Recent findings regarding the role of AR in breast cancer (7,12–17) suggest that estimating AR activity may have some clinical utility since trials with both Enzalutamide and Enobosarm (48) in metastatic ER + disease resistant to other endocrine and chemotherapy treatments. Here we identified PSA, ZAG and PIP as candidate biomarkers reflecting tumor AR activity in breast cancer. These factors are all secreted proteins with potential to serve as serum biomarkers (49–53).
We selected three genes encoding secreted proteins most responsive to DHT in vivo analysis: KLK3, AZGP1 and PIP in an AR + PDX. In AR-ChIP-seq analysis of MDA-MB-453 cells, AR binding was demonstrated at androgen response elements upstream of these genes. Other candidate genes among seven androgen responsive genes included FASN (fatty acid synthase), CD9 (CD9) and THBS1 (thrombospondin 1), which also showed significant AR binding at their gene loci (data not shown). The majority of AR-regulated genes expression data are from prostate cancer cell lines and PSA is a well-known AR-regulated protein (44,54,55). In breast cancer, previous studies show that DHT can induce PSA expression in cell lines (56–58). Also, there is evidence in the literature supporting our determination of ZAG (59,60) and PIP (46,61,62) as androgen responsive in breast cancer. In this study, candidate protein expressions were analyzed in 9 cell lines, showing that some are more responsive to androgen and some are less so (Fig. 3, 4). GSEA analysis (Fig. 5a-c) suggests that these candidate factors correlate with AR in a considerable number of breast cancer patients.
In the analysis of anti-proliferative responses of cell lines to Enza, it effectively inhibited growth in all cell lines tested, and IC50 for each cell line averaged 43.8 ± 12.9 µM which is a clinically achievable concentration (20). These results suggest that AR + breast cancer cells rely on AR function for survival, to some extent, regardless of subtype or proliferative response to DHT (Supplementary Fig. 2a-d). These observations are consistent with previous reports showing the pro-tumor roles of AR in multiple breast cancer subtypes (12–14,16,18–22,63,64). We theorized that high expression of candidate proteins indicates a higher androgen dependence and responsiveness to Enza. We analyzed correlations between Enza IC50 and expression of AR or the candidate proteins in in vitro models (Supplementary Fig. 2e), and while statistical significance was not achieved, it is a future direction to test this correlation in clinical trials using AR-targeted therapy and would be particularly useful if PSA, AZGP1 or PIP in serum correlated with AR dependency/response to AR targeting agents.
As shown above, with regard to KLK3 and AZGP1, the response in MDA-MB-453 to DHT and Enza is consistent in the individual cells where they are expressed at all. MDA-MB-453 has such an abundance of PIP protein that the reactivity to DHT and Enza cannot be evaluated properly, but previous reports from other groups using breast cancer cell lines showed that gene and protein expression are linked, with regard to PSA and PIP (46,58,65). We therefore examined the significance of these candidate genes in publically available data sets. KLK3 and AZGP1 expression were not strongly correlated with AR expression in all breast cancer subtypes (Supplementary Fig. 5a, b). However, AR activity cannot be measured solely by AR expression, and it is known that activity can be regulated by ligand binding and competing ER (66–70). However, consistent with our hypothesis, the results of GSEA using the androgen responsive gene set demonstrated that the expression of candidate genes accurately reflects a larger gene signature representing tumor AR activity in all breast cancer subtypes (Fig. 5a-c). Since the candidate genes were identified from the androgen response genes in HCI-009 PDX and GSEA was performed using this androgen response gene set, it is not surprising that candidate genes correlate with this androgen-responsive gene set. However, we also validated this analysis using another androgen-responsive gene set derived from an AR-positive breast cancer cell line MDA-MB-453 (63). With the exception in KLK3 expression in some subtypes, candidate genes showed a significant positive correlation with this androgen-responsive gene set (Supplementary Fig. 7). When the same analysis was performed on the less strongly expressed -candidate genes, (FASN, CFH, CD9 and THBS1), only FASN showed a correlation with the androgen responsive gene set (data not shown). Interestingly, AZGP1 and PIP were particularly high in the LAR TNBC subtype (Fig. 6c); thus, serum levels may be useful as markers for predicting LAR TNBC AR dependency with high accuracy.
Although it is difficult to accurately quantify the protein levels in conditioned medium because there are no good internal standards, relative levels suggest that the secreted candidate proteins in the conditioned medium are regulated by AR in many of the cell lines (Fig. 4). Serum PSA is an established biomarker of tumor burden in prostate cancer (71). Although there is some controversy regarding the differences in PSA expression between normal and breast cancer tissues (72), several groups, including us, have reported that serum PSA levels are higher in breast cancer patients than in healthy women, indicating that tumor derived PSA is detectable in serum of breast cancer patients (52,55,73,74). Recently, we found that serum PSA levels positively correlated with AR expression in primary tumors (52). Combined with the present findings, the future clinical use of serum PSA as a biomarker for tumor AR activity in breast cancer is promising. However, in this study, PSA could not be detected in the conditioned medium of most cell lines examined (Fig. 4). In our previous analysis, the positive rate of serum PSA was 36.1% for metastatic breast cancer and 13.3% for early-stage breast cancer. (52), suggesting considerably low levels of serum PSA in most breast cancer cases. Therefore, establishing a more sensitive assay for PSA quantification is necessary for full clinical translation. ZAG expression in breast cancer has been documented and is considered as a potential biomarker for breast carcinoma (50,75–78) because its expression was detected exclusively in patients with ductal carcinoma when compared to the normal breast tissue of healthy women. Serum ZAG is also significantly higher in breast cancer patients than healthy control patients as well as correlating with disease burden in breast cancer patients (50). PIP, also known as gross cystic disease fluid protein 15 (GCDFP-15), is commonly used in the clinic as a breast cancer biomarker (79–82) to assist in characterizing metastases of unknown origin. There has also been controversy over the difference in PIP expression between normal and breast cancer tissues. In early studies, PIP was shown to be absent in normal breast epithelium, whereas in breast cancers PIP is frequently expressed (80,81). Others have shown that PIP is frequently present in uninvolved breast tissue (83). However, since highly increased levels of PIP have been detected in the peripheral plasma from patients with primary and metastatic breast cancer were detected in comparison to normal subjects, its significance as a potential serum marker in breast cancer is promising (51,81,84). Taken together, serum levels of these candidate protein may reflect tumor AR activity. Thus, further studies are needed to determine the relationship between serum levels of these factors and tumor biology. It is possible that these proteins might not have a predictive advantage over the general AR gene signature of tumor tissue itself. However, breast cancer cells change their biological properties as they develop resistance to treatment (reviewed in T. Hanamura et al (7)). Clinically, in the advanced or metastatic setting, systemic therapy is thought to induce alterations to tumor biology as well (85). Because gene signature monitoring requires tissue collection and cannot be done easily or often, these candidate secreted proteins are suggested to be useful as less invasive biomarkers that can be assessed at any time point to help monitor drug response or resistance.
Assessments of tumor AR signaling by liquid biopsy have been reported by other groups. Both AR mRNA and protein in circulating tumor cells was investigated and shown to be evaluable in blood samples (86–88). In recent years, deep-sequencing techniques applied to blood samples have shown that AR pathways are activated in circulating tumor cells from bone-predominant breast cancer (89). Other groups found that 47% of all AR variants in cell-free DNA of breast cancer patients were pathogenic or likely pathogenic (90). Although, as our three candidate proteins, the predictive value of these potential biomarkers remains to be evaluated in further trials.
AR signals are known to have immunosuppressive effects in in-vivo models of various autoimmune diseases, follicular thyroid cancer and colon cancer (91). ZAG has structural similarities to MHC class I, and analysis of various disease models has shown that it may suppress immune response, but its function in the cancer microenvironment is not clear (78). PIP plays multiple roles in biology, including fertility, immuno-regulation, anti-microbial activity, and tumor progression (92). Interestingly, ZAG and PIP can form a complex with each other, suggesting a cooperative role between these two proteins (93), although their biological significance / activity in breast cancer remains to be determined. Thus, we are currently conducting further pre-clinical and clinical evaluations of these proteins and their immune-modulatory potential in breast cancer.