PTEN loss or PI3K pathway activation represents one of the most frequent genetic alterations found in human CaPs (Taylor et al., 2010
). Dysregulation of the PTEN/PI3K pathway has also been associated with resistance to conventional anti-androgen therapies (Ham et al., 2009
). Despite these clinical observations, the consequence of PTEN loss and how its loss influence androgen/AR signaling axis and CRPC development are unclear. By genetically deleting Pten
in the prostatic epithelium and analyzing human CaP samples, our studies support the hypothesis that PTEN loss or PI3K pathway activation may function in a cell autonomous manner to promote androgen/AR-independent CaP progression and CRPC development. Although Pten
null epithelial cells remain sensitive to androgen withdrawal or AR ablation, the resulting activated PI3K/AKT pathway is sufficient to compensate androgen/AR signaling blockage, mobilize basal and transient amplifying stem/progenitor cells and promote cell proliferation. Therefore, in CaPs initiated by PTEN loss or PI3K activation, the overall outcome of cancer development, especially CRPC development, depends on the balance of androgen-dependent cell survival/differentiation and androgen-independent cell proliferation. PTEN loss enhances the expressions of EGR1, c-JUN and EZH2, which in turn suppresses AR transcription factor activity and output. Inhibiting FKBP5/PHLPP-mediated negative feedback to AKT activation, as a result of castration or AR inhibition, may further enhance the strength of the PI3K/AKT pathway and tilt the balance toward androgen-independent growth (). Importantly, results derived from our genetically engineered mouse models, including PTEN controlled AR transcription output and AR-FKBP5-PHLPP regulatory loop, can also be observed in human CaP samples. Given commonly observed heterogeneous PTEN
deletion/mutation and AR expression patterns within individual human CaPs, shown in our study and published by others (Attard et al., 2009b
; Taylor et al., 2010
), we would expect to observe a greater range of phenotypic correlating between PTEN loss, AR expression and CRPC development in human patients.
PTEN loss promotes CRPC development by two collaborative mechanisms
Using genetically engineered animal models, our findings may provide potential insight to the clinical settings. Although hormone therapy immediately after radical prostatectomy improves survival and reduces the recurrence (Messing et al., 1999
), it is not clear whether early androgen deprivation therapy (ADT) will prevent cancer progression and CRPC development. That early castration of the Pten
null CaP model does not significantly impede carcinoma development and castration-resistant growth suggests that patients with PTEN loss or PI3K pathway activation may not benefit from aggressive early hormone treatment. While neoadjuvant ADT may partially reduce tumor load and PSA levels, it may select for cells with activated compensatory cell survival and proliferating signaling pathways, such as those PTEN
null or PI3K/AKT-activated, and ultimately facilitate resistance to anti-androgen therapy.
An important finding from our study is that PTEN loss suppresses AR transcription factor activity and androgen-responsive gene expression in both murine models and human prostate samples. Interestingly, AR itself is not down-regulated in Pten
null CaPs, even after castration (data not shown), similar to human cancers after short-term castration or after 9 months of neoadjuvant ADT (Mostaghel et al., 2007
). Previous studies suggest that the PI3K pathway can either activate or suppress AR activity (Kaarbo et al., 2010
; Lei et al., 2006
; Lin et al., 2004
; Lin et al., 2003
). Our analyses demonstrate that PTEN, by regulating the expressions and activities of EGR1 and c-JUN transcription factors as well as the level of EZH2, controls AR transcription output, and thereby, render PTEN
null CaP cells less or completely independent of signaling provided by androgens and epithelial AR, hence promote castration-resistant growth. However, how PTEN/PI3K pathway controls EGR1, c-JUN and EZH2 expression and activities requires further study.
The mosaic pattern of AR deletion observed in our in vivo model also mimic the heterogeneous AR expression observed in our human CaP studies and those reported by others (Attard et al., 2009b
). After castration of Pb-Cre+;PtenL/L;ArL/Y
mutants, we observed significant outgrowth of Pten−
cancerous regions, accompanied by elevated P-PAKT and cell proliferation. Adjacent to these outgrowth are AR+
regions. Therefore, it is possible that within heterogeneous malignant CaPs, those with low or negative AR expression fail to respond to conventional anti-androgen treatment and continue to proliferate and survive as a consequence of PTEN loss and PI3K/AKT activation. While such cells would not constitute PSA producing cancer cells, they would contribute towards overall tumor load.
An intriguing finding of our study is the elevated P-AKT (S473) levels despite genetic deletion of Ar
and surgical castration. The recently defined relationship between FKBP5 and the AKT phosphatase, PHLPP (Gao et al., 2005
) (Brognard et al., 2007
) (Pei et al., 2010
), provides a potential mechanism as how PI3K/AKT signaling can be activated upon inhibition of the AR/androgen signaling axis. Specifically, while in normal cells PHLPP levels are high, which keeps P-AKT levels low, down-regulation of FKBP5 in Pten-negative cells that have undergone AR/androgen ablation would lead to a reduction of PHLPP’s association with P-AKT and consequently enhance AKT activity. Although it is difficult to assess P-AKT levels in all human CaP samples due to various sample harvesting and preparation procedures used, we did observed statistically significant correlation between the levels of PTEN and PHLPP as well as AR and FKBP5. Importantly, when a fresh resected CaP sample was analyzed, we found that regions with low or no AR expression have lower levels of FKBP5 and PHLPP and enhanced P-AKT and P-S6 staining. Besides the AR-FKBP5-PHLPP feedback loop, we also observed changes in other scaffold proteins and mediators of the PI3K pathway (Table S1
). It will be interesting for future studies to ascertain whether other AR/androgen regulated genes, such as FKBP11 and IGFBP3/6, also have the capacity to modulate PI3K/AKT signaling in a manner similar to FKBP5.
Increasing evidence suggests that different cellular compartments may contribute differently towards cancer initiation. For example, the prostate epithelial AR may confer some suppressor function while the stromal AR has been postulated to be mitogenic (Cunha et al., 2002
; Simanainen et al., 2009
), thus raising the possibility that stromal AR plays a dominant role in Pten null cancer initiation or during the development of CRPC. In fact, using the TRAMP CaP model, a previous report has shown that reduction of stromal AR content may lead to reduced tumor progression (Niu et al., 2008b
). However, extrapolating these data to human disease is challenging because neuroendocrine carcinoma observed in the TRAMP model, a phenotype which is rare in human disease (Abbas et al., 1995
; Tetu et al., 1987
). It is also possible that while AR loss or degradation in differentiated luminal epithelium results in cell death, AR loss in basal or transient amplifying initiating cells is well tolerated. In fact, recent reports have shown that the basal “LSC” stem/progenitor cells either from the Pten null CaP model (Mulholland et al., 2009
) or from primary human tissue that has been transformed (Goldstein et al., 2010
) are sufficient for tumor reconstitution. Thus, it would be interesting and potentially clinically relevant to ascertain if AR-null stem progenitor cells would be capable of cancer initiation. Interestingly, cells with high N-cadherin expression, a marker associated epithelial-mesenchymal transition and cancer metastasis at late stage disease are also AR-null or low and forced expression of N-cadherin results in reduced AR expression and elevated P-AKT in human CaPs (Tanaka et al., 2010
Previous studies have shown that mTOR inhibition alone is relatively ineffective at reducing overall tumor load in Pb-Cre+;PtenL/L
mutants (Kinkade et al., 2008
) (Zhang et al., 2009
). To test the hypothesis that combined AR and mTOR targeting may be more effective where single agent use is not, we evaluated the impact of total AR/androgen ablation in conjunction with mTOR inhibition. When comparing Pb-Cre+;PtenL/L;ArL/Y
mutants under castration alone to those with rapamycin, we observed marked inhibition of cell proliferation and reduction of tumor load. Thus, these pre-clinical data suggests that complete abolishment of the AR/androgen signaling axis combined with mTOR inhibition is superior to single agent use, most likely by inhibiting the crosstalk between the two pathways. Thus, more effective blockade of the androgen/AR axis with new generation inhibitors such as abiraterone and MDV3100 in combination with mTOR or PI3K/mTOR dual inhibitors may prove to be further advantageous in treating CRPC cases initiated by alterations of PTEN/PI3K pathway.