Abnormality of selective autophagic degradation of tenascin-C promotes resistance to T cell-mediated immune attack in triple negative breast cancer

Introduction: Accumulating data has demonstrated the crucial role of immunity in TNBC biology, suggesting the potential involvement of immunotherapy to treat this malignancy. A variety of different immuno[[Unsupported Character - Codename s]]therapeutic strategies, such as anti-PD-1 or anti-PD-L1, are currently being tested in preclinical studies in TNBC patients. However, the response rate (RR) to PD-1 or PD-L1 antibody remains low in TNBC. Autophagy regulates the antitumor immune response. The mechanism that autopahgy stimulates or limits the T cell immune system’s attack on tumor cells has not been well understood. Here, we examine the complex role of autophagy in TNBC tumor immunity. Methods: We depleted Atg5, Atg7 and Beclin1 expression using specific single-guide RNAs (sgRNAs) to construct autophagy-deficient TNBC cell lines. The caspase-3 cleavage assay and lactat dehydrogenase-release (LDH) assay were performed to measuring T-cell-mediated cytotoxicity. SILAC and high-resolution MS procedures were implemented to quantify protein variations between autophagy-competent and autophagy- incompetent cells. Flow cytometry analysis was performed to detect the proliferation and activity of T cells. IHC was performed with antibody against Tenascin-C (TNC), LC3, CD8 in paraffin blocks of human TNBC breast lesions. The blockade of TNC alone or in combination with immunotherapy was determined. Statistical analyses were conducted using GraphPad Prism, SPSS software and JavaGSEA Desktop Application. A p value of Results: We found that only in basal tumors but not in Luminal and Her2 tumors, Atg5 and Beclin 1 expression were found to be positively related with the abundance of B cells, CD4 T cells, CD8 T cells, neutrophils, and dendritic cells. The failure of autophagy contributed to the limitation of T-lymphocytes immune system’s attack on TNBC cells in vitro and in vivo. We also identified a novel role for TNC as a key regulator of autophagy-deficient-mediated immunosuppression, in which TNC was Lys63 ubiquitinated by the E3 ligase SKP2, thus promoting its recognition by the autophagy receptorp62 and leading to its selective degradation. The degradation of TNC by the autophagy-lysosome pathway was physiologically significant and clinically relevant in TNBC patients. More importantly, we demonstrated that blockade of TNC sensitized T cell-mediated tumor killing and improved the antitumor effects by PD1 blockade in autophagy-impaired TNBC tumors. Conclusion: Our studies identify a crosstalk between autophagy and immune compartments as a novel therapeutic strategy for TNBC patients with poor immunotherapy response. Our findings indicates that blockade of TNC may be an effective adjuvant treatment with immunotherapy as a novel management strategy for TNBC patients, especially with autophagy deficiency. Note: This abstract was not presented at the meeting. Citation Format: Rong Deng, Zhi-ling Li, Hai-Liang Zhang, Xuan Li, Jia Mia, Li-Huan Zhou, Yun Huang, Yan Yu, Jun Tang, Xiao-Feng Zhu. Abnormality of selective autophagic degradation of tenascin-C promotes resistance to T cell-mediated immune attack in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4950.