Celastrol inhibits the immune escape and proliferation of triple - negative breast cancer (TNBC) through the LKB1 - AMPK - PD-L1/MYC signaling pathway

Background: Celastrol can effectively inhibit tumor proliferation and interfere with the process of tumor immune escape, but the specific regulatory pathways remain unclear. This study explored the regulatory mechanisms related to celastrol in the treatment of TNBC and might provide potential therapeutic targets.

Methods: The proliferation ability of tumor cells was analyzed using growth curves and colony - formation assays. The invasion and migration abilities of tumor cells were assessed through transwell and cell - scratch assays. To determine the expression levels of related proteins, Western blot analysis was conducted. qPCR (Real - time quantitative PCR) was utilized to detect the mRNA (Messenger RNA) levels of relevant molecules. RNA-seq (RNA Sequencing) was carried out to identify the related molecular signaling pathways involved. To analyze the interactions between protein partners, Co -IP (Co-Immunoprecipitation) was performed. Fluorescence confocal experiments were executed to examine the co-localization between molecules. For gene knockdown, siRNA (Small interfering RNA) was applied. Finally, IHC (Immunohistochemistry) was used to detect the expression of related molecules within tissues. This set of experiments provided a multi - dimensional understanding of tumor cell behavior and the underlying molecular basis. Flow cytometry is used to analyze changes in the cell cycle.

Results: Celastrol could significantly inhibit the proliferation, invasion, migration, and immune - escape abilities of TNBC cells. When the proliferation ability of triple - negative breast Cancer was inhibited alone, its invasion and migration abilities were also significantly inhibited. RNA seq showed that after treatment with celastrol, the LKB1 (Liver Kinase B1) - AMPK (AMP-activated protein kinase) signaling pathway was significantly activated, while the PD-L1 (Programmed Death-Ligand 1) pathway was significantly inhibited. The results of Western blot indicated that celastrol activated the phosphorylation levels of LKB1 and AMPK and decreased the PD-L1 protein expression, which was consistent with the qPCR results. After over-expressing LKB1, the protein expression of PD-L1 was significantly inhibited, which was consistent with the results after celastrol treatment. The results of co - immunoprecipitation showed that there was an interaction between PD-L1 and MYC (MYC proto-oncogene). The extracellular domain of PD-L1 and the degron domain of MYC mediate their functional interaction, and the fluorescence confocal experiment suggested that there was co - localization between PD-L1 and MYC. After knocking out PD-L1, the protein expression of MYC was significantly decreased. PD - L1 increased the protein stability of MYC in the form of K63 - linked ubiquitination (Lysine 63-linked ubiquitination). Moreover, when MYC was inhibited, the proliferation ability of tumor cells was significantly reduced. The results of animal experiments also showed that celastrol could significantly inhibit the proliferation of subcutaneous xenograft tumors in mice, specifically manifested as a decrease in both tumor volume and weight. The results of immunohistochemistry indicated that celastrol could activate the phosphorylation levels of LKB1 and AMPK in tissues and reduce the protein expressions of PD-L1 and MYC.

Conclusion: Celastrol inhibits the proliferation and immune - escape abilities of TNBC by activating the LKB1 - AMPK - PD-L1/MYC signaling pathway, providing potential therapeutic targets for further treatment of TNBC.