinoma cells. We have also demonstrated that the syncytiotrophoblast in choriocarcinoma is invasive and that invasion is reduced when ATP6AP2 is knocked down. We propose that ATP6AP2 may, therefore, be associated with the invasive potential of choriocarcinomas, because it is involved in choriocarcinoma cell differentiation, from cytotrophoblast cells to invasive syncytiotrophoblast. We have shown that BeWo cells secrete sATP6AP2 and that this reflects both ATP6AP2 levels and syncytialisation. We, therefore, propose that sATP6AP2 could be used as a biomarker to measure the invasive potential of choriocarcinoma (i.e. the higher the levels the more invasive the tumour). Finally, we have shown that FURIN, and not MBTPS1, is responsible for sATP6AP2 cleavage in choriocarcinoma but other proteases (inhibited by DEC-RVKR-CMK) may also be involved.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/REP-20-0650.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Health and Medical Research Council, Australia (APP1043537 and APP1161957) and the John Hunter Charitable Trust. K G P was supported by an Australian Research Council Future Fellowship (FT150100179). S K M was supported by an Australian Government Research Training Programme Scholarship and is now supported by Hunter Medical Research Institute researcher bridging funds.
Author contribution statement
S K M made substantial contributions to experimental design and data acquisition, analysis, and interpretation. S K M wrote the initial drafts of the manuscript. S J D, R G S K, and C C M made substantial contributions to experimental design, data acquisition, analysis and interpretation, and in subsequent revising of drafts of the manuscript. K G P and E R L had substantial contributions to the experimental conception and design, interpretation of data, and revising drafts of the manuscript. All authors approved the final version of the article to be published.
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