Fig 2: INHBA promotes lung cancer cell invasion via inhibiting the Hippo pathway. (A) Protein complex of Merlin, FRMD6 and WWC1 activates the Hippo pathway, which increases the phosphorylation of YAP and prevents its translocation and binding to TEAD in the nucleus. (B) INHBA inhibits the Hippo pathway by downregulating Merlin expression, which decreases the phosphorylation of YAP, and promotes its translocation and binding to TEAD in the nucleus. INHBA, inhibin βA; YAP, yes-associated protein; WWC1, WW and C2 domain-containing 1; FRMD6, FERM domain-containing 6; MST, mammalian sterile 20-like 1/MOB kinase; LATS, large tumor suppressor kinase; p, phosphorylated; TEAD, TEA domain; CTGF, connective tissue growth factor; Cyr61, cysteine rich angiogenic inducer 61.

Fig 3: INHBA negatively regulates Hippo signaling by downregulating Merlin protein expression levels in lung cancer cells. (A) Western blotting revealed that only Merlin protein expression levels were decreased in H1299 cells transfected with the INHBA overexpression plasmid. (B) Western blotting revealed the efficacy of Merlin overexpression plasmids in H1299 cells. (C) Rescue experiments showed that overexpression of Merlin reversed the effect of INHBA on activating YAP in H1299 cells. (D) Reverse transcription-quantitative PCR indicated no change in NF2 (encoding Merlin) mRNA expression levels after H1299 cells were transiently transfected with the INHBA plasmid. (E) Immunoprecipitation analysis revealed that INHBA could bind to Merlin. *P<0.05, **P<0.01, ***P<0.001. INHBA, inhibin βA; YAP, yes-associated protein; WWC1, WW and C2 domain-containing 1; FRMD6, FERM domain-containing 6; NF2, neurofibromin 2.

Fig 4: INHBA activates YAP and induces its nuclear translocation in lung cancer cells. (A and B) Western blotting revealed that the expression levels of pYAP, pLATS1/2, and the ratio of pYAP/YAP and pLATS1/2/LATS1/2, were decreased in H1299 cells transfected with the INHBA overexpression plasmid and increased in A549 cells transfected with si-INHBA; total YAP and LATS1/2 levels were not affected. (C-G) Subcellular fractionation indicated that, after INHBA overexpression in cytoplasm, YAP accumulation in the cytoplasmic fraction decreased and YAP accumulation in the nuclear fraction increased. RT-qPCR indicated that H1299 cells transiently transfected with the INHBA plasmid exhibited increased (H) CTGF and (I) CYR61 mRNA expression levels. RT-qPCR demonstrated that A549 cells transiently transfected with si-INHBA exhibited decreased (J) CTGF and (K) CYR61 mRNA expression levels. (L-N) Western blotting indicated that H1299 cells transiently transfected with the INHBA plasmid exhibited increased CTGF and CYR61 protein expression levels. (O-Q) Western blotting demonstrated that A549 cells transiently transfected with si-INHBA exhibited decreased CTGF and CYR61 protein expression levels. *P<0.05, **P<0.01. INHBA, inhibin βA; YAP, yes-associated protein; si, small interfering; NC, negative control; LATS, large tumor suppressor kinase; p, phosphorylated; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; RT-qPCR, reverse transcription-quantitative PCR.

Fig 5: Representative photomicrographs of the results of a human tissue microarray for INHBA and YAP in 238 tumor tissues and 30 normal lung tissues. In normal lung tissues, INHBA expression was (A) negative and (B) positive YAP nuclear expression was limited to type II pneumocytes. Under conditions of (C) low INHBA protein expression levels, (D) nuclear YAP protein expression levels were low and the expression levels of cytoplasmic YAP were high. Under conditions of (E) high INHBA protein expression levels, (F) cytoplasmic YAP protein expression levels were low and nuclear YAP protein expression levels were high. Original magnification, ×40 or ×200. INHBA, inhibin βA; YAP, yes-associated protein; NSCLC, non-small cell lung cancer.