Fig 2: Blocking the ZFP36L1-SDC4-TGF-β loop inhibited osteosarcoma EMT and lung metastasis.In bone lesions, the high expression of ZFP36L1 expedited SDC4 mRNA degradation and reduced SDC4 interacted with TGFBR3, leading to increased free TGFBR3 cleavage by MMP. The increased soluble TGFBR3 (sTGFBR3) subsequently inhibited activation of TGF-β signaling pathway via blocking TGF-β1 binding to TGFBR1/2. Conversely, in metastatic OS cells and lung metastases, the low expression of ZFP36L1 reduced SDC4 mRNA degradation, leading to an abnormal accumulation of SDC4. The interaction between SDC4 and TGFBR3 protected against TGFBR3 cleavage by MMP, resulting in a decrease in extracellular sTGFBR3. The decrease in sTGFBR3 facilitated the activation of the TGF-β signaling pathway by enhancing the binding of TGF-β1 to TGFBR1/2, ultimately resulting in osteosarcoma EMT and lung metastasis. Intriguingly, activation of the TGF-β signaling pathway further upregulated SDC4 expression, which caused a vicious circle. Targeting the ZFP36L1-SDC4-TGF-β loop with MK2 inhibitor III or SB431542 effectively inhibited osteosarcoma EMT and lung metastasis.

Fig 3: TGFBR3 gene expression levels are elevated in the mesenchymal stem-like (MSL) subtype of TNBC. A) Heat map representation of gene expression for 587 TNBC tumors for each TGF-β ligand and receptor. B) Quantification of average TGFBR3 gene expression across TNBC tumor subtypes, average based on individual TNBC tumor probe intensity values (**P = 0.004; ***P <0.0003 for a two-tailed Student’s t-test). C) Heat map of TGFBR3 mRNA expression in TNBC representative cell lines. D) Quantification of TGFBR3 gene expression across representative TNBC cell lines (**P = 0.004; for a two-tailed Student’s t-test, ns = not significant). E) qRT-PCR analysis of TGFBR3 average mRNA expression (2-ΔΔCt) [28] from representative TNBC cell lines; graph bars represent the mean of three replicates with SEM error bars. F) Immunoblot analysis of TβRIII protein expression in TNBC representative cell lines, results representative of two independent experiments. SEM, standard error of the mean; TNBC, triple negative breast cancer; TβRIII, type III transforming growth factor-beta receptor.

Fig 4: Interaction between SDC4 and TGFBR3 protected against TGFBR3 cleavage by MMP.WB (A) and SBE-luciferase activity assays (B) were conducted to determine the role of SDC4 in regulating the TGF-β signaling pathway via the extracellular pathway in 143B cells. C ELISA tests were performed to measure the level of sTGFBR3 in the CM of oeSDC4 or shSDC4 143B cells. IP assays were carried out to reveal the interaction between SDC4 and TGFBR3 in exogenous (D) and endogenous (E) ways in 143B cells. F Immunofluorescence assay showed the colocalization of SDC4 and TGFBR3 on the cell surface in 143B cells or U2OS cells. Upper scale bar, 10 μm. Lower scale bar, 1 µm. G ELISA was conducted to measure TGFBR3 cleavage regulated by TAPI2 or DAPT in 143B cells. WB (H) and SBE-luciferase activity (I) assays were conducted to elucidate the role of TAPI2 in the modulation of the TGF-β signaling pathway attenuated by oeZFP36L in 143B cells. The data are shown as means ± SEMs; *P < 0.05, **P < 0.01.