Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Accumulating evidence suggests that autophagy influences cellular immune responses. However, the autophagy-dependent mechanisms of tumor cells regulating the immune attack of T cells remain unclear. Our lab had been working on the role of autophagy in cancer for several years. Our previous work has demonstrated that impaired autophagy leads to abnormalities of the selective autophagic degradation of HK2, which contributes to the substantial concomitant enhancement of glycolysis in liver cancer1. We also found that CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id protein and neuroblastoma cell differentiation2. Breast cancer is one of the first diseases genetically linked to a deficiency in autophagy 3-5. In this study, we tried to determine the suppressive factors that are minimally required for governing the balance between pro-tumorigenic and anti-tumorigenic immune responses in TNBC. We examined the complex role of autophagy in TNBC tumor immunity from the perspective of abnormal autophagic protein degradation.
The in-silico analysis of The Cancer Genome Atlas (TCGA) data showed that only in basal-like breast cancer, tumor-infiltrating CD4+ and CD8+ lymphocytes were positively related to Atg5 and Beclin1 expression. Inspired by this result, we developed a defective autophagy model in vitro and in vivo by using specific single-guide RNAs (sgRNAs) or RNAi to perturb the expression of genes required for the activation of autophagy. We validated that autophagy defects inhibited T cell-mediated killing in TNBC tumors in vitro and in vivo. As the autophagic potential may be impaired in TNBC tumors, this finding may at least partially explain why most TNBC patients are resistant to immune checkpoint inhibitors.
A SILAC assay was adopted to further explore the mechanisms involved in autophagy-deficiency-mediated immunosuppression in TNBC tumors. We found that Tenascin-C (TNC), an extracellular matrix glycoprotein, was selectively targeted for lysosomal degradation by an autophagy-dependent mechanism that involved the autophagy cargo receptor p62. Autophagy-deficient TNBC cells displayed increased expression of TNC, and this increase was blunted by the restoration of autophagy. Besides, knockdown of TNC in autophagy-deficient TNBC cells restored the cells’ susceptibility to killing by cytotoxic T cells in vitro and in vivo. Notably, inhibition of TNC in autophagy-impaired TNBC cells improved the anti-tumor effects of immune checkpoint inhibitors in vitro and in vivo. TNC has been subjected to extensive investigation due to its selective expression in the breast cancer6,7. Therefore, the prevalent upregulation of TNC provides a rationale target in TNBC patients. In this study, our findings identified the crosstalk between autophagy and immune compartments as a targetable component for potential therapeutic strategies for TNBC patients with poor response to immune checkpoint blockade therapy. According to Kaplan-Meier analysis of relapse-free survival based on TNC levels, TNBC patients with the basal-like 1, basal-like 2, immunomodulatory, mesenchymal, and mesenchymal stem-like subtypes, especially those with the basal-like and immunomodulatory subtypes, may benefit from a combination strategy using inhibition of TNC and immune checkpoint inhibitors.
1 Jiao, L. et al. Regulation of glycolytic metabolism by autophagy in liver cancer involves selective autophagic degradation of HK2 (hexokinase 2). Autophagy 14, 671-684, doi:10.1080/15548627.2017.1381804 (2018).
2 Li, X. et al. CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation. Nature communications 8, 1159, doi:10.1038/s41467-017-01272-2 (2017).
3 Liang, X. H. et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 402, 672-676, doi:10.1038/45257 (1999).
4 Tang, J. et al. Low expression of ULK1 is associated with operable breast cancer progression and is an adverse prognostic marker of survival for patients. Breast cancer research and treatment 134, 549-560, doi:10.1007/s10549-012-2080-y (2012).
5 Tang, H. et al. Decreased mRNA Expression in Human Breast Cancer is Associated with Estrogen Receptor-Negative Subtypes and Poor Prognosis. EBioMedicine 2, 255-263, doi:10.1016/j.ebiom.2015.01.008 (2015).
6 Howeedy, A. A. et al. Differential distribution of tenascin in the normal, hyperplastic, and neoplastic breast. Laboratory investigation; a journal of technical methods and pathology 63, 798-806 (1990).
7 Guttery, D. S., Shaw, J. A., Lloyd, K., Pringle, J. H. & Walker, R. A. Expression of tenascin-C and its isoforms in the breast. Cancer metastasis reviews 29, 595-606, doi:10.1007/s10555-010-9249-9 (2010).