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CACNA2D1 regulates the progression and influences the microenvironment of colon cancer

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Abstract

Background

Calcium voltage-gated channel auxiliary subunit alpha 2/delta 1 (CACNA2D1), a gene encoding a voltage-gated calcium channel, has been reported as an oncogene in several cancers. However, its role in colon cancer (CC) remains unclear. This study aimed to investigate the function of CACNA2D1 and its effect on the microenvironment in CC.

Methods

Immunohistochemistry (IHC) analysis was performed on samples collected from 200 patients with CC who underwent curative colectomy. Knockdown experiments were performed using CACNA2D1 siRNA in the human CC cell lines HCT116 and RKO, and cell proliferation, cycle, apoptosis, and migration were then analyzed. The fibroblast cell line CCD-18Co was co-cultured with CC cell lines to determine the effect of CACNA2D1 on fibroblasts and the relationship between CACNA2D1 and the cancer microenvironment. Gene expression profiles of cells were analyzed using microarray analysis.

Results

IHC revealed that high CACNA2D1 expression was an independent poor prognostic factor in patients with CC and that CACNA2D1 expression and the stroma are correlated. CACNA2D1 depletion decreased cell proliferation and migration; CACNA2D1 knockdown increased the number of cells in the sub-G1 phase and induced apoptosis. CCD-18Co and HCT116 or RKO cell co-culture revealed that CACNA2D1 affects the cancer microenvironment via fibroblast regulation. Furthermore, microarray analysis showed that the p53 signaling pathway and epithelial–mesenchymal transition-associated pathways were enhanced in CACNA2D1-depleted HCT116 cells.

Conclusions

CACNA2D1 plays an important role in the progression and the microenvironment of CC by regulating fibroblasts and may act as a biomarker for disease progression and a therapeutic target for CC.

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Acknowledgements

This work was supported by a Grants-in-Aid for Scientific Research (C) (23K08218, 23K06654, 23K08115, 22K08832, 21K08689) and Early-Career Scientists (22K16518) from the Japan Society for the Promotion of Science.

Author information

Author notes

  1. Hiroyuki Inoue and Atsushi Shiozaki contributed equally to this work.

Authors and Affiliations

  1. Division of Digestive Surgery, Department of Surgery, Kyoto Prefectural University of Medicine, 465 Kajii-Cho, Kamigyo-Ku, Kyoto, 602-8566, Japan

    Hiroyuki Inoue, Atsushi Shiozaki, Toshiyuki Kosuga, Hiroki Shimizu, Michihiro Kudou, Tomohiro Arita, Hirotaka Konishi, Shuhei Komatsu, Yoshiaki Kuriu, Takeshi Kubota, Hitoshi Fujiwara & Eigo Otsuji

  2. Department of Surgical Pathology, Kyoto Prefectural University of Medicine, Kyoto, Japan

    Yukiko Morinaga & Eiichi Konishi

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  1. Hiroyuki Inoue
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  3. Toshiyuki Kosuga
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Contributions

All authors contributed to the study conception and design. HI acquired data and performed experiments. HI, AS, TK, HS, and MK contributed to the analysis and interpretation of data. EK and YM instructed the evaluation of immunohistochemical scores. HI and AS wrote the manuscript. TA, HK, SK, YK, TK, HF, and EO made critical revisions. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Atsushi Shiozaki.

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Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by the Medical Ethics Review Committee of the Kyoto Prefectural University of Medicine (approval no. ERB-C-2423 and no. M2023-528). Informed consent to be included in the study, or the equivalent, was obtained from all patients.

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Supplementary Information

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Supplementary file1 (DOCX 55 KB)

535_2024_2095_MOESM2_ESM.tif

Supplementary file2 Supplementary Fig. S1. a. Hematoxylin and eosin staining of normal colon tissue. b. Immunohistochemistry (IHC) staining of normal colon tissue using anti-CACNA2D1 antibodies. In addition to cancer, some glandular tissues and vascular endothelial cells were also positively stained. c, d, e. IHC staining of primary human colon cancer samples treated with (c) anti-CACNA2D1 antibody, (d) isotype control antibody, or without (e) antibodies. Magnification, ×100. Scale bars, 200 µm (TIF 6230 KB)

535_2024_2095_MOESM3_ESM.tif

Supplementary file3 Supplementary Fig. S2. CACNA2D1 expression levels in human colon cancer (CC) samples and overall survival analysis following curative resection based on CACNA2D1 expression levels. Patients were classified into the low-CACNA2D1 expression group (IHC score < 0.4, n=96, blue line) and high-CACNA2D1 expression group (IHC score ≥ 0.4, n=104, red line) (TIF 203 KB)

535_2024_2095_MOESM4_ESM.tif

Supplementary file4 Supplementary Fig. S3. Survival analysis according to the expression level of CACNA2D1 based on various databases. a, b. Gene Expression Profiling Interactive Analysis (GEPIA) data showing the low CACNA2D1 expression group (n=176, blue line) and high CACNA2D1 expression group (n=95, red line). c. UALCAN data showing the low CACNA2D1 expression group (n=208, blue line) and high CACNA2D1 expression group (n=71, red line) (TIF 369 KB)

535_2024_2095_MOESM5_ESM.tif

Supplementary file5 Supplementary Fig. S4. a. CACNA2D1 siRNA effectively reduced CACNA2D1 mRNA levels in SW480 cells. b, c. Effects of CACNA2D1 downregulation on the SW480 cell proliferation. In (b), cell counting was performed at 48, 72, and 96 h after siRNA transfection.; in (c), cell viability was assessed with a colorimetric water-soluble tetrazolium salt-8 assay at 0, 24, 48, 72, and 96 h after siRNA transfection. d. Effects of CACNA2D1 downregulation on the proportions of early and late apoptotic SW480 cells. Control or CACNA2D1 siRNA-transfected cells were stained with propidium iodide (PI) and annexin V and subjected to flow cytometry analysis. Data are presented as the mean ± SEM; n=3. *p<0.05 vs. the control siRNA (TIF 378 KB)

535_2024_2095_MOESM6_ESM.tif

Supplementary file6 Supplementary Fig. S5. The off-target effects were evaluated using CACNA2D1 siRNA other than that used in Figures 2–5. a. Evaluation of knockdown efficiency, b. Proliferation assays were performed (TIF 265 KB)

535_2024_2095_MOESM7_ESM.tif

Supplementary file7 Supplementary Fig. S6. a, b. Anti-tumor effects of amlodipine. a. Cell viability assay. (b.) IC50. c. Effects of amlodipine on the proportions of early and late apoptotic HCT116 and RKO cells. Control or amlodipine-treated cells were stained with propidium iodide (PI) and annexin V and subjected to a flow cytometric analysis. Data are reported as the mean ± SEM; n=3. *p<0.05 vs. the control (TIF 456 KB)

535_2024_2095_MOESM8_ESM.tif

Supplementary file8 Supplementary Fig. S7. Fibroblast proliferation assay upon co-culture with HCT116 and RKO cells (TIF 143 KB)

535_2024_2095_MOESM9_ESM.tif

Supplementary file9 Supplementary Fig. S8. Analysis of the expression of cancer-associated fibroblast (CAF) markers after fibroblasts were co-cultured with either fibroblasts or cancer cell lines (HCT116 or RKO). Data are presented as the mean ± SEM; n=3. *p<0.05 vs. the control (fibroblasts co-cultured with fibroblasts) (TIF 226 KB)

535_2024_2095_MOESM10_ESM.tif

Supplementary file10 Supplementary Fig. S9. Correlation analysis of CACNA2D1 and epithelial-mesenchymal transition (EMT)-related gene expression in cBioPortal (TIF 1998 KB)

535_2024_2095_MOESM11_ESM.tif

Supplementary file11 Supplementary Fig. S10. Assessment of overexpression 1. a. CACNA2D1 HaloTag plasmid effectively increased the CACNA2D1 mRNA levels in SW620 and DLD-1 cells. b, c. Effects of CACNA2D1 overexpression on the proliferation of SW620 and DLD-1 cells. In (b), cell counting was performed at 48 and 72 h after siRNA transfection.; in (c), cell viability was assessed with a colorimetric water-soluble tetrazolium salt-8 assay at 0, 24, 48, and 72 h after plasmid transfection. d, e. Effect of CACNA2D1 overexpression on (d) the number of cells in the sub-G1 phase in SW620 and DLD-1 cells and (e) early and late apoptotic cells. f. Effect of CACNA2D1 overexpression on wound closure in SW620 and DLD-1 cells. Mean ± SEM; n=3. *p<0.05 (significantly different from the control HaloTag plasmid) (TIF 3708 KB)

535_2024_2095_MOESM12_ESM.tif

Supplementary file12 Supplementary Fig. S11. Assessment of overexpression 2. a. Effect of CACNA2D1 overexpression significantly increased fibroblast cell migration. b. Effect of CACNA2D1 overexpression on the secretion of PDGF-BB to the culture medium in SW620 and DLD-1 cells. c. Quantitative reverse transcription-polymerase chain reaction of the relative expression of genes associated with the p53 signaling and EMT-associated pathways following CACNA2D1 plasmid transfection in SW620 and DLD-1 cells. Gene expression levels were normalized to those of ACTB. Mean ± SEM; n=3. *p<0.05 (significantly different from the control HaloTag plasmid) (TIF 1085 KB)

535_2024_2095_MOESM13_ESM.tif

Supplementary file13 Supplementary Fig. S12. In vivo experiments using CACNA2D1-depleted HCT116 cells by siRNA. a, b. Tumor sizes and volumes of the two groups at the end of the experiment. c. Tumor growth curves of the negative control and CACNA2D1 knockdown groups of nude mice. Mean ± SEM; n=5. *p<0.05 (significantly different from the control siRNA) (TIF 1612 KB)

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Inoue, H., Shiozaki, A., Kosuga, T. et al. CACNA2D1 regulates the progression and influences the microenvironment of colon cancer. J Gastroenterol (2024). https://doi.org/10.1007/s00535-024-02095-x

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