Immune suppression is a hallmark of cancer, and reinvigorating anti-tumor immune responses is a key focus in cancer treatment. PD-1/PD-L1 immune checkpoint inhibition (ICI) is a promising therapy, but resistance is common. PD-L1 internalization following antibody binding leads to some lysosomal degradation, but significant recycling back to the cell membrane also occurs, mediating resistance. Therefore, promoting lysosomal degradation and inhibiting PD-L1 recycling is a promising strategy to overcome this resistance. CMTM6 aids PD-L1 recycling, and its depletion promotes lysosomal degradation. However, the detailed mechanism of PD-L1 lysosomal degradation remains unclear. Hsc70 (heat shock protein family A (Hsp70) member 8), a cytoplasmic chaperone protein, plays a crucial role in endosomal microautophagy (eMI) and chaperone-mediated autophagy (CMA). Approximately 40% of mammalian proteins contain KFERQ-like motifs, serving as Hsc70 substrates. Hsc70 delivers cargo proteins to lysosomes via CMA or eMI, with eMI playing a significant role in degrading proteins in a semi-aggregated state or with post-translational modifications that prevent CMA degradation. This study investigates the role of Hsc70 in PD-L1 degradation.

Extensive research has explored PD-L1 degradation, primarily focusing on the proteasome pathway. Studies have shown that inhibiting PD-L1 glycosylation or deubiquitination promotes proteasomal degradation. Other studies have shown that HIP1R targets PD-L1 for lysosomal degradation. CMTM6 has been identified as a key regulator of PD-L1 recycling to the cell membrane, highlighting the importance of the endosomal-lysosomal pathway. However, detailed mechanisms involving lysosomal degradation remain understudied. The study of PD-L1 degradation in the endosomal lysosomal pathway is scarce. The role of Hsc70, a key chaperone in both CMA and eMI, in PD-L1 degradation has not been fully elucidated.

The study used mass spectrometry to identify Hsc70 as a major PD-L1 interacting protein. Hsc70 overexpression in MCF-7 and PANC1 cells was used to investigate its effect on PD-L1 expression, showing decreased total and surface PD-L1 protein levels. Lysosome inhibitors (Leupeptin + NH4Cl or E64D), proteasome inhibitor (MG132), and autophagy inhibitor (3-MA) were used to determine the mechanism of PD-L1 degradation. The role of CMA was investigated by manipulating Lamp2a expression and using CMA inducers Spautin-1 and AC220. eMI involvement was investigated by knocking down TSG101 and VPS4 (key regulators of eMI), and by using the eMI inhibitor U18666A. A mutant form of Hsc70 (Hsc70-3KA) was used to study the importance of its C-terminal domain interaction with phosphatidylserine. The competitive binding of Hsc70 and CMTM6 to PD-L1 was investigated using co-immunoprecipitation and various PD-L1 truncations. The role of TFG, identified via mass spectrometry, was investigated through overexpression and knockdown studies. In vivo studies used 4T1 murine breast cancer cells in immunocompetent and immunodeficient mice. AUY-922, an Hsp90 inhibitor, was used to investigate its effect on Hsc70 expression and PD-L1 degradation. Combination therapy studies with AUY-922 and anti-PD-L1 or anti-CTLA4 antibodies were also performed. Standard techniques such as western blotting, flow cytometry, immunofluorescence, immunohistochemistry, and qRT-PCR were employed. Detailed mass spectrometry methods are described, including sample preparation, digestion, and data analysis.

Mass spectrometry identified Hsc70 as a PD-L1 interacting protein. Hsc70 overexpression significantly reduced total and surface PD-L1 levels, primarily through lysosomal degradation via eMI, not CMA. Hsc70 competes with CMTM6 for PD-L1 binding. Knockdown of CMTM6 enhanced Hsc70-PD-L1 interaction and PD-L1 degradation. TFG, a COPII-mediated vesicle transport protein, was identified as a mediator of Hsc70-induced PD-L1 degradation. In vivo studies showed that Hsc70 overexpression suppressed tumor growth in immunocompetent mice, increasing intratumoral CD8+ T cells and reducing PD-L1 levels. The Hsp90 inhibitor AUY-922 increased Hsc70 levels and promoted PD-L1 degradation through eMI, enhancing anti-tumor immunity in vivo. AUY-922 treatment significantly enhanced the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 therapies.

This study elucidates a novel mechanism of Hsc70-mediated PD-L1 degradation via eMI, offering potential therapeutic strategies. Hsc70's competitive binding with CMTM6 for PD-L1 is key to this mechanism. The finding that AUY-922, an Hsp90 inhibitor, upregulates Hsc70 and enhances PD-L1 degradation provides a promising therapeutic target. The in vivo data strongly support the anti-tumor effects of Hsc70 upregulation and the potential for combination therapy with AUY-922 and ICI. The identification of TFG as a mediator in the Hsc70 pathway provides further mechanistic insights into eMI regulation in PD-L1 degradation.

This research reveals a crucial role for Hsc70 in promoting anti-tumor immunity by targeting PD-L1 for lysosomal degradation through eMI. The Hsp90 inhibitor AUY-922 is identified as a potential therapeutic agent to enhance this process. Future research could focus on further elucidating the role of TFG and other factors involved in eMI regulation, developing more specific Hsc70 modulators, and exploring the application of this mechanism in other cancers and diseases.

The study primarily focused on breast cancer cell lines and a murine model. Further investigations are needed to validate these findings across a wider range of cancer types and in human clinical trials. While the study strongly suggests Hsc70's role in PD-L1 degradation is primarily through eMI and not CMA, additional experiments could further solidify this conclusion. The specific interactions between Hsc70, CMTM6, TFG, and PD-L1 warrant more detailed investigation at the molecular level.