Research ArticleExperimental Studies

The Complement C3a–C3a Receptor Axis Regulates Epithelial-to-Mesenchymal Transition by Activating the ERK Pathway in Pancreatic Ductal Adenocarcinoma

REI SUZUKI, YOSHINORI OKUBO, TADAYUKI TAKAGI, MITSURU SUGIMOTO, YUKI SATO, HIROKI IRIE, JUN NAKAMURA, MIKA TAKASUMI, TSUNETAKA KATO, MINAMI HASHIMOTO, RYOUICHIRO KOBASHI, TAKUTO HIKICHI and HIROMASA OHIRA
Anticancer Research March 2022, 42 (3) 1207-1215; DOI: https://doi.org/10.21873/anticanres.15587

Abstract

Background: We aimed to clarify the role of complement C3a and its receptor C3aR in progression of pancreatic ductal adenocarcinoma (PDAC). Materials and Methods: We evaluated the serum levels of C3 and C3a in patients with PDAC. C3aR expression in tissue was assessed using a tissue microarray. To confirm the protumoral effects of C3a in PDAC, we conducted in vitro experiments using PDAC cell lines (Panc-1 and MiaPaca-2) that exhibit high C3aR expression. Results: Serum levels of both C3 and C3a were higher in 26 patients with PDAC than in 28 nontumor-bearing controls. In the tissue microarray, we observed increased expression of C3aR in PDAC cells, especially in cases with metastatic lesions. In vitro experiments showed that C3a facilitated tumor cell proliferation, migration and invasion by activating the extracellular-regulated kinase signaling pathway and inducing epithelial-to-mesenchymal transition. Inhibition of the C3a-C3aR axis by pharmacological blockade and short-hairpin RNA-mediated knockdown of C3aR alleviated its protumoral effect. Conclusion: These findings provide a new approach for the development of treatments targeting the C3a–C3aR axis.

Key Words:

Pancreatic ductal adenocarcinoma (PDAC) is the seventh leading cause of cancer-associated death worldwide (1). The incidence of PDAC continues to increase but improvement in oncological outcomes of advanced PDAC is limited (2, 3). To develop new treatments, researchers have focused on exploring the mechanisms essential for the progression of PDAC.

Systemic and focal inflammation have been considered significant factors in the progression of PDAC (4). The complement system is acknowledged as a factor involved in innate and acquired immunity that play a role in the first-line defense in the detection and removal of invading pathogens. It exists in its inactive form under normal conditions but becomes activated by pathogens via three different cascades (classical, alternative and lectin). The most important complement proteins, C3a and C5a, are generated during cleavage and act as anaphylatoxins, which interact with their respective G protein-coupled signaling receptors (C3aR and C5aR), promote immune cell migration, and activate cell-specific effector functions (5).

Recently, new aspects of complement systems have drawn attention. Cancer cells have been shown to express complement receptors on their surface, and C3a and C5a can act as cytokines to induce cancer progression. Previous studies have demonstrated protumoral effects of the C5a– C5aR axis in gastric cancer, lung cancer and others (6-9), and others have evaluated the role of the C3a–C3aR axis in cancer progression (10-12). With regard to pancreatic cancer, we were only able to find one study reported by Aykut et al. In their study, they illustrated that fungal microbes promoted pancreatic oncogenesis and tumor progression via activation of mannose-binding lectin–C3a–C3aR axis utilizing transgenic mice: however, the role of the C3a–C3aR axis in tumor progression in human cancer cell and related cell signaling pathway was not described (13). Therefore, we aimed to clarify the role of complement C3a and its receptor C3aR in pancreatic cancer progression in this study.

Materials and Methods

Patients and sample collection. We enrolled 26 patients with PDAC and 28 nontumor controls (seven patients with autoimmune pancreatitis, two with chronic pancreatitis and 10 biliary stones) in our study (Table I). All patients with PDAC underwent endoscopic ultrasound-guided fine-needle aspiration biopsy to confirm the diagnosis. The study protocol was approved by the Institutional Review Board of Fukushima Medical University (#2387). All participants provided written informed consent.

View this table:
Table I.

Comparison of the clinical and demographic parameters of the patients.

Patient clinical and demographic data, including age; white blood cell, neutrophil and lymphocyte counts; serum C-reactive protein level; serum carcinoembryonic antigen level; and serum cancer antigen 19-9 (CA19-9) level, were extracted from electronic medical records. Enzyme-linked immunosorbent assay kits were used to measure serum levels of C3 (HK366-01; Hycult Biotech, Uden, The Netherlands) and C3a (HK354-01; Hycult Biotech) according to the manufacturer’s protocols.

The TNM stage was determined according to the American Joint Committee on Cancer/Union for International Cancer staging system, version 8 (14, 15). For survival analysis, the overall survival of 16 patients with PDAC treated with the combination of gemcitabine and nab-paclitaxel was defined as the period from the date of initiation of chemotherapy to the date of either death or the last follow-up examination.

For serum collection, 8 ml of blood was collected and incubated at room temperature for at least 60 minutes to allow clotting. Samples were then centrifuged at 1,000 × g for 10 minutes. The serum was collected and stored in aliquots at –80°C.

Cell lines and reagents. Commercially available cell lines (HPNE, Panc-1, MiaPaca-2 and BxPC-3) were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in RPMI 1640 containing the same supplements. All cell lines were cultured in a humidified atmosphere containing 5% CO2 at 37°C and were grown to 70% to 80% confluence in 10-cm culture dishes prior to their use in experiments. Human recombinant C3a protein was purchased from R&D Systems (Minneapolis, MN, USA), and the C3aR antagonist SB290157 was purchased from Cayman Chemical Industry (Ann Arbor, MI, USA).

Tissue microarray (TMA) and immunohistochemical staining. A TMA containing 29 pancreatic cancer tissues was purchased from US Biomax (catalog number: HPanA060CS03, Derwood, MD, USA). A 2-mm core with a 4-μm thickness was obtained from pancreatic cancer and adjacent normal pancreatic tissues from one patient and arrayed onto a glass slide. Immunohistochemical staining was performed according to a procedure described previously (16). The TMA slides were deparaffinized, rehydrated, and heated in a pressure cooker filled with sodium citrate buffer (pH 6.0) for antigen retrieval, after which the slides were treated with 3% H2O2 for 30 minutes at 37°C to block endogenous peroxidase activity. Immunohistochemistry was performed using antibodies targeting C3aR (dilution 1:100; Bioss Antibodies, Beijing, PR China). The percentages of tumor cells and normal pancreatic duct cells exhibiting immunoreactivity TMA slide were evaluated at 400× magnification. The staining intensity was defined based on the percentage of positively stained PDAC cells and normal pancreatic duct epithelium with 0, 1, 2 and 3, denoting negative, weak, moderate and strong staining, respectively. The final histoscore was calculated as follows: (1× % weakly positive tumor cells) + (2× % moderately positive tumor cells) + (3× % strongly positive tumor cells), with a maximum histoscore of 300 (17).

Cell proliferation assay and viability staining. Panc-1 and MiaPaca-2 cells were seeded at a density of 3,000 cells in 200 μl of medium supplemented with 10% fetal bovine serum (FBS) in a 96-well plate. Cells in each well were treated with combinations of 0.1 or 0.2 μM C3a, in the presence or absence of C3aR1 short-hairpin RNA (shRNA) treatment or with 1 μM SB290157. After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg/ml) was added to each well, and the optical density of each well at 590 nm was measured with a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) (18).

Cell migration and invasion assay. Migration and invasion assays were conducted as previously described (19). The cell migration assay was performed using 8.0-μm pore size Millicell Hanging Cell Culture Inserts (Millipore, Volketswil, Switzerland). After preincubation with RPMI 1640 medium for 30 minutes at 37°C, the lower chambers were filled with 650 μl of RPMI 1640 medium containing 10% FBS. Subsequently, PDAC cells (Panc-1, MiaPaca-2) at a density of 2.5×104 cells per well in 500 μl of serum-free RPMI-1640 medium were seeded into the upper chambers of the inserts and cultured for 24 hours at 37°C in 5% CO2. Invasion assays were conducted in 24-well cell culture dishes using specialized inserts containing a polycarbonate membrane with an 8-μm pore size (CORNING Inc., Corning, NY, USA) that was coated with Matrigel. PDAC cells (2.5×104 per well) in 500 μl of serum-free RPMI-1640 medium were seeded in the upper chambers, and the lower chambers were filled with 650 μl of RPMI-1640 medium containing 10% FBS. The cells were allowed to invade the Matrigel for 48 hours. In both experiments, the cells in each well were treated with 0.1-0.2 μM C3a in the presence or absence of either C3aR1 shRNA or 1 μM SB290157. After incubation, cells remaining in the upper chamber were scraped off the membrane with cotton swabs, and migrating and invading cells were fixed in menthol, stained with crystal violet, and counted in three adjacent microscopic fields for each membrane at a magnification of ×200.

Lentivirus-mediated transfection of pancreatic cancer cell lines. Enhanced Green Fluorescent Protein (EGFP)-tagged short-hairpin RNA (shRNA) expression vectors targeting C3aR1 were generated and cloned into recombinant lentivirus particles [pLV(shRNA)-EGFP:T2A: Puro-U6>hC3aR1 (shRNA1), pLV(shRNA)-EGFP:T2A:Puro-U6>h C3aR1 (shRNA2) and pLV(shRNA)-EGFP:T2A:Puro-U6>hC3aR1 (shRNA3)] (VectorBuilder, Guangzhou, ROC). As a negative control, we used pLV[shRNA]-EGFP:T2A:Puro-U6>Scramble (vector ID: VB010000-0009mxc). The indicated cell lines were transfected with shRNA using lentivirus containing the expression vectors and polybrene (5 μg/ml). We purchased all related reagents from VectorBuilder. After overnight incubation, the medium containing the lentivirus particles was removed, and puromycin (1.5 μg/ml) was added to select stably infected cells. After 48 hours, the infection efficiency was determined by visualizing the expression of the fluorescent marker GFP. Final confirmation of the down-regulation of expression of C3aR was conducted by polymerase chain reaction (PCR) and flow cytometry.

Extraction of total RNA. Total RNA was extracted from cultured cells using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and total RNA (including miRNAs) was extracted using an miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocols (18). cDNA was prepared from 500 ng of total RNA using an iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). Sample purity was determined by measuring the ratio of absorbance at 260/280 nm, the values of which for all total RNA samples were >1.8.

Quantitative real-time PCR. One microliter of cDNA was used as a template in a 20-μl PCR. The PCR products were amplified using specific primers (TaqMan MiRNA Assay) and TaqMan Universal PCR Master Mix II (Applied Biosystems, Foster City, CA, USA) and detected using the StepONE Plus Real Time PCR System (Applied Biosystems). Each sample was run in triplicate. TaqMan probes for miRNA detection of the following genes in cultured cells were obtained from Applied Biosystems: C3aR1 (Hs00269693_s1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs02786624_g1). The results were analyzed using the comparative cycle threshold (ΔΔCt) method.

Western blot analysis. Cells were washed twice with phosphate-buffered saline and scraped from the plates in RIPA buffer (50 mM Tris HCl (pH 8), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) containing a protease inhibitor cocktail (Thermo Fisher Scientific). Proteins were electrophoresed in sample buffer through acrylamide gels and then transferred to polyvinylidene difluoride membranes (Clear Blot Membrane-P; ATTO Co., Ltd., Tokyo, Japan). Membranes were blocked with 0.5% Tris buffered saline with Tween 20 containing 3% nonfat milk, incubated with primary antibodies (1:1,000) overnight at 4°C, and then incubated with horseradish peroxidase-conjugated anti-rabbit or mouse secondary antibodies (1:3000; Sigma–Aldrich, St. Louis, MO, USA). Blots were visualized using the ECL Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). β-Actin and vinculin served as loading controls (18). Band densitometric analysis was performed with National Institutes of Health ImageJ software (20). Antibodies against C3aR (BS-2955R) were purchased from Bioss, and antibodies against phospho-AKT serine/threonine kinase 1 (AKT) (#9271S), total AKT (#9272), phospho-extracellular-signal regulated kinase 1/2 (p-ERK1/2) (#197G2), total ERK1/2 (#137F5), p-c-rapidly accelerated fibrosarcoma kinase RAF (#9421T), total-c-RAF (#9422T), phosphomitogen-activated protein kinase kinase 1/2 (MEK1/2) (#9154T), total MEK1/2 (#8727T), snail family transcriptional repressor 1 (SNAI1) (#3879T), α-smooth muscle actin (#14968), vinculin (#4650S) and β-actin (#3700) were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma–Aldrich.

Flow cytometry. After lentivirus-mediated shRNA transduction, the cells were harvested in 10-cm dishes at a cell density of 1-2×106 cells/well. Harvested cells were trypsinized and resuspended in 500 μl of FACS buffer (RPMI 1640 without phenol red+3% fetal calf serum + 0.1% sodium azide). The cell pellets were washed three times with FACS buffer, and anti-human C3aR antibody conjugated with phycoerythrin/cyanin7 (#345808, BioLegend, San Diego, USA) was added to the suspension. After another 3 washes, C3aR expression on the cell surface was analyzed by flow cytometry (FACS Canto II; BD Biosciences, San Jose, CA, USA).

Analysis of RNA-seq data. The mRNA-seq data for pancreatic tissue from 178 patients were obtained from the Cancer Genome Atlas (TCGA) database (https://tcga-data.nci.nih.gov/tcga/). Normalized mRNA expression data (the calculated expression for all reads aligning to a particular mRNA per sample) were collected from TCGA Data Portal using the Subio platform version 1.21 (Kagoshima, Japan). Clinical data, including prognosis and disease stage, were also downloaded for each patient.

Statistical analysis. All results are presented as raw data or as medians with ranges for nonparametric data and means±SD for parametric data. To compare continuous variables, Student’s t-test and the Mann–Whitney U-test were performed. Correlations between the serum levels of C3 and C3a and other clinical characteristics were evaluated using Spearman’s correlation analysis. The median overall survival after initial chemotherapy was calculated using the Kaplan–Meier method and compared with the log-rank test. All statistical analyses were conducted in GraphPad Prism 9.1.2 (GraphPad, San Diego, CA, USA). A two-tailed p-value of less than 0.05 was considered statistically significant.

Results

High serum C3 and C3a levels in patients with PDAC. There were no significant differences between PDAC and nontumor controls with respect to age, white blood cell counts, lymphocyte counts and C-reactive protein, but the serum neutrophil counts and levels of carcinoembryonic antigen and CA19-9 were higher in patients with PDAC (Table I). Serum levels of C3 and C3a were higher in patients with PDAC than in the nontumor controls (p=0.04 and p=0.009, respectively; Figure 1A). With regard to metastasis, serum levels of C3 and C3a were higher in cases with metastasis (p=0.02 and p=0.03; Figure 1B). In the survival analysis of 16 patients who underwent palliative chemotherapy with the combination of gemcitabine and nab-paclitaxel, those with high median serum levels of C3 had a poorer prognosis than those with low levels (12 vs. undefined months, p=0.02) (Figure 1C). In the correlation analysis, serum levels of C3 and C3a were negatively related to age (C3: r=–0.55, p=0.003; C3a: r=–0.62, p=0.0007), while no significant correlation was observed for other indices of systemic inflammation and tumor markers (Table II).

Figure 1.
Figure 1.

Serum levels of complement C3 and C3a in the patients. A: Serum levels of C3 and C3a were higher in patients with pancreatic ductal adenocarcinoma (PDAC) than in nontumor control patients. B: Among patients with PDAC, serum levels of C3 and C3a were higher in cases with metastasis (M+) than cases without metastasis (M–). C: The serum level of C3, but not C3a, predicted the overall survival of patients who underwent systemic chemotherapy. The data are presented as the means±standard deviation. Significantly different at *p<0.05 and **p<0.01.

View this table:
Table II.

Correlation between the complement levels and other clinicopathological data in 26 patients with pancreatic ductal adenocarcinoma.

C3aR expression in PDAC tissue. In a TMA comprising pancreatic cancer tissue, we observed that the intensity of C3aR expression was higher in PDAC tissues than in normal adjacent tissues (p=0.0004) (Figure 2A). Moreover, the expression intensity score was higher in patients with metastasis than in those without metastasis (p=0.01; Figure 2B).

Figure 2.
Figure 2.

C3a receptor (C3aR) expression in pancreatic ductal adenocarcinoma (PDAC). A: Representative images of C3aR expression in a tissue microarray of PDAC tissue (magnification, ×200). B: Tissue expression of C3aR was higher in PDAC tissues than in normal adjacent tissues and in cases with metastasis. Expression of C3aR was detected by real-time polymerase chain reaction (C) and western blot (D). Treatment with human recombinant complement C3a enhanced the proliferation (E), migration (F) and invasion (G) of PDAC cells (magnification, ×200). H: C3a enhanced expression of extracellular regulated kinase (ERK), α-smooth actin (α-SMA) and snail family transcriptional repressor 1 (SNAI1). Significantly different at *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. The data are presented as the means±SD.

To clarify the role of C3aR expression and its ligand, C3a, in tumor progression, we conducted in vitro analysis using pancreatic cancer cell lines. In the PCR and western blot analyses, Panc-1 and MiaPaca-2 cells showed higher expression of C3aR than did the other cell lines (Figure 2C and D). Subsequently, our studies focused on Panc-1 and MiaPaca-2 cells, which we then treated with recombinant human C3a and evaluated its effects on tumor proliferation, migration and invasion. In the proliferation assay, we found that C3a promoted tumor proliferation in a dose-dependent manner in Panc-1 cells and MiaPaca-2 cells (Figure 2E). Similarly, C3a enhanced tumor migration and invasion (Figure 2F and G).

We further evaluated cell signaling pathways influenced by C3a stimulation and found that the activity of the ERK, but not the AKT, was enhanced by C3a stimulation. Furthermore, C3a induced expression of SNAI1 and α-smooth muscle actin. These results suggested that C3a plays a role in tumor growth and epithelial-to-mesenchymal transition (EMT) by potentiating ERK activation (Figure 2H).

The role of the C3a–C3aR axis in PDAC progression. To elucidate whether C3a activated the signaling pathway via C3aR in pancreatic cancer progression, we established cells with stable C3aR knockdown using lentivirus-mediated shRNA transduction; both cell lines exhibited successful C3aR knockdown (Figure 3A and B). C3aR knockdown reduced the effect of C3a on cell proliferation, migration and invasion (Figure 3C-E). Additionally, we utilized the C3aR antagonist SB290157 to evaluate whether pharmacological blockade of C3aR induced similar effects as those of C3aR shRNA knockdown. The results suggest that 1 μM SB290157 itself did not have the ability to influence tumor progression but did block the effect of C3a (Figure 3). These results demonstrated that the C3a–C3aR axis might play a role in the progression in pancreatic cancer.

Figure 3.
Figure 3.

The role of the C3a-C3a receptor (C3aR) axis in pancreatic ductal adenocarcinoma (PDAC) progression. A and B: Cells with stable C3aR knockdown via lentivirus-mediated shRNA transduction were established, and the reduction in C3aR expression was confirmed by real-time polymerase chain reaction and flow cytometry. C3aR knockdown reduced the effect of C3a on cell proliferation (C), migration (D) and invasion (E) (magnification, ×200). SB290157 (1 μM), a C3aR antagonist, blocked the effect of C3a (0.2 μM) on cell proliferation (F), migration (G) and invasion (H) (magnification, ×200). ns; not significant, CTL; nontreated cell control. Significantly different at *p<0.05, **p<0.01 and ***p<0.001. The data are presented as the means±SD.

Discussion

In the present study, we discovered that C3aR expression and C3a stimulation promoted the proliferation, migration and invasion of pancreatic cancer cells via activation of the ERK signaling pathway, which increases the expression of SNAI1 and eventually induces EMT. To the best of our knowledge, this is the first study to clarify the biophysiological role of the C3a–C3aR axis in pancreatic cancer progression. Additionally, shRNA knockdown or blockade of C3aR alleviated the effect of C3a in cancer progression, which might serve as a basis for the development of novel treatments targeting the C3a–C3aR axis.

The complement system is renowned for its immune response against invading pathogens (21). Considering its role in the immune system, a number of studies have illustrated its role as a defense mechanism against cancer (22). The complement system is considered to cause damage to cancer cells by direct or indirect mechanisms. Activated complement cascades can cause cleavage of C5 to form C5a and C5b, the latter of which forms a complex with other complements and generates the membrane attack complex. Deposition of an adequate number of membrane attack complexes on the cell surface can disrupt the phospholipid layer of the cell membrane and result in cell lysis. Moreover, complement can facilitate phagocytosis of opsonized cancer cells by macrophages and neutrophils. However, accumulated evidence has highlighted an opposing role of the complement system in cancer. Complement proteins (e.g., C3, C3d, C5b-9, C1q, mannan-binding lectin, mannose-binding lectin-associated serine protease, and factor H) are detectable in tumors and blood and have been suggested to be related to tumor progression (23-28). More recently, the interaction between the anaphylatoxin C5a and its receptor C5aR were found to elicit a protumor effect by promoting tumor cell growth, increasing tumor cell motility and invasiveness, and recruiting immunosuppressive cells (e.g., myeloid-derived suppressor cell) to the tumor microenvironment (29, 30). Regarding C3 and C3a, several studies have reported similar protumoral roles in various cancer types, including melanoma, ovarian cancer and colorectal cancer (31-33). In pancreatic cancer, Andoh et al. first reported that pancreatic cancer cells produced C3 under the regulation of transforming growth factor-beta (34, 35), and Chen et al. evaluated the correlation between intratumoral C3 expression and clinical characteristics (36). In this study, they assessed the expression levels of C3 and other complement proteins in the various stages of pancreatic cancer using immunohistochemistry, PCR and western blotting. They found that the expression levels of complement C3 were higher in PDAC than in normal pancreatic tissues. More recently, Aykut et al. found that mannose-binding lectin–C3a–C3aR axis was activated by Malassezia species in mouse PDAC. However, to our knowledge, no study has ever addressed the role of C3a and C3aR in human PDAC or elucidated related cell signaling pathways for tumor progression (13).

The present study has limitations. Firstly, this study was conducted at a single institution with a small number of patients, which suffers from a lack of variety in the clinical background of patients in the nontumor control group. Secondly, the TMA slides were purchased from a vendor, and the only clinical information available with the slides was tumor stage. Finally, we did not conduct animal experiments to confirm our hypothesis speculated from the results of the clinical and in vitro experiments. Therefore, these data need to be verified with a study comprising a large number of patients with various clinical backgrounds and with in vivo animal experiments.

In conclusion, we provide the first evidence that the C3a– C3a receptor axis regulates EMT by activating the ERK pathway in pancreatic cancer. The findings provide a new approach for the development of treatments targeting the C3a–C3aR axis.

Acknowledgements

The present study was supported by JSPS KAKENHI Grant Number 20K16419.

Footnotes

  • Authors’ Contributions

    RS conceived the study idea and designed the study. RS and YO conducted the experiments. RS, YO, TT, MS, YS, HI, JN, MT, TK, MH, RK, TH and HO took part in patient care and provided important intellectual input. All Authors contributed to article revision, read, and approved the submitted version.

  • Conflicts of Interest

    The Authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

  • Received December 28, 2021.
  • Revision received January 14, 2022.
  • Accepted January 18, 2022.

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The Complement C3a–C3a Receptor Axis Regulates Epithelial-to-Mesenchymal Transition by Activating the ERK Pathway in Pancreatic Ductal Adenocarcinoma
REI SUZUKI, YOSHINORI OKUBO, TADAYUKI TAKAGI, MITSURU SUGIMOTO, YUKI SATO, HIROKI IRIE, JUN NAKAMURA, MIKA TAKASUMI, TSUNETAKA KATO, MINAMI HASHIMOTO, RYOUICHIRO KOBASHI, TAKUTO HIKICHI, HIROMASA OHIRA
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