Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Apr 27:12:889159.
doi: 10.3389/fonc.2022.889159. eCollection 2022.

Extracellular Matrix Protein 1 Regulates Colorectal Cancer Cell Proliferative, Migratory, Invasive and Epithelial-Mesenchymal Transition Activities Through the PI3K/AKT/GSK3β/Snail Signaling Axis

Sirui Long  1   2 , Jie Wang  1   2 , Fanbin Weng  1   2 , Debing Xiang  1   2 , Guiyin Sun  1   2
Affiliations

Extracellular Matrix Protein 1 Regulates Colorectal Cancer Cell Proliferative, Migratory, Invasive and Epithelial-Mesenchymal Transition Activities Through the PI3K/AKT/GSK3β/Snail Signaling Axis

Sirui Long et al. Front Oncol. .

Abstract

In prior reports, extracellular matrix protein 1 (ECM1) upregulation has been reported in colorectal cancer (CRC) patient tumor tissues, and has been suggested to be related to the metastatic progression of CRC, although the underlying mechanisms have yet to be clarified. In this study, we found that ECM1 was overexpressed in both CRC tissues and cell lines. Upregulation of ECM1 was correlated with tumor size, lymph node status and TNM stage in CRC patients. Knocking down ECM1 suppressed CRC cell growth, migration and invasion, in addition to reducing the expression of Vimentin and increasing E-cadherin expression. The overexpression of ECM1, in contrast, yielded the opposite phenotypic outcomes while also promoting the expression of p-AKT, p-GSK3β, and Snail, which were downregulated when ECM1 was knocked down. Treatment with LY294002 and 740 Y-P reversed the impact upregulation and downregulation of ECM1 on CRC cell metastasis and associated EMT induction. In vivo analyses confirmed that ECM1 overexpression was able to enhance EMT induction and CRC tumor progression. In conclusion, ECM1 influences CRC development and progression in an oncogenic manner, and regulates CRC metastasis and EMT processes via the PI3K/AKT/GSK3β/Snail signaling axis.

Keywords: Extracellular matrix protein 1; PI3K/AKT/GSK3β/Snail signaling pathway; colorectal cancer; epithelial-mesenchymal transition; metastasis.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Analysis of ECM1 expression in CRC tumors and cell lines. (A) ECM1 levels in CRC patient tumors and adjacent normal tissue samples were analyzed using the Oncomine database. (B) Representative IHC staining results for CRC patient tumor and paracancerous tissues. (C) ECM1 mRNA expression in CRC tumors and paracancerous tissues. (D) ECM1 protein levels in CRC tumors and paracancerous tissues. (E) ECM1 mRNA levels in CRC and colonic epithelial cell lines. (F) ECM1 protein levels in CRC (SW480, SW620, HT29, HCT116, HCT15) and colonic epithelial (NCM460) cell lines. Data are means ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 2
Figure 2
ECM1 regulates proliferative activity in CRC cells. (A, B). Western blotting and qPCR were performed to assess ECM1 knockdown. (C, D). Western blotting and qPCR were employed to assess ECM1 expression in CRC cells following its overexpression. CCK-8 and colony formation assays were used to assess cellular proliferation, revealing that (E, G) ECM1 knockdown suppressed HCT15 cell proliferation relative to control cells, while (F, H) ECM1 overexpression enhanced HCT116 cell proliferation. Data are means ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 3
Figure 3
ECM1 regulates the progression of the cell cycle and apoptotic death in CRC cells. Following ECM1 knockdown or overexpression, apoptosis and cell cycle progression were analyzed via flow cytometry. (A, B) Changes in the progression of the cell cycle and apoptotic cell death following the knockdown of ECM1. (C, D) Changes in the progression of the cell cycle and apoptotic cell death following ECM1 overexpression. Data are means ± SD (**P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 4
Figure 4
ECM1 regulates CRC cell invasion and migration. CRC cell migratory and invasive activity were scrutinized through Transwell and wound healing assessments following ECM1 knockdown (A–C) or upregulation (D–F). Data are means ± SD (***P < 0.001, ****P < 0.0001).
Figure 5
Figure 5
ECM1 regulates CRC cell EMT induction. EMT-related protein expression was analyzed following the knockdown or upregulation of ECM1. (A–C) Vimentin and E-cadherin protein levels were assessed in HCT15, HCT15-NC, and HCT15-siECM1 cells. (D–F) Vimentin and E-cadherin protein levels were investigated in HCT116, HCT116-NC, and HCT116-ECM1 cells. Data are means± SD (**P < 0.01, ***P < 0.001).
Figure 6
Figure 6
ECM1 regulates signaling via the PI3K/AKT/GSK3β/Snail axis within CRC cells. Western blotting was used to assess AKT, p-AKT, GSK, pGSK3β, and Snail levels in CRC cells following (A, B) ECM1 knockdown or (C, D) ECM1 overexpression. Data are means ± SD (**P < 0.01, ***P< 0.001, ****P < 0.0001).
Figure 7
Figure 7
PI3K/AKT agonist and inhibitor treatment can reverse the effects of altered ECM1 expression on CRC cells. HCT116-ECM1 were treated for 24 h with LY294002 (50 or 75 uM), or HCT15-siECM1 were treated 24 h with 740 Y-P (30 or 50 uM), after which invasive and migratory activity were assessed through Transwell and wound healing assays (E–G, I–K), while Western blotting was used to assess AKT, p-AKT, GSK, pGSK, Snail, E-cadherin, and Vimentin expression (A–D, H,L). Data are means ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, ****P <0.0001).
Figure 8
Figure 8
ECM1 promotes in vivo CRC tumor growth. (A–C) ECM1 enhanced in vivo tumor growth, as determined through measurements of tumor weight and volume at 25 days post-implantation. (D–G) ECM1, Vimentin, and E-cadherin expression levels in xenograft tumors were assessed via Western blotting. Data are means ± SD (**P < 0.01, ***P < 0.001)
See this image and copyright information in PMC

References

    1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global Cancer Statistics 2018: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin (2018) 68(6):394–424. doi: 10.3322/caac.21492 - DOI - PubMed
    1. Oki E, Ando K, Nakanishi R, Sugiyama M, Nakashima Y, Kubo N, et al. . Recent Advances in Treatment for Colorectal Liver Metastasis. Ann Gastroenterol Surg (2018) 2(3):167–75. doi: 10.1002/ags3.12071 - DOI - PMC - PubMed
    1. Mongiat M, Fu J, Oldershaw R, Greenhalgh R, Gown AM, Iozzo RV. Perlecan Protein Core Interacts With Extracellular Matrix Protein 1 (ECM1), a Glycoprotein Involved in Bone Formation and Angiogenesis. J Biol Chem (2003) 278(19):17491–9. doi: 10.1074/jbc.M210529200 - DOI - PubMed
    1. Gao D, Ma X, Lian P, Zhou S, Chen J. Pathogenetic Mechanism of Lipoid Proteinosis Caused by Mutation of the Extracellular Matrix Protein 1 Gene. Mol Med Rep (2018) 17(6):8087–90. doi: 10.3892/mmr.2018.8928 - DOI - PubMed
    1. Wang Z, Zhou Q, Li A, Huang W, Cai Z, Chen W. Extracellular Matrix Protein 1 (ECM1) is Associated With Carcinogenesis Potential of Human Bladder Cancer. Onco Targets Ther (2019) 12:1423–32. doi: 10.2147/OTT.S191321 - DOI - PMC - PubMed