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Transcriptome analysis of an AKT inhibitor-resistant endometrial cancer cell line

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Abstract

Background

Drug resistance in endometrial cancer (EC) is a serious problem and a barrier to improving prognosis. The PI3K/AKT/mTOR pathway is highly activated in EC and can serve as a potential therapeutic target. Inhibitors against AKT have been developed, but resistance to these inhibitors is a concern. This study aimed to establish AKT inhibitor resistant cell lines and identify differentially expressed genes (DEGs) between parental and AKT inhibitor resistant cell lines to understand the mechanism of drug resistance to AKT inhibitors in EC.

Methods

The sensitivity of eight EC cell lines to AKT inhibitor was analyzed. One of them was used to establish a drug-resistant cell line. DEGs were examined using RNA sequencing (RNA-seq). Furthermore, DEGs were comprehensively analyzed to identify hub genes. Hub genes were evaluated using quantitative real-time polymerase chain reaction.

Results

RNA-seq identified 617 DEGs. Hub genes were selected using bioinformatics analysis. The top 10 hub genes were TNF, CDH1, CCND1, COL1A1, CDH2, ICAM1, CAV1, THBS1, NCAM1, and CDKN2A. Relative mRNA expression was significantly upregulated for TNF, CDH1, CCND1, THBS1, p16INK4a, and p14ARF and significantly downregulated for CDH2, ICAM1, and NCAM1 in borussertib-resistant EC cell line.

Conclusions

Drug resistance to AKT inhibitors may depend on genes related to cell adhesion-mediated resistance and transforming growth factor β signaling.

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Data availability

The datasets generated and/or analyzed during this study are available from the corresponding author upon reasonable request.

Abbreviations

ABC transporter:

ATP-binding cassette transporter

BP:

Biological process

CAM-DR:

Cell adhesion-mediated drug resistance

CC:

Cellular components

CCK-8:

Cell counting kit-8

DAVID:

Database for Annotation, Visualization, and Integrated Discovery

DEGs:

Differentially expressed genes

DMSO:

Dimethyl sulfoxide

EC:

Endometrial cancer

EMT:

Epithelial-mesenchymal transition

FDR:

False discovery rate

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

MF:

Molecular function

PPI:

Protein–protein interaction

qPCR:

Quantitative real-time polymerase chain reaction

TGF-β:

Transforming growth factor β

TNF:

Tumor necrosis factor

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Funding

This work was supported by funding from Kyoto Tachibana University (grant number: 23007).

Author information

Authors and Affiliations

  1. Department of Medical Technology and Sciences, Faculty of Health Sciences, Kyoto Tachibana University, 34 Yamada-cho, Oyake, Yamashina-ku, Kyoto, 607-8175, Japan

    Takafumi Onishi, Hirokazu Odashima, Yukihiko Osawa & Manabu Hattori

  2. Research Center for Life and Health Sciences, Kyoto Tachibana University, Kyoto, Japan

    Takafumi Onishi, Hirokazu Odashima, Yukihiko Osawa & Manabu Hattori

  3. Department of Pathology, Osaka University Graduate School of Medicine, Osaka, Japan

    Tsuyoshi Takashima

  4. Department of Medical Life Sciences, School of Medical Life Sciences, Kyushu University of Health and Welfare, Miyazaki, Japan

    Kazuki Shibahara

  5. Cancer Cell Institute, Kyushu University of Health and Welfare, Miyazaki, Japan

    Kazuki Shibahara

  6. Department of Medical Life Science, College of Life Science, Kurashiki University of Science and the Arts, Okayama, Japan

    Shoji Takagi

  7. Kake Institute of Cytopathology, Okayama, Japan

    Shoji Takagi

  8. Department of Medical Technology, Kawasaki University of Medical Welfare, Okayama, Japan

    Shinichi Tanaka

  9. Department of Health and Nutrition, Faculty of Health Science, Kio University, Nara, Japan

    Michihiro Mori

  10. Graduate School of Health Science, Kio University, Nara, Japan

    Michihiro Mori

Authors
  1. Takafumi Onishi
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  2. Tsuyoshi Takashima
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  3. Kazuki Shibahara
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  4. Shoji Takagi
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  5. Shinichi Tanaka
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  6. Michihiro Mori
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  7. Hirokazu Odashima
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  8. Yukihiko Osawa
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  9. Manabu Hattori
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Contributions

TO: conceptualization, methodology, investigation, analysis, writing, and review; TT: conceptualization, methodology, writing, and review; KS: conceptualization and review; ST: conceptualization, methodology, investigation, analysis, and review; ST: conceptualization, analysis, and review; MM: conceptualization, methodology, writing, and review; HO: methodology and review; YO: methodology and review; MH: methodology and review.

Corresponding author

Correspondence to Takafumi Onishi.

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The authors declare no conflicts of interest.

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Supplementary Information

Below is the link to the electronic supplementary material.

43440_2024_581_MOESM1_ESM.xlsx

Supplementary file1 Supplemental Table 1. Primer sequences for quantitative real-time polymerase chain reaction. (XLSX 10 KB)

43440_2024_581_MOESM2_ESM.xlsx

Supplementary file2 Supplemental Table 2. List of biological processes extracted using Gene Ontology analyses. p-values < 0.05 were considered significant. Count indicates the number of genes. Gene is the gene symbol associated with the term. (XLSX 19 KB)

43440_2024_581_MOESM3_ESM.xlsx

Supplementary file3 Supplemental Table 3. List of cellular components extracted using Gene Ontology analyses. p-values < 0.05 were considered significant. Count indicates the number of genes. Gene is the gene symbol associated with the term. (XLSX 17 KB)

43440_2024_581_MOESM4_ESM.xlsx

Supplementary file4 Supplemental Table 4. List of molecular functions extracted using Gene Ontology analyses. p-values < 0.05 were considered significant. Count indicates the number of genes. Gene is the gene symbol associated with the term. (XLSX 15 KB)

43440_2024_581_MOESM5_ESM.xlsx

Supplementary file5 Supplemental Table 5. List of pathways activated by differentially expressed genes detected in the KEGG pathway analysis. p-values < 0.05 were considered significant. Count indicates the number of genes. Gene is the gene symbol associated with the term. KEGG, Kyoto Encyclopedia of Genes and Genomes. (XLSX 11 KB)

43440_2024_581_MOESM6_ESM.pptx

Supplementary file6 Supplemental Figure 1. Images of soft-agar colony-formation assay. Images were taken using a phase-contrast microscope (SZX10) with a 4x objective lens and a digital camera (AdvanCam-HD1080P II), with imaging software (AdvanView). Borussertib-resistant SNG-M cells were treated with 10 µM borussertib. Parental SNG-M cells were treated with the same amount of dimethyl sulfoxide as the borussertib treatment. Cultures were incubated continuously for 1 month in an incubator at 37°C and 5% CO2. Scale = 400 µm. N = 3. (PPTX 2607 KB)

43440_2024_581_MOESM7_ESM.pptx

Supplementary file7 Supplemental Figure 2. Images of parental and borussertib-resistant SNG-M cells. Images were taken using a phase-contrast microscope (SZX10) with a 20x objective lens and a digital camera (AdvanCam-HD1080P II), with imaging software (AdvanView). Scale = 100 µm. N = 3. (PPTX 13782 KB)

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Onishi, T., Takashima, T., Shibahara, K. et al. Transcriptome analysis of an AKT inhibitor-resistant endometrial cancer cell line. Pharmacol. Rep 76, 379–389 (2024). https://doi.org/10.1007/s43440-024-00581-w

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