Title : Polyphyllin D inhibits chaperone-mediated autophagy by targeting the HSC70-LAMP2A interaction in non-small cell lung cancer

Background and Purpose: Chaperone-mediated autophagy (CMA) is a selective degradation pathway that is highly active in various malignancies, including non-small cell lung cancer (NSCLC), and contributes to tumor progression. Currently, no targeted chemical inhibitors of CMA are available. This study aims to investigate the role of CMA in NSCLC and to identify a specific small-molecule inhibitor targeting the core CMA components HSC70 and LAMP2A.

Experimental Approach: CMA activity in human NSCLC tissue samples was assessed using tyramide signal amplification dual immunofluorescence. High-content screening was performed to identify CMA inhibitors from a natural product library. The direct targets of the candidate compound were identified using drug affinity responsive target stability coupled with mass spectrometry (DARTS-MS) and validated through cellular thermal shift assay (CETSA), microscale thermophoresis (MST), and LC-MS/MS binding site analysis. The molecular mechanisms of CMA inhibition were investigated in vitro and in vivo using xenograft mouse models.

Results: High expression and co-localization of HSC70 and LAMP2A in NSCLC tissues correlated with poor patient prognosis. Polyphyllin D (PPD) was identified as a potent CMA inhibitor that disrupts the HSC70-LAMP2A interaction. PPD directly binds to the E129 and T278 residues at the nucleotide-binding domain of HSC70 and the C-terminal domain of LAMP2A, respectively, thereby blocking substrate translocation into lysosomes. PPD also inhibited LAMP2A homomultimerization. Mechanistically, PPD suppressed the HSC70-LAMP2A-eIF2α signaling axis, leading to unfolded protein accumulation and ROS generation. Notably, PPD also blocked the compensatory activation of macroautophagy by inhibiting the STX17-SNAP29-VAMP8 complex, thereby impairing autophagosome-lysosome fusion. In vivo, PPD significantly suppressed NSCLC tumor growth without notable toxicity.

Conclusions and Implications: PPD is a first-in-class targeted CMA inhibitor that simultaneously blocks HSC70-LAMP2A interaction and LAMP2A multimerization. By suppressing CMA without inducing macroautophagy compensation, PPD represents a promising therapeutic strategy for NSCLC treatment.

Keywords: Chaperone-mediated autophagy; HSC70; LAMP2A; Polyphyllin D; Non-small cell lung cancer

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