Abstract PD10-05: PD10-05 Neoadjuvant tamoxifen therapy reactivates tumor suppressor protein p53 in luminal breast cancer patients: Results from a window-of-opportunity clinical trial
Authors: Das Gokul M. , Kulkarni Swati A. , Oturkar Chetan , Rosario Spencer , Edge Stephen B. , Wang Jianmin , Swetzig Wendy M. , Hutson Alan D. , Kaipparettu Benny , Groman Adrienne , Adjei Araba , Goey Andrew K. , Morrison Carl D. , Kumar Shicha 
Published: March 2023

Abstract
Therapeutic effect of tamoxifen (Tam) against ER+ breast cancer (BC) is known to be mediated by its binding to estrogen receptor-alpha (ERά/ESR1) and inhibiting estrogen signaling leading to altered gene expression. Besides this canonical mode of function, our pre-clinical studies had revealed a novel mechanism wherein ESR1 directly binds wild type p53 (wt TP53) resulting in repression of its tumor suppressor functions, and Tam blocks this inactivation of TP53. Although patients with Luminal Tumors expressing wt TP53 are known to be more responsive to tamoxifen therapy, the underlying mechanism in tumors has remained unknown. To test the hypothesis that abrogation of the ESR1-mediated functional inactivation of TP53 is one of the major mechanisms that underlie the early effects of Tam therapy, we conducted a window of opportunity clinical trial in newly diagnosed luminal breast cancer patients undergoing surgical therapy. Methods: 59 women with ER+ invasive BC were randomized to 20mg Tam daily for 28 days prior to surgery or standard of care (SOC). TP53 status was confirmed by massively parallel sequencing. ER+ wt TP53 tumors were included in the study. IHC was performed on FFPE tissue from tumors to compare expression of ESR1 and TP53 along with their selected downstream targets. ESR1–TP53 interaction in situ was determined by Proximity Ligation Assay (PLA).17β-estradiol and Tam metabolites were measured in the plasma, tumor, and surrounding normal tissue using LC-MS/MS. Global transcriptome analysis in tumors was conducted by RNA-seq. Proteome expression in resected tumors was analyzed by reverse phase protein array (RPPA) with 216 proteins. Findings: Importantly, IHC on tumor tissues showed that the levels of ESR1 and TP53 were not altered in response to Tam therapy, whereas ESR1–TP53 interaction was considerably disrupted by Tam (in situ PLA data). Differential gene expression (DGE) analysis using DESeq2 R package followed by GSEA pathway analysis showed that 307 genes were differentially expressed (p&lt; 0.05) (log FC&gt;1.5) in tumors from Tam-treated versus untreated patients in response to Tam therapy. Pathways representing TP53 signaling, stem cells, and low-grade luminal breast cancer were upregulated in the Tam treated group while those representing adipogenesis, invasive breast cancer, estradiol response, ras signaling, and E2F targets were downregulated. “Master Regulators” identified by iRegulon included several p53 targets. Integration of RNA-seq and RPPA data revealed that DEGs fall into three categories: (i) regulated by TP53, (ii) regulated by ESR1, and (iii) regulated by both TP53 and ESR1. Together, the data demonstrated that in addition to its conventional effects mediated by its binding to ESR1 and inhibiting estrogen signaling leading to altered gene expression, Tam disrupted the ESR1–TP53 interaction leading to functional reactivation of TP53 and reprogramming of gene expression. Conclusions: Our data 1) support ESR1–TP53 crosstalk in tumors as a novel mechanism underlying endocrine therapy response of luminal BC patients, and 2) highlight the importance of factoring TP53 into therapeutic strategies for ER+ BC patients, and 3) have implications in stratifying ER+ BC patients to those who will or will not be responsive to Tam therapy.
Citation Format: Gokul M. Das, Swati A. Kulkarni, Chetan Oturkar, Spencer Rosario, Stephen B. Edge, Jianmin Wang, Wendy M. Swetzig, Alan D. Hutson, Benny Kaipparettu, Adrienne Groman, Araba Adjei, Andrew K. Goey, Carl D. Morrison, Shicha Kumar. PD10-05 Neoadjuvant tamoxifen therapy reactivates tumor suppressor protein p53 in luminal breast cancer patients: Results from a window-of-opportunity clinical trial [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 PD10-05.