Transfer of miRNA in tumor-derived exosomes suppresses breast tumor cell invasion and migration by inducing M1 polarization in macrophages

Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11–amino acid sequence between amino acids 717 and 727 of Tiam1 (717GEGTDAVKRS727L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1). Using an Escherichia coli–derived calmodulin-binding peptide (CBP)–tagged recombinant Tiam1 (amino acids 393–728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393–738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1–ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)–tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1–ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin–Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.

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MENA promotes esophageal squamous cell carcinoma cell invasion and migration via MMP-2, MMP-9 and AKT activation

10.21203/rs.3.rs-19339/v1 ◽

2020 ◽

Author(s):

Yueheng Li ◽

Na Gao ◽

Jing Li ◽

Zhengfan Gao ◽

Zhenzhen Yang ◽

...

Keyword(s):

Tumor Cell ◽

Cell Invasion ◽

Tumor Cell Invasion ◽

Wound Healing Assay ◽

Transwell Assay ◽

Invasion And Migration ◽

Akt Activation ◽

Qrt Pcr ◽

And Migration ◽

Migration Capacity

Abstract Background: Esophageal squamous cell carcinoma (ESCC) is a fatal disease with poor prognosis. The predominant reason for ESCC-related death is metastasis caused by tumor cell invasion. Human MENA protein is a member of Ena/Vasp family, which plays a critical role during tumor cell invasion. However, the biological effect of MENA in ESCC cell lines remains unclear Methods: In this study, fluorescent quantitative real-time PCR (qRT-PCR) were conducted to detect the mRNA expression of MENA in tumor and para-cancer tissue, CCK-8 assay and clone formation assay were conducted to evaluate cell proliferation activity, Transwell assay and wound-healing assay were conducted to detect the changes of cell invasion and migration capacity, siRNA and MENA expression vector were constructed to explore biological function of MENA in ESCC cell lines. Western blot analysis were conducted to detect the expressions of MENA , molecular markers of epithelial-mesenchymal transition (EMT), Akt, p-Akt, MMP-2 and MMP-9 respectively in ESCC cell line. Results: The qRT-PCR experiment results showed that MENA expression in ESCC tissue of 35 patients was relatively higher than that in tissue adjacent to cancer. CCK-8 assay suggested that tumor cell proliferation capacity was suppressed followed by the knockdown of MENA expression in Mena high ESCC cell TE13 and was potentiated by the overexpression of MENA in Mena low ESCC cell TE1. Transwell assay and wound healing assay demonstrated that interfering in MENA could inhibit TE13 cells invasion and migration capacity by affecting the expressions of Matrix metalloproteinase-2(MMP-2) and Matrix metalloproteinase-9 (MMP-9), in contrast, overexpression of MENA in Mena low ESCC cell TE1 could promote invasion and migration by up-regulated expression of MMP-2 and MMP-9. Western blot analysis indicated that interfering of MENA expression could affect EMT-related molecular markers (E-cadherin, N-cadherin, Snail, Slug), Akt and p-Akt Conclusions: Our study reveal that MENA could promote the ESCC cell invasion and migration by upregulate MMP-2, MMP-9 expression and Akt activation. Meanwhile, interfering of MENA expression could affect EMT in ESCC cells. This indicated that MENA may be a potential molecular therapeutic target for ESCC metastasis

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