RHEBL1 expression is activated by the Oct4-Sox2 complex in oral squamous cell carcinoma

RHEBL1 (RHEB2), a member of the Ras superfamily, has established roles in tumor-promoting signaling pathways including mTOR activation and NF-κB transcription; however, its specific role in oral squamous cell carcinoma (OSCC) and its regulation by the stem cell transcription factors Oct4 and Sox2 have not been previously characterized. This study aimed to elucidate the function and mechanism of RHEBL1 in OSCC development and analyze the regulatory influence of Oct4 and Sox2 on RHEBL1 expression. Immunohistochemistry and immunofluorescence assessed RHEBL1, Oct4, and Sox2 expression in normal and precancerous tissues. RHEBL1-overexpressing and knockout cell lines were created for in vitro assessment of proliferation and self-renewal; mice models were used to evaluate tumor formation in vivo. Bioinformatics analyses predicted Oct4 and Sox2 binding sites within the RHEBL1 promoter, validated by dual-luciferase reporter assays and ChIP-PCR. RHEBL1 showed high expression in OSCC and adjacent tissues, with linear arrangement of positive cells in the basal layer. High levels of RHEBL1, Oct4, and Sox2 were observed at the tumor invasion front and the basal layer of adjacent oral epithelia. RHEBL1 overexpression enhanced sphere formation and induced subcutaneous tumor-like lesions in immunodeficient mice, characterized histologically by invasive growth patterns and vascular structures, whereas control cells showed no such phenomenon. Furthermore, RHEBL1 knockout significantly reduced in vitro sphere formation and in vivo tumorigenicity. Oct4-Sox2 complexes bound two sites in the RHEBL1 promoter; mutations in these sites reduced transcriptional activation. Thus, this study demonstrated that Oct4 and Sox2 promote OSCC initiation and proliferation by regulating RHEBL1 expression. Clinical trial number: not applicable.