Exosomal annexin A6 induces gemcitabine resistance by inhibiting ubiquitination and degradation of EGFR in triple-negative breast cancer

Ting Li,Yiqun Du,Biyun Wang,Jian Zhang,Zhonghua Tao,Leiping Wang,Xiaojia Liu,Xichun Hu,Jun Cao,Yihui Zhu

doi:10.1038/s41419-021-03963-7

Abstract

Exosomes are carriers of intercellular information that regulate the tumor microenvironment, and they have an essential role in drug resistance through various mechanisms such as transporting RNA molecules and proteins. Nevertheless, their effects on gemcitabine resistance in triple-negative breast cancer (TNBC) are unclear. In the present study, we examined the effects of exosomes on TNBC cell viability, colony formation, apoptosis, and annexin A6 (ANXA6)/EGFR expression. We addressed their roles in gemcitabine resistance and the underlying mechanism. Our results revealed that exosomes derived from resistant cancer cells improved cell viability and colony formation and inhibited apoptosis in sensitive cancer cells. The underlying mechanism included the transfer of exosomal ANXA6 from resistant cancer cells to sensitive cancer cells. Isobaric peptide labeling–liquid chromatography–tandem mass spectrometry and western blotting revealed that ANXA6 was upregulated in resistant cancer cells and their derived exosomes. Sensitive cancer cells exhibited resistance with increased viability and colony formation and decreased apoptosis when ANXA6 was stably overexpressed. On the contrary, knockdown ANXA6 restored the sensitivity of cells to gemcitabine. Co-immunoprecipitation expression and GST pulldown assay demonstrated that exosomal ANXA6 and EGFR could interact with each other and exosomal ANXA6 was associated with the suppression of EGFR ubiquitination and downregulation. While adding lapatinib reversed gemcitabine resistance induced by exosomal ANXA6. Moreover, ANXA6 and EGFR protein expression was correlated in TNBC tissues, and exosomal ANXA6 levels at baseline were lower in patients with highly sensitive TNBC than those with resistant TNBC when treated with first-line gemcitabine-based chemotherapy. In conclusion, resistant cancer cell-derived exosomes induced gemcitabine resistance via exosomal ANXA6, which was associated with the inhibition of EGFR ubiquitination and degradation. Exosomal ANXA6 levels in the serum of patients with TNBC might be predictive of the response to gemcitabine-based chemotherapy.

Highlights

Breast cancer, especially triple-negative breast cancer (TNBC), is the most common malignancy in women and a serious threat to public health
Our results revealed that exosomal Annexin A6 (ANXA6) derived from gemcitabine-resistant cancer cells interacted with EGFR and induced gemcitabine resistance by inhibiting EGFR ubiquitination and degradation
These biological interactions between exosomal ANXA6 and EGFR in TNBC were mirrored by statistical evidence that exosomal ANXA6 levels in serum from patients with TNBC who received first-line gemcitabine-based chemotherapy were predictive of therapeutic response

Summary

INTRODUCTION

Especially triple-negative breast cancer (TNBC), is the most common malignancy in women and a serious threat to public health. Immunohistochemistry and scoring Using an institution review board-approved sample collection protocol at RNA isolation, reverse-transcription, and qPCR Total RNA was extracted from cells using a TRIzol kit (ThermoFisher Scientific, Waltham, MA, USA). The resin was collected by centrifugation and washed three times with lysis buffer, and the amount of HA-EGFR bound to Annexin A6 beads was detected by western blot analysis. The following antibodies were used: anti-ANXA6 (1:500, 12542-1-AP, Proteintech, USA), anti-ANXA1 (1:1000, 21990-1-AP, Proteintech, USA), anti-EGFR (1:1000, ab52894, Abcam, USA), anti-TFPI (1:2000, 66842-1-Ig, Proteintech, USA), anti-COL8A1 ELISA The levels of exosomal ANXA6 from patient serum were detected using a human Annexin A6 ELISA kit (Shanghai Enzyme-linked Biotechnology Co., Ltd, China) following the manufacturer’s instructions. All statistical analyses were performed using the GraphPad Prism® 7.0a software and the Statistical Package for Social Sciences Version 22.0 (SPSS 16.0, USA)

RESULTS

DISCUSSION

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