Study On The Anti-metastatic Melanoma By Targeting The Hippo-YAP Signaling Pathway

Melanoma is a common skin tumor caused by uncontrolled cell proliferation and has strong metastastic capability and high degree of malignancy with increasing incidence worldwide in recent years.Ultraviolet radiation with excessive intense sunlight is the main cause of melanomas.Most malignant melanomas originate from melanocytes of normal skin.Early and un-metastatic melanomas are usually cured by surgical resection,with a five-year survival rate of 93%;however,if the melanoma is not treated in time,it is prone to metastasis and enters advanced stages.For the advanced metastatic melanoma,there is still no effective treatment method in the world,and the five-year survival rate of the patients is only 4.6%,thus metastatic melanoma is one of the highest lethal diseaes.Metastatic melanoma is highly tolerant to radiotherapy and is prone to drug resistance.General antitumor drugs have poor therapeutic effect on the metastatic melanoma.The lack of effective anti-metastatic melanoma drug is a major cause of very low efficacy in the treatment of the disease.Therefore,how to find new targets and develop effective new drugs against the metastatic melanoma is an urgent scientific question to be solved.Recent studies have shown that the abnormality of Hippo-YAP signaling pathway plays an indispensable role in the pathogenesis of various malignant tumors.The upstream of Hippo signaling pathway is composed of the tumor suppressor genes SAV1/2,MST1/2,MOB1A,and LATS1/2,while the downstream of the pathway is consisted of the oncogenes YAP/TAZ.Interestingly,it is extremely rare that there are two types of function distinct signal molecules in a single signaling pathway in cells.When the Hippo signaling pathway is activated,the expression of the upstream tumor suppressors SAV1/2,MST1/2,MOB1A,and LATS1/2 is increased,and the key signaling proteins LATS1/2 form a complex with the oncogenic YAP/TAZ,thus keeping the latter localized in the cytoplasm where the YAP/TAZ protein is phosphorylated by LATS1/2 and then degraded in lysosomes,eliminating the tumorigenic effect of the oncogene YAP/TAZ.In a variety of malignant tumors,the upstream of the Hippo signaling pathway tumor suppressor gene such as LATS1/2 is defective,which leads to the oncogene YAP/TAZ free,and enters the nucleus to promote overexpression of multiple tumorigenic genes,thereby promoting tumorigenesis and tumor malignant progression.In short,the function of the Hippo signaling pathway in melanoma cells is mainly to inhibit melanoma cell proliferation and metastasis,but this pathway is defective in various malignant tumors including melanoma.So far,there are no effective drug targeting the Hippo-YAP signaling pathway in the clinical setting.Therefore,how to enhance the tumor suppressive effect such as LATS1/2 and inhibit the oncogenic effect such as YAP in the Hippo signaling pathway in tumor cells has become a key scientific question to be solved in Hippo signaling pathway targeted melanoma treatment.In China,using Chinese herbal medicine to treat tumors has a long history.The traditional Chinese herbal medicine is a treasure house derived from our ancestors,providing rich experiences in treating tumors.Based on the above scientific issues,we propose that the presence of an active component that may activate the Hippo signaling pathway and exert anti-melanoma effect in the traditional Chinese herbal medicine.After extensive screening 120 traditional Chinese herbal medicines,we found triptonide,an active component purified from the traditional Chinese herbal medicine,has a strong inhibitory effect on various tumors with low toxicity.In this study,we unexpectedly found that triptonide has a very strong and selective anti-metastatic melanoma effect,with an IC50 of 5.56 nM which is much lower than the dose used in other tumors,and interestingly,it has no obvious toxic effect on normal skin cells at the effective doses.Cell biology studies showed that triptonide increased the size of melanoma cells,reduced proliferation,increased apoptosis,and effectively inhibited the migration and invasion of melanoma cells;More importantly,triptonide effectively inhibited melanoma metastasis in mice.Accordingly,we raised a scientific question:Does triptonide inhibit the melanoma metastasis by activating the tumor suppressive Hippo signaling pathway?In light of that Hippo signaling pathway is aberrant in melanoma,we boldly proposed a scientific hypothesis that triptonide inhibits the downstream oncogene YAP in Hippo signaling pathway by activating the tumor suppressor component that in the upstream of the Hippo signaling pathway,thereby inhibiting the metastasis of melanoma cells.Our molecular mechanism studies revealed that after triptonide was applied to melanoma cells,the tumor suppressive Hippo signaling pathway is enhanced,particularly,the expression of SAV1,LATS1,and other tumor suppressor genes were increased.Subsequently,we examined the gene expression of the most terminal effector molecule YAP in the downstream of the Hippo signaling pathway in melanoma cells after triptonide treatment.The results showed that triptonide did not affect the transcription of YAP gene.Western blotting revealed that triptonide markedly enhanced the lysosomal degradation of YAP,and reduced YAP protein levels in tumor cells,thereby reducing the growth and metastasis of melanoma cells.In addition,we also found that the levels of p-AKT protein were also significantly reduced after triptonide treatment.In summary,we found that triptonide selectively increases the expression of the tumor suppressor SAV1 and LATS1 in the Hippo signaling pathway,promotes the downstream of Hippo signaling pathway YAP protein lysosomal degradation,and simultaneously reduces the protein levels of p-AKT,therefore inhibiting the expression of downstream tumorigenic genes and ultimately inhibiting the metastasis of melanoma.Our findings provide novel strategy and new drug candidate for the treatment of metastatic melanoma,inheriting and carrying forward the traditional Chinese medicine.