Abstract P1-13-19: ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in HER2 positive breast cancer

Abstract Background: Trastuzumab combined with pertuzumab is the main therapy for HER2 positive breast cancer, but poor patient response due to drug resistance remains a clinical challenge. Organoids derived from tumor patients can highly maintain the heterogeneity of the original tumor, and have been used in drug sensitivity testing and new drug development. To screen for genes that associated with trastuzumab or pertuzumab drug resistance, this study used HER2-positive breast cancer organoids to establish a model of trastuzumab combined with pertuzumab and conducted high-throughput sequencing to screen the differential genes. We carried out further research to explore the effect of ESAM on drug sensitivity of HER2-targeted therapy and clarify the specific regulatory mechanisms and potential therapeutic targets of ESAM. Method: 8 HER2 positive breast cancer organoids were cultured. Immunohistochemistry, hematoxylin-eosin staining and Immunofluorescence were performed to identify the consistency between organoids and primitive tumors. According to drug sensitivity results of organoids to trastuzumab combined with pertuzumab, organoids were divided into a relatively sensitive group and a relatively insensitive group. Trastuzumab combined with pertuzumab was regularly used to stimulate the two groups. After 3 cycles, high-throughput transcriptome sequencing was used to evaluate their gene expression to find a gene responsible for trastuzumab and pertuzumab resistance. METABRIC and our center database were used to determine the prognostic value of ESAM. ESAM overexpression and knockdown stably transfected cell lines were constructed in SK-BR-3 and BT474 breast cancer cell lines by lentivirus. Colony formation assay, CCK-8 assay, organoid model and mouse xenograft model were conducted to examine the influence of ESAM on proliferation and trastuzumab or pertuzumab sensitization in vitro and in vivo. RNA-seq and GSEA analysis were performed to investigate the downstream pathways of ESAM. Western blot was used to confirm the relationship between ESAM and mTOR pathway. Small interfering RNA and mTOR/PI3K pathway inhibitors were used to confirm the function of mTOR in ESAM-induced drug resistance of trastuzumab. Results: HER2 positive breast cancer organoids can maintain the pathological characteristics of primitive tumors. The results of high-throughput transcriptome show that ESAM is significantly up-regulated in the relatively insensitive group compared to the relatively sensitive group. METABRIC and our center database suggest that high ESAM mRNA expression was associated with poor OS, RFS, DFS and PFS of HER2-positive breast cancer patients. CCK-8 and cell colony formation assay confirm that ESAM can promote the proliferation of HER2-positive breast cancer cells and inhibit the drug sensitivity of HER2-positive breast cancer cells to trastuzumab and pertuzumab. GSEA analysis shows high ESAM can activate mTORC1 signaling pathway. Western blotting analysis proves the expression of ESAM is positively correlated with mTOR pathway. Inhibition of mTOR pathway in ESAM overexpression cells can suppress ESAM-mediated proliferation and reverse the drug sensitivity of cells to trastuzumab and pertuzumab. In vivo, overexpression of ESAM can promote the proliferation of mouse mammary tumors and reduce the sensitivity of mouse mammary tumors to trastuzumab, combined use of PI3K inhibitor could reverse drug sensitivity of mouse mammary tumors to trastuzumab. Conclusion: High ESAM expression is associated with poor prognosis in patients with HER2 positive breast cancer. ESAM can activate mTOR pathway, promote cell proliferation and reduce the sensitivity of HER2 positive breast cancer cells to trastuzumab and pertuzumab, which can be inhibited by the application of PI3K/mTOR inhibitors. Keywords: ESAM; HER2 positive breast cancer; HER2 targeted therapy; mTOR; drug sensitivity. Citation Format: Jiong Wu, Xujie Zhou, Yayun Chi, Jingyan Xue. ESAM reduces anti-HER2 therapy sensitivity by activating mTOR pathway in 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 P1-13-19.