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ARF6 (adenosyl diphosphate ribosylation factor 6) is a special subtype of the ARFs family and is widely expressed in tissues and organs such as brain, breast, liver, and prostate. It is mainly distributed in cell membranes and endosomes at the cellular level. ARF6 plays an important role in endocytosis, extracellular secretion, endosomal circulation, cell division, microvesicle trafficking and release, formation of intercellular adhesion junctions, and actin cytoskeletal remodeling. In addition, ARF6 is associated with the clathrin-dependent pathway and the clathrin-independent pathway of endocytosis, such as transferrin receptor, β2 adrenergic receptor, M2 cholinergic receptor, and cadherin.
In recent years, it has been found that ARF6 is highly expressed in various tumor tissues such as breast cancer, melanoma, glioma, lung cancer and prostate cancer, and is closely related to the invasion and metastasis of tumor cells. ARF6 mainly affects the invasion and migration ability of tumor cells through the following aspects: (1) Destruction of adhesion formation between tumor cells, promoting cell invasive phenotype, enhancing tumor cell movement and invasion ability; (2) Regulating cell muscle protein and cytoskeletal remodeling promotes the formation of synaptic structures associated with tumor cell migration and invasion, such as lamellipodia, membrane ruffles, and invadopodia; (3) Promoting the release of microvesicles containing various proteolytic enzymes in tumor cells, and enhance the ability of tumor cells to degrade extracellular matrix proteins.
Figure 1. A model of Arf6-mediated signals in cancer. (Li, et al. 2017).
ARF6 Regulates the Formation of Intercellular Adhesions
The correct establishment of intercellular adhesion junction plays a key role in maintaining the polarity of epithelial cells, and the destruction of intercellular adhesion junction is an important link for epithelial tumor cells to obtain invasion , migration phenotype, achieve tumor cell invasion, and metastasis. ARF6 inhibits cell-cell junctions and promotes tumor cell invasion and migration by regulating the expression and distribution of protein molecules associated with intercellular adhesions, including cadherin, catenin, and integrins.
Cadherin is the basis for the formation of adhesion junctions between epithelial cells and one of the polar marker proteins of epithelial cells. Under physiological conditions, ARF6 regulates the polarization of E-cadherin in polarized epithelial cells in the basement membrane and intracellular early interstitial, maintaining the dynamic stability of adhesion. Allaire et al. found that Rab35 is activated by acting on its downstream effector molecule, cyanidin-β2 [ACAP2 /centaurin - β2 (ACAP2 is ARF6-specific GAP)], inhibiting ARF6 activity and enhancing cadherin on tumor cell surface, promoting the expression level of intercellular adhesion junctions and inhibiting the invasion and migration of tumor cells. Xu et al. used siRNA interference technology and overexpression of ARF6 (T27N) to further confirm that EGF activates ARF6 in breast cancer cells mainly by promoting E-cadherin internalization of breast cancer cells, leading to destruction of intercellular adhesion junctions and enhancing invasion and migration ability of breast cancer cells.
β-catenin acts as a key molecule in the intercellular adhesion junction and forms a complex with cadherin to participate in the construction of cell adhesion junctions. ARF6 induces the depolymerization of β-catenin and cadherin complex by regulating β-catenin internalization, leading to destruction of intercellular adhesion junctions and enhancing tumor cell invasion. Grossmann et al found that WNT5A binds to the Frizzled 4 - LDL 6 complex in melanoma cells. Activation of intracellular ARF6 by GEP100 promotes the internalization of β-catenin, which leads to the depolymerization of β-catenin and N-cadherin complex, destroys the intercellular adhesion junction and enhances the invasion ability of tumor cells. The targeting factor SLIT2 binds to the melanoma cell surface receptor ROBO1, which can effectively inhibit ARF6 activity, thereby stabilizing the intercellular β-catenin and N-cadherin complex, and inhibit cell migration and invasion induced by ARF6 activation.
Intergrins are important molecules that recognize and adhere to each other between cells and extracellular matrix proteins. Enhancing the internalization of integrin and the circulation between the cell basement membrane and the serosa can significantly enhance the migration and invasive ability of tumor cells. It was found that β1 integrin, transferrin and major histocompatibility class I (MHCI) were co-localized in the circulating endosomes regulated by ARF6. ARF6 affects the invasion and migration of tumor cells by regulating intracellular integrin internalization and circulation.
ARF6 Regulates Cytoskeletal Remodeling
The formation of functional dendritic pseudopods on the cell adhesion surface is the first synaptic structure formed during tumor invasion and migration, and is also one of the important markers for tumor cells to obtain an invasive phenotype. Endogenous ARF6 is located in the dendritic pseudopodia of breast cancer and melanoma cells. Eades et al. found in breast cancer cells that ARF6 overexpression can be induced by inducing cell-invasive pseudopods, resulting in an invasive phenotype in non-invasive breast cancer MCF-7 cells. This suggests that ARF6-induced dendritic pseudopod formation plays an important regulatory role in the invasion of breast cancer cells. Marchesin et al. found that the ARF6 extremely active mutant ARF6 (T157N) was overexpressed in breast cancer MDA-MB-231 cells. Activation of ARF6 leads to enhanced Rac1 activity. Rac1 can increase the expression of intracellular actin monomer and activate pseudopodia on the surface of the membrane by activating Arp2 /3 and down-regulating the activity of F - actin depolymerization factor.
ARF6 Regulates Microvesicle Release
Studies have found that tumor cells can also degrade the extracellular matrix by releasing microvesicles containing multiple proteases from tumor cells to the surrounding cells and tissues, enhancing the invasion and migration ability of tumor cells and achieving distant invasion and metastasis of the tumor. The study found that there are microvesicles with positive ARF6 expression in human prostate cancer tissues. The expression of ARF6-positive microvesicles in peripheral blood of nude mice model of prostate cancer metastasis increase significantly compared with the control group, suggesting that ARF6 regulates the release of microvesicles from tumor cells and plays an important role in tumor invasion and migration.