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Insulin-like growth factor-1 activates different catalytic subunits p110 of PI3K in a cell-type-dependent manner to induce lipogenesis-dependent epithelial–mesenchymal transition through the regulation of ADAM10 and ADAM17

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Abstract

The activation of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) is critical for the induction of epithelial–mesenchymal transition (EMT) by growth factors, including insulin-like growth factor 1 (IGF-1). The activation of intracellular lipogenesis provides proliferative and survival signals for cancer cells. In this study, we investigated the connection between lipogenesis-related EMT processes and IGF-1-mediated PI3K p110 isoform activation in primary (SW480 cells) and metastatic (SW620) colon carcinoma cells. We also examined the underlying signaling pathway that promotes fatty acid synthesis in IGF-1-activated colon cancer cells. IGF-1 stimulation upregulated the expression of lipogenic enzymes as well as the activation of Nardilysin (N-arginine dibasic convertase, NRD1) and its downstream targets, a disintegrin and metalloproteases 10 (ADAM10) and ADAM17. The upregulation of the Lyn/Syk-mediated PI3K p110δ isoform in SW480 cells and the Lyn-dependent PI3K p110α isoform in SW620 cells triggered fatty acid production and cell motility in IGF-1-activated colon cancer cells. Pharmacological inhibition with A66 (PI3K p110α specific inhibitor) and CAL-101 (PI3K p110δ specific inhibitor) efficiently inhibited EMT in colon cancer cells by blocking the NRD1/ADAM family protein signaling pathway. Gene silencing of NRD1 and ADAM family proteins attenuated the generation of intracellular fatty acid and the migratory activity of colon cancer cells. Our results suggest that the different isoforms of the PI3K p110 subunit could be therapeutic targets for primary and metastatic colon cancer and that regulation of the NRD1/ADAM signaling pathway controls lipogenesis-mediated EMT in IGF-1-stimulated colon cancer cells.

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Abbreviations

ACC:

Acetyl-CoA carboxylase

AceCS1:

Acetyl-CoA synthetase 1

ACLY:

ATP-citrate lyase

ACSL1:

Mammalian long-chain acyl-CoA synthetase 1

ADAM:

A disintegrin and metalloproteinase

α-SMA:

Alpha(α)-smooth muscle actin

CRC:

Colorectal cancer

EMT:

Epithelial–mesenchymal transition

ERK:

Extracellular signal-regulated kinase

FASN:

Fatty acid synthase

IGF-1:

Insulin-like growth factor-1

MMP:

Matrix metalloproteinase

NRD1:

N-arginine dibasic convertase

PI3K:

Phosphoinositide 3-kinase

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Acknowledgments

This study was supported by the Basic Science Research Program of Ministry of Education (NRF-2015R1D1A1A01056672) and the Ministry of Science, ICT & Future Planning (NRF-2015R1C1A2A01053732) through the National Research Foundation (NRF) of Republic of Korea.

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Authors and Affiliations

  1. Department of Biochemistry, Kosin University College of Medicine, Busan, 49267, Republic of Korea

    Ga Bin Park

  2. Department of Anatomy, Inje University College of Medicine, 75, Bokji-ro, Busanjin-gu, Busan, zip code: 47392, Republic of Korea

    Daejin Kim

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11010_2017_3148_MOESM1_ESM.tif

Supplemental Figure 1. IGF-1-mediated Syk kinase activation regulates colon cancer cell migration. (A) Cells (2 × 105/6-well) were treated with 100 pg/ml IGF-1 for 24 h. Total protein was subjected to Western blot analysis with the indicated antibodies. (B-D) To inhibit Syk phosphorylation, cells were pre-exposed to 200 nM Syk inhibitor Bay 61-3606 for 2 h and then treated with 100 pg/ml IGF-1 for 24 h. Total cell lysates were immunoblotted with antibodies against (B) p-Syk (Tyr323), p-Syk (Tyr525/526), Syk, or p110δ; (C) NRD1, ADAM10, ADAM17, E-cadherin, N-cadherin, Vimentin, ZO-1, or α-SMA. Data are representative of three independent experiments. β-actin served as an internal control. (D) The migratory activity and invasiveness of SW620 cells were detected by the tumor transendothelial migration assay kit and the BME cell invasion assay kit, respectively, as described in the Materials and Methods section. Each value is the mean ± standard deviation of 3 determinations. *, p<0.05. **, p<0.05. Data are representative of three independent experiments. Supplementary material 1 (TIFF 9202 kb)

11010_2017_3148_MOESM2_ESM.tif

Supplemental Figure 2. The expression of intracellular lipogenic enzymes in IGF-1-treated colon cancer cells at various conditions. Total protein was subjected to Western blot analysis with antibodies against AceCS1, p-ACLY, ACLY, ACSL1, p-ACC, ACC, or FASN protein were performed. β-actin served as an internal control. (A) Cells were transfected with Lyn-siRNA (200 nM) or control-siRNA for 36 h prior to experiments and then treated with 100 pg/ml IGF-1 for 24 h. (B) Cells were pre-exposed to 10 μM PI3K/Akt inhibitor LY294002 for 2 h and then treated with 100 pg/ml IGF-1 for 24 h. (C) Cells were transfected with NRD1-siRNA (200 nM) or control-siRNA for 36 h prior to experiments and then 100 pg/ml IGF-1 for 24 h. Supplementary material 2 (TIFF 8192 kb)

11010_2017_3148_MOESM3_ESM.tif

Supplemental Figure 3. NRD1 silencing prevents EMT processes in IGF-1-treated colon cancer cells. (A) Cell motility was increased by IGF-1 as measured by a wound healing assay. Cells were wounded (0 h) and maintained for 24 h in complete medium. Dotted lines indicate the edges of the wounds. Wound closure (measured after 24 h) was faster in cells treated with control-siRNA than in those treated with NRD1-siRNA. (B and C) The migratory activity and invasiveness of SW480 (B) or SW620 (C) cells were detected by the tumor transendothelial migration assay kit and the BME cell invasion assay kit, respectively, as described in the Materials and Methods section. Each value is the mean ± standard deviation of three determinations. *, p<0.05. **, p<0.05. Data are representative of three independent experiments. Supplementary material 3 (TIFF 9545 kb)

11010_2017_3148_MOESM4_ESM.tif

Supplemental Figure 4. Intracellular triglyceride production of IGF-1-activated colon cancer cells. (A) Cells (2 × 105/6-well) were treated with 100 pg/ml IGF-1 for 24 h. (B) Cells were pre-exposed to 10 μM PI3K/Akt inhibitor LY294002 for 2 h and then treated with 100 pg/ml IGF-1 for 24 h. (C) Cells were transfected with ADAM10-siRNA (200 nM), ADAM17-siRNA (200 nM), or control-siRNA for 36 h prior to experiments and then treated with 100 pg/ml IGF-1 for 24 h. Post treatment lipid was extracted and total triglyceride was estimated. Accumulation of cellular triglyceride was assayed using a colorimetric assay according to the manufacturer’s instruction. The amount of triglyceride present in the samples may be determined from the standard curve, which obtained from the appropriate triglyceride standards. Each value is the mean ± standard deviation of 3 determinations. Data are representative of three independent experiments. Supplementary material 4 (TIFF 9815 kb)

11010_2017_3148_MOESM5_ESM.tif

Supplemental Figure 5. Intracellular cholesterol production of IGF-1-activated colon cancer cells. (A) Cells (2 × 105/6-well) were treated with 100 pg/ml IGF-1 for 24 h. (B) Cells were pre-exposed to 10 μM PI3K/Akt inhibitor LY294002 for 2 h and then treated with 100 pg/ml IGF-1 for 24 h. (C) Cells were transfected with ADAM10-siRNA (200 nM), ADAM17-siRNA (200 nM), or control-siRNA for 36 h prior to experiments and then treated with 100 pg/ml IGF-1 for 24 h. After post-treatment with IGF-1, lipid was extracted and then total cholesterol was estimated. Accumulation of cellular cholesterol was assayed using a colorimetric assay according to the manufacturer’s instruction. The amount of cholesterol present in the samples may be determined from the standard curve, which obtained from the appropriate cholesterol standards (in Kit). Each value is the mean ± standard deviation of 3 determinations. Data are representative of three independent experiments. Supplementary material 5 (TIFF 9804 kb)

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Park, G.B., Kim, D. Insulin-like growth factor-1 activates different catalytic subunits p110 of PI3K in a cell-type-dependent manner to induce lipogenesis-dependent epithelial–mesenchymal transition through the regulation of ADAM10 and ADAM17. Mol Cell Biochem 439, 199–211 (2018). https://doi.org/10.1007/s11010-017-3148-0

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