Yuanjie Niu

Androgen receptor is a tumor suppressor and proliferator in prostate cancer.

Targeting androgens/androgen receptor (AR) functions via androgen deprivation therapy (ADT) remains the standard treatment for prostate cancer. However, most tumors eventually recur despite ADT. Here we demonstrate that the prostate AR may function as both a suppressor and a proliferator to suppress or promote prostate cancer metastasis. Results from orthotopically recombining stromal WPMY1 cells with epithelial PC3 prostate cancer cells in mice demonstrated that restoring AR in epithelial PC3 cells or knockdown of AR in stromal WPMY1 cells suppressed prostate cancer metastasis. Knockdown of the AR in epithelial CWR22rv1 prostate cancer cells also resulted in increased cell invasion in vitro and in vivo. Restoring AR in PC3 cells (PC3-AR9) results in decreased invasion in bone lesion assays and in vivo mouse models. Mice lacking the prostate epithelial AR have increased apoptosis in epithelial luminal cells and increased proliferation in epithelial basal cells. The consequences of these two contrasting results led to the expansion of CK5/CK8-positive intermediate cells, and mice developed larger and more invasive metastatic tumors in lymph nodes and died earlier than wild-type littermates. Mechanistic dissection suggested that androgens/AR might directly or indirectly modulate metastasis-related genes and suppression of TGFβ1 signals results in the partial inhibition of AR-mediated metastasis. Collectively, our understanding of these opposing roles of prostatic AR may revolutionize the way we combat prostate cancer, and allow the development of new and better therapies by targeting only the proliferative role of AR.

Increased prostate cell proliferation and loss of cell differentiation in mice lacking prostate epithelial androgen receptor

Developmental studies of the prostate have established that ductal morphogenesis, epithelial cytodifferentiation, and proliferation/apoptosis are regulated by androgens acting through stromal androgen receptor (AR). Here, we found mice lacking epithelial AR within the mature prostate (pes-ARKO) developed prostate tissue that was less differentiated and hyperproliferative relative to WT littermates. Epithelial AR protein was significantly decreased in 6-week-old mice and was nearly absent by ≥24 weeks of age. Circulating levels of testosterone, external genitalia, or fertility were not altered in pes-ARKO mice. A significant (P < 0.05) increase in bromo-deoxyuridine-positive epithelia was observed in ventral and dorsal-lateral prostates of pes-ARKO mice at 24 weeks of age. Less differentiation was observed as indicated by decreased epithelial height and glandular infolding through 24 weeks of age, differentiation markers probasin, PSP-94, and Nkx3.1 were sig nificantly decreased, and epithelial sloughing and luminal cell apoptosis increased from 6 to 32 weeks of age in pes-ARKO mice. Gain of function occurred by crossing pes-ARKO to the T857A transgenic mice containing constitutively activated AR. In T857A-pes-ARKO mice prostates were of normal size, contained glandular infoldings, and maintained high secretory epithelium, and the appropriate prostatic epithelial proliferation was restored. Collectively, these results suggest that prostatic epithelial AR plays an important role in the homeostasis of the prostate gland. These data support the hypothesis that epithelial AR controls prostate growth by suppressing epithelial proliferation in the mature gland.

Targeting the stromal androgen receptor in primary prostate tumors at earlier stages

To differentiate roles of androgen receptor (AR) in prostate stromal and epithelial cells, we have generated inducible-(ind)ARKO-TRAMP and prostate epithelial-specific ARKO TRAMP (pes-ARKO-TRAMP) mouse models, in which the AR was knocked down in both prostate epithelium and stroma or was knocked out in the prostate epithelium, respectively. We found that loss of AR in both mouse models resulted in poorly differentiated primary tumors with expanded intermediate cell populations. Interestingly, knockdown of both epithelial and stromal AR in ind-ARKO-TRAMP mice at earlier stages resulted in smaller primary prostate tumors with lower proliferation rates, and knockout of AR in pes-ARKO-TRAMP mice resulted in larger primary prostate tumors with higher proliferation rates. The differential proliferation rates, yet with similarly expanded intermediate cell populations, indicated that the prostate stromal AR might play a more dominant role than the epithelial AR to promote primary tumor proliferation at an early stage of tumor. Tissue recombination of human prostate stromal cell lines (WPMY1-v or WPMY1-ARsi) with human prostate cancer epithelial cell lines (PC3-v or PC3-AR9) further demonstrated that the AR might function as a suppressor in epithelial cells and a proliferator in stromal cells in the primary prostate tumors. The dual roles of the AR in prostate epithelium and stroma may require us to reevaluate the target and timing of androgen-deprivation therapy for prostate cancer patients and may suggest a need to develop new drugs to selectively target stromal AR in the primary prostate tumors at earlier stages.

Differential androgen receptor signals in different cells explain why androgen-deprivation therapy of prostate cancer fails

Prostate cancer is one of the major causes of cancer-related death in the western world. Androgen-deprivation therapy (ADT) for the suppression of androgens binding to the androgen receptor (AR) has been the norm of prostate cancer treatment. Despite early success to suppress prostate tumor growth, ADT eventually fails leading to recurrent tumor growth in a hormone-refractory manner, even though AR remains to function in hormone-refractory prostate cancer. Interestingly, some prostate cancer survivors who received androgen replacement therapy had improved quality of life without adverse effect on their cancer progression. These contrasting clinical data suggest that differential androgen/AR signals in individual cells of prostate tumors can exist in the same or different patients, and may be used to explain why ADT of prostate cancer fails. Such a hypothesis is supported by the results obtained from transgenic mice with selective knockout of AR in prostatic stromal vs epithelial cells and orthotopic transplants of various human prostate cancer cell lines with AR over-expression or knockout. These studies concluded that AR functions as a stimulator for prostate cancer proliferation and metastasis in stromal cells, as a survival factor of prostatic cancer epithelial luminal cells, and as a suppressor for prostate cancer basal intermediate cell growth and metastasis. These dual yet opposite functions of the stromal and epithelial AR may challenge the current ADT to battle prostate cancer and should be taken into consideration when developing new AR-targeting therapies in selective prostate cancer cells.

New therapy targeting differential androgen receptor signaling in prostate cancer stem/progenitor vs. non-stem/progenitor cells

The androgen deprivation therapy (ADT) to systematically suppress/reduce androgens binding to the androgen receptor (AR) has been the standard therapy for prostate cancer (PCa); yet, most of ADT eventually fails leading to the recurrence of castration resistant PCa. Here, we found that the PCa patients who received ADT had increased PCa stem/progenitor cell population. The addition of the anti-androgen, Casodex, or AR-siRNA in various PCa cells led to increased stem/progenitor cells, whereas, in contrast, the addition of functional AR led to decreased stem/progenitor cell population but increased non-stem/progenitor cell population, suggesting that AR functions differentially in PCa stem/progenitor vs. non-stem/progenitor cells. Therefore, the current ADT might result in an undesired expansion of PCa stem/progenitor cell population, which explains why this therapy fails. Using various human PCa cell lines and three different mouse models, we concluded that targeting PCa non-stem/progenitor cells with AR degradation enhancer ASC-J9 and targeting PCa stem/progenitor cells with 5-azathioprine and γ-tocotrienol resulted in a significant suppression of the tumors at the castration resistant stage. This suggests that a combinational therapy that simultaneously targets both stem/progenitor and non-stem/progenitor cells will lead to better therapeutic efficacy and may become a new therapy to battle the PCa before and after castration resistant stages.

Tissue Prostate-Specific Antigen Facilitates Refractory Prostate Tumor Progression via Enhancing ARA70-Regulated Androgen Receptor Transactivation

Despite being well recognized as the best biomarker for prostate cancer, pathophysiologic roles of prostate-specific antigen (PSA) remain unclear. We report here that tissue PSA may be involved in the hormone-refractory prostate cancer progression. Histologic analyses show that the increased tissue PSA levels are correlated with lower cell apoptosis index and higher cell proliferation rate in hormone-refractory tumor specimens. By stably transfecting PSA cDNA into various prostate cancer cell lines, we found that PSA could promote the growth of androgen receptor (AR)-positive CWR22rv1 and high-passage LNCaP (hormone-refractory prostate cancer cells) but not that of AR-negative PC-3 and DU145 cells. Surprisingly, the protease activity of PSA is not crucial for PSA to stimulate growth and promote AR transactivation. We further showed that increased PSA could enhance ARA70-induced AR transactivation via modulating the p53 pathway that results in the decreased apoptosis and increased cell proliferation in prostate cancer cells. Knockdown of PSA in LNCaP and CWR22rv1 cells causes cell apoptosis and cell growth arrest at the G(1) phase. In vitro colony formation assay and in vivo xenografted tumor results showed the suppression of prostate cancer growth via targeting PSA expression. Collectively, our findings suggest that, in addition to being a biomarker, PSA may also become a new potential therapeutic target for prostate cancer. PSA small interfering RNA or smaller molecules that can degrade PSA protein may be developed as alternative approaches to treat the prostate cancer.

Differential Androgen Deprivation Therapies with Anti-androgens Casodex/Bicalutamide or MDV3100/Enzalutamide versus Anti-androgen Receptor ASC-J9® Lead to Promotion versus Suppression of Prostate Cancer Metastasis

Background: Androgen deprivation therapy (ADT) suppresses prostate cancer (PCa) growth, yet its effects on PCa metastasis remain unclear. Results: ADT with MDV3100/enzalutamide or Casodex/bicalutamide versus ASC-J9® led to enhanced versus suppressed PCa metastasis. Conclusion: Casodex/MDV3100 induces PCa metastasis via modulation of TGF-β1/Smad3/MMP9 signaling. Significance: Targeting androgen receptor with ASC-J9® is better than targeting androgens with Casodex/MDV3100 to better battle PCa metastasis. Despite the fact that androgen deprivation therapy (ADT) can effectively reduce prostate cancer (PCa) size, its effect on PCa metastasis remains unclear. We examined the existing data on PCa patients treated with ADT plus anti-androgens to analyze ADT effects on primary tumor size, prostate-specific antigen (PSA) values, and metastatic incidence. We found that the current ADT with anti-androgens might lead to primary tumor reduction, with PSA decreased yet metastases increased in some PCa patients. Using in vitro and in vivo metastasis models with four human PCa cell lines, we evaluated the effects of the currently used anti-androgens, Casodex/bicalutamide and MDV3100/enzalutamide, and the newly developed anti-AR compounds, ASC-J9® and cryptotanshinone, on PCa cell growth and invasion. In vitro results showed that 10 μm Casodex or MDV3100 treatments suppressed PCa cell growth and reduced PSA level yet significantly enhanced PCa cell invasion. In vivo mice studies using an orthotopic xenograft mouse model also confirmed these results. In contrast, ASC-J9® led to suppressed PCa cell growth and cell invasion in in vitro and in vivo models. Mechanism dissection indicated these Casodex/MDV3100 treatments enhanced the TGF-β1/Smad3/MMP9 pathway, but ASC-J9® and cryptotanshinone showed promising anti-invasion effects via down-regulation of MMP9 expression. These findings suggest the potential risks of using anti-androgens and provide a potential new therapy using ASC-J9® to battle PCa metastasis at the castration-resistant stage.

REST mediates androgen receptor actions on gene repression and predicts early recurrence of prostate cancer

The androgen receptor (AR) is a key regulator of prostate tumorgenesis through actions that are not fully understood. We identified the repressor element (RE)-1 silencing transcription factor (REST) as a mediator of AR actions on gene repression. Chromatin immunoprecipitation showed that AR binds chromatin regions containing well-characterized cis-elements known to mediate REST transcriptional repression, while cell imaging studies confirmed that REST and AR closely co-localize in vivo. Androgen-induced gene repression also involves modulation of REST protein turnover through actions on the ubiquitin ligase β-TRCP. Androgen deprivation or AR blockage with inhibitor MDV3100 (Enzalutamide) leads to neuroendocrine (NE) differentiation, a phenomenon that is mimicked by REST inactivation. Gene expression profiling revealed that REST not only acts to repress neuronal genes but also genes involved in cell cycle progression, including Aurora Kinase A, that has previously been implicated in the growth of NE-like castration-resistant tumors. The analysis of prostate cancer tissue microarrays revealed that tumors with reduced expression of REST have higher probability of early recurrence, independently of their Gleason score. The demonstration that REST modulates AR actions in prostate epithelia and that REST expression is negatively correlated with disease recurrence after prostatectomy, invite a deeper characterization of its role in prostate carcinogenesis.

Altered prostate epithelial development in mice lacking the androgen receptor in stromal fibroblasts.

Androgens and the androgen receptor (AR) play important roles in the development of male urogenital organs. We previously found that mice with total AR knockout (ARKO) and epithelial ARKO failed to develop normal prostate with loss of differentiation. We have recently knocked out AR gene in smooth muscle cells and found the reduced luminal infolding and IGF‐1 production in the mouse prostate. However, AR roles of stromal fibroblasts in prostate development remain unclear.

Altered prostate epithelial development and IGF-1 signal in mice lacking the androgen receptor in stromal smooth muscle cells †‡§

Androgens and the androgen receptor (AR) play critical roles in the prostate development via mesenchymal–epithelial interactions. Smooth muscle cells (SMC), differentiated from mesenchyme, are one of the basic components of the prostate stroma. However, the roles of smooth muscle AR in prostate development are still obscure.