Fig 2: PAK3 enhances the JNK-Jun signal pathway.a PK and SPK cells were analyzed for the expression of Smad4, PAK3, and JNK-Jun signaling-related factors by Western blotting and the experiment was repeated three times independently with similar results. b Western blots showing the effects of Smad4 overexpression in SPK cells and the experiment was repeated three times independently with similar results. c IHC staining detected the expression of Smad4, PAK3, p-JNK, and p-Jun in mouse lung tumors of PK and SPK mice. Scale bar, 25 µm (magnification, ×40). d Quantification of the average percentage of Smad4, PAK3, p-JNK, and p-Jun positive cells in mouse lung tumors of PK and SPK mice. Data presented are the mean ± SEM from three biologically independent samples (n = 3); *p = 0.001802131; **p = 0.007103699; ***p = 0.001259358; ****p = 0.000180321; as determined by two-tailed Student’s t-test.

Fig 3: Smad4 negatively regulates PAK3 via transactivation of miR-495 and miR-543 expression.a A luciferase reporter fused to PAK3 3' UTRs was constructed in a pcDNA3.1-Luc-MCS plasmid. b Luciferase activities of PK and SPK cells transfected with PAK3 3'UTR-Luc plasmid confirmed increased PAK3 expression level SPK cells. Data presented are the mean ± SD from three biologically independent samples (n = 3); *p = 0.023816011, as determined by two-tailed Student’s t-test. c Luciferase reporter assay showing that overexpression of Smad4 decreased PAK3 expression level in SPK cells. Data presented are the mean ± SEM from three biologically independent samples (n = 3); ***p = 1.58613E-05, as determined by a two-tailed Student’s t-test. d RT-PCR detected expression of PAK3 correlated miRNAs in PK, SPK, and SPK overexpressing Smad4 cells. Data presented are the mean ± SEM from three biologically independent samples (n = 3); *p = 0.012761254; **p = 0.00456168; ?p = 0.005190084; ?p = 0.015974559; ?p = 0.024995116; as determined by two-tailed Student’s t-test. e, f Expression levels of miR-495 and miR-543 were determined in PK and SPK mouse lung tumors. n = 9, data represent means ± SEM; p < 0.05, as determined by two-tailed Student’s t-test. g, h The interaction between Smad4 and miR-495 (g) or miR-543 (h) in SPK cells were verified by ChIP assay. Data presented are the mean ± SEM from three biologically independent samples (n = 3); ***(h)p = 6.64287E-06; ***(g)p = 0.00021899; as determined by two-tailed Student’s t-test. i qRT-PCR analysis gene expression in PK cells. Data presented are the mean ± SEM from three biologically independent samples (n = 3). ***p = 5.32332E-07; **p = 0.000179381.

Fig 4: MiR-495 and miR-543 directly bind to the PAK3 3' UTR and attenuate the metastatic potential of lung cancer cells in vitro and in vivo.a, b The expression of PAK3 was detected at both mRNA and protein levels after SPK cells were transfected with miR-495 or miR-543 mimics. Data presented are the mean ± SD from three biologically independent samples (n = 3). ***p = 0.000448037; **p = 0.007514536; *p = 0.009195246. c The expression of PAK3 in SPK cells was detected following the transfection with miR-495 or miR-543 inhibitors (antagomiR-495 or antagomiR-543) and the experiment was repeated three times independently with similar results. d, e PAK3 3'UTR and its mutation luciferase report system were constructed. The activity of the luciferase reporter bearing the WT 3' UTR of PAK3 was measured in H1299 cells transfected with miR-495 or miR-543 mimics. Luciferase activity means the expression level of PAK3 3' UTR. Data presented are the mean ± SEM from three biologically independent samples (n = 3); **p = 0.007393441 (d); *p = 0.018265986 (d); **p = 0.001333882 (e); *p = 0.002263372 (e); as determined by two-tailed Student’s t-test. f Cells were made a wound between the two red dashed lines, the area of the two red dashed lines represent the level of wound healing and the cell migration activity. Cell migration ability was detected by wound healing assay in PK, SPK, SPK miR-495, and SPK miR-543 cells, and the experiment was repeated three times independently with similar results. Scale bar, 100 µm. g Cell migration ability was detected by wound healing assay after antagomiR-495 or antagomiR-543 was introduced into SPK cells, and the experiment was repeated three times independently with similar results. Scale bar, 100 µm. h–j Six-eight weeks SPK mice were treated by adeno-Cre and then injected NC agomir (n = 9) or miR-495 agomir (n = 10) after 4 weeks, once every 2 weeks. After 8 weeks, all mice were sacrificed to check lung cancer metastasis (j) and collected lung tumors to test the expression level of PAK3, p-JNK, and p-Jun, these representative mouse samples were shown by IHC (h), and Western blotting (i). Scale bar, 25 µm (magnification, ×40).

Fig 5: Correlation between Smad4 and PAK3/JNK/Jun expression in human lung cancer samples.a Representative IHC images displaying an inverse correlation between Smad4, PAK3, p-JNK, and p-Jun in human normal lung tissues, lung tumors, and lung cancer metastatic samples. Scale bar, 25 µm (magnification, ×40). b Statistics of positively stained percentages in human normal lung tissues (n = 15) and lung cancer metastatic tissues: early tumor (n = 15); advanced tumor (n = 12); lymph nodes metastases (n = 22); bone metastases (n = 8). c Pearson correlation analysis of protein expressions in Fig. 7a, b. d A proposed model for the role of Smad4 in lung cancer metastasis.