Abstract P3-05-02: Upregulation of canonical Wnt signaling may underlie an aggressive disease course in androgen receptor-negative triple negative breast cancer
Authors: Bhattarai Shristi , Garlapati Chakravarthy , Arora Komal , Mittal Karuna , Klimov Sergey , Krishnamurti Uma , Li Xiaoxian B , Wali Deepika , Gupta Meenakshi , Manne Upender , Kapoor Shobhna , Cavalli Luicane , Aneja Ritu 
Published: March 2020

Background: Based on the expression of androgen receptor (AR), triple-negative breast cancer (TNBC) is subdivided into AR-positive and AR-negative subtypes. Although AR is a potential therapeutic target for AR-positive TNBC, its prognostic role in TNBC remains controversial. Lack of AR expression is associated with a more aggressive disease course. Whereas AR-positive TNBC responds well to AR antagonists, there are, beyond chemotherapy, no targeted drugs available for AR-negative patients. Thus, we aimed to examine the impact of AR loss on the prognosis for TNBC and to uncover molecular pathways/drivers that can be therapeutically targeted in AR-negative TNBC.

Methods: We evaluated AR expression immunohistochemically in annotated, formalin-fixed paraffin-embedded samples from 420 TNBC patients obtained from various institutions in the US. Samples with ≥1% of AR-positive nuclei were deemed AR-positive. We next performed genome-wide copy number profiling (array-CGH analysis) to determine whether losses and/or deletions of the AR gene contributed to tumor aggressiveness of AR-negative TNBC. Finally, using a combination of in silico analysis, in vitro assays, and RNA-sequencing, we found molecular drivers/pathways that were upregulated in AR-negative TNBC.

Results: AR was expressed in approximately 25% of the total cases, and loss of AR was associated with poor overall survival of TNBC patients (p=0.03; n=316 for AR-negative, n=104 for AR-positive) among all treated patients as well as among the subset of adjuvant chemotherapy-treated patients (p=0.06; n=230 for AR-negative, n=75 for AR-positive). Further, the array-CGH analysis showed that AR-negative TNBC presented higher levels of copy number alterations compared to AR-positive TNBC (28.5±4.40 and 13.79±3.48, P&lt;0.01, respectively). These alterations affected mainly the cytobands 1p36.33-p36.32, 1q21.1-q44, 9p24.3-p13.1, 10p15.3-p11.21, 11p15.5-p11.2, 12p13.33-p11.21, 19p13.2-p12, 20q11-q13.33, and 22q11.1-q12.3 cytobands, which were present in higher numbers of AR-negative TNBC.

Next-generation RNA-sequencing of TNBC cases and gene set enrichment analysis revealed upregulation of the Wnt/β-catenin axis in AR-negative cases. Proteomic data from TCGA showed upregulation of β-catenin and Dvl3 expression-evidence of overexpression of Wnt signaling in AR-negative TNBC. Furthermore, protein levels of β-catenin were higher in AR-negative TNBCs compared to AR-positives (p&lt;0.05), suggesting aberrant activation of Wnt/β-catenin signaling in AR-negative TNBC. Using reporter gene assays in cultured cells, we confirmed the activation of Wnt signaling and elevated levels of Wnt target genes (Axin2, CD44, and Cyclin D1) in AR-negative TNBC cells relative to AR-positives. Enhanced nuclear accumulation of β-catenin in AR-negative TNBC indicated reduced β-catenin phosphorylation. Knockdown of AR in AR-positive TNBC cells showed features of AR-negative phenotype in AR-positive cells, in that we observed reduced expression of E-cadherin (suggesting enhanced epithelial-mesenchymal transition) and higher β-catenin expression. Concomitantly, β-catenin target genes were also upregulated resulting in enhanced proliferation, migration and invasion capacities (p&lt;0.05).

Conclusion: Our results suggest an association of AR loss with poor clinical outcome in TNBC. The data support the dysregulation of the oncogenic Wnt/β-catenin pathway in AR-negative cancer cells. Finally, we elucidated a previously unknown link of AR loss to a more aggressive disease course and discovered actionable targets in AR-negative TNBCs.

Citation Format: Shristi Bhattarai, Chakravarthy Garlapati, Komal Arora, Karuna Mittal, Sergey Klimov, Uma Krishnamurti, Xiaoxian B Li, Deepika Wali, Meenakshi Gupta, Upender Manne, Shobhna Kapoor, Luicane Cavalli, Ritu Aneja. Upregulation of canonical Wnt signaling may underlie an aggressive disease course in androgen receptor-negative triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-02.