Discovery of the late autophagy inhibitor FZU‑0045‑053 and its anti‑breast cancer and immunomodulatory effects.

Breast cancer is characterized by notable heterogeneity and remains one of the leading causes of cancer‑related death among women. Autophagy, a process by which cells use lysosomes to degrade cytoplasmic proteins and damaged organelles, is not only associated with chemotherapy resistance, but is also involved in immune‑mediated tumor cell killing and immune evasion, making it a promising target for cancer therapy. Pharmacological inhibition of autophagy in breast cancer cells suppresses tumor progression. In the present study, the small molecular compound FZU‑0045‑053 (053) was identified, which exhibited autophagic and immunomodulatory effects. The effect of 053 on autophagy regulation in breast cancer cells was evaluated using transmission electron microscopy, an mRFP‑GFP‑ microtubule‑associated protein 1 light chain 3 (LC3) tandem fluorescent adenovirus, the CYTO‑ID Autophagy Detection Kit and western blot analysis. Cell viability was subsequently assessed with proliferation assay and ATP assay kits. Apoptosis induction and the expression of immune‑related molecules were measured by flow cytometry. Furthermore, a triple‑negative breast cancer mouse model was established to validate the antitumor and autophagy‑modulating effects of 053 in vivo using immunofluorescence and immunohistochemical staining. Finally, a 4T1 syngeneic mouse model was utilized to corroborate the immunomodulatory effects of 053 in vivo through immunohistochemistry and flow cytometric analysis. The findings indicated that 053 regulated autophagy in the breast cancer cell lines MDA‑MB‑231 and MCF‑7, similar to the late autophagy inhibitor chloroquine. This regulation resulted in the accumulation of autophagic substrates, specifically LC3‑II and sequestosome 1, by blocking autophagic flux. By blocking autophagy flux, 053 suppressed proliferation, induced apoptosis and ultimately restored chemosensitivity in MDA‑MB‑231 cells. In addition, the MDA‑MB‑231 xenograft model indicated that 053 inhibited autophagy by blocking autophagic flux, which lead to the accumulation of LC3 and sequestosome 1. 053 also negatively regulated the expression of programmed death‑ligand 1 (PD‑L1) in tumor cells. The 4T1 xenograft model showed that 053 had a notable immune‑promoting effect, whereby it not only negatively regulated the expression of PD‑L1 in tumor cells but also modulated T cell activation and proliferation by downregulating the expression of co‑inhibitory molecules (T‑cell immunoglobulin and mucin‑domain containing‑3 and programmed cell death protein 1) on T cells and upregulating co‑stimulatory molecules (4‑1BB, OX40 and inducible T‑cell co‑stimulator). In vivo xenograft models demonstrated that 053 had notable antitumor effects and high biosafety, with improved antitumor efficacy when combined with the chemotherapy drug gemcitabine. In summary, 053 can block autophagy and promote antitumor immune responses, showing promise as a new generation of adjuvant drugs for tumor chemotherapy and immunotherapy.