Zhenfei Li

Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer

Prostate cancer resistance to castration occurs because tumours acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signalling by the androgen receptor and the development of castration-resistant prostate cancer. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly requires enzymatic reactions by 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A) and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival. We hypothesized that abiraterone is converted by an enzyme to the more active Δ4-abiraterone (D4A), which blocks multiple steroidogenic enzymes and antagonizes the androgen receptor, providing an additional explanation for abiraterone’s clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3βHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive androgen receptor antagonism by D4A is comparable to the potent antagonist enzalutamide. D4A also has more potent anti-tumour activity against xenograft tumours than abiraterone. Our findings suggest an additional explanation—conversion to a more active agent—for abiraterone’s survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment.

Redirecting abiraterone metabolism to fine-tune prostate cancer anti-androgen therapy

Abiraterone blocks androgen synthesis and prolongs survival in patients with castration-resistant prostate cancer, which is otherwise driven by intratumoral androgen synthesis. Abiraterone is metabolized in patients to Δ4-abiraterone (D4A), which has even greater anti-tumour activity and is structurally similar to endogenous steroidal 5α-reductase substrates, such as testosterone. Here, we show that D4A is converted to at least three 5α-reduced and three 5β-reduced metabolites in human serum. The initial 5α-reduced metabolite, 3-keto-5α-abiraterone, is present at higher concentrations than D4A in patients with prostate cancer taking abiraterone, and is an androgen receptor agonist, which promotes prostate cancer progression. In a clinical trial of abiraterone alone, followed by abiraterone plus dutasteride (a 5α-reductase inhibitor), 3-keto-5α-abiraterone and downstream metabolites were depleted by the addition of dutasteride, while D4A concentrations rose, showing that dutasteride effectively blocks production of a tumour-promoting metabolite and permits D4A accumulation. Furthermore, dutasteride did not deplete the three 5β-reduced metabolites, which were also clinically detectable, demonstrating the specific biochemical effects of pharmacological 5α-reductase inhibition on abiraterone metabolism. Our findings suggest a previously unappreciated and biochemically specific method of clinically fine-tuning abiraterone metabolism to optimize therapy.

Aberrant corticosteroid metabolism in tumor cells enables GR takeover in enzalutamide resistant prostate cancer

Prostate cancer is driven by androgen stimulation of the androgen receptor (AR). The next-generation AR antagonist, enzalutamide, prolongs survival, but resistance and lethal disease eventually prevail. Emerging data suggest that the glucocorticoid receptor (GR) is upregulated in this context, stimulating expression of AR-target genes that permit continued growth despite AR blockade. However, countering this mechanism by administration of GR antagonists is problematic because GR is essential for life. We show that enzalutamide treatment in human models of prostate cancer and patient tissues is accompanied by a ubiquitin E3-ligase, AMFR, mediating loss of 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which otherwise inactivates cortisol, sustaining tumor cortisol concentrations to stimulate GR and enzalutamide resistance. Remarkably, reinstatement of 11β-HSD2 expression, or AMFR loss, reverses enzalutamide resistance in mouse xenograft tumors. Together, these findings reveal a surprising metabolic mechanism of enzalutamide resistance that may be targeted with a strategy that circumvents a requirement for systemic GR ablation. DOI: http://dx.doi.org/10.7554/eLife.20183.001

Development and validation of a novel LC-MS/MS method for simultaneous determination of abiraterone and its seven steroidal metabolites in human serum: Innovation in separation of diastereoisomers without use of a chiral column

Abiraterone acetate (AA), the prodrug of abiraterone, is FDA-approved for the treatment of castration-resistant prostate cancer. Abiraterone is metabolized in patients to a more potent analogue, D4A. However, we have recently reported that this analogue is further metabolized to additional metabolites in patients treated with AA. Here, we present a liquid chromatography-tandem mass spectrometry method developed to resolve and detect abiraterone and its seven metabolites in human serum using an AB Sciex Qtrap 5500 mass analyzer coupled with a Shimadzu Nexera UPLC station. Analytes and the internal standard (abiraterone-d4) were extracted from human serum using the liquid-liquid extraction procedure. The analytes were separated using a Zorbax Eclipse Plus C18 150×2.1mm, 3.5μm column at 40°C and an isocratic mobile phase 35% A (0.1% formic acid in water), 65% B (0.1% formic acid in methanol:acetonitrile; 60:40). Electrospray ionization in positive mode was applied with multiple reaction monitoring in a total run time of 13min. Abiraterone detection was linear in the range 2-400ng/mL and all metabolites from 0.1-20ng/mL. The method was validated following US FDA guidelines for bioanalytical method validation, and all the metabolite results were within the acceptance limits. Despite the similarity in structure and mass transition between the metabolites, the validated method separated all the metabolites, including diastereomers, to allow accurate identification and quantitation of each compound.

Clinical significance of D4A in prostate cancer therapy with abiraterone

Androgen deprivation therapy (ADT), either by surgical or medical castration depletes testicular production of testosterone (T) and has long been considered as the mainstay of upfront treatment for advanced prostate cancer. Despite the initial response to this treatment, disease progression to castration-resistant prostate cancer (CRPC) usually occurs. The adrenal production of androgen precursor steroids, such as dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEA-S), permits intratumoral production of T and 5α-dihydrotestosterone (DHT), activation of androgen receptor (AR) and AR-dependent gene expression. Abiraterone (Abi; given as Abi acetate) and enzalutamide (Enz) are 2 of the agents that target different parts of the androgen pathway by blocking androgen production and signaling, ultimately improving overall survival for patients with metastatic CRPC.1,2 Abi is a potent inhibitor of 17α-hydroxylase/17,20-lyase (CYP17A1), which is a key enzyme required for DHEA and DHEA-S production. Synthesis of DHT, the most potent androgen, from DHEA necessitates a series of enzymes, including 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A), and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes. This process enables the conversion of ADT-responsive prostate cancer into CRPC. Multiple retrospective reviews have shown the limited efficacy of Enz after Abi treatment failure and vice versa.3,4 To date there are very little data on the appropriate sequence of Enz and Abi utilization, which might thereby maximize the potential efficacy and duration of response to treatment with these agents. Our recently published research demonstrates that Δ4, 3-keto-abi (D4A) is a metabolite of Abi that is formed in patients with prostate cancer and has CYP17A1 inhibition activity that is comparable to Abi.5 Furthermore, D4A inhibits 3βHSD (a key enzyme involved in CRPC progression) more potently than Abi and at higher concentrations it demonstrates significant SRD5A inhibitory effects as well. Moreover, although Abi has been shown to have AR antagonistic activity,6 we observed more potent AR inhibition activity with D4A that is comparable to Enz in our experiments.5 As a result, by targeting multiple steps of the AR signaling pathway, D4A showed a significant increase in progression free survival compared to Abi and Enz in our xenograft models, suggesting that it might play an important role in the clinical activity of Abi in CRPC patients. Abi is converted to D4A by 3βHSD. Increased 3βHSD enzymatic function, with the HSD3B1(1245C) genetic variant encoding a missense in 3βHSD1 as one known mechanism, also increases intratumoral flux to DHT that may result in the development of treatment resistance.7 Therefore, D4A conversion ratio could serve as an indicator of 3βHSD activity and ultimately may predict resistance to hormonal therapy for prostate cancer. On the other hand, higher 3βHSD activity would translate into a higher D4A concentration, which is a more potent metabolite than Abi as described above. Although the Abi to D4A conversion ratio is low (at about 5%) in most patients, it appears to be quite variable. A higher conversion ratio might mimic conditions of treatment with Enz, particularly given the AR antagonist activity of D4A. In other words, 3βHSD activity level in a patient might be helpful in predicting the potential response to Abi and ultimately a patient with higher concentration of D4A with Abi treatment might be less likely to benefit from subsequent Enz treatment. Conversely, increasing D4A levels by increasing Abi concentrations or the conversion to D4A might increase the clinical benefit of treatment with Abi. In addition, because 3βHSD is required for mineralocorticoid synthesis, 3βHSD inhibition by D4A might reduce mineralocorticoid production that could translate into a better side effect profile (less hypertension and hypokalemia) and potentially lower dose of prednisone requirement in patients with higher D4A levels. These questions have yet to be answered in clinical studies. Finally, it is possible that steroidogenic metabolites of Abi are not limited to D4A, and that other metabolites with clinically relevant biochemical activity contribute to response and resistance to treatment with Abi.

Abstract A17: Abiraterone metabolism and a novel therapeutic strategy for castration resistant prostate cancer

Prostate cancer is the most common cancer in men in the United States. Abiraterone (abi) is approved by the FDA for treatment of castration resistant prostate cancer (CRPC). However, ~30% of patients have limited or no response to abi. The underlying mechanism of resistance is elusive in these cases, and the best treatment plan for such patients is not clear. Thus we sought to determine a potential mechanism of resistance by analyzing abi metabolism. We found that abi undergoes metabolism catalyzed by steroidogenic enzymes. Abi is converted sequentially to delta-4-abi (D4A), then undergoes C5 reduction to produce several metabolites, including 5α-abi and 5 other metabolites. D4A has more potent anti-tumor activity than abi itself because it inhibits multiple enzymes and antagonizes the androgen receptor (AR) directly. D4A inhibits cell proliferation and xenograft growth better than Abi. However, 5α-Abi has more limited effects on steroidogenic enzymes and is a modest AR agonist, which might promote abi resistance. We suggest that abi metabolism could be a biomarker for abi response, and patients with limited or no response to abi might have less D4A or more 5α-Abi. Furthermore, we demonstrate that dutasteride, a 5α-reductase inhibitor, suppresses the conversion from D4A to 5α-Abi in both prostate cancer cell lines and patients, which might enhance the clinical response to abi. Taken together, these data reveal a novel metabolic pathway of abi that can be exploited for treatment, specifically as a biomarker. It is achievable in clinic to fine tune abi metabolism to obtain greater concentrations of D4A, the most potent anti-tumor abi metabolite, in patients by supplementing abi treatment with dutasteride. Citation Format: Zhenfei Li, Mohammad Alyamani, Jianneng Li, Mary-Ellen Taplin, Nima Sharifi. Abiraterone metabolism and a novel therapeutic strategy for castration resistant prostate cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A17.

Loss of dihydrotestosterone-inactivation activity promotes prostate cancer castration resistance detectable by functional imaging

Androgens such as testosterone and dihydrotestosterone are a critical driver of prostate cancer progression. Cancer resistance to androgen deprivation therapies ensues when tumors engage metabolic processes that produce sustained androgen levels in the tissue. However, the molecular mechanisms involved in this resistance process are unclear, and functional imaging modalities that predict impending resistance are lacking. Here, using the human LNCaP and C4-2 cell line models of prostate cancer, we show that castration treatment–sensitive prostate cancer cells that normally have an intact glucuronidation pathway that rapidly conjugates and inactivates dihydrotestosterone and thereby limits androgen signaling, become glucuronidation deficient and resistant to androgen deprivation. Mechanistically, using CRISPR/Cas9-mediated gene ablation, we found that loss of UDP glucuronosyltransferase family 2 member B15 (UGT2B15) and UGT2B17 is sufficient to restore free dihydrotestosterone, sustained androgen signaling, and development of castration resistance. Furthermore, loss of glucuronidation enzymatic activity was also detectable with a nonsteroid glucuronidation substrate. Of note, glucuronidation-incompetent cells and the resultant loss of intracellular conjugated dihydrotestosterone were detectable in vivo by 18F-dihydrotestosterone PET. Together, these findings couple a mechanism with a functional imaging modality to identify impending castration resistance in prostate cancers.

The association of D4, 3-keto-abi (D4A) and response to abiraterone in castration-resistant prostate cancer (CRPC).

245 Background: Abiraterone (Abi), a potent inhibitor of 17α-hydroxylase/17,20-lyase (CYP17A1), is a standard treatment for men with metastatic CRPC. Abi is converted to D4A by 3β-hydroxysteroid dehydrogenase (3βHSD). D4A inhibits CYP17A1, 3βHSD, and steroid-5α-reductase (SRD5A) and has direct androgen receptor antagonist activity, which together make it a more potent agent than Abi in xenograft models. It is not known if conversion to D4A in patients (pts) correlates with response or resistance to Abi. Methods: Blood was collected (single time point on Abi) from CRPC pts who started Abi during 2011-2015. Abi and D4A were extracted from serum and analyzed by mass spectrometry. The purpose of this ongoing study is to assess the potential correlation between D4A and response to treatment. Results: 32 patients with CRPC had blood collected. 4 pts (12.5%) received ketoconazole and 6 (18.8%) chemotherapy prior to Abi. Median pre-Abi prostate-specific antigen (PSA) was 14.3 ng/ml (0.6-646.1). Median time from i...