The Endocrine Society Centennial: Hormones and Apoptosis in the Prostate Gland… Live and Let Die

In society, the act of suicide is considered antithetical to life itself. In the microcosm of the cell, however, suicidal death is a creative element of normal homeostasis. As such, programmed cell death deregulation leads to diseases of many organs including the prostate gland.

The prostate gland is a male reproductive tract organ that entirely depends on androgens for normal growth and function throughout life. Furthermore, this gland is susceptible to a high incidence of growth disorders with aging, both benign and malignant. Manipulation of the androgen sensitivity of the prostate gland has been a primary tool for therapeutic treatment of these diseases in men for the past 75 years. Indeed, Nobel laureates Charles Huggins (1, 2) and Andrew Schally (3) demonstrated, using different approaches, that the interruption of androgen action led to the involution of prostate tumors and considerable therapeutic benefit. The molecular basis for the tissue involution, however, was not clear for several decades.

In the early 1970s, Kerr and colleagues (4, 5) morphologically described that castration-induced involution of the rat prostate gland proceeded via the newly defined phenomenon of apoptosis, a Greek term originally coined by the father of medicine, Hippocrates, to describe the slow degradation of bones with aging. Careful observations using electron microscopy revealed a novel form of cell death initiated with condensation of cytoplasm and nuclear chromatin into small round bodies surrounded by an envelope, frequently forming surface protuberances as the cells condensed. This apparently preprogrammed process involved cell turnover in normal cells and, in endocrine-dependent tissues, was under hormonal regulation. It was thus proposed that apoptosis served a general homeostatic function in regulating cell turnover in both normal and pathological conditions (6).

It was not until 1988 that the seminal discoveries of Natasha Kyprianou and John Isaacs (7), published in Endocrinology, laid the foundations of programmed cell death (apoptosis) as the molecular mechanism driving prostate involution in response to androgen deprivation therapy (Figure 1). At that time, it was well established that androgens, specifically DHT, engaged the androgen receptor (AR) to elicit biological responses essential for prostate homeostasis and that AR was rapidly depleted upon castration, preceding tissue involution (8–11). Unknown, however, was the molecular mechanism that triggered cellular apoptosis upon androgen withdrawal. To address this possibility, Kyprianou and Isaacs used an in vivo model of the rat ventral prostate so that the findings were physiologically relevant and clinically applicable. The first task was to show that whereas circulating T levels precipitously fell to 3% of their original levels within 6 hours after castration, the prostatic DHT levels followed a more gradual decline that correlated with a complete loss of nuclear AR by 12 hours. Once established that these key signaling factors were rapidly depleted, the team turned their attention to the impact of this depletion on cellular DNA content and integrity. Although DNA content began to decline at 48 hours after castration and was progressively lost over 7 days, they discovered that this was not through proteolytic degradation but rather by its specific fragmentation into nucleosomal oligomers in discrete multiples of approximately 180 bp, a process initiated within 12–24 hours after castration. This could not be replicated by treating cells with proteases but rather required the specific activation of Ca+2-Mg+2-dependent endonucleases upon AR loss, further detailed in a separate publication (12). They concluded that DNA fragmentation is a key event in the apoptotic process, temporally preceding the chromatin condensation described by Kerr et al (5). The use of gel electrophoresis to identify this DNA fragmentation led to the first documentation of the characteristic DNA laddering that subsequently became the basis for verifying true apoptosis in multiple systems. It further provided the biochemical basis of the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, also known as TUNEL, the gold standard used for apoptotic detection today. Of importance, these authors astutely observed, using sophisticated flow cytometry, that DNA fragmentation did not occur in a linear fashion after rapid AR depletion, rather a subset of cells fragmented all of their DNA, whereas the rest fragmented none of their DNA. In other words, not all cells die at once. Thus, they proposed that additional regulatory factors other than nuclear AR may control activation of apoptosis.

Figure 1.

The Centennial Papers chosen were the first to describe the molecular events that initiated cellular apoptosis, also known as programmed cell death, in the prostate gland upon hormone depletion. The first paper discovered that within 24 hours after androgen withdrawal by castration, rat prostate tissue levels of DHT reached a nadir, nuclear AR was depleted, and Ca+2-Mg+2-dependent endonucleases were activated, which triggered DNA fragmentation into approximately 180-bp nucleosomal oligomers. Shortly thereafter, the second paper by the same team (Kyprianou and Isaacs [7]) provided the original description of TGFβ and TGFβR in the prostate, discovering that they were under negative androgen regulation. Importantly, the study determined that TGFβ was a key effector of apoptosis, thus identifying a hitherto unknown role for this recently discovered cytokine. Together a signaling cascade was identified whereby androgen deprivation leads to the loss of the nuclear AR axis, increasing expression of TGFβ and TGFβR in prostate epithelium, which together triggers apoptosis and leads to prostatic involution. These pioneering discoveries provided a mechanistic framework for the future development of targeted therapies for both benign and malignant prostate diseases.

Kyprianou next questioned whether neighboring cells influenced the initiation of apoptosis by potentially secreting paracrine factors that enhance the suicide of their neighbors. The answer to this question turned out to be an unequivocal yes. In two papers published 6 months later in Endocrinology (13) and the following year in our sister journal Molecular Endocrinology (14), the Kyprianou and Isaacs team identified TGFβ as an intermediary factor that additionally regulated prostatic apoptosis. At that time, TGFβ was a known cytokine with bifunctional regulatory capacity for cell growth as both a proliferative inhibitory or stimulatory factor in a cell context-dependent manner (15). In addressing its potential role in prostate homeostasis, the Johns Hopkins team provided the first evidence that TGFβ was transiently induced at the mRNA and protein level in the prostate after androgen depletion. Importantly, this immediately preceded the DNA fragmentation at 24 hours after castration, suggesting mechanistic involvement of TGFβ signaling in apoptosis. To assess this directly, they exposed prostate glands in vivo to orthotopic TGFβ implants, which triggered an apoptotic response in the prostate epithelium of intact rats and accelerated apoptosis in castrated rats, indicating that TGFβ itself was sufficient to initiate apoptosis.

To define the signaling pathway driving this effect, they next identified, for the first time, the presence of TGFβ receptors (TGFβRs) in prostate epithelial cells. Furthermore, expression of both the ligand and its cognant receptor were found to be under the direct negative regulation by androgens. This key finding thus identified a signaling cascade whereby androgen deprivation leads to a loss of the nuclear AR axis, increasing expression of TGFβ and TGFβR in prostate epithelium, which together triggers apoptosis and leads to prostatic involution. Subsequent studies identified prostate stromal cells as a major source of TGFβ, thus expanding the neighbor cells that triggered this process to include stomal-epithelial interactions between apoptosis effectors (16). Several key findings were uncovered in these early classic papers including the following: (1) TGFβR, a cell membrane receptor, was identified in prostate tissue; (2) functional cross talk between AR and TGFβ was discovered; and (3) TGFβ was established as a key effector of prostatic apoptosis. Whereas prior evidence had established a critical role for TGFβ as a positive or negative regulator of cell proliferation (15, 17), the implications of this Endocrinology publication were substantial because it first identified a role for TGFβ in inducing apoptosis, the other side to the balance of the tissue growth equation, thus defining a new dimension for TGFβ in tissue homeostasis.

Together these citation classics published in Endocrinology in 1988 provided impactful discoveries not only in the field of endocrinology but also in cancer therapeutics. This formative work provided the mechanisms for future development of targeted therapies, a platform made possible by subsequent discoveries over the next 2 decades that unearthed key players in the apoptotic cascade that can be targeted not only for androgen-dependent tumors but also for castration-resistant prostate cancer, the ultimate clinical challenge. These include Bcl2- and clusterin-antisense oligonucleotides, SMAC (second mitochondrial-derived activator of caspase) mimetics, caspase activation, and TGFβ inhibitors (18, 19), all currently in clinical trials for advanced prostate cancer (20–23).

What are the remaining challenges? All prostate cancer patients eventually relapse after androgen deprivation therapy. Thus, therapeutic resistance remains the ultimate challenge. An understanding of the phenotypic and molecular landscape of the resistant populations will enable direct development of apoptotic-based therapies that effectively kill the cells. Surviving prostate cancer cells have the potential to meet their apoptotic fate through a myriad of pathways that do not require the androgen trigger. It is up to the next wave of scientific discoveries to find the new cell executioners. Autophagy, anyone?