Prognostic role of Androgen Receptor splice variant 7 (AR-V7) in the pathogenesis of breast cancer

Abstract

Background: The Androgen Receptor (AR) has emerged as an endocrine therapy target in Breast Cancer, exhibiting up to 80% expression in clinical cases. AR-V7, a constitutively activated splice variant of AR with a truncated ligand-binding domain (LBD), demonstrates ligand-independent transcriptional activity and resistance to nonsteroidal antiandrogens like Bicalutamide or Enzalutamide, targeting the LBD. In metastatic prostate cancer, elevated AR-V7 levels lead to therapeutic resistance and increased metastasis.

Methods: In this study, we evaluated the expression of AR and AR-V7 in cell lines and a cohort of 89 patients undergoing surgical intervention for treatment-naïve breast cancer. Further clinicopathological correlations and survival analysis were performed to evaluate the relationship between the AR and AR-V7 expression and clinical outcomes.

Results: AR-V7/AR-FL ratio was elevated in the TNBC cell line and downregulation of AR-FL upon AR antagonists' treatment led to a compensatory increase in AR-V7. Clinical samples showed significantly elevated expression of AR and AR-V7 in tumors compared to control cases. Further clinicopathological correlation revealed aggressive clinical traits, higher pathological grades, and poor survival with AR-V7 expression.

Conclusions: Our study unravels AR-V7 as a marker for poor clinical outcomes, predicting breast cancer aggressiveness, and encourages consideration of AR-V7 as a probable target for therapeutic intervention.

Keywords: AR-V7; Androgen receptor; Biomarker; Breast cancer; Splice variant.

Fig. 1

Transcript structures for AR full-length and the splice variants. The human AR gene consists of 8 canonical exons out of which exon 1 encodes for NTD (blue), exons 2 and 3 for DBD (orange), a hinge region (purple), and exons 4–8 for LBD (green). Notably, AR-V7 retains exons 1–3, followed by cryptic exon CE3b (brown). Splicing pattern for AR-2, AR-TV4, AR-TV5, AR-TV6, and AR-V567es is also represented. Labelling of peptides is in accordance with the latest Human Genome Reference Consortium GRCh38.p14 (hg38). (Map not to scale). Created in BioRender. Srivastava, T. (2024) BioRender.com/k18q250

Fig. 2

Relative mRNA and protein expression of AR, AR-FL, and AR-V7 its breast cancer cells. The upper panel, A, shows qRT-PCR of AR (targeting all primary transcripts), AR-FL, and AR-V7 in three cell lines. B. Western blotting of breast cancer cell lines along with prostate cancer cell line, LNCaP indicated expression of AR-FL in all but MDA-MB-231 cells. However, AR-V7 was detected in this cell line more prominently than AR-FL

Fig. 3

Elevation in the AR-V7 levels by treatment of breast cancer cell lines with AR antagonists. It can be observed that while AR-FL consistently goes down with the antagonists’ treatment, there is an increase in the AR-V7 expression

Fig. 4

Normalized expression of AR-Vs in clinical samples. A. Upper panel shows qRT-PCR data of AR, AR-FL, and AR-V7 along with MKI67 genes. B. Lower panel denotes cumulative densitometry data of western blotting. Representative immunoblots are attached below. P value < 0.05 was considered statistically significant through the Mann–Whitney U test

Fig. 5

Immunohistochemical staining of AR-FL and AR-V7 in different subtypes of breast cancer tissues. IHC of AR exhibited strong nuclear staining while AR-V7 was weakly stained. It was interesting to note that in the attached TNBC, AR was negative while AR-V7 shows mild positivity for the same case. Images at the magnifications 10X, 20X and 40X, respectively

Fig. 6

Kaplan − Meier curve depicting the relationship between AR and AR-V7 expression and probability of overall and disease-free survival. The KM curve shows pronounced association with the risk of events with AR-V7 positivity. Tick marks indicate censored data