Research ArticleExperimental Studies

Intracellular C3 Modulates EMT via the Akt/Smad Pathway in Pancreatic Cancer Cells

REI SUZUKI, TADAYUKI TAKAGI, MITSURU SUGIMOTO, YUKI SATO, HIROKI IRIE, YUKI YOMOGIDA and HIROMASA OHIRA
Anticancer Research December 2022, 42 (12) 5743-5750; DOI: https://doi.org/10.21873/anticanres.16081

Abstract

Background/Aim: We aimed to explore the role of intracellular C3 in pancreatic ductal adenocarcinoma (PDAC). Materials and Methods: We evaluated C3 expression in PDAC using a gene expression database and tissue microarray. To clarify the role of C3 expression in PDAC, we conducted knockdown experiments using C3 short hairpin RNA (shRNA) in BxPC-3 cells. Differences in protein expression and cell behaviours were analysed. Results: C3 was highly expressed in PDAC and correlated with cancer metastasis. In vitro experiments using BxPX-3 cells showed that C3 and its active form, C3a, were expressed in tumour cells. C3 knockdown reduced cell migration and invasion by inhibiting Akt/Smad pathway activation. TNF-α, not IL-6, enhanced C3 expression in this PDAC cell line. Conclusion: Intracellular C3 may regulate epithelial-mesenchymal transition in pancreatic cancer.

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 improvements in the oncologic outcomes of patients with advanced PDAC have been limited (2, 3). To develop new treatments, researchers have focused on exploring the mechanisms essential for the progression of PDAC.

Systemic and local inflammation have been considered significant factors in the progression of PDAC (4, 5). The complement system is part of the innate and acquired immunity, where it plays a role in the first line of defence against pathogens. It exists in its inactive form under normal conditions but becomes activated by pathogens via three different pathways. Due to its defensive role against pathogens, the complement system has been considered to have an antitumor effect against cancer progression. However, recent studies have revealed protumour effects of the complement system in various cancers (6, 7). In our recent study, we reported novel roles of C3a and the C3a receptor (C3aR) in PDAC. In this study, we found that the C3a-C3aR axis might be related to the activation of the Erk pathway and epithelial-to-mesenchymal transition (EMT) (8). More recently, several studies reported a multifunctional role of “intracellular” complement in cancer (9-11). However, the biophysiological roles of intracellular C3 expression in PDAC remain unknown. Therefore, in this study, we aimed to clarify the role of intracellular complement C3 in PDAC progression.

Materials and Methods

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 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with heat-inactivated 10% fetal bovine serum (FBS) (12). All cell lines were cultured in a humidified atmosphere containing 5% CO2 at 37°C and grown to 70% to 80% confluence in 10-cm culture dishes prior to their use in experiments (13).

Extraction of total RNA. Total RNA was extracted from cultured cells using a RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocols. cDNA was prepared from 500 ng of total RNA using an iScript™ Advanced cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Sample purity was determined by measuring the A260/A280 ratio, which was >1.8 for all total RNA samples (14).

Quantitative real-time PCR. One microlitre of cDNA was used as the template in a 20-μl reaction volume for PCR. 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 a StepONE Plus Real Time PCR System (Applied Biosystems). Each sample was run in triplicate. TaqMan probes for detection of the following mRNAs in cultured cells were obtained from Applied Biosystems: C3 (Hs00163811_m1), IL-6 (Hs00174131_m1), TNF-α (Hs00174128_m1) and GAPDH (Hs02786624_g1). Relative expression levels were calculated using the comparative cycle threshold (ΔΔCt) method (15).

Western blot analysis. Cells were washed twice with phosphate-buffered saline (PBS) and harvested by scraping the plates in RIPA buffer [50 mM Tris HCl (pH 8), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS] containing protease inhibitor cocktail (Thermo Fisher Scientific, Carlsbad, CA, USA). Proteins were electrophoresed in sample buffer on acrylamide gels and then transferred to polyvinylidene difluoride membranes (Clear Blot Membrane-P; ATTO Co., Ltd., Tokyo, Japan). The membranes were blocked with 0.5% PBS with Tween 20 (PBS-T) containing 3% non-fat milk, incubated with primary antibodies (1:1,000) overnight at 4°C, and then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies (1:3,000; Sigma–Aldrich). The blots were visualized using an ECL western blotting Detection System (GE Healthcare, Buckinghamshire, UK). GAPDH served as a loading control (13). Band densitometric analysis was performed with NIH ImageJ software. Antibodies against p-Akt (#9271S), total Akt (#9272), p-Erk1/2 (#4377S), total Erk1/2 (#4695S), p-mTor (#8828S), total mTor (#2983S), p-Smad2/3 (#8828S), and total Smad2/3 (#8685S) were purchased from Cell Signaling Technology (Boston, MA, USA). The anti-β-actin antibody (#MA5-15739) was purchased from Invitrogen (Gaithersburg, MD, USA). Anti-mouse and anti-rabbit secondary antibodies were purchased from Sigma–Aldrich.

Tissue microarray and immunohistochemical staining. A tissue microarray (TMA) containing 29 pancreatic cancer tissues was purchased from US Biomax (catalogue number: HPanA060CS03, Rockville, MD, USA). A 2-mm core with a thickness of 4 μm was obtained from pancreatic tumour and adjacent normal pancreatic tissues from each patient and arrayed onto a glass slide. Immunohistochemical staining was performed according to a previously described procedure (16). The TMA slides were deparaffinized, rehydrated, and heated in a pressure cooker filled with sodium citrate buffer (pH 6.0) for antigen retrieval and were then treated with peroxide (3% H2O2) for 30 min at 37°C to block endogenous peroxidase activity. Immunohistochemical staining was performed using an antibody specific for C3 (1:100 dilution; Abcam, ab200999). The percentages of tumour cells and normal pancreatic duct cells exhibiting immunoreactivity on the TMA slides 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 scores of 0, 1, 2 and 3, denoting negative, weak, moderate, and strong staining, respectively. The final histoscore was calculated as follows: (1× % weakly positive tumour cells) + (2× % moderately positive tumour cells) + (3× % strongly positive tumour cells). The maximum histoscore was thus 300 (17).

Immunocytochemical staining. BxPC-3 cells (2.5×104 cells per well) were cultured in a 4-well chamber in RPMI 1640 medium supplemented with 10% FBS. After a 24-h incubation, the cells were fixed with 4% paraformaldehyde solution for 10 min at room temperature. The slides were washed twice with PBS-T, and the cells were permeabilized with 0.1% Triton X-100 for 10 min. After two more washes with PBS-T, the cells were blocked with PBS-T containing 1% bovine serum albumin (BSA) for 30 min (13). Then, the cells were incubated overnight at 4°C with primary antibodies specific for C3 (ab200999, 1:100), C3a (AF3677-SP, 1:100) and β-actin (#MA5-15739, 1:100) diluted in PBS-T with 1% BSA. Anti-goat Alexa Fluor® 647 (ab150135), anti-rabbit Alexa Fluor® 488 (ab150077), and anti-mouse Alexa Fluor® 488 (ab150113) secondary antibodies were added after decanting the solution and washing the cells with PBS three times. For nuclear counterstaining, DAPI (ab104139) solution was applied to the slides for 5 min. Images were acquired using fluorescence digital microscopy (BZ-X810, Keyence Corporation, Osaka, Japan).

Cell migration and invasion assays. Migration and invasion assays were conducted as previously described (8). The cell migration assay was conducted by using Millicell Hanging Cell Culture Inserts with an 8.0-μm pore size membrane (Millipore). The invasion assay was conducted in 24-well cell culture dishes using specialized inserts containing a Matrigel-coated polycarbonate membrane with an 8.0-μm pore size (Corning). After preincubation with RPMI 1640 medium for 30 min at 37°C, the lower chambers were filled with 650 μl of RPMI 1640 medium containing 10% FBS. Subsequently, BxPC-3 PDAC cells (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 h at 37°C in 5% CO2. After incubation, the non-migrated and invaded cells remaining in the upper chamber were scraped off the membrane with cotton swabs, and the migrated and invaded cells were fixed with methanol, stained with crystal violet, and counted in three adjacent microscopic fields per membrane at 200× magnification.

Lentivirus-mediated shRNA transduction in pancreatic cancer cell lines. EGFP-tagged short hairpin RNA (shRNA) expression vectors targeting C3 were generated and cloned into recombinant lentiviral particles (pLV(shRNA)-EGFP:T2A:Puro-U6>hC3 (shRNA1), pLV(shRNA)-EGFP:T2A:Puro-U6>hC3 (shRNA2)). As a negative control, we used pLV(shRNA)-EGFP:T2A:Puro-U6>Scramble (Vector ID: VB010000-0009mxc). BxPC-3 cells were transduced with these shRNAs using lentivirus containing the corresponding expression vectors in the presence of polybrene (5 μg/ml). We purchased all related reagents from VectorBuilder (18). After overnight incubation, the medium containing the lentiviral particles was removed, and puromycin (1.5 μg/ml) was added to select stably transduced cells. After 48 h, the transduction efficiency was determined by visualizing the expression of the fluorescent marker GFP. Final confirmation of the down-regulated expression of C3 was conducted by PCR.

Gene expression analysis. Expression profiles by microarrays were obtained from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) (19). The mRNA-seq data for pancreatic tissue from 178 patients were obtained from The Cancer Genome Atlas (TCGA) database (20). Normalized mRNA expression data (the calculated expression values for all reads aligning to a particular mRNA in each sample) were collected from the TCGA Data Portal using Subio Platform version 1.21 (Kagoshima, Japan). Clinical data, including prognosis and disease stage, were also downloaded for each patient (14).

Statistical analysis. All results are presented as raw data or as medians with ranges for nonparametric data and means±SDs for parametric data. To compare continuous variables, Student’s t-test and the Mann–Whitney U-test were performed. Correlations between C3 expression and cytokine expression were evaluated using Spearman correlation analysis. Median overall survival times obtained by analysis of TCGA data were 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 <0.05 was considered statistically significant (21).

Results

C3 expression in PDAC tissue. Analysis of C3 gene expression levels in the GDS4103 dataset (PDAC and paired normal tissue samples from 36 patients) showed higher median C3 expression in PDAC tissue than that in normal tissue (12.3 vs. 9.2, p<0.001) (Figure 1A). In a TMA containing pancreatic cancer tissues, we observed that the intensity of C3 expression was higher in PDAC tissues than that in normal adjacent tissues (p=0.03) (Figure 1B). Moreover, the expression intensity score was higher in patients with metastasis than that in those without metastasis (p=0.04). Survival analysis using TGCA data showed that the median survival time in the group with high C3 expression (C3 high; 20.1 months) was worse than that in the group with low C3 expression (C3 low; 24.6 months), but the difference was not statistically significant (p=0.06) (Figure 1C).

Figure 1.
Figure 1.

Expression of C3 in pancreatic ductal adenocarcinoma (PDAC). (A) C3 gene expression was higher in PDAC tissues than that in normal adjacent tissues. (B) The C3 expression intensity score in the TMA was higher in PDAC tissues than that in normal adjacent tissues. Additionally, the C3 expression intensity score was higher in PDAC tissues in patients with metastasis than that in those without metastasis. The TMA contained samples from 29 cases of PDAC with matched normal adjacent tissues. (C) Prognostic analysis using TCGA data.

Localization and biophysiological roles of C3 in PDAC. To clarify the role of C3 expression in tumour progression, we conducted in vitro analyses using the BxPC-3 PDAC cell line. In the PCR and western blot analyses, BxPC-3 showed higher expression of C3 than did the other cell lines, including the immortalized normal human pancreatic ductal cell line (HPNE) (Figure 2A and B). Immunocytochemical staining revealed intracellular expression of C3 and its activated fragment C3a in the cytoplasm of BxPC-3 cells (Figure 2C). To elucidate the effect of intracellular C3 on signalling pathways during pancreatic cancer progression, we established BxPC-3 cells with stable C3 knockdown via lentivirus-mediated shRNA transduction (Figure 3A). In the cell migration and invasion assays, C3 knockdown reduced the cellular migration and invasion abilities (Figure 3B). We further evaluated cell signalling pathways influenced by C3 knockdown and found that the activity of Akt and Smad pathways but not the Erk pathway was reduced by C3 knockdown (Figure 3C).

Figure 2.
Figure 2.

C3 expression in pancreatic ductal adenocarcinoma (PDAC) cell lines. (A&B) The BxPC-3 PDAC cell line showed higher C3 expression than the other cell lines by PCR and western blot analysis. (C) Immunocytochemical staining showed intracellular C3 expression and cytoplasmic C3a expression in the BxPC-3 cell line. ns: Not significant; ****p<0.0001.

Figure 3.
Figure 3.

Effect of C3 knockdown in the BxPC-3 pancreatic ductal adenocarcinoma (PDAC) cell line. (A) PCR showed successful knockdown of C3 expression in shRNA-transduced BxPC-3 cells. (B) C3 knockdown reduced cell migration (upper panels) and invasion (lower panels). (C) Western blot analysis revealed that C3 knockdown reduced the protein levels of phosphorylated Akt and Smad2/3. (D&E) While the tumour growth curves did not differ significantly between the two groups, the final tumour volume and tumour weight were reduced with C3 knockdown. shRNA: short hairpin RNA. *p=0.01-0.05; **p=0.01-0.001.

Regulation of C3 expression in BxPC-3 cells. We further investigated the regulatory mechanism of C3 expression in PDAC. Using TCGA data, we found that the expression of IL-6 and TNF-α was positively related to that of C3 (r=0.43, p<0.0001 and r=0.41, p=0.001, respectively) (Figure 4A). To confirm the relationship between C3 and both cytokines, we harvested BxPC-3 cells treated with recombinant IL-6 (50 and 100 ng/ml) or TNF-α (20 and 40 ng/ml) for 72 h and extracted RNA from the lysates. TNF-α treatment significantly increased the expression of C3 in BxPC-3 cells, whereas treatment with IL-6 did not (Figure 4B).

Figure 4.
Figure 4.

Regulatory mechanism of C3 expression in pancreatic ductal adenocarcinoma (PDAC). (A) Analysis of gene expression in TCGA data revealed a positive association between the expression of C3 and cytokines. (B) Treatment with TNF-α significantly increased C3 expression, whereas treatment with IL-6 did not. TCGA: The Cancer Genome Atlas.

Discussion

In the present study, we found that complement C3 was highly expressed in PDAC tissue and might be related to metastasis. Subsequent experiments revealed the intracellular localization of C3, and knockdown of C3 expression by shRNA was found to be associated with reduced cancer cell migration and invasion. To the best of our knowledge, this study is the first to clarify the expression of intracellular C3 in a PDAC cell line and describe its biophysiological roles. These findings may provide a further understanding of the nature of PDAC and might serve as a foundation for the development of novel treatments targeting intracellular C3.

C3 is the most abundant complement protein in serum and plays a pivotal role in complement cascades (22-24). C3 cleavage fragments generated during complement activation mediate anaphylaxis, the production of chemotactic factors, target opsonization, modification of B- and T-cell responses, and elimination of immune complexes. Although C3 is primarily synthesized in the liver, it is also synthesized by many other cell types, suggesting that complement activation may occur in situ during inflammation before the influx of serum complement. Recently, researchers have revealed the unique role of intracellular C3 in various cell types. Liszewski et al. reported that complement C3 was activated intracellularly in human T cells and mediated cell survival and Th1 induction (25). Additionally, King et al. found that C3 was highly expressed in human pancreatic islets and prevented β cell death via ATG16L1 interaction and autophagy regulation (26). Several studies have revealed unique roles of intracellular C3 in cancer cells. Zha et al. found that intracellular activation of C3 in lung cancer cells led to resistance to PD-L1 antibody treatment by modulating tumour-associated macrophages (11). Yuan et al. reported that intracellular C3 over-expression activated the JAK2/STAT3 pathway and correlated with gastric cancer progression (10). In pancreatic cancer, Chen et al. reported high C3 expression in pancreatic cancer tissue compared with that in adjacent normal pancreatic tissue; however, no significant relation with prognosis or disease progression was shown (27). Moreover, no studies have explored the origin of C3 in PDAC and clarified its pathophysiological roles in tumours.

The regulatory mechanism of C3 expression varies by cell type. In skin mast cells, TNF-α in combination with either IL-4 or IL-13 was found to synergistically enhance C3 expression (28). In hepatocytes, IL-1β and IL-6 might be involved in the regulation of C3 expression (29). Regarding pancreatic cells, Andoh et al. reported that C3 mRNA expression was up-regulated by either IL-1β or TNF-α in Panc-1 and MiaPaca-2 cell lines (30). While we selected BxPC-3 cells, which exhibit higher C3 expression than those two cell lines, the regulatory mechanism is believed to be similar across PDAC cell lines.

The present study has limitations. First, we utilized only one cell line for experiments, since among the PDAC cell lines evaluated, only BxPC-3 cells showed high C3 expression. Second, the TMA slides were purchased from a vendor, and the only clinical information available related to the slides was tumour stage. Detailed clinical information, such as prognosis and treatment response, was not provided. Third, technical challenges may arise in the establishment of a subcutaneous xenograft tumour model, since the tumour growth curves exhibited considerable differences. Considering that such large variations were not observed in the final tumour volume and weight, the cancer cells were believed to be inoculated deeply into the submucosal tissue, which made accurate measurement of the tumours with an external calliper difficult.

Conclusion

Intracellular C3 regulates EMT by activating the Smad pathway in pancreatic cancer. These findings provide a new approach for the development of treatments targeting intracellular C3.

Acknowledgements

The Authors greatly appreciate Ms. Chikako Sato and Ms. Rie Hikichi for their skilful assistance in the experiments. The present study was supported by JSPS KAKENHI grant number 20K16419.

Footnotes

  • Authors’ Contributions

    RS conceived, designed, and performed the experiments. RS, TT, MS, YS, HI, YY and HO analysed the data and wrote the paper. All Authors read and approved the final manuscript.

  • Conflicts of Interest

    The Authors declare that they have no competing interests in relation to this study.

  • Received October 6, 2022.
  • Revision received October 17, 2022.
  • Accepted October 18, 2022.

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Intracellular C3 Modulates EMT via the Akt/Smad Pathway in Pancreatic Cancer Cells
REI SUZUKI, TADAYUKI TAKAGI, MITSURU SUGIMOTO, YUKI SATO, HIROKI IRIE, YUKI YOMOGIDA, HIROMASA OHIRA
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