Chemotherapy induces ACE2 expression in breast cancer via the ROS-AKT-HIF-1α signaling pathway: a potential prognostic marker for breast cancer patients receiving chemotherapy
Authors: Zuo Xiaoyan , Ren Sixin , Zhang He , Tian Jianfei , Tian Ruinan , Han Baoai , Liu Hui , Dong Qian , Wang Zhiyong , Cui Yanfen , Niu Ruifang , Zhang Fei 
Published: November 2022

Abstract
Background
Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system and a well-known functional receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into host cells. The COVID-19 pandemic has brought ACE2 into the spotlight, and ACE2 expression in tumors and its relationship with SARS-COV-2 infection and prognosis of cancer patients have received extensive attention. However, the association between ACE2 expression and tumor therapy and prognosis, especially in breast cancer, remains ambiguous and requires further investigation. We have previously reported that ACE2 is elevated in drug-resistant breast cancer cells, but the exact function of ACE2 in drug resistance and progression of this malignant disease has not been explored.

Methods
The expression of ACE2 and HIF-1α in parental and drug-resistant breast cancer cells under normoxic and hypoxic conditions was analyzed by Western blot and qRT-PCR methods. The protein levels of ACE2 in plasma samples from breast cancer patients were examined by ELISA. The relationship between ACE2 expression and breast cancer treatment and prognosis was analyzed using clinical specimens and public databases. The reactive oxygen species (ROS) levels in breast cancer cells were measured by using a fluorescent probe. Small interfering RNAs (siRNAs) or lentivirus-mediated shRNA was used to silence ACE2 and HIF-1α expression in cellular models. The effect of ACE2 knockdown on drug resistance in breast cancer was determined by Cell Counting Kit 8 (CCK-8)-based assay, colony formation assay, apoptosis and EdU assay.

Results
ACE2 expression is relatively low in breast cancer cells, but increases rapidly and specifically after exposure to anticancer drugs, and remains high after resistance is acquired. Mechanistically, chemotherapeutic agents increase ACE2 expression in breast cancer cells by inducing intracellular ROS production, and increased ROS levels enhance AKT phosphorylation and subsequently increase HIF-1α expression, which in turn upregulates ACE2 expression. Although ACE2 levels in plasma and cancer tissues are lower in breast cancer patients compared with healthy controls, elevated ACE2 in patients after chemotherapy is a predictor of poor treatment response and an unfavorable prognostic factor for survival in breast cancer patients.

Conclusion
ACE2 is a gene in breast cancer cells that responds rapidly to chemotherapeutic agents through the ROS-AKT-HIF-1α axis. Elevated ACE2 modulates the sensitivity of breast cancer cells to anticancer drugs by optimizing the balance of intracellular ROS. Moreover, increased ACE2 is not only a predictor of poor response to chemotherapy, but is also associated with a worse prognosis in breast cancer patients. Thus, our findings provide novel insights into the spatiotemporal differences in the function of ACE2 in the initiation and progression of breast cancer.