LC3B upregulation by NANOG promotes immune resistance and stem-like property through hyperactivation of EGFR signaling in immune-refractory tumor cells

Immune selection drives tumor cells to acquire refractory phenotypes. We previously demonstrated that cytotoxic T lymphocyte (CTL)-mediated immune pressure enriches NANOG⁺ tumor cells with stem-like and immune-refractory properties that make them resistant to CTLs. Here, we report that the emergence of refractory phenotypes is highly associated with an aberrant macroautophagic/autophagic state of the NANOG⁺ tumor cells and that the autophagic phenotype arises through transcriptional induction of MAP1LC3B/LC3B by NANOG. Furthermore, we found that upregulation of LC3B expression contributes to an increase in EGF secretion. The subsequent hyperactivation of EGFR-AKT signaling rendered NANOG⁺ tumor cells resistant to CTL killing. The NANOG-LC3B-p-EGFR axis was preserved across various types of human cancer and correlated negatively with the overall survival of cervical cancer patients. Inhibition of LC3B in immune-refractory tumor models rendered tumors susceptible to adoptive T-cell transfer, as well as PDCD1/PD-1 blockade, and led to successful, long-term control of the disease. Thus, our findings demonstrate a novel link among immune-resistance, stem-like phenotypes, and LC3B-mediated autophagic secretion in immune-refractory tumor cells, and implicate the LC3B-p-EGFR axis as a central molecular target for controlling NANOG⁺ immune-refractory cancer. Abbreviations: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A₁; CASP3: caspase 3; CFSE: carboxyfluorescein succinimidyl ester; ChIP: chromatin immunoprecipitation; CI: confidence interval; CIN: cervical intraepithelial neoplasia; CSC: cancer stem cell; CTL: cytotoxic T lymphocyte; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; FIGO: International Federation of Gynecology and Obstetrics; GFP: green fluorescent protein; GZMB: granzyme B; HG-CIN: high-grade CIN; IHC: immunohistochemistry; LG-CIN: low-grade CIN; LN: lymph node; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCL1: myeloid cell leukemia sequence 1; MLANA/MART-1: melanoma antigen recognized by T cells 1; MUT: mutant; NANOG: Nanog homeobox; PDCD1/PD-1: programmed cell death 1; PMEL/gp100: premelanosome protein; RTK: receptor tyrosine kinase; TMA: tissue microarray; WT: wild type.