Genetic Analysis of Triple‐Negative Breast Cancer Cell Lines and the Therapeutic Role of Akt/Nrf2 Crosstalk in Resistance to EGFR Inhibitors
Authors: Kaboli Parham Jabbarzadeh , Chen Yao , Xiang Shixin , Xiao Zhangang 
Published: April 2020

Introduction

Triple‐Negative Breast Cancer (TNBC) is the most incurable subtype of breast cancer. Tang and Epidermal growth factor receptor (EGFR), which is a well‐known tyrosine kinase receptor, is expressed in 50% of TNBC cells. Chemotherapy with tyrosine kinase inhibitors (TKIs) has been recognized as the most important therapeutic method against TNBCs. TNBC patients showed resistance to EGFR targeted therapy. Recently, we studied the antitumor effects of berberine in MDA‐MB231 TNBC and MCF‐7 cells
(1). We would like to report our unexpected results and the predicted association between different pathways and resistance to chemotherapy.

Material and methods

To explain the mechanism of resistance to lapatinib and other EGFR inhibitors, we analyzed genetic profiles of ten TNBC cell lines. We selected 35 genes from different pathways which are involved in resistance including Akt signaling, Nrf2 anti‐oxidant signaling, and TGF‐β/SMADs pathway together with PD‐L1, CD28, CD80, and CD86.

Results

We clearly observed that MDA‐MB‐231 cells showed resistance to berberine and lapatinib as compared to MCF‐7. We tested lapatinib against Akt activation in MDA‐MB231 cells. We observed that lapatinib could strongly increase the level of Akt activation compared to untreated MDA‐MB231 cells. We then analyze the genetic profile of ten TNBC cell lines including MDA‐MB‐231. We observed that sixteen genes significantly upregulated or downregulated in TNBC cells. Nuclear factor erythroid 2‐related factor (NFE2L2; Nrf2) and DNMT1 were significantly overexpressed in all TNBC cells (Figure
1). Nrf2 is also activated by Akt through which several anti‐oxidant genes are expressed
(2). Nrf2 reduces reactive oxygen species (ROS) and inhibits ROS‐dependent apoptosis
(3). In addition, GSK3B was increased in TNBC cells. In contrast, EGFR reduced in the TNBC cells (except BT‐20 and MDA‐MB‐468). We suggest that a core of three proteins is involved in resistance to EGFR inhibitors. Akt, Nrf2, and GSK3B activations affected by DNMT1 and SIRT1 epigenetic regulations leading to resistance to therapy which need further evaluation (Figure
2).

Conclusion

Based on our preliminary finding, we suggest Akt as a crossroad for resistance to EGFR inhibitors. We now plan to study the mechanism of epigenetic regulation of Akt/Nrf2 axis much deeper, and discovering of potent drugs against DNMT1 and Akt in combination with EGFR targeted therapy seems to be more effective than the use of EGFR inhibitors alone in TNBC cells.

Support or Funding Information

This project was supported by the Southwest Medical University.

Gene expression analysis of Akt signaling.

Akt is significantly expressed in almost all TNBC cells. EGFR is expressed low compared with Akt and KRAS, two EGFR downstream signaling molecules. Nrf2 which activates anti‐oxidant and drug resistance genes is very high in all studied TNBC cells. DNMT1 involved in methylation of DNA is also significantly increased in TNBC cell lines. Gene expression lower than 4 TPM considered as low expression based.

Figure 1

Suggested mechanism of resistance to therapy promoted by Akt signaling.

We compared the effects of berberine and lapatinib on the Akt activation in MDA‐MB231. We observed that berberine inhibited both EGFR and Akt, but lapatinib enhanced Akt activation showing the role of Akt in resistance to TKIs such as lapatinib. The interaction of Akt with GSK3B and Nrf2 may cause resistance to TKIs. This picture shows the possible mechanism of resistance.

Figure 2