AR deregulation as a key pathological driver of PFS?

Induced Wild Type AR Overexpression and the potential relevance of SBMA to PFS

As discussed, as the CAG trinucleotide sequence extends in the N-terminal domain of the AR, there is a consequent functional decline in transcriptional efficiency which is seemingly associated with a compensatory increase in androgen levels. However, at longer repeat lengths, high androgen levels can exert a deleterious and ultimately toxic response. Crucially, polyglutamine expansion is not the only way ligand-dependent toxicity can be conferred to the AR protein, and overexpression of the wild type AR can cause a paradoxical loss of function and toxic gain of function. This is reflective of evidence in other polyglutamine diseases that point to gain of native protein function underlying pathology ​(Paulson et al., 2017)​. It is now appreciated that balanced gene expression is vital for homeostasis, and overexpression of wild-type proteins causes disease states in humans ​(Ohshima et al., 2017; Shastry, 1995)​. Multiple studies demonstrate that, while seemingly paradoxical, sufficient increases in AR expression converge with loss of function phenotypes, with an inverse U‐shaped curve representative of AR gene dose response in tissues. The pathological consequence of overexpression of the AR is therefore coherent with Prelich’s observation that overexpression of proteins mimics a loss of function and interferes with its function antimorphically. The mechanisms by which overexpression causes a mutant phenotype is therefore of great importance to further understand ​(Prelich, 2012)​.

Generating mice overexpressing AR solely in skeletal muscle, Monks et al. reported the striking and seemingly counter-intuitive observation that overexpression of the Wild-Type androgen receptor recapitulates the pathological consequence of polyglutamine expansion despite a polyglutamine repeat tract comprised of 22 glutamines. Decreased viability was observed in males of all seven transgene lines but not in females. Interestingly, administration of flutamide to pregnant dams enhanced perinatal survival, suggesting prenatal androgen activation of the overexpressed AR, not the overexpression per se, is causative of death. Two transgenic mouse lines of differing WT AR copy number (L78 < L141) were characterised. L141 males exhibited a far more severe phenotype, corresponding to a significantly higher AR expression at the mRNA and protein level. Surviving L78 males were functionally comparable to wild type despite a lower body weight. However, L141 males exhibited a marked phenotype of lower body weight, curvature of the thoracic spine, severe deficits in motor function and muscle strength, and early death. Castration dramatically restored function in L141 mice, illustrating the androgen dependency of the toxicity. Remarkably, although L141 females were apparently unaffected by AR overexpression per se, when administered testosterone to the approximate circulating level of male mice, they rapidly developed a comparable disease phenotype to male L141 mice including motor dysfunction and muscle atrophy. Over 9 days of T treatment was fatal to female L141 mice. L78 female mice did not become symptomatic or atrophic with T treatment, even for prolonged periods. This parallels the asymptomatic L78 male. This is strongly indicative that the degree of overexpression dictates severity of androgen-mediated toxicity and, as Monks et al. observe in several contexts, that overexpressed AR confers toxicity once activated by hormonal ligand ​(Monks et al., 2007)​.

Monks et al. compared differentially regulated genes in myogenic transgene mice and the SBMA AR97 and AR113Q models. Gene expression in the transgene AR-overexpressing muscle revealed similar deregulation to AR Knock-out muscle, further suggesting that a paradoxical loss of AR function results from overexpression of the androgen receptor. The finding of overexpression of WT AR reproducing a phenotype comparable to polyglutamine expansion was noted to be surprising and puzzling considering SBMA is associated with a loss of AR function whereas overexpression of the AR would typically be expected to enhance the function of androgen signaling ​(Mo et al., 2010)​. Further striking findings were provided through investigation of the contributions of native AR interactions to polyglutamine-expanded AR toxicity in Drosophila models. Nedelsky et al. determined that native interactions at AF-1 of the AR modify toxicity while AF-2 coregulator interaction and function is essential for toxicity. Expressing AR in the photoreceptor neurons and accessory pigment cells of the eyes of the developing flies, they demonstrated a polyglutamine length and ligand-dependent degenerative phenotype. While flies reared on normal food did not demonstrate pathology, a degenerative phenotype in the posterior margin of the eye occurred in flies reared on food containing DHT. This androgen and polyQ length dependent degenerative phenotype of atrophy and functional deficit was further demonstrated in larval tissues including salivary glands and motor neurons. Crucially, Nedeslky et al. reported that wild-type AR of a 12Q polyglutamine-length, when expressed at very high levels, resulted in an degenerative phenotype indistinguishable from that caused by expansion of the AR polyglutamine tract ​(Nedelsky et al., 2010)​. This reflected the dose dependency and pathological consequence of wild-type AR overexpression well reported by Monks et al. Furthermore, though generally weaker, expression analysis revealed a similar dysregulation in both AR12Q+DHT and AR52Q+DHT files, lending further support to a link between an amplification of native function and the toxicity induced by polyglutamine expansion and is supportive of a conserved mechanism. Interestingly, quantitative analysis did not reveal a correlation between the amount of high molecular weight species and neurodegeneration in their Drosophila model. This is in line with the lack of AR positive aggregates reported in transgenic mice that recapitulated the SBMA phenotype ​(Monks et al., 2007)​. The presence of aggregates was in previous decades presumed to be a driving factor in pathogenesis of SBMA, however this is no longer the case and a direct mechanistic involvement is controversial ​(Todd & Lim, 2013)​. Providing another parallel between the effect of polyglutamine expansion and wild type overexpression, Halievski et al. demonstrated that in mice expressing a human androgen receptor of 97 CAGs and the wild-type overexpressing myogenic mice, several common transcriptional effects were seen, such as robust downregulation of BDNF and NT-4 transcripts. Remarkably, similar effects were seen indistinctly across both synaptic and extrasynaptic domains, suggesting a broad effect and involvement of common deleterious AR-mediated mechanisms across cell types ​(Halievski et al., 2019)​.

While it might be expected AR overexpression would result in a hyper-masculine socio-sexual phenotype, Swift-Gallant et al. demonstrated significant reductions of male-typical aggressive and sexual behaviours in transgene AR overexpressing mice. This non-linear androgen response was curiously reflective of loss of AR function. Interestingly, same-sex anogenital investigation was increased and male-typical preferences for female olfactory cues were disrupted in globally overexpressing mice but not mice only overexpressing AR in neural tissue, suggesting a direct role of non-neuronal AR in mediation of socio-sexual behaviours. A decrease in testosterone production is not a sufficient explanation for the mechanistic consequences of overexpression on masculine physiological and behavioural phenotypes and the many convergences with loss of function models, and reduced testosterone was not routinely observed in models of overexpression ​(Swift-Gallant et al., 2016)​. Monks and Swift-Gallant considered a uniform global loss of AR function unlikely, proposing a cellular mechanism that would be differentiated according to affected neurological or physiological tissue and system. This would implicate regional variations, possibly including site-specific cofactor influences and differential transcriptional effects resulting from regional epigenetic changes. Additionally, overexpression of AR has been suggested as a plausible mechanistic route to alteration in neurosteroid synthesis ​(Monks & Swift-Gallant, 2018)​.

Considerable evidence exists to support an overlapping androgen dependent toxicity in the contexts of AR polyglutamine tract expansion and overexpression of the wild-type AR, and a loss of function coincident to both insufficient and excessive AR signaling. It is therefore highly significant that both hypogonadism ​(Seftel, 2005)​ and the multi-systemic symptom profile of SBMA ​(Querin et al., 2017)​ bear a clear resemblance to the broad symptomatology of PFS. However, it is important to consider that there are notable areas of presentation and progression in which the disease states of PFS and SBMA differ. Neurocognitive symptoms are profoundly more severe in PFS than are reported in SBMA, although these domains of disease involvement are not without overlap as we have illustrated. Tongue atrophy is not reported in PFS. These differences are likely inherent to the aetiologies of the respective diseases: An endocrine disruption leading to epigenetic dysregulation in PFS and a genetic glutamine repeat sequence as causative factor in SBMA. While SBMA is a characteristically slow progressing condition, PFS can, in many cases, onset extremely rapidly with the discussed “crash”. After this onset, an initial period of weeks or months during which the pathology is often rapidly progressive to what patients refer to as a “baseline” state occurs. Atrophy of androgen dependent tissue and physiological changes are often reported over this time. Beyond this, PFS is not always markedly progressive, with some patients experiencing improvement or stabilisation of their symptoms to variable points over subsequent months or years. As we will discuss, exogenous testosterone can sometimes cause symptomatic intensification, and significant and rapid phenotypical deterioration with additional symptomatic physiological domains can occur following exposure to further antiandrogenic endocrine disrupting substances. Previously discussed as the “crash”, the majority patient experience of a intensification or development of symptoms after cessation of the drug may reflect the return of 5a-dihydrotestosterone to physiological levels in the presence of the newly uninhibited 5-alpha reductase enzymes. In the myogenic models discussed, when male physiological levels of androgens were administered to female L141 mice a severe disease state is rapidly induced, while the L78 mice were largely asymptomatic. A site-specificity and expression level-dependency of induced AR overexpression therefore serves as a compelling explanation for the large variation in the toxic post-drug phenotype, manifesting as either a continuation of on-drug side effects or, more commonly, the crash, which can vary from an onset of sexual dysfunction, libido loss, anxiety and depression to a devastating and disabling physiological and psychological alteration including cognitive impairment of executive function, derealisation, anhedonia, panic attacks, memory loss, total insomnia, dysautonomia, atrophy of androgen-responsive tissue and metabolic changes.

A “malignancy switch” for the androgen receptor?

In the absence of serum endocrine or other toxicological findings that could account for the pathological features of PFS ​(Irwig, 2014; Melcangi et al., 2017)​ we suggest a biological event during use of finasteride is causing an often permanent change in the ordinary metabolic function of cells through epigenetic alteration. Although this is controversial to suggest, the potential severity of the disease cannot be overstated, and in a a significant number of cases the health problems are severe, progressive, do not resolve with time and entail a peculiar endocrine fragility. We hypothesise underlying pre-existing genetic and/or epigenetic factors differentiate those who are prone to developing PFS, and this predisposition effects deleterious epigenetic modifications by means of a conserved mechanism upon significant reduction of intracellular androgen-dependent transactivation through various modes of action including but not limited to 5alpha reductase inhibition. These vectors include downregulation of AR mRNA, an induced increase of protein degradation, upregulation of enzymes capable of reducing endogenous AR ligand to inactive androgen metabolites and suppression of steroidogenic enzymes. We further suggest the necessary exposure and severity of symptomatic outcomes are dependent on interindividual differences within this/these underlying predisposing factor(s) and the resulting degree of persistent dysregulation of the androgen receptor on a site-specific basis.

The epigenetically determined fate of somatic cells is not terminal. Epigenetic barriers preservative of cellular integrity were famously visualised by Conrad Waddington’s epigenetic landscape, which described a ball running down valleys in determination of its ultimate differentiated state ​(Slack, 2002)​. However, these can be overcome given the correct stimuli, and the past decades have seen rapid advancements in cellular reprogramming methods ​(MacArthur et al., 2009)​. As chemicals are capable of inducing reversal of cell lineage, Kanherkar et al. investigated the possibility of permanent epigenetic alterations occurring following exposure to pharmacological agents. HEK-293 cells cultured in the SSRI antidepressant citalopram revealed significant differential methylation in hundreds of genes. They proposed the term “pharmaceutical reprogramming” to describe a partial dysdifferentiation event resulting from drug-induced methylation changes that consequently alter cellular function and integrity ​(Kanherkar et al., 2018)​. Evidence demonstrates adult sex typical behaviour can be altered in mammals under certain conditions and may be a function of epigenetic maintenance and gene expression with behavioural impacts ​(McCarthy, 2019)​. In relation to androgen signalling, significant recent work has suggested that biologically meaningful differences that directly influence behaviour and function pertaining to sexual traits can arise from epigenetic alteration to the program of the androgen receptor ​(Schuppe et al., 2020)​.

As well as fibrotic changes in the penis, Enatsu et al. reported a reduction of AR and an increase in ER expression in the prostate of young rats administered dutasteride, speculating that an improper response to androgens upon restoration could underlie the sexual dysfunction in PFS owing to altered local receptor expression ​(Enatsu et al., 2016)​. This study demonstrated a deleterious influence exerted by 5alpha reductase inhibition in young rats that entailed morphological alterations to sexual organs and epigenetic remodelling that trended towards the effect of castration. However, many factors exclude the typical response to prolonged 5alpha reductase inhibition from being an applicable model for the behaviour of PFS. These include the rarity of PFS amongst 5ari users, the clinical picture of PFS including pathological development and/or progression of the disease following cessation, a prevalence in younger men using a lower dose, the brevity of exposure in some of the most severely affected cases, the commonly reported responses of PFS patients to trialled therapies, and the previously reported determination of persistent and significant upregulation of the AR in prepuce tissue of PFS patients. Nevertheless, the parabolic nature of AR expression would suggest Enatsu’s hypothesis of an induced dysfunction in local androgen response owing to epigenetic remodelling is plausible. Finasteride has previously been shown to upregulate prostate epithelial AR significantly in BPH patients after 30 days of exposure ​(Hsieh et al., 2011)​. Corradi et al. demonstrated that Finasteride induced a persisting overexpression of the AR and important alterations in the tissue microenvironment of the prostate gland in young gerbils. Across three stages of postnatal development, the content and intensity of AR immunostaining were noticeably elevated, particularly in epithelial cell nuclei. Both the tissue changes and AR overexpression proved persistent. Interestingly, when contrasted with their respective control groups, a greater increase in AR nuclear intensity could be observed in the young (8% to 61.5%) Finasteride administered experimental group as opposed to the old Finasteride administered experimental group (66% to 72.5%) at the conclusion of the post-treatment phase ​(Corradi et al., 2009)​.

Coskuner et al, reviewing literature on persistent sexual symptoms in a subset of 5alpha reductase inhibitor users, considered tissue-specific epigenetic effects likely given the persistence of symptoms ​(Coskuner et al., 2019)​. In considering the mechanistic origins of the development of PFS following endocrine disruption with Finasteride, Traish proposed that androgen deprivation and depletion of the substrate precursors for the 3α-hydroxy-steroid dehydrogenases causative of a block in neurosteroidgenesis, attenuating the function of steroid and neurotransmitter receptors and inducing changes in the expression of a host of gene products, eliciting epigenetic changes manifested in histone acetylation, DNA methylation and upregulation of the AR. Traish thus suggests these changes, together with the consequent depletion of neurosteroids, manifest in the development of PFS in susceptible individuals ​(Traish, 2018)​. Di Loreto et al had previously suggested that it was tempting to speculate that PFS patients have triggered processes associated with advanced age by pharmaceutical androgen deprivation ​(Di Loreto et al., 2014)​. The natural decline of testosterone values with ageing has been well established ​(Kaufman & Vermeulen, 2005)​. PFS may thus represent an aberration of such processes, resulting as an adaptive epigenetic response to the pre-receptor disruption of androgen signaling during finasteride use.

Our stated hypothesis for PFS as an epigenetic adaption induced by pharmaceutically interrupted androgen signalling accounts for a deregulated epigenome and the onset and/or symptomatic intensification following finasteride withdrawal, often after a brief resolution of symptoms, which standardised questionnaires including our own data indicate is an intrinsic feature of the syndrome (Propeciahelp Post-Drug Syndrome Survey: Data not provided). Cessation of finasteride will result in a surge in androgen production owing to the newly uninhibited 5a-reductase enzyme. Presuming a 60% reduction of basal DHT levels during finasteride use, cells epigenetically adapted to a depletion of androgenic signaling owing to the pharmacological reduction of DHT would be exposed to a 300% increase in DHT upon cessation. As molecular level investigation has revealed a persistent elevation in expression of the androgen receptor in symptomatic tissue of a PFS cohort, this may entail a deleterious ligand-dependent effect in alignment with the demonstrated in vitro and in vivo models discussed. Application of such a conceptual framework to the pathology of PFS is not unprecedented. Professor Charles Ryan explained the tissue response to testosterone in terms of a “bell curve” in his book The Virility Paradox. He wrote of PFS: “I think this is what we are seeing here. With a greater concentration of receptors, the organ becomes more sensitive to testosterone and at a certain point, paradoxically, that sensitivity may shut down” ​(Ryan, 2018)​.

We hypothesise that a loss of function and toxic gain of function manifests tissue specifically in a broad spectrum of clinical endpoints, from functional impairment to atrophy in affected tissues. In consideration of this, we would expect future gene expression analysis of symptomatic tissue in severely affected patients to reveal widespread dysregulation of gene expression. A consideration of how a dysregulation of the AR and associated epigenetic remodelling might occur as an aberrant result of antiandrogenic endocrine disruption, and how it may influence broader gene expression, is therefore necessary. This can be contextualised via known molecular mechanisms.

The most well recognised epigenetic adaptations occurring as a result of androgen deprivation therapy is in the context of castration resistant prostate cancer. As a driver of epithelial cell growth and proliferation as well as a fundamental aspect of prostate cancer progression, the androgen receptor axis has been the predominant therapeutic target in prostate cancer for over 75 years ​(Kim & Ryan, 2012; Takeda et al., 2018)​. Patients develop resistance to androgen deprivation therapy after a period of this first line treatment, a state with very poor prognosis known as castration resistant prostate cancer. Second generation antiandrogen treatments have been developed, however nearly all men also develop resistance to this, suggestive of a mechanistic response irrespective of the agent ​(Robinson et al., 2015)​. Although not always observed, amplification of the AR is the most common mechanism of castration resistance ​(Takeda et al., 2018)​ and is the only consistent gene expression change associated with hormone refractory disease ​(Chen et al., 2003)​. The amplification of the AR occurs during androgen deprivation therapy ​(Friedlander et al., 2011; Visakorpi et al., 1995)​ or antiandrogen treatment ​(Coutinho et al., 2016)​ and represents an adaptive response to the low androgen environment ​(Perner et al., 2015; Ruggero et al., 2018; Teply et al., 2018)​ that sensitizes cells to lower levels of hormone ​(Waltering et al., 2009)​. Interestingly, low, rather than high, endogenous testosterone levels have been associated with poor prognostic features in prostate cancer and disease reclassification during active surveillance ​(Amadi et al., 2018; San Francisco et al., 2014)​. Several lines of evidence suggest low levels of androgen may predispose to more aggressive tumours ​(Swerdloff et al., 2017)​. Gravina et al. provided evidence that epigenetic mechanisms can contribute to castration resistant phenotypes, demonstrating that pca cell models in androgen-deprived medium or bicalutamide progressively increased DNMT expression, which increased in proportion to AR upregulation. These findings were verified in patient tissue. DMNT was additionally shown to be regulated by AR, as siRNA AR interference greatly reduced DNMT modulation ​(Gravina et al., 2011)​.

Chen et al. hypothesised that any one of a number of primary molecular events that alter AR activity and increase AR mRNA could represent a common final pathway for castration resistance in PCa. In support of this, it was demonstrated that LNCaP cells altered to express a threefold greater level of AR grew in low androgen concentrations whereas LNCaP cells did not unless supplemented with androgen, confirming that AR overexpression alone confers castration resistance. In addition, they demonstrated that the androgen receptor must bind its ligand to confer hormone-refractory growth. LBD mutant LNCaP constructs did not exhibit hormone-refractory growth beyond vector controls even at ten-fold increases of AR expression levels. Interestingly, AR antagonists, in the circumstance of overexpression, induced certain androgen regulated genes ​(Chen et al., 2003)​. This paradoxical response is reflected in the apparent vulnerability CRPC cells exhibit to supraphysiological androgens. Teply et al. demonstrated clinical response and short-lived resensitisation to enzalutamide through bipolar androgen therapy using exogenous testosterone ​(Teply et al., 2018)​. Similarly, Christensen et al. reported a remarkable clinical and prostate-specific antigen response to a combination of high doses of testosterone and radium 223 in a patient with metastatic CRPC whose disease had progressed while receiving a number of antiandrogenic therapies ​(Christensen et al., 2019)​. ctDNA consistently showed a high degree of AR amplification. These findings suggest that the switch to a hormone refractory state entails a markedly different response to ligand.

Large scale sequencing studies have shown over 90% of cases of advanced CRPC exhibit overexpressed or altered AR, alongside significant alteration to genes involved in histone rearrangement and chromatin modification ​(Barbieri et al., 2012; Braadland & Urbanucci, 2019; Grasso et al., 2012; Robinson et al., 2015; Taylor et al., 2010)​. Chromatin structure is at least partially definitive of a cell’s transcriptional program, and determines vast networks of regulatory elements tissue-specifically ​(Pihlajamaa et al., 2015)​. Chromatin relaxation is part of an adaptive response that increases the probability of genomic access and transcription, and enables continued function in a situation in which sufficient androgens and androgen signaling are therapeutically reduced ​(Braadland & Urbanucci, 2019)​. Patterns of open chromatin differ in CRPC to BPH or PCa samples, with large interindividual variance in CRPC ​(Alfonso Urbanucci et al., 2017)​. Braadland and Urbanucci suggest that selective or adaptive remodelling occurs mainly upon treatment challenge with AR-targeted therapies ​(Braadland & Urbanucci, 2019)​. Sequencing in independent AR overexpressing models by Urbanucci et al. revealed genome wide increases in open confirmations of chromatin and an increased opening at androgen responsive binding sites. Androgens further increased this chromatin opening, suggesting ligand potentiates an AR-driven chromatin remodelling in the context of AR overexpression ​(Alfonso Urbanucci et al., 2017)​. This represents a potential “feed forward” mechanism in which the overexpressed AR further facilitates chromatin remodelling that allows the AR greater access and increased binding at the genome ​(Braadland & Urbanucci, 2019)​. Additionally, progression to CRPC entails a significant reprogramming of the AR cistrome ​(Pomerantz et al., 2015; Sharma et al., 2013)​.

Mechanistic alteration of master regulators of the epigenome have been established to play a key role via increasing AR transcriptional activity ​(Ruggero et al., 2018)​, and their behaviour can be context sensitive. The chromatin remodelling enzyme lysine-specific demethylase 1 has emerged as having a dual role given its context-sensitive promotive or repressive effects on AR ​(Cai et al., 2011; Metzger et al., 2005)​. High androgen levels have been demonstrated to cause AR-mediated recruitment of LSD1 to facilitate gene silencing via negative autoregulation of the AR gene ​(Cai et al., 2011)​, while in the context of CRPC this feedback loop is apparently broken given that low androgen levels drive AR overexpression ​(Ruggero et al., 2018)​. LSD1 coactivator or corepressor activity is influenced by post-transcriptional modifications, such as its phosphorylation status which can switch the enzymes substrate ​(Metzger et al., 2007; Shi et al., 2004)​. The tyrosine kinase Src, upregulated in CRPC ​(Siu et al., 2016)​, inactivates the AR corepressor LCoR that ordinarily downregulates AR in response to ligand. This subsequently activates AR at the chromatin level in CRPC ​(Asim et al., 2011)​. A large number of micro-RNAs have been identified to act as post-transcriptional regulators of the AR ​(Perner et al., 2015)​. The miRNA miR137 regulates an androgen-mediated feedback loop that inhibits a large network of crucial AR coregulators in normal prostate epithelia, while epigenetic loss of miR137 in CRPC leads to coregulator and, consequently, AR overexpression ​(Nilsson et al., 2015)​.

It is notable that, in contrast with other DNA binding elements, the AR is able to initiate epigenetic modification of chromatin by itself ​(Tewari et al., 2012)​. Higher AR levels increase AR’s genome-wide binding to chromatin upon stimulation with low concentration of ligand ​(A Urbanucci et al., 2011)​. AR overexpression recruits AR and the basic epigenetic machinery to the chromatin to alter histones at AR binding sites and favour chromatin accessibility in the presence of low androgen levels ​(Alfonso Urbanucci et al., 2011)​. Chromatin remodeling proteins such as FOXA1 and HOXB13 are also known to co-localise with AR ​(Stelloo et al., 2017)​ and are capable of recruiting acetylating and methylating coregulators including CBP/p300 and MLL ​(Braadland & Urbanucci, 2019)​. Many coregulators of the AR exert chromatin remodelling effects themselves ​(Bannister & Kouzarides, 2011)​, and there is evidence that the AR upregulates a number of its coregulators gene-specifically through varied mechanisms, including AIB1, CBP, MAK, BRCA1, β-catenin, ATAD2, and MID1 ​(Perner et al., 2015; Alfonso Urbanucci et al., 2008, 2017)​. Several coregulators of the AR including p300, CBP and TIF2 have been shown to increase as a result of androgen deprivation ​(Agoulnik et al., 2006; Comuzzi et al., 2004; Heemers et al., 2007)​. Even a modest overexpression of AR can alter expression and amounts of AR coregulators ​(Chen et al., 2003)​, many of which are histone acetylating ​(Alfonso Urbanucci et al., 2011)​. Key bromodomain proteins, which locus-specifically affect chromatin opening, are androgen regulated and upregulated in AR overexpressing cells. These proteins participate in an AR deregulation-driven feedback loop that increases AR chromatin accessibility ​(Alfonso Urbanucci et al., 2017)​. The Jumonji C KDM4 histone lysine demethylases are overexpressed in CRPC, and KDM4B expression has been significantly correlated with AR. KDM4B influences chromatin and may induce relaxation in conditions of androgen deprivation that are relevant to progression to CRPC ​(Duan et al., 2019)​.

Gritsina et al. reviewed current knowledge regarding the function of AR signaling in driving target gene repression and silencing by regulation of the epigenetic machinery. Ligand-bound AR binds to the enhancers and/or promoter elements of target genes and mediates assembly and recruitment of the repressive complexes, including histone deacetylases, lysine-specific demethylase 1, and enhancer of zeste homolog 2. AR directly and indirectly induces cascades involving the stabilisation of protein-protein interactions and recruitment of complexes responsible for the removal of acyl groups, demethylation, inhibition of transcriptional activators, and trimethylation, resulting in chromatin modifications that render gene regulatory elements inaccessible or silenced ​(Yu et al., 2019)​.

Taken together, research has identified a clear role for AR expression in genome-wide epigenetic status, along with the ability of the AR to recruit and drive the basic elements of the epigenetic machinery. Additionally, it is apparent that a refractory response to antiandrogenic treatment can occur irrespective of agent. With consideration to these findings, a potential feed-forward mechanism of AR overexpression, potentiated by androgens, may have significant mechanistic implications for the onset and progressive worsening of PFS with the “crash” after cessation of the medication, during which time the multi-systemic symptoms and physiological effects of the condition become apparent or intensify with significant interindividual variability in severity. This occurrence is most usually in a time frame of days or weeks, a timeframe correlating to the return and increase of endogenous DHT levels as the newly functional 5-alpha reductase enzyme is replenished. DHT has been demonstrated to alter the regulation of a number of AR coactivators gene-specifically depending on the level of the receptor, suggesting plausible involvement of coactivator regulation in a feedback loop potentiating increased AR signaling ​(Alfonso Urbanucci et al., 2008)​.

illustration of a proposed mechanism underlying pfs
fig. A proposed basic mechanism in pathologically affected cells
underlying the development of PFS.

As the transition to CRPC results from androgen deprivation or androgen-axis targeted treatment, an induction of AR deregulation could have relevance to the increased incidence of higher Gleason score prostate cancers in 5ari patients ​(Sarkar et al., 2019; Theoret et al., 2011; Traish et al., 2014; Van Rompay et al., 2018)​. It is of significance that, following three years of use and then cessation, finasteride has been demonstrated to accelerate the progression of male pattern hair loss significantly. Using technologies unavailable at the time of finasteride’s clinical approval, Van Neste recently reported the first evidence of what they describe as a “drug dependency” of terminal scalp hair follicles in AGA patients and a “post-finasteride rebound phenomenon” in patients who had stopped finasteride after 3 years of successful maintenance. During 3 years of finasteride use, 99%‐100% terminal hair counts were recorded suggesting effective maintenance. However, while terminal hair was maintained on drug, within 30 months “off-drug” androgenic alopecia had significantly worsened, only 5.8% of terminal hair could be measured, with 94% having miniaturised and become unproductive. This is far in excess of the expected regression rates that were previously established in these patients and robustly predicted at 6% per year ​(Van Neste, 2019)​. It was previously reported that vertex dermal papilla cells in balding samples were 1.9 fold higher in AR expression than those from the occipital scalp ​(Kwon et al., 2004)​, and frontal follicles are 40% higher in AR expression in males compared with women ​(Sawaya & Price, 1997)​. Increased DNA methylation of the AR promoter in occipital follicles from men with AGA is suggestive of toxicity mediated by receptor levels ​(Cobb et al., 2011)​. In agreement, AGA models support an AR-mediated pathological process. Transgenic mice overexpressing human AR in the skin exhibit impaired hair regeneration when exposed to DHT, while hydroxyflutamide can abolish this effect ​(Crabtree et al., 2010)​. An adaptive increase in AR expression following androgen deprivation is therefore a plausible a mechanistic explanation for an increase in hormone sensitivity causative of the dramatic finasteride-induced progression of male pattern hair loss observed by Van Neste. Similarly, epigenetic amplification in PFS could reflect the common reports of a significant acceleration in MPB following development of the condition.

Therapeutic responses to androgens and antiandrogens in PFS

There is no known therapeutic approach for PFS ​(Than et al., 2018)​ and no consistently safe or effective therapy has emerged from two decades of patient self-experimentation. Owing to the common PFS symptom profile ostensibly pointing towards decreased androgenic activity and low or hypogonadal levels of testosterone in some cases of PFS, many patients have undergone treatment with exogenous androgens. While this can be of benefit in some patients, it is very rare that this is complete or consistently effective even if a temporary improvement is observed in some symptoms. Remarkably, symptoms can be exacerbated by administration of androgens. This is well reported even in patients in whom PFS has caused a clinical hypogonadism. Patients receiving testosterone replacement therapy prior to PFS have reported a dramatic intolerance to exogenous androgens following the onset of the condition. Testosterone is ordinarily associated with a decrease in depression an improved verbal memory ​(Cherrier et al., 2014)​ as well as anxiolytic effect in men, women and animals ​(McHenry et al., 2014)​. The reverse has been well reported in PFS patients, even when hypogonadal. Beyond cognitive symptoms, sexual dysfunction and physical symptoms such as muscle wastage can be exacerbated. This is highly remarkable and paradoxical. A patient who since committed suicide reported further rapid penile shrinking upon local application of topical DHT gel at a dosage of 5g per day with therapeutic intent. This is striking and paradoxical with consideration as to the known effect of DHT in increasing penile size ​(Arteaga-Silva et al., 2008; Becker et al., 2016; Choi et al., 1993)​. Patients will often report feeling no response at all to high doses of testosterone. Interindividually variable “saturation” points with regard to androgen response ​(Morgentaler & Traish, 2009; Zitzmann, 2009)​ have been hypothesised, and this may be of relevance to the therapeutic failure of testosterone in PFS. A threshold at which androgen-mediated toxicity reaches saturation has been observed with regard to the degree of symptoms seen in SBMA models ​(Chevalier-Larsen & Merry, 2011)​, in the toxic effect of DHT in SBMA motor neurons ​(Sheila et al., 2019)​, and in prostate cancer, in which testosterone therapy does not accelerate the disease progression despite androgen dependence ​(Morgentaler & Traish, 2009)​. In PFS, this reaction to exogenous ligand could plausibly be reflective of the degree of AR overexpression per site and per patient, and offers an explanation as to why more favourable partial responses to androgens are sometimes seen, while other patients can often rapidly worsen with raising androgen levels. Of note, it has been reported that an SBMA patient exhibited a notably similar reversible deterioration with androgen administration ​(Kinirons & Rouleau, 2008)​. Importantly, this would be in keeping with the observed responses of female transgenic mice overexpressing WT AR in skeletal muscle to exogenous testosterone equivalent to circulating male levels, which caused striking differences in deleterious physiological effects depending on the degree of AR overexpression ​(Monks et al., 2007)​.

Across the history of the propeciahelp forum, the most consequentially profound responses described entail significant modulation of symptoms by further exposure to substances that lower androgens through mechanisms including 5 alpha reductase inhibition, or substances that reduce concentrations of or inhibit AR. While rapid and severe worsening can occur, patients have equally often reported the dramatic return of function in the domains affected by PFS, usually temporarily. These are nearly always taken in the absence of the knowledge they are taking pharmaceuticals or natural extracts with antiandrogenic properties and are frequently sought out based upon their purported benefits in marketing and health editorials concerning relief of symptoms or through online reports from other patients. These have included zinc, quercetin, resveratrol, milk thistle, licorice root, turmeric/curcumin, sulforaphane, DIM, sodium butyrate, saw palmetto, tribulus terrestris, polyphenol rich products such as cacao nibs or pomegranate, and soy and soy isoflavones including genistein, all of which are notably antiandrogenic through various mechanisms ​(Agarwal et al., 2006; Boam, 2015; Cicero et al., 2019; De Amicis et al., 2019; Hiipakka et al., 2002; Jang et al., 2019; Kampa et al., 2017; Le et al., 2003; Sabbadin et al., 2019; Samykutty et al., 2013; Sandeep et al., 2015; Shiota et al., 2011; Xing, 2001)​. A remarkable overlap can be noted with nutraceuticals that are of increasing interest in the treatment of AR-mediated conditions and with substances or extracts causing patients to develop and present with PFS, as we have noted. Patients have independently described significant and remarkable multi-domain relief following use of AR antagonists including bicalutamide ​(Rice et al., 2019)​, and drugs with an antiandrogenic effects such as ibuprofen, paracetamol, dexamethasone, omeprazole, leuprolide acetate and mifepristone ​(Hoda et al., 2016; Inder et al., 2009; Kortenkamp, 2020; Kristensen et al., 2010; Song et al., 2004; Sørensen et al., 2016)​, and even finasteride itself. Recently, truvada, an antiretroviral medication combining tenofovir disoproxil and emtricitabine, has been reported to improve some PFS patients significantly in multiple symptom domains. Marketed as PrEP, truvada is a reverse transcriptase inhibitor. RTI drugs have been considered for potential therapeutic efficacy in hormonally refractive prostate cancer due to in vitro results suggesting the capability of Nevirapine to induce extensive reprogramming of gene expression, resensitizing cells to stimulation by extracellular ligand and consequentially re-establishing the efficacy of antiandrogen treatment with bicalutamide ​(Landriscina et al., 2009)​​.

These common reports are highly remarkable and of relevance to the potential of a pathologic link between PolyQ toxicity and deleterious consequences of site-specific overexpression of the wild type androgen receptor. This would appear to be in alignment with functional rescue in SBMA models targeting the androgen pathway ​(Cortes & La Spada, 2018; Katsuno et al., 2003; Minamiyama, 2004; Nedelsky et al., 2010; Rinaldi et al., 2015)​, and the molecular level responses to androgens and antiandrogens in AR overexpressing CRPC as discussed.

It is of the utmost importance to establish that antiandrogenic therapeutic strategies are dangerous for PFS patients. Patients can persistently exacerbate or develop further symptoms in multiple domains of the condition upon rechallenge or subsequent exposure to substances with antiandrogenic effect. This often occurs after a dramatic improvement of existing multisystemic symptoms. In 2018, a PFS patient who had taken supplementary resveratrol described a profound reversal of symptoms including insomnia, erectile dysfunction, libido loss and fatigue shortly before taking his own life. We note a key vulnerability of this cohort to what we believe to be an aberrant epigenetic response following exposure to antiandrogenic substances. This vulnerability appears significantly exacerbated following initial development of PFS, and even phenol or isoflavone-rich foods have resulted in clear reports of persistent worsening or the triggering of further symptoms. PFS patients most at risk of this are, in our experience, those who present with severe phenotypes after short use of finasteride or a causative antiandrogenic substance. Therefore, until more is known regarding the molecular mechanisms underlying the development of PFS, we strongly urge physicians dealing with PFS patients to be aware of this unique and peculiar vulnerability to therapeutic substances or medicines with antiandrogenic modality. This is of relevance to both prescribed therapies such as SRI antidepressants and to self-driven “natural” therapeutic attempts that can involve high dose phenolic compounds or vitamins marketed as health supplements. Owing to the sometimes profound endocrine sensitivity induced by PFS, safely managing the condition can be a significant burden for patients.

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