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Transcriptomic profiling on localized gastric cancer identified CPLX1 as a gene promoting malignant phenotype of gastric cancer and a predictor of recurrence after surgery and subsequent chemotherapy

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Abstract

Background

Localized gastric cancer (GC) becomes fatal once recurring. We still have room for improving their prognoses.

Methods

Transcriptomic analysis was done on surgically resected specimens of 16 patients with UICC stage III GC who underwent curative gastrectomy and adjuvant oral fluoropyrimidine monotherapy. Four of them were free from disease for longer than 5 years, and the others experienced metachronous metastasis within 2 years after surgery. Quantitative RT-PCR determined mRNA expression levels of primary gastric cancer tissues, which were collected from 180 patients who underwent gastric resection for stage II–III GC without preoperative treatment between 2001 and 2014. We tested alteration of malignant phenotypes including drug resistance of GC cell lines by siRNA and shRNA-mediated knockdown and forced expression experiments.

Results

CPLX1 was identified as a candidate biomarker for GC recurrence among 57,749 genes. Inhibiting and forced expression experiments indicated that CPLX1 promotes proliferation, motility, and invasiveness of GC cells, and decreases apoptosis and sensitivity to fluorouracil. Subcutaneous xenograft mouse models revealed that shRNA-mediated knockdown of CPLX1 also attenuated tumor growth of MKN1 cells in vivo. Overexpression of CPLX1 in gastric cancer tissue correlated with worse prognosis and was an independent risk factor for peritoneal recurrence in subgroups receiving adjuvant chemotherapy.

Conclusions

CPLX1 may represent a biomarker for recurrence of gastric cancer and a target for therapy.

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Abbreviations

EMT:

Epithelial–mesenchymal transition

mRNA:

Messenger RNA

OD:

Optical density

qRT-PCR:

Quantitative reverse-transcription polymerase chain reaction

RNA-seq:

RNA-sequencing

siRNA:

Small interfering RNA

shRNA:

Small hairpin RNA

VC:

Vesicular cycle

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Acknowledgements

We thank Taiho Pharmaceutical Co. Ltd for technical support, and advice on this project through discussion on our result.

Funding

This study was funded by Taiho Pharmaceutical Co. Ltd.

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Authors and Affiliations

  1. Department of Gastroenterological Surgery (Surgery II), Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Haruyoshi Tanaka, Mitsuro Kanda, Dai Shimizu, Chie Tanaka, Yoshikuni Inokawa, Norifumi Hattori, Masamichi Hayashi, Goro Nakayama & Yasuhiro Kodera

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  1. Haruyoshi Tanaka
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  2. Mitsuro Kanda
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Contributions

Study concept and design; MK and YK. Acquisition of data; MK, HT, DS, and YK. Analysis and interpretation of data; HT, MK, and YK. Drafting of the manuscript; HT, MK, and YK. Critical revision of the manuscript for important intellectual content; HT, MK, SD, CT, NH, YI, MH, GN, and KY. Statistical analysis; HT and MK. Obtained funding; MK and YK. Technical, or material support; HT, MK, DS, and CT. Study supervision; YK.

Corresponding author

Correspondence to Mitsuro Kanda.

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The authors declare no conflicts of interest.

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Brief description: Transcriptomic analysis identified CPLX1 gene as a novel oncogene candidate for gastric cancer. CPLX1 may promote epithelial–mesenchymal transition and evading apoptosis of gastric cancer cells even under a cytotoxic agent, and also be a predictor for recurrence after surgery for UICC Stage II–III gastric cancer.

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535_2022_1884_MOESM4_ESM.pdf

Supplementary file4 Supplemental Fig. S1. Kaplan–Meier plot panel of overall survival, and progression-free survival using external datasets grouped by the median value of each gene expression level to extract 12 candidate genes up-regulated. The left panels present overall survival and the right present recurrence-free survival. Data were retrieved from the GSE62254, GSE15459, GSE22377, GSE62254, GSE29272, and GSE38749, of which UICC Stages were limited to II–III. Supplemental Fig. S2. (a) Conventional immunoblot assay detecting CPLX1 expression of KATOIII cells. (b) Whole picture of digital immunoblot of seven gastric cancer cells (two left panels) and (c) of cells transfected with siRNAs and overexpression vector compared with control siRNA and empty vector, respectively. (d) CPLX1-knockdown efficiencies of N87, OCUM1, and NUGC2 cells. (e) Proliferation assay comparing KATOIII cells, KATOIII cells transfected with siControl, and siCPLX1, and comparing NUGC2 cells transfected with siControl and siCPLX1. Supplemental Fig. S3. (a) Apoptotic cell detection by staining with annexin-V (See Fig. 2e). Phase-contrast and fluorescence images were merged to be presented. (b) Proportions of annexin-V positive stained KATOIII cells untreated, and transfected with siControl and siCPLX1. (c) Drug sensitivity tests to fluorouracil (5-FU) on seven gastric cancer (GC) cell lines. GR values indicate values calculated by normalized growth rate inhibition (GR) metrics. (d) Scatter plot between CPLX1 mRNA expression (CPLX1/GAPDH) and area under the dose–response curves (AUC) to 5-FU of seven GC cell lines. A correlation was tested with the Spearman test. (e) PCR-based EMT-related 84 genes profiling to test co-expression with CPLX1 among 14 GC cell lines. The significances of coefficients were represented by rho (ρ) values. In this panel, other genes generally considered to be related to epithelial–mesenchymal transition (EMT) were presented than the genes presented picked out in Fig. 3. (f) Whole raw pictures of the sandwich ELISA array comparing parental KATOIII cells, that mediated by siControl, and siCPLX1. (g) The whole picture of digital immunoblot of MKN1cells transfected with shCPLX1 compared with the control shRNA (shControl). Supplemental Fig. S4. (a) Histogram of CPLX1 mRNA expression values of 180 samples. The upper quantile value of the present cohort was 0.00682 and indicated by the vertical line. (b) Representative immunohistochemistry of gastric cancer tissue with weak CPLX1 expression and adjacent gastric tissue. (PDF 59442 KB)

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Tanaka, H., Kanda, M., Shimizu, D. et al. Transcriptomic profiling on localized gastric cancer identified CPLX1 as a gene promoting malignant phenotype of gastric cancer and a predictor of recurrence after surgery and subsequent chemotherapy. J Gastroenterol 57, 640–653 (2022). https://doi.org/10.1007/s00535-022-01884-6

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  • DOI: https://doi.org/10.1007/s00535-022-01884-6

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