As mentioned above, the presence of AR expression in breast cancer has always been appreciated but the ability to utilize this information effectively for therapy remains elusive. Although androgen therapy lost its status as a modality for managing breast cancer in the 1970s, laboratory investigation into the presence of AR in breast cancer continued and through the years began to gather momentum [50
]. Aided by new, more sensitive methods to detect AR, studies demonstrated that AR, somewhat paradoxically, is the most commonly expressed nuclear hormone receptor in breast cancer [52
]. Despite this fact, AR levels are not routinely assessed because they have not been shown to predict responses to currently used therapies. In contrast, assays for ER and progesterone receptor (PR) are performed routinely and have been shown to predict for response to currently approved endocrine therapies.
The success of ER/PR and HER2 targeted therapies has shifted interest in AR to those breast cancers that lack ER/PR and/or HER2 expression, so called triple negative disease. In addition, AR targeted therapies may also be important for breast cancers that have developed resistance to current hormone and HER2 directed therapies. Interestingly, AR is expressed in the majority of ER negative breast cancers with apocrine differentiation [57
]. These tumors often have amplification of HER2 making them amenable to HER2 targeted therapies such as trastuzumab [1
]. Recent work detailing the gene expression of triple negative breast cancers supports these findings [59
]. However, it has also been shown both in vitro
and in vivo
that combinatorial therapy targeting both the MAP kinase pathway and AR is an effective means of reducing tumor cell viability and tumor burden [60
]. Whether used as a singular modality or in combination with other systemic agents, AR directed therapies could be a valuable treatment for a large proportion of breast cancers.
Perhaps the most cogent argument for targeting AR is that approximately 10-35% of tumors without ER/PR expression or HER2 amplification (triple negative tumors) express AR [61
]. A study by Moinfar et al. examined sex hormone receptor status as well as HER2 amplification in various grades of carcinoma in situ and invasive cancer [63
]. They found an inverse correlation between histopathological grade and the expression of all sex hormone receptors in breast tumors. As tumor grade progressed from 1 to 3, AR expression decreased from 95% to 76% in DCIS and 88% to 47% in invasive carcinoma. In the same analysis, ER expression decreased from 100% to 8% in DCIS and to 9.5% in invasive carcinoma, with increasing tumor grade. These findings have been validated by another independent report [54
]. In addition, it has been shown that, albeit at a low percentage (25%), AR is the sole sex hormone receptor expressed in distant breast cancer metastases [52
]. As stated, although AR expression decreases with higher tumor grade, the levels of expression remain significantly higher than ER, making AR a potentially valuable target for new therapies.
Interestingly, in the study by Moinfar et al., HER2 expression increases as tumor grade increases – 0% in grade 1 to 84% and 42% in grade 3 DCIS and invasive carcinoma respectively [63
]. It is well known that this heightened HER2 expression is often a result of gene amplification [64
], but gene amplification of other important mediators of breast carcinogenesis, such as nuclear hormone receptors, is less well-documented. Recently our lab examined the possibility of AR amplification as a possible etiologic factor of breast cancer as has been implied with ER amplification [65
]. Examination of a tissue microarray consisting of multiple cores from 18 separate samples showed no amplification of AR [40
]. Replicates of these samples were similarly analyzed for AR expression; 62% of samples (21 of 34) had high levels of AR expression. Therefore, AR is overexpressed in the majority of breast tumors but its overexpression is not a result of gene amplification as is commonly seen with HER2. Consistent with this finding, recent reports did not find a high rate of ER amplification in breast cancers in contrast to the original report from Holst et al. [67
]. Regardless, the frequency of AR expression in primary breast cancer samples justifies its consideration as a potential therapeutic target.
Although the ability to target AR for breast cancer therapy has yet to be effectively deployed, many studies have looked at the utility of AR in breast cancer as a prognostic or predictive marker in breast cancer. AR expression has been linked favorably to response to the synthetic progesterone analogue MPA, with high levels of AR expression predicting for remission of tumors treated with MPA [72
]. Whether this is a result of the drug acting directly through AR is unclear, however. In another report by Rakha et al., AR was shown to be the most valuable prognostic factor when compared to a number of other markers including ER, PR, HER2, EGFR, and p53. Expression of AR in lymph-node positive tumors was prognostic for a better disease-free interval and overall survival, and loss of AR expression correlated closely with higher tumor grade, higher recurrence, and metastases [74
]. Agoff et al. also found that expression of AR correlated with longer time to relapse of disease [75
]. These studies focused on triple negative breast cancers, but the same correlation was found when ER positive tumors were examined. Castellano et al. determined in an ER positive cohort of patients that AR positivity was a prognostic marker for longer time to relapse and disease specific survival [76
]. Collectively, these recent studies continue to support data from more historic reports demonstrating that AR expression in tumors correlates with good prognosis [51
Because of the potential prognostic importance of AR, there also has been a desire to identify more assessable surrogate biomarkers for AR expression in breast cancer. For example, prostate specific antigen (PSA) is a well-known androgen- responsive gene and detection in the blood is used as a biomarker for prostate cancer. Secretion of PSA by breast cells, either benign or malignant, has been evaluated [77
]. Also, correlation of PSA levels with breast cancer risk has been determined using feasible bioassays on samples collected via minimally invasive procedures such as collection of nipple aspirate fluid [78
] or patient serum [79
]. In the latter study, there is an association between higher detectable levels of secreted PSA and histological grade of the breast tumor. However, other reports present conflicting data about this association [80
]. Further studies are warranted before the routine use of PSA as reliable marker for breast cancer detection can be implemented.
While useful as a prognostic factor, AR lacks a causative association between its expression and carcinogenesis. This has led researchers to explore the correlation of other factors in breast carcinogenesis with AR. One report did find a non-significant increase in breast cancer incidence in women taking a testosterone patch for increased libido over control patients, but the study lacked conclusive evidence to link increased androgens with breast cancer risk [82
]. A series of studies tried to determine a causative role of AR in breast tumorigenesis involving the BRCA1 and BRCA2 tumor suppressor genes. BRCA genes require inactivation of both alleles for a phenotypic effect. In individuals with a germline mutation of one allele, other genes can modify the susceptibility of the second wild type BRCA allele to inactivation, rendering it more permissive to alterations that lead to its loss or silencing. In the case of AR, the length of a trinucleotide repeat (CAG) in the N-terminus of the gene has been linked to increased risk of developing breast cancer in BRCA mutation carriers; AR therefore may have a role in breast carcinogenesis in these kindreds [83
]. This initial report was bolstered by a subsequent discovery that BRCA1 enhances AR activity by binding to the activation function domain in the N-terminal portion of the protein [84
]. However, the validity of this hypothesis has been challenged [85
]. As another example, Gonzalez-Angulo et al. explored the relationship in breast cancer between AR levels and mutations in the PI3-kinase alpha catalytic subunit, PIK3CA. They found AR tends to be more highly expressed in cancers with a particular PIK3CA mutation in the kinase domain of the protein [88
], a finding further supported by recent studies by Pietenpol and colleagues [59
]. Importantly, this correlation also holds in the subset of cancers that are negative for expression of ER and PR. This information leads to the intriguing idea of coupling androgen-based therapy with therapies targeting other important pathways.