Utility of ankyrin 3 as a prognostic marker in androgen-receptor-positive breast cancer

Androgen receptor (AR) and AR signaling pathways are thought to play a role in breast cancer (BC) and are potentially related to treatment responses and outcomes. Ankyrin 3 (ANK3) is associated with AR stability in cancer cells. In the present study, we investigated the clinicopathological utility of ANK3 expression with emphasis on AR and its associated signalling pathway at transcriptomic and proteomic phases. The Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) cohort (n = 1980) and The Cancer Genome Atlas (TCGA) dataset (n = 1039) were used to assess the expression and significance of ANK3 mRNA and other AR signalling pathway-associated gene signature. Using immunohistochemistry, ANK3 protein expression was evaluated in large (n = 982) cohort of early-stage BC with long-term follow-up and compared with clinicopathological characteristics and its prognostic value in the whole cohort and the subgroups stratified by AR protein expression. An AR-related gene signature was developed, comprising 20 genes, which included ANK3. This AR-related gene signature was significantly associated with AR mRNA expression, oestrogen receptor, human epidermal growth factor receptor 2 (HER2) status and the patients’ outcomes. In tumours with high AR protein expression (n = 614), high ANK3 protein expression was significantly associated with progesterone receptor positivity and it was independently associated with the good outcomes (p = 0.025). This study indicates that ANK3 is related to AR signalling pathway and is associated with BC prognosis.

The mRNA expression data of these genes, including ANK3, together with the clinicopathological characteristics and outcomes of patients with BC, were collected from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset [24,25] (n = 1,980) and the Cancer Genome Atlas (TCGA) [26] dataset (n = 1,039) provided by cBioPortal [27].
The normalisation method of mRNA expression in the METABRIC cohort was previously described [24]. TCGA mRNA data was log2-transformed prior to cluster analysis. For cluster analysis [28] and heat mapping construction, Cluster 3.0 and Java Treeview was used [29].
Data were filtered to remove all genes that did not have at least one observation with absolute values greater than 2.0 or whose maximum minus minimum values were less than 2.0.

ANK3 Protein Expression
A total of 982 BC patients who underwent surgery at Nottingham City Hospital in the UK between 1987 and 1998 (referred to as the Nottingham Primary Breast Cancer Series) were included in this study. All patients had undergone breast-conserving surgery or modified radical mastectomy without any neoadjuvant treatment. The availability and assessment of hormone receptors (AR, ER and progesterone receptor [PR]), HER2 and Ki67 were described in previous studies [7,[30][31][32][33][34][35][36][37]. The cohort was stratified on the basis of AR expression [7], with 614 patients (62.5%) with high and 368 patients (37.5%) with low AR expression (Supplementary Table 1).

Statistical Analysis
Statistical analyses were conducted using SPSS v24.0 (IBM, Armonk, NY, USA). The relationship between ANK3 mRNA with ANK3 protein expression and AR mRNA expression was examined using Pearson's correlation coefficient test. In order to assess the associations between AR mRNA expression and groups stratified by the AR-related gene signature, the Mann-Whitney U test was used. The chi-square test as univariate analysis and the logistic regression test as multivariate analysis were used to assess several clinicopathological factors, including tumour size, lymph node status, histological grade, ER, PR, HER2 and molecular subtypes, stratified by groups based on AR-related gene signature and levels of ANK3 protein expression. In order to assess the prognostic utility of ANK3 expression, Kaplan-Meier survival curves was used. In univariate and multivariate analyses, to assess the associations between clinicopathological factors, including ANK3 expression, and prognosis, 95% confidence intervals (CIs) were assessed using the Cox proportional hazards regression model. In these survival analyses, the median value (H-score = 120) was used as a cut-off point to divide the samples into high and low expression groups.

! 6
ANK3 mRNA Expression and AR Signaling Pathway Gene Signature High ANK3 mRNA expression was significantly associated with high AR mRNA expression  Table 2).
Using the dendrogram of cluster analysis, the METABRIC and TCGA cohorts were stratified into two groups on the basis of the AR-signaling-pathway-associated genes [Figs. 1(a) and 1(b)], where tumours in group 1 had significantly lower AR mRNA expression than that in Group 2 (p < 0.0001). Group 1 tumours included 899 (45%) from the METABRIC and 541 (52%) from TCGA cohort.
In the METABRIC and TCGA cohorts, multivariate analysis indicated that the AR-related gene signature in group 2 was significantly associated with lower grade (p = 0.0070, and p = 0.0093 respectively), ER positivity (p < 0.0001, and p < 0.0001 respectively), and HER2 positivity (p < 0.0001 and p < 0.0001; Table 1). In the METABRIC cohort, the AR-related gene signature was significantly associated with molecular subtype (p < 0.0001), with 83% of the basal-like tumours in group 1 and 90% of the luminal B tumours in group 2 (Table 1).
Although the expression of ANK3 and AR mRNA was not a significant independent In 198 cases in the METABRIC dataset, which overlapped with the Nottingham Primary Series, ANK3 mRNA and ANK3 protein expression were significantly correlated (r = 0.15, p = 0.039). In the Nottingham series, 579 (59%) tumours had low ANK3 expression (H-score < 120) and 403 (41%) had high ANK3 expression (H-score > 120). High AR expression was present in 614 (63%) tumours and low AR expression was present in 368 (37%). Among those with high AR expression, 250 (41%) also had high ANK3 expression. A similar proportion (153, 42%) had high ANK3 expression in the low AR expression group (n = 368).
AR expression was not associated with ANK3 expression on proteomic analysis (p = 0.79).
When all 982 cases were combined (i.e. not stratified according to AR expression), ANK3 was not a significant prognostic factor (Supplementary Figure 2).

DISCUSSION
AR expression is a crucial factor in the progression of BC, as it controls the expression of various genes and proteins through a genomic pathway [5,6]. In this pathway, AR mediates intracellular steroid hormone-related signaling pathways to regulate the transcription of target genes in conjunction with other transcription factors, such as signal transducers and activators of transcription [43,44]. As a mechanism involved in the development of BC, AR expression might be involved in the crosstalk with epidermal growth factor receptor pathways, such as human epidermal growth factor receptor 1 (EGFR) and HER2 signaling [45]. In this study, there were a significant correlation between ANK3 and AR mRNA and ANK3 was one of the gene component of the AR-related gene signature. When BC was classified into 2 groups ! 9 based on the expression of AR-related gene signature, the group 2 gene signature, which was associated with high AR mRNA expression and present in 90% of luminal B tumours, was a significant prognostic factor indicating poor outcomes in BC. This finding suggests that aberrant AR-related oncogenic pathway activation is associated with a number of factors that portend a poor BC outcome.
In a previous study using microarray gene expression analysis, the downregulation of ANK3 ANK3 knockdown exhibit significant increases in cell invasion through an AR-dependent mechanism as a regulator of AR protein stability [16]. In the present study, the association between ANK3 protein expression and outcomes was highly significant in BC with high AR expression. In addition, high ANK3 protein expression was associated with PR positivity.
These findings suggest that ANK3 may play an important role in the maintenance of hormonal activity, and AR stabilisation by ANK3 may therefore be related to the improved outcomes in BC patients with high AR expression. A proportion of ER-negative BC are generally considered to retain active AR signaling [6,52]. Several prospective clinical trials of AR-targeted therapies have been conducted on TNBC with high AR expression. These trials indicated that treatment with an AR inhibitor is feasible, with a clinical benefit rate of ! 10 approximately 20% in TNBC [53 -55]. The upregulation of ANK3 may increase AR stability and improve the response to an AR inhibitor in TNBC. Further functional and translational research is necessary in order to explore the association of ANK3 with AR stability with the efficacy of treating BC with an AR inhibitor.
In conclusion, the AR signaling pathway and ANK3 mRNA expression are associated with AR mRNA expression and BC prognosis. High ANK3 protein expression is an independent prognostic factor in BC with high AR expression. Overall, these findings indicate that ANK3 may play an important role in breast tumour progression and, in conjunction with AR, may be related to BC outcomes.