TP53 Status as a Determinant of Pro- vs Anti-Tumorigenic Effects of Estrogen Receptor-Beta in Breast Cancer

Utpal K. Mukhopadhyay; Chetan C. Oturkar; Christina Adams; Nadi Wickramasekera; Sanjay Bansal; Rajesh Medisetty; Austin Miller; Wendy M. Swetzig; Laxmi Silwal-Pandit; Anne-Lise Børresen-Dale; Chad J. Creighton; Jun Hyoung Park; Santhi D. Konduri; Alka Mukhopadhyay; Alexander Caradori; Angela Omilian; Wiam Bshara; Benny Abraham Kaipparettu; Gokul M. Das

Disclosures

J Natl Cancer Inst. 2019;111(11):1202-1215. 

In This Article

Abstract and Introduction

Abstract

Background: Anti-tumorigenic vs pro-tumorigenic roles of estrogen receptor-beta (ESR2) in breast cancer remain unsettled. We investigated the potential of TP53 status to be a determinant of the bi-faceted role of ESR2 and associated therapeutic implications for triple negative breast cancer (TNBC).

Methods: ESR2-TP53 interaction was analyzed with multiple assays including the in situ proximity ligation assay. Transcriptional effects on TP53-target genes and cell proliferation in response to knocking down or overexpressing ESR2 were determined. Patient survival according to ESR2 expression levels and TP53 mutation status was analyzed in the basal-like TNBC subgroup in the Molecular Taxonomy of Breast Cancer International Consortium (n = 308) and Roswell Park Comprehensive Cancer Center (n = 46) patient cohorts by univariate Cox regression and log-rank test. All statistical tests are two-sided.

Results: ESR2 interaction with wild-type and mutant TP53 caused pro-proliferative and anti-proliferative effects, respectively. Depleting ESR2 in cells expressing wild-type TP53 resulted in increased expression of TP53-target genes CDKN1A (control group mean [SD] = 1 [0.13] vs ESR2 depletion group mean [SD] = 2.08 [0.24], P = .003) and BBC3 (control group mean [SD] = 1 [0.06] vs ESR2 depleted group mean [SD] = 1.92 [0.25], P = .003); however, expression of CDKN1A (control group mean [SD] = 1 [0.21] vs ESR2 depleted group mean [SD] = 0.56 [0.12], P = .02) and BBC3 (control group mean [SD] = 1 [0.03] vs ESR2 depleted group mean [SD] = 0.55 [0.09], P = .008) was decreased in cells expressing mutant TP53. Overexpressing ESR2 had opposite effects. Tamoxifen increased ESR2-mutant TP53 interaction, leading to reactivation of TP73 and apoptosis. High levels of ESR2 expression in mutant TP53-expressing basal-like tumors is associated with better prognosis (Molecular Taxonomy of Breast Cancer International Consortium cohort: log-rank P = .001; hazard ratio = 0.26, 95% confidence interval = 0.08 to 0.84, univariate Cox P = .02).

Conclusions: TP53 status is a determinant of the functional duality of ESR2. Our study suggests that ESR2-mutant TP53 combination prognosticates survival in TNBC revealing a novel strategy to stratify TNBC for therapeutic intervention potentially by repurposing tamoxifen.

Introduction

Triple negative breast cancer (TNBC), most of which is composed of a basal-like breast cancer (BC) molecular subtype, does not express estrogen receptor-alpha (ESR1), progesterone receptor, or human epidermal growth factor 2 receptor (HER2). Therefore, currently available targeted therapies for BC are not effective against these very aggressive tumors. This, coupled with the long-term ineffectiveness of cytotoxic chemotherapy, makes it urgent to discover new therapeutic targets and strategies to treat TNBC. Although ESR1 is not expressed, ESR2 is expressed in about 60%–80% of TNBC.[1–3] Furthermore, unlike luminal BCs where TP53 is wild type (WT) in the majority of cases, TP53 is mutated in about 80% of TNBC.[4–6] ESR1's role as a pro-tumorigenic factor in BC is well established, and our studies have shown that ESR1 is capable of binding and functionally inactivating WT TP53 in luminal BC.[7–10] ESR1-TP53 crosstalk resulting in cooperation[11] or antagonism[12] has also been reported. However, the role of ESR2 in BC has been elusive.[13,14] Although an anti-proliferative role for ESR2 has been proposed primarily based on overexpression of exogenous ESR2 cDNA in cancer cell lines,[15–17] including overexpression in a TNBC cell line to show antagonism toward mutant TP53,[18,19] data from other studies do not fit this paradigm. For example, treatment of ovariectomized mice with an ESR2-specific agonist resulted in increased cell proliferation to a similar extent that was observed upon treatment with 17β-estradiol.[20] Further, markers of cell proliferation co-localize with ESR2 in mammary epithelial cells[21] and in TNBC.[22] Both of these observations are inconsistent with an anti-proliferative role for ESR2. More recently, ESR2 has been shown to have a pro-proliferative role in BC stem cells.[23] On the other hand, ESR2 is reported to alleviate the inhibitory effect of ESR1 on TP53-mediated transcriptional regulation.[24] Although some retrospective studies on BC tissues showed that ESR2 is an indicator of favorable prognosis,[25–31] pro-tumorigenic functions of ESR2 were observed in other studies.[32–36] Only some of these divergent effects could be attributed to expression of specific isoforms and their cellular location.[37] Such inconsistent observations suggest that ESR2 may have bi-faceted functions depending on the cellular context.[1,13,38,39] However, the mechanisms underlying such bi-faceted functions of ESR2 remain unknown.

Somatic mutations in TP53 are very frequent and are clonally dominant compared with other genes in TNBC.[5,40] In addition to losing tumor suppressor properties and exerting dominant-negative regulation over any remaining WT TP53, certain mutant TP53s are known to acquire oncogenic gain-of-function properties. Cellular functions mediated by mutant TP53 are context dependent and include increased invasiveness, angiogenesis, abnormal epigenetic regulation, enhancement of "stemness," and therapeutic resistance.[41–49] In this study, we have analyzed the interaction between ESR2 and TP53 signaling and addressed whether TP53 status could be a determinant of pro- vs anti-proliferative functions of ESR2 affecting clinical outcome in basal-like TNBC patients.

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