Inhibin ?A(INHBA) Is An Independent Prognostic Factor And Promotes Invasion Via Hippo Signaling In Non-small Cell Lung Cancer

Objective: Non-small cell lung cancer(NSCLC)is one of the most common malignant tumors and the leading cause of cancer-related deaths.Although tremendous progress has been made in surgery,radiotherapy,chemotherapy and targeted therapy over the past years,the 5-year survival rate of lung cancer is still less than 15%.The main reason is that most patients had advanced lung cancer or metastasis when they diagnosed.There were important clinical significances for guiding clinical diagnosis and treatment by searching for markers for predicting the development of lung cancer and exploring the molecular mechanism of lung cancer development.Inhibin ?A(INHBA),a disulfide bond homodimer of activin A(activin A),is a member of transforming growth factor(TGF)-?(TGF-?)superfamily.Multiple studies had confirmed that INHBA has two diametrically opposed biological effects of promoting cancer and suppressing cancer.There wee also individual studies suggesting that INHBA is associated with poor prognosis of lung cancer,but the underlying molecular mechanism is still unclear.Hippo signaling pathway is a highly conserved signaling pathway discovered in recent years.It can participate in the regulation of organ size,cell proliferation,apoptosis and other pathophysiological processes through its core transcriptional regulator Yes-associated protein(YAP).The research of our group also confirmed that the Hippo pathway was involved in the occurrence and development of lung cancer,and the increase of YAP expression in the nucleus is associated with poor prognosis of lung cancer.We are also observed that more and more studies showed TGF-? and Hippo pathway were related.As a member of the TGF-? superfamily,in-depth study of the role and molecular mechanism of INHBA in regulating the Hippo pathway will help to analyze the regulatory network of the two major signal pathways of TGF-? and Hippo in NSCLC.Therefore,we intend to detect the expression of INHBA in NSCLC,and combine in vitro functions and molecular experiments to explore the biological functions and potential molecular mechanisms of INHBA in NSCLC,so as to provide important clues for us to understand the mechanism of NSCLC progress and targeted intervention.Methods: For the study of human NSCLC tissue specimens,we collected paraffin specimens including 238 cases of cancer and adjacent tumors and 30 pairs of primary tumors and lymph node metastases,and 10 pairs of fresh specimens of cancer and adjacent tumors.Immunohistochemistry(IHC),Western blot or Real-time PCR were used to detect the expression of INHBA to analyze its correlation with clinicopathological factors,lymph node metastasis and prognosis;(2)For the study of INHBA on the biological functions of NSCLC,we first used Western blot to detect the expression of INHBA in NSCLC cell lines and normal bronchial epithelial HBE cells,and screened relatively high and low expression cell lines of INHBA for further studies in vitro;INHBA high expression cell line was transfected with si RNA,INHBA low expression cell line were transfected with overexpression plasmid,Western blot was used to detect the Epithelial-mesenchymal transition(EMT)related proteins,Transwell migration and Invasion experiments was used to detect cell migration and invasionese to evaluate the effects of INHBA on the biological functions of lung cancer cells;(4)In the study of INHBA promoted NSCLC invasionese by regulating Hippo pathway,we first used Western blot or Real-time PCR to detect the expression of YAP and LATS1/2,as well as the phosphorylated proteins p YAP,p LATS1/2,and the downstream transcription factors CTGF and CYR61 by up or down regulating the NHBA expression,to evaluate the effect of INHBA on the Hippo pathway;IHC was used to detect Whether the expression of INHBA in the specimen is related to the subcellular localization of YAP protein 238 cases of NSCLC paraffin specimens;Subcellular fractionation experiment were used to detect the changes in the expression of YAP of cytoplasm and nucleus,to further clarify whether INHBA affects its subcellular localization;Verteporfin(VP),the Hippo pathway inhibitor,was used in the Rescue experiment on the INHBA over-expressing cell line,Western blot was used to detect EMT-related proteins,and the Transwell migration and invasion experiment was used to detect the cell migration and invasion ability to clarify whether INHBA exerts its biological function through the Hippo pathway.(5)In the study of the potential molecular mechanism of INHBA regulating the Hippo pathway,we first used Western blot to detect the upstream regulatory proteins(FRMD6,Merlin and WWC1)of Hippo pathway to find the protein regulated by INHBA;Rescue experiment was performed to determine whether INHBA regulates Hippoy pathway activity by down-regulating Merlin expression;Co-immunoprecipitation(Co-IP)was used to determine whether INHBA binds to Merlin protein.Results:(1)In the study of clinical significance of INHBA,we found that the expression of INHBA protein in 238 cases of NSCLC was significantly higher than that of normal alveolar epithelium(P<0.001);the protein and m RNA expression levels of INHBA in 24 fresh NSCLC tissues were both significantly higher than the mathed adjacent tissues(all P value <0.001);the INHBA protein expression in 30 lymph node metastatic tissues were significantly higher than that in the primary tumor(P=0.0015);statistical analysis showed that the positive expression of INHBA were significantly related with tumor staging(TNM II-IV;P=0.017)and poor differentiation(P=0.016)in 238 NSCLC;Kaplan–Meier survival curve showed that INHBA positive expression was associated with shorter overall survival(OS)and disease-free survival(DFS)(P values were 0.002 and 0.001);multivariate Cox regression analysis showed that INHBA is an independent prognostic indicator of NSCLC(P=0.019).These results indicated that INHBA is associated with poor prognosis and a potential predictive prognostic marker in NSCLC.(2)In the study of the biological function of INHBA on NSCLC cells,we found that overexpression of INHBA inhibited E-cadherin expression and increased the N-cadherin,vimentin and snail expression,while knockdown of INHBA increased E-cadherin expression and increased N-cadherin,vimentin and snail expression;Transwell migration and invasion experiments showed overexpression of INHBA enhanced the migration and invasion ability of H1299 cells,while knockdown INHBA expression reduced the migration and invasion ability of A549 cells.These results indicated that INHBA promoted the invasion of NSCLC cells.(3)In the study of whether INHBA promotes the invasion of NSCLC through the Hippo pathway,IHC results showed INHBA was negatively correlated with the YAP positive expression in the cytoplasm,and positively with YAP positive expression in the nucleus(P =0.014 and 0.045,respectively;the protein levels of p-YAP and p-LATS1/2 were significantly reduced by up-regulating INHBA,while increased by down-regulating INHBA expression;in addition,overexpression of INHBA up-regulated the downstream transcription factors CTGF and and CYR61 expression,while knocking down INHBA down-regulated their expression;subcellular fractionation experiment showed that the expression level of YAP in the nucleus was significantly increased,while the expression in the cytoplasm was significantly decreased by up-regulating INHBA expression;rescue experiment showed that VP could reverse the role of INHBA in promoting the invasion of NSCLC.These results indicated that INHBA enhanced the invasion ability of NSCLC by inhibiting the Hippo pathway.(4)In the study of the potential molecular mechanism of INHBA in regulating the Hippo pathway,we found that only Merlin protein of the upstream regulatory proteins of the Hippo pathway decreased by up-regulating INHBA expression;Rescue experiments showed overexpression of Merlin significantly reversed the inhibitory effect of INHBA on YAP phosphorylation;more interestingly,INHBA had no effect on Merlin m RNA levels,and the Co-IP results showed that INHBA could bind to Merlin protein.These results indicated that INHBA inhibited Hippo pathway activity by down-regulating Merlin expression at the protein level.Conclusion:(1)We confirmed that the INHBA expression is related to the poor prognosis of NSCLC,and is an independent prognostic factor of NSCLC;(2)we revealed that INHBA inhibits the Hippo pathway by down-regulating Merlin expression at the protein level,and promotes YAP transfering to nuclearthe to up-regulate the downstream transcription factors CTGF and CYR61 expression,which in turn promotes the invasion of NSCLC.