TJP2 inhibits clear cell renal cell carcinoma progression by up-regulating TIMM21 to modulate the SPRY4-ERK1/2 signaling pathway

Currently, numerous studies have clearly established the tumor-suppressive role of Tight Junction Protein 2 (TJP2). Although our previous research confirmed the down-regulation of TJP2 in ccRCC, its biological functions and underlying regulatory mechanisms remain poorly understood. In this study, we characterized the biological functions of TJP2 using cell phenotypic assays and mouse orthotopic renal tumor growth model. Subsequently, we identified mitochondrial import inner membrane translocase subunit Tim21 (TIMM21) as the downstream target protein of TJP2 through TMT (Tandem Mass Tags)-based proteomic profiling and co-immunoprecipitation (co-IP) experiment. Furthermore, we elucidated the downstream signaling mechanisms by which the TJP2/TIMM21 axis inhibits ccRCC progression using a combination of IP, Immunofluorescence, Western blotting, qRT-PCR, and RNA-seq analyses. Our experimental results confirmed that TJP2 over-expression significantly inhibits the growth, migration, and invasion of ccRCC cells and promotes cell apoptosis both in vitro and in vivo. TIMM21 is under-expressed in ccRCC, and its decreased expression correlates with unfavorable pathological characteristics and poor patient prognosis. Mechanistically, TJP2 directly binds to TIMM21 protein, promoting its expression and inhibiting its ubiquitination levels. TJP2 over-expression up-regulates SPRY4 expression and inhibits the ERK1/2 signaling pathway. Notably, knockdown of TIMM21 in TJP2-overexpressing cells significantly reverses TJP2-induced cellular phenotypic inhibition, while suppressing SPRY4 expression and promoting pERK1/2 expression. Consistently, knockdown of SPRY4 similarly rescues the inhibited cellular phenotypes caused by TIMM21 over-expression and restores pERK1/2 levels. Our study demonstrates the mechanism by which the TJP2/TIMM21 axis accelerates SPRY4 expression and restrains the ERK1/2 signaling pathway, thereby suppressing ccRCC progression.