Transglutaminase‐2, RNA‐binding proteins and mitochondrial proteins selectively traffic to MDCK cell‐derived microvesicles following H‐Ras‐induced epithelial–mesenchymal transition

Abstract

Epithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Here, we report that shed microvesicles (also termed microparticles/ectosomes) secreted from MDCK cells following oncogenic H-Ras-induced EMT (21D1-sMVs) are biochemically distinct from exosomes and parental MDCK-sMVs. The protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorageindependent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1-sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation.

The updated role of exosomal proteins in the diagnosis, prognosis, and treatment of cancer

“…Proteomic analyses of the secretomes of MDCK and 21D1 cells showed the remodeling of extracellular matrix (ECM) proteins, including decreased levels of basic membrane components (e.g., collagen type IV) and proteases (e.g., matrix metalloproteinase 1 (MMP-1)) 60 . Proteomic analyses of 21D1 cell-derived exosomes showed a decrease in some epithelial biomarkers (e.g., E-cadherin and EpCAM) but an increase in mesenchymal biomarkers (e.g., vimentin) and proteinases (e.g., MMP-1) 61 . Therefore, a thorough recognition of the function of exosomal proteins in EMT is needed.…”

The Biological and Biomechanical Role of Transglutaminase-2 in the Tumour Microenvironment

“…This was dependent on the simultaneous transfer of fibronectin, which is cross-linked within the microvesicle and is required for fibroblast activation [ 245 , 250 ]. The enrichment of TG2 in MVs following h-RAS-induced EMT provides a potential link between EVs and the ability of TG2 to mediate EMT and subsequent invasive behaviour [ 251 ].…”

A novel anoikis-related gene signature predicts prognosis in patients with head and neck squamous cell carcinoma and reveals immune infiltration

“…Besides, MTDH-dependent anoikis resistance is activated by the PI3K/Akt pathway, and anoikis resistance is obtained by inhibiting caspase-3 activation and activating CXCR4 expression levels (Zhou et al, 2014), targeting MTDH can limit PDAC metastasis (Suzuki et al, 2017). Recently, a study suggested that FN1 promotes cellular aggregate formation conferring anoikis resistance to tumor cells (Han et al, 2021), and that pretreatment of exosomes with anti-FN1 antibodies attenuates the invasive ability of fibroblasts (Shafiq et al, 2021). PRDX4 is overexpressed in a variety of tumor tissues (Jia et al, 2019), and inhibits anoikis resistance through the β-catenin/ ID2 pathway, thereby promoting the growth and metastasis of hepatocellular carcinoma cells (Wang et al, 2019).…”