Fig 2: ZEB1 regulates ETAR expression through the repression of miR-200b/c.a, b Expression of miR-200b/c and ETAR, ZEB1 proteins in HEY cells transfected for 72 h with siRNA control (si-Ctr) or si-ZEB1 as determined by qRT-PCR (a) and WB (b). U6 and β-actin are used to normalize miRNA and protein expression, respectively. Values are the mean ± SD (n = 3; *p < 0.0001 vs. Ctr). c, d Luciferase activity (c) in HEY cells co-transfected for 48 h with the indicated mimic-miRNAs, in the presence or in the absence of ZEB1 plasmid together with the ETAR 3′UTR reporter plasmid. qRT-PCR (d) of the miR-200b/c expression in cells transfected as above and normalized to U6. Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.01 vs. Ctr; **p < 0.001 vs. ZEB1-transfected cells). e Expression of ETAR and ZEB1 proteins in lysates from cells treated as in c analyzed by WB. β-actin is used as loading control. f, g Luciferase activity (f) in HEY cells co-transfected for 48 h with the indicated anti-miRNAs, in the presence or absence of si-ZEB1 together with the ETAR 3′UTR reporter plasmid. qRT-PCR (g) of the miR-200b/c expression, in cells transfected as above and normalized to U6. Values are the mean ± SD expressed as fold induction (n = 3; *p < 0.01 vs. Ctr; **p < 0.001 vs. si-ZEB1-transfected cells). h Luciferase activity in HEY cells co-transfected for 48 h with si-Ctr or si-ZEB1 together with the reporter plasmid containing the 3’UTR region of ETAR (ETAR 3′UTR) or its triple mutant (mut/ΔETAR 3′UTR). Values are the mean ± SD expressed as fold induction (n = 3; *, p < 0.001 vs. ETAR 3′UTR-transfected Ctr cells). i Schematic model of the ETAR/ZEB1 and miR-200b/c feedback circuit.

Fig 3: Particulate matter PM10 increases the protein levels of EMT markers in A549 cells after 48 h of treatment. Particulate matter exposure increases the protein levels of Snail, Slug, ZEB1, N-cadherin, α-SMA, and vimentin, and decreases the protein levels of E-cadherin, while TGF-β treatment increases the protein levels of Snail, Slug, ZEB1, N-cadherin, α-SMA, and vimentin, and decreases the protein levels of E-cadherin in cultures of A549 cells transfected with non-targeting siRNA. These effects are prevented by TGFB1 siRNAs only in Slug, ZEB1, N-cadherin, α-SMA, and vimentin. Cells were exposed to PM10 (10 µg/cm2) and TGF-β (5 ng/mL) for 48 h. The quantitative results were expressed after normalization using GAPDH as a loading control. Data are reported as the means ± SD of three independent experiments. The images are representative of the data obtained. (*) indicates statistically differences between treatments. Densitometry was performed using arbitrary units p < 0.05.

Fig 4: Requirement of the Arf6-based mesenchymal pathway in both mesenchymal-type and amoeboid-type cancer invasion. Several receptor tyrosine kinases (RTKs), including those for epidermal growth factor (EGF) and hepatocyte growth factor (HGF), as well as G-protein-coupled receptors (GPCRs) for lysophosphatidic acid (LPA), are known to activate RhoA and Rac1 to promote cancer invasiveness; in which RhoA is preferentially required for amoeboid-type invasion, whereas Rac1 is required for mesenchymal-type invasion. We show that the Arf6-AMAP1-EPB41L5 pathway, activated either by EGF or by LPA, is essential for both mesenchymal-type and amoeboid-type invasion. EPB41L5 is a mesenchymal-specific protein primarily induced by ZEB1 in breast cancer. ZEB1, as well as EPB41L5, are also necessary for both mesenchymal-type and amoeboid-type invasion. We propose that, for cancer therapeutics, blocking Arf6, such as by inhibition of the mevalonate pathway, might be more effective than blocking RhoA or Rac1. It should be noted that blocking Arf6 may also effectively decrease the drug resistance of cancer cells, if cells overexpress the Arf6-based pathway (see text).

Fig 5: Representative immunohistochemical staining images of (A) bladder cancer negative staining control; (B) normal epithelium stained for HIF-1α; (C) normal epithelium stained for ZEB1; grade (D) G1, (E) G2 and (F) G3 tumor tissue stained for HIF-1α; grade (G) G1, (H) G2 and (I) G3 tumor tissue stained for ZEB1. Certain positively stained cells are indicated by black arrows. Scale bar, 50 µm. HIF-1α, hypoxia inducible factor-1α; ZEB1, zinc-finger E-box binding homeobox 1.