Metformin Combining PD-1 Inhibitor Enhanced Anti-Tumor Efficacy in STK11 Mutant Lung Cancer Through AXIN-1-Dependent Inhibition of STING Ubiquitination

Zhiguo Wang; Conghua Lu; Kejun Zhang; Caiyu Lin; Fang Wu; Xiaolin Tang; Di Wu; Yuanyao Dou; Rui Han; Yubo Wang; Chao Hou; Qin Ouyang; Mingxia Feng; Yong He; Li Li

doi:10.3389/fmolb.2022.780200

Metformin Combining PD-1 Inhibitor Enhanced Anti-Tumor Efficacy in STK11 Mutant Lung Cancer Through AXIN-1-Dependent Inhibition of STING Ubiquitination

Front Mol Biosci. 2022 Feb 23:9:780200. doi: 10.3389/fmolb.2022.780200. eCollection 2022.

Authors

Zhiguo Wang¹, Conghua Lu¹, Kejun Zhang², Caiyu Lin¹, Fang Wu³, Xiaolin Tang¹, Di Wu¹, Yuanyao Dou¹, Rui Han¹, Yubo Wang¹, Chao Hou¹, Qin Ouyang⁴, Mingxia Feng¹, Yong He¹, Li Li¹

Affiliations

¹ Department of Respiratory Disease, Daping Hospital, Third Military Medical University (Army Medical University), Chongqing, China.
² Department of Outpatients, Daping Hospital, Third Military Medical University (Army Medical University), Chongqing, China.
³ Department of Oncology, Hunan Key Laboratory of Tumor Models and Individualized Medicine, Hunan Key Laboratory of Early Diagnosis and Precision Therapy in Lung Cancer, The Second Xiangya Hospital, Central South University, Changsha, China.
⁴ School of Pharmacy, Third Military Medical University (Army Medical University), Chongqing, China.

Abstract

Background: Non-small-cell lung cancer (NSCLC) with STK11 mutation showed primary resistance to immune checkpoint inhibitors (ICIs). The glucose-lowering drug metformin exerted anti-cancer effect and enhanced efficacy of chemotherapy in NSCLC with KRAS/STK11 co-mutation, yet it is unknown whether metformin may enhance ICI efficacy in STK11 mutant NSCLC. Methods: We studied the impact of metformin on ICI efficacy in STK11 mutant NSCLC in vitro and in vivo using colony formation assay, cell viability assay, Ki67 staining, ELISA, CRISPR/Cas9-mediated knockout, and animal experiments. Results: Through colony formation assay, Ki67 incorporation assay, and CCK-8 assay, we found that metformin significantly enhanced the killing of H460 cells and A549 cells by T cells. In NOD-SCID xenografts, metformin in combination with PD-1 inhibitor pembrolizumab effectively decreased tumor growth and increased infiltration of CD8⁺ T cells. Metformin enhanced stabilization of STING and activation of its downstream signaling pathway. siRNA-mediated knockdown of STING abolished the effect of metformin on T cell-mediated killing of tumor cells. Next, we found that CRISPR/Cas9-mediated knockout of the scaffold protein AXIN-1 abolished the effect of metformin on T cell-mediated killing and STING stabilization. Immunoprecipitation and confocal macroscopy revealed that metformin enhanced the interaction and colocalization between AXIN-1 and STING. Protein-protein interaction modeling indicated that AXIN-1 may directly bind to STING at its K150 site. Next, we found that metformin decreased K48-linked ubiquitination of STING and inhibited the interaction of E3-ligand RNF5 and STING. Moreover, in AXIN-1 ^-/- H460 cells, metformin failed to alter the interaction of RNF5 and STING. Conclusion: Metformin combining PD-1 inhibitor enhanced anti-tumor efficacy in STK11 mutant lung cancer through inhibition of RNF5-mediated K48-linked ubiquitination of STING, which was dependent on AXIN-1.

Keywords: AXIN-1; STING; STK11; immunotherapy; lung cancer; metformin.