Scientists claim blocking testosterone in advanced prostate cancer may not be the best therapeutic strategy against the disease. They found that production of 5α-dihydrotestosterone (DHT), which drives tumor growth, involves a dominant pathway that bypasses testosterone completely.
University of Texas Southwestern Medical Center researchers evaluated human castration-resistant protate cancer (CRPC) cell lines, fresh tissue obtained from human tumor metastases, and mouse xenograft models of CRPC growth. They found that while DHT synthesis in CRPC is widely assumed to involve a pathway that hinges on 5α-reduction of testosterone, the dominant route of DHT synthesis in CRPC actually requires 5α-reduction of androstenedione (AD) by steroid 5α-reductase isoenzyme-1 (SRD5A1) to 5α-androstanedione, which is then converted to DHT.
Nima Sharifi, at UT Southwestern’s Harold C. Simmons Comprehensive Cancer Center, and colleagues report that their findings suggest both blocking the conversion of AD to testosterone won’t significantly inhibit DHT synthesis and also that testosterone may not be the best marker for monitoring the intratumoral response or resistance of tumors to upstream inhibitors of adrenal steroid synthesis such as abiraterone acetate. The team reports its findings in PNAS in a paper titled “Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer.”
Androgen-deprivation therapy with depletion of gonadal testosterone represents the mainstay of treatment for advanced prostate cancer, Dr. Sharifi et al note. While therapy is usually effective initially, metastatic disease almost always eventually acquires resistance to gonadal testosterone depletion. A critical mechanism in driving the progression of CRPC is a gain-of-function in the androgen receptor (AR).
In CRPC androgen ligand is generated within the tumor, and multiple clinical studies have shown that intratumoral concentrations of testosterone and DHT sufficient to activate AR-dependent transcription are maintained in CRPC despite suppression of serum testosterone. DHT, rather than testosterone, is the principal androgen bound to AR in the prostate cell nucleus.
Intratumoral synthesis of DHT is generally believed to require 17-keto reduction of AD to testosterone, followed by 5α-reduction of testosterone to DHT. This conventional pathway assumes that testosterone is the obligate precursor of DHT. An alternative pathway to DHT synthesis, however, has been identified that circumvents testosterone and instead requires 5α-dione as the precursor.
To investigate which pathway for DHT synthesis is dominant in CRPC, the authors analyzed six established human prostate cancer cell lines obtained from patients with CRPC. In all six cell lines, flux from AD→5α-dione occurred earlier and more rapidly than flux from AD→testosterone, as evidenced by 5α-dione and testosterone detection by HPLC.
To establish proof-of principle for the preferred route of AD metabolism in clinical samples, biopsies of metastatic tumors from two men with CRPC were investigated. As in the six CRPC cell lines, the dominant route of AD metabolism was again found to be 5α-reduction to 5α-dione, and in one patient there was apparently no conversion from AD to testosterone.
To test the requirement of the conventional pathway (AD→testosterone→DHT) versus the alternative pathway (AD→5α-dione→DHT) for CRPC growth in vivo, the authors analyzed subcutaneous xenografts of LAPC4 and LNCaP tumors in surgically orchiectomized mice supplemented with testosterone and AD. They found that both LAPC4 and LNCaP tumors in mice supplemented with AD grew to a greater size, more quickly, than those in animals supplemented with testosterone.
The increased SRD5A1 expression evident in CRPC tumors is generally thought to reflect its role in converting testosterone to DHT in the conventional pathway, Dr. Sharifi and colleagues add. To test whether SRD5A1 might actually catalyze flux from AD→5α-dione in the alternative pathway, both SRD5A1 and SRD5A2 were stably knocked down in LNCaP and LAPC4 cells using lentiviral shRNAs.
Analyses confirmed that synthesis of 5α-dione, DHT, and total 5α-reduced steroids was largely blocked by silencing SRD5A1 using two independent shRNAs. Inhibiting SRD5A2 had no such effect. “Notably, blocking flux from AD→5α-dione by silencing SRD5A1 diverts AD instead to increased testosterone,” they write.
To determine the effect of blocking flux from AD→5α-dione by silencing SRD5A1 expression on AR-dependent transcription, the researchers treated SRD5A1-silenced cells with AD, and assessed prostate-specific antigen expression. As expected, they found that AD-induced PSA expression was significantly reduced in the absence of SRD5A1.
They then investigated whether the role of SRD5A1 expression in the alternative pathway is critical for driving CRPC growth. To this end, surgically orchiectomized mice supplemented with sustained-release AD pellets were injected subcutaneously with cells expressing either an SRD5A1 shRNA or a control nonsilencing shRNA.
The results all suggested that cells lacking RD5A1 were disadvantaged in terms of growth. Conversely, xenograft growth in 5α-dione supplemented mice was nearly identical among animals given either the control shRNAs, or the SRD5A1-silencing shRNAs, “further demonstrating that the specific role of SRD5A1 in the alternative pathway and CRPC growth is the conversion of AD→5α-dione,” the researchers stress.
“In contradistinction to widely held assumptions about the major pathway that drives CRPC progression, our findings show that the major metabolic pathway from adrenal precursor steroids to DHT in CRPC circumvents testosterone as an obligate precursor and that the transformation of AD→5α-dione by SRD5A1 is a required step for DHT synthesis and tumor progression,” they conclude. “These findings reframe the fundamental metabolic pathway that drives CRPC progression and shed light on the development of new therapeutic strategies.”