Prostate-Specific Antigen and Female Breast Cancer—Revisited

Prostate-specific antigen (PSA) is one of the best known and clinically useful cancer biomarkers. It is widely used for screening, diagnosis, and management of prostate cancer. PSA (also known as kallikrein-3, KLK3) belongs to a family of 15 genes called “tissue kallikreins “or “tissue kallikrein-related peptidases” and is a secreted serine protease that has chymotrypsin-like activity (1). Its physiological function in males was established many years ago (2). PSA digests seminal gel proteins in the ejaculate and facilitates semen liquefaction, thus promoting spermatozoan motility and enhanced fertilization. Although PSA is a valuable cancer biomarker, its possible role in the initiation and progression of prostate cancer has not been established (3). Consequently, it is considered a biomarker which has no, or unknown, relevance to prostate cancer initiation and progression. Since women have no prostate (4), it was assumed for many years from its discovery, that PSA is a protein that is only found in the prostate, but not in other male tissues, and certainly not in female tissues. The given name of this antigen/protein underlines this belief. Our group has serendipitously discovered that PSA is also produced in other tissues in the male and the female, and especially in the female breast (4, 5). While this observation was initially met with skepticism, due to the use of immunological methods for female PSA characterization, it was later confirmed by molecular analysis that indeed the female breast produces PSA in relatively large amounts and secrets it into the ductal system of the breast (6). Subsequently, PSA was found in all breast secretions, such as the milk of lactating women, nipple aspirate fluid, and breast cyst fluid, and it was established that its expression in the breast is highly up-regulated by androgens and progestins (7). Tissue culture systems using breast cancer cell lines confirmed these observations (7). These original findings have now been confirmed by others (reviewed in (8, 9)). The measurement of PSA in clinical practice is facilitated by sensitive and specific immunological assays that are based on the sandwich principle (ELISAs). Most of the assays that were released for clinical use in the 1980s and 1990s were not highly sensitive, but were able to detect PSA at a level of 0.1 ng/mL or higher (these assays are also known as second-generation assays). In the 2000s, our group, and others, developed more sensitive PSA assays (known as third-generation assays) with sensitivities in the 1 pg/mL range (10, 11). These ultrasensitive assays facilitated earlier detection of relapse in prostate cancer patients and initiated studies on the levels of PSA in female fluids, especially serum. At that time, it was established that the levels of PSA in female serum are extremely low, close to, or just below 1 pg/mL, but the lack of more sensitive techniques precluded the investigation of PSA in female serum as a possible biomarker of female breast cancer. In contrast, PSA was easily measurable in breast tissue extracts and various studies have established that PSA is a prognostic indicator in breast cancer, generally associated with better prognosis in patients who have higher levels of tissue PSA (12). Parallel studies in nipple aspirate fluid—which contains large amounts of PSA—indicated that women with higher PSA in nipple aspirate fluid were less prone to developing breast cancer, although some results were contradictory (13). A major advance in the investigation of PSA as a biomarker for female breast cancer was facilitated by the development of fifth-generation assays by companies such as Mesoscale (Rockville, MD, USA), which allowed for the measurement of complexed PSA down to 6 fg/mL and of free PSA down to 100 fg/mL (14). These assays were able to quantify PSA in all female sera and opened the door for studies to evaluate if PSA has any value as a biomarker for sporadic breast cancer. Preliminary studies by Mesoscale have shown that there was not much difference in serum of complexed or free PSA between women with or without breast cancer (15). Thus, the interest in using PSA as a biomarker for breast cancer declined. Nevertheless, the measurement of PSA in female serum has revealed that this could be a good biomarker for hyperandrogenism in women and that women produce less PSA as they age (16). The possible role of PSA in breast cancer initiation and progression has not been studied extensively and there is no clue as to what the physiological role of PSA in the female breast is. Unpublished observations and conference discussions in the 2000s prompted us to put forward the hypothesis that PSA may have a role in the breast as a proteolytic enzyme that digests luminal secretions from the breast epithelial cells, thus facilitating cleaning of the ducts and removal of possible endogenous or exogenous mutagenic or carcinogenic compounds that may accumulate there. According to our knowledge, there is no published data regarding the PSA degradome in secretions found in breast ducts. But a proteomic study of nipple aspirate fluid revealed about 800 proteins (17). Another possible suggestion includes a role of PSA in activating proteinase activated receptors (PARs), thus initiating cascade reactions related to cellular inflammation and proliferation. PAR activation has been shown for some other members of the KLK family (1). Previous immunohistochemical studies confirmed PSA localization in the lumen of breast ducts. One of the best ways to study the possible pathogenic role of PSA in breast cancer is to identify natural knockouts of the PSA gene and their propensity to develop breast cancer. Recently, groups in Germany and Iran identified a large family of patients with familial breast cancer. Among the approximately 40 members, several of them had breast cancer (18). By sequencing the germline DNA of a proband, and subsequently of additional members, it was identified that 4 patients (sisters) with familial breast cancer co-segregated a mutation in the PSA gene (and a mutation in another gene, RASSF1 [Ras association domain family member 1]), which predictably does not allow for PSA protein to be synthesized, thus representing a natural knockout (in heterozygous state). This mutation is a 13-base pair deletion in the 5′ region of the PSA gene, which includes the translation initiation codon, ATG (Fig. 1). This mutation will predictably affect the synthesis of PSA protein (although this was not experimentally demonstrated), thus rendering these carriers incapable of producing physiological amounts of PSA. The almost perfect co-segregation of this PSA gene mutation (and the RASSF1 gene mutation) with breast cancer suggested that germline variations in RASSF1 and KLK3 are candidate contributors to familial breast cancer development, in addition to other genes, and that PSA qualifies as a breast cancer tumor suppressor. Our mining of the Genome Aggregation Database (gnomAD) did not identify the mentioned 13-base pair deletion but identified a rare single-nucleotide polymorphism (SNP) in the translation start codon (estimated allele frequency 1:250 000) that predictably causes the same effect as the 13-base pair deletion (Fig. 1). The latter SNP has not as yet been reported in any familial breast cancer patients. Mutations in the PSA gene affecting the translation initiation codon. Upper panel: wild-type sequence indicating the translation initiation codon ATG (methionine). Lower-left panel: 13-base pair deletion identified in the Iranian family Eklund et al. (15), which abolishes the translation initiation codon. Lower-right panel: a single nucleotide substitution (T > C) abolishing the translation initiation codon. This mutation was found once in the Genome Aggregation Database (gnomAD) but we are unaware if this mutation, which predictably has the same functional consequence as in the left panel, is associated with familial breast cancer. This figure was created with BioRender.com and is used by permission of the copyright owner. The observation that some patients who cannot produce physiological amounts of PSA may be susceptible to breast cancer development is highly important, even if this mutation is extremely rare, since it has so far been found in a single family. Understanding the role that PSA plays in the female breast and the connection of PSA with other molecules that are secreted in the breast lumen, or co-segregate with the disease (like the RASSF1 gene), may provide insights into the role of PSA in breast cancer initiation and progression. It may also facilitate therapeutic or preventative interventions which could aim to restore/enhance the amount of PSA that is produced in the breast, and thus reduce the risk of breast cancer. We already know that at least some compounds, such as androgens and progestins, facilitate increased transcription of PSA in the breast and in breast cancer cell lines, and this may form the basis of a preventative strategy for breast cancer. All in all, it appears that PSA that is produced by the epithelial cells of the breast and is secreted into the lumen, as we showed by immunohistochemistry many years ago, has a role to play in either initiating cascade reactions through proteinase-activated receptors, or the digestion of other proteins in the lumen of the breast ducts to facilitate removal of cell debris and other secretions which are potentially mutagenic or carcinogenic (19, 20). We conclude that the role of PSA in the female breast should be further investigated in the hope that the accumulated knowledge may facilitate novel preventative interventions. The recent trend of sequencing the germline and tumor DNA of all women with breast cancer will undoubtedly contribute to the understanding of this and other mutations that lead to PSA-null individuals and to associate these individuals with the risk of familial, and probably sporadic, breast cancer. Human Genes: KLK3, kallikrein-3; PSA, prostate specific antigen; RASSF1, Ras association domain family member 1. Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors’ Disclosures or Potential Conflicts of Interest:Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:Employment or Leadership: M. Stengelin is an MSD employee. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared.

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