Abstract P1-13-20: Reprogramming of oncogenic signal pathways and metabolism during trastuzumab resistance formation of HER2 positive breast cancer
Authors: Duan Ningjun , Hua Yijia , Hu Shuang , Yin Yongmei 
Published: March 2023

Abstract
Background The formation of resistance against trastuzumab deeply affects the treatment of HER2 positive breast cancer. Although studies have demonstrated several possible reasons that cause the trastuzumab resistance, the precise changes of cell process as well as the interaction between cancer cells and tumor microenvironment during the formation of resistance are still poorly understood. Here we figured out several crucial changes including oncogenic signal pathways as well as metabolism processes especially amino acids and polyunsaturated fatty acids (PUFAs) during the establishment of trastuzumab resistance. We further suggested that the inducers of ferroptosis may be promising reagents for trastuzumab resistant HER2 positive breast cancer. Methods The trastuzumab resistant cell was generated from a sensitive cell line SKBR3. To simulate the adaptation process of HER2 positive tumor to anti-tumor drug, the concentration of trastuzumab was changed gently to prevent cell death. After 8 weeks treatment, trastuzumab concentration in medium was raised from 1μg/ml to 6μg/ml, and a “persister” cell line SKBR3_HP was obtained. The original cell line SKBR3 was cultured for 8 weeks as well to exclude any changes that caused by culture condition. And each cell line includes 3 biological repeats. The viability of two cell lines is measured by CCK8 cell proliferation test with different concentration of trastuzumab. Total RNA of both SKBR3 and SKBR3_HP cells were prepared for sequencing. Activity scores of oncogenic signal pathways as well as metabolism processes were defined and calculated as the relative gene expression value averaged over all genes in this pathway in certain cell type. Flow cytometry was applied for reactive oxygen species measurement with BODIPY-C11. Results After 8 weeks treatment of trastuzumab, the persister cell line SKBR3_HP showed a significant higher viability than original sensitive cell line SKBR3. Oncogenic signal pathway analysis revealed that RTK-Ras, MYC and HIPPO pathways turn into a more active state in SKBR3_HP cells, as well as the upregulation of several cell cycle genes, which are all the response to the blockage of HER2 signal cascade and together maintains cell proliferation. Beside signal pathway, the reprogramming of metabolism is also the consequence of cell adaptation to trastuzumab. Minor changes in energy metabolism, such as glycolysis, citrate cycle and oxidative phosphorylation were observed in SKBR3_HP cells. However, amino acid metabolism, including the synthesis of phenylalanine, histidine, arginine, proline, cysteine and methionine, was largely enhanced in SKBR3_HP cells. Fatty acid, specifically the metabolism of PUFAs, such as linoleic acid, alpha-linolenic acid and arachidonic acid, was activated during the formation of trastuzumab resistance, which was confirmed by the lipid metabolomics data that most n-3/n-6 PUFAs decreased in SKBR3_HP cells. As cysteine and PUFAs metabolism might closely associate with cellular redox balances, erastin and RSL3 were applied to interrupt the intake of cysteine and potentiate the lipid peroxidation process, respectively. A situation of higher ferroptosis sensitivity accompanies raising peroxidated lipids could be detected in SKBR3_HP cells. Conclusion By analyzing the transcriptome and metabolomics data of trastuzumab sensitive and persister cell lines, we pointed out that the changes of oncogenic signal pathway, together with metabolism variation, particularly amino acids and PUFAs, are all the cellular adaptations to high trastuzumab environment. And the higher ferroptosis sensitivity of persister cells could be a valuable treatment target.
Citation Format: Ningjun Duan, Yijia Hua, Shuang Hu, Yongmei Yin. Reprogramming of oncogenic signal pathways and metabolism during trastuzumab resistance formation of HER2 positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-20.