CDK6/4 inactivates BAP1 deubiquitinase destabilizing VHL to promote metastatic colonization in liver

Single organ metastasis to liver typically occurs in uveal melanoma (UM) and colorectal carcinoma (CRC). Colonization in the host organ is a beachhead step of the metastasis process. The governing pathways of the seeding cells are far away clarified. This study aimed at deciphering governing pathways of the seeding cells that required for metastatic colonization and developing novel strategies against metastasis. Mass spectrum analysis of Co-IP pellets was applied to identify the kinase and the substrates of BAP1. Findings were further validated using GST-pull down assay and the proximity ligation assay. The function of CDK6/4-BAP1-VHL signaling axis in promoting liver metastasis was investigated by UM and CRC mouse model. Mass spectrum analysis of Co-IP pellets with anti-BAP1 revealed that VHL bound with BAP1. Their physical interaction was further confirmed by GST-pull down assay and proximity ligation assay. We mapped the interacting domains between BAP1 and VHL. In vivo ubiquination assay showed that BAP1 deubiquitinated VHL at K48-linked poly-ubiquitin chain and stabilized VHL protein. Further, exosomal CDK6/4 serine/threonine kinases were identified to phosphorylate BAP1 at S369 to inactivate BAP deubiquitinationase, forming CDK6/4-BAP1-VHL signaling axis. Functionally, BAP1 inhibited CSCs, EMT and metastatic colonization to liver in uveal melanoma and colorectal carcinoma via upregulating VHL protein, which was, however, reversed by targeting CDK6/4. In conclusion, VHL is a novel substrate of BAP; and BAP1 suppresses CSCs and metastatic colonization to liver via substrate VHL. These findings may shed lights on the mystery substrates of BAP1 and the underlying mechanism of colonization in liver and intervention targets. VHL protein is a novel substrate of BAP1 deubiquitinase. The interacting domains between BAP1 and VHL are mapped. BAP1 deubiquitinates and stabilizes VHL in K48-linked poly ubiquitin chain. BAP1 deubiquitinationase activity is inhibited by the S369 phosphorylation of BAP1 that is catalyzed by CDK6/4 kinases, forming CDK6/4-BAP1-VHL signaling axis. CDK6/4-containing tumor derived exosomes secreted by EMT cells and CSCs may further increase CSCs and EMT by an autocrine- or paracrine-like mechanism, forming a positive feedback loop. Targeting CDK6/4 is an effective way to release BAP1 tumor suppressive activity. BAP1 functionally inhibits CSCs and metastatic colonization to liver in uveal melanoma and colorectal carcinoma via stabilizing VHL protein. BAP1 and VHL are negatively correlated with liver metastasis in patients with uveal melanoma and colorectal carcinoma.