RPL21 Interacts With LAMP3 To Promote Colorectal Cancer Invasion And Metastasis By Regulating Focal Adhesion Formation

Colorectal cancer(CRC)is one of the most common malignancies in our country.Metastasis is the leading cause of death among CRC patients.Therefore,it is important to explore the molecular mechanisms of metastasis to develop effective therapeutic targets for CRC.In the present study,we hypothesized that ribosomal protein L21(RPL21)promotes CRC metastasis;however,its underlying mechanism remains to be investigated further.Quantitative reverse transcription polymerase chain reaction(qRT-PCR),Western Blot and immunohistochemistry(IHC)were performed to measure the expression of RPL21 in CRC cells and tissues,and we found that RPL21 was highly expressed in CRC,contributing to tumor invasiveness and poor patient prognosis.Functionally,wound healing and Transwell migration and invasion assays were performed to study the migration and invasion of cultured CRC cells.An orthotopic CRC mouse model was developed to investigate the metastatic ability of CRC tumor cells,which confirmed that RPL21 could promote the migration and invasion of CRC cells in vitro and tumor metastasis in vivo.Moreover,lysosome-associated membrane protein 3(LAMP3)was identified by transcriptome sequencing as a highly related gene of RPL21 and was essential in promoting migration and invasion of CRC cells.Mechanistically,the phosphorylation level and the subcellular localization of transcription factor EB(TFEB)and dual-luciferase reporter gene assays revealed that RPL21 activated the transcriptional function of TFEB to upregulate LAMP3 expression.GST/His pulldown assays demonstrated that RPL21 directly bound to the aa 341-416 domain of LAMP3 via its aa 1-40 and aa 111-160 segments.The combination of RPL21 and LAMPS enhanced the stability of the RPL21 protein by suppressing the degradation of ubiquitin-proteasome system.Furthermore,the results of cell adhesion assay,immunofluorescence and Western Blot showed that RPL21 and LAMP3 promoted the formation of immature focal adhesions and reduced the adhesion ability of cells to extracellular matrix by activating the FAK/Paxillin/ERK signaling pathway,thereby enhancing the distant spreading of CRC cells.This study highlighted the prometastatic effect of RPL21 by regulating focal adhesion formation in a LAMP3-dependent manner during CRC progression.The interaction between RPL21 and LAMP3 may function as a potential therapeutic target against CRC.