Ubiquitin Specific Peptidase 24‐mediated Interleukin 6 Expression in Tumor‐associated Microenvironment Promotes Tumor Malignancy

Ubiquitin‐specific peptidases (USPs), belong to a large family of cysteine proteases, and are deubiquitinases (DUBs) that specifically recognize and remove ubiquitin molecules from proteins. Previously, we discovered ubiquitin‐specific peptidase 24 (USP24) was upregulated in late stage lung cancer patients due to its genomic variants caused by single nucleotide polymorphism (SNP) or RNA variants caused by RNA editing. The up‐regulated USP24 can promote lung cancer cell metastasis by activating several metastatic related genes through modifying histone methylation. Meanwhile, in the immunohistochemistry results, we also found leukocytes infiltrating in tumors expressed higher USP24 level than surrounding lung cancer cells in lung cancer patient specimens. The role of USP24 in microenvironment remains unclear and this finding implied us that USP24 might correlate to tumor associated macrophages (TAMs) and is related to tumor‐associated microenvironment. We chose monocyte‐differentiated M2 macrophages for further studies since most macrophages located at tumor‐associated microenvironments are M2 macrophages. We found USP24 level was significantly increased when THP‐1 monocytes differentiated into M2 macrophages. Knockdown USP24 in M2 macrophages can reduce the migratory and chemotaxic activity of lung cancer cells and angiogenetic ability of human umbilical vein endothelial cells (HUVEC) induced by M2 derived conditional medium. In vivo experiments also showed USP24 knockdown decreased M2 induced lung cancer cells metastasis and angiogenesis. We analyzed several well‐known metastasis related factors, including interleukin 6 (IL‐6), interleukin 8 (IL‐8), interleukin 10 (IL‐10), and several C‐C motif chemokine ligands (CCLs), and we found IL‐6 mRNA and protein level was significantly decreased in USP24 knockdown M2 macrophages and conditional medium derived from USP24 knockdown M2. IL‐6 replenishment can rescue migratory and chemotaxic ability of conditional medium derived from USP24 knockdown M2 suggesting that USP24 can induce cancer cells metastasis through promoting IL‐6 expression. Transcription factors such as nuclear factor kappa B (NF‐kB), activator protein 1 (AP‐1), and signal transducer and activator of transcription 3 (Stat3) have been reported to activate IL‐6 promoter activity, and we found NF‐kB mRNA and protein level were decreased in M2 macrophages after USP24 knockdown. When we analyzed the promoter region of NF‐kB, we found p300, a target protein of USP24 which was identified in our previous study has several putative binding sites in NF‐kB promoter region. Meanwhile, we found USP24 can interact with p300 and knockdown UPS24 can decrease p300 level but increase the ubiquitin status of p300 in M2 macrophages, just like in lung cancer cells. Therefore, we hypnotize that when monocytes differentiate into M2 macrophages, USP24 level is increased and induce NF‐kB expression through stabilizing p300. The up‐regulated NF‐kB then can activate IL‐6 expression and secretion, and result in the promotion of lung cancer metastasis. More experiments needed to be done to support our hypothesis. We think by understanding the detail correlation between USP24 and IL‐6 in tumor‐associated macrophages can provide an insight into the prevention of metastasis in the future. Support or Funding Information We are grateful for the support from the Tissue Bank, Research Center of Clinical Medicine, National Cheng Kung University Hospital. This work was supported by the Program for Promoting Academic Excellence and Developing World Class Research Center of National Cheng Kung University, with grant (103‐2320‐B‐006‐035‐MY3) obtained from the Ministry of Science and Technology, Taiwan.