Khellin Modulates DDIT4 to Regulate Autophagy in Triple-Negative Breast Cancer

Objective: To investigate the potential anti-tumor mechanism and clinical impact of Khellin in triple-negative breast cancer (TNBC). Methodology: MDA-MB-231 cells were treated with 20 μM Khellin for 24 hours, and total RNA was extracted for transcriptome sequencing. Differentially expressed genes (DEGs) were screened, followed by functional and pathway enrichment analyses. Survival analysis, gene set enrichment analysis, and tumor immune microenvironment (TIME) analysis were employed to study the role of key genes. Subsequently, DDIT4 expression and changes in autophagy marker proteins were assessed using qRT-PCR and Western blot. Results: A total of 158 DEGs were discovered, with high enrichment in signaling pathways such as PI3K-Akt. DDIT4 was considerably elevated after Khellin treatment, and higher expression was associated with a better prognosis in TNBC patients. Immune infiltration and single-cell transcriptome analysis demonstrated that elevated DDIT4 expression improves anti-tumor immune activity in the tumor microenvironment. Khellin administration increased the LC3B-II/I ratio and decreased P62 protein production, which could be reversed by autophagy inhibitors. Conclusion: Khellin upregulates DDIT4 to activate autophagy in TNBC cells and remodel the tumor immune microenvironment. This study elucidates the critical function of DDIT4 in mediating Khellin-induced autophagy and immune modulation, laying the groundwork for a better understanding of its antitumor effects.