The phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt (PI3K/Akt) pathway is a key pathway activated in castrate-resistant prostate cancer (CRPC). This preclinical study evaluates targeting of Akt with AZD5363 alone and in combination with enzalutamide (ENZ) to prevent and delay resistance. Our results demonstrate AZD5363 has significant proapoptotic, antiproliferative activity as monotherapy in ENZ-resistant cell lines in vitro and significantly decreased tumour growth in ENZ-resistant xenograft. The combination of AZD5363 and ENZ showed synergistic decreases in cell proliferation and induced cell-cycle arrest and apoptosis in prostate cancer cell lines LNCaP and C4-2. Notably, the combination of AZD5363 and ENZ resulted in an impressive regression of castrate-resistant LNCaP xenograft tumours without any recurrence demonstrated, whereas progression occurred with both monotherapies. Serum prostate-specific antigen (PSA) levels were also continuously suppressed, and nadir PSA levels were lower in the combination arm compared to ENZ alone. Combination AZD5363 and ENZ at time of castration similarly resulted in significant regression of tumours, with greater relative suppression of PSA compared to when administered to castrate-resistant xenografts. In summary, combination AZD5363 and ENZ significantly delays the development of ENZ resistance in preclinical models through synergistic increases in apoptosis and cell cycle arrest. Our results also suggest greater efficacy may be seen with earlier combination treatment. This study provides preclinical data to support evaluation of combination targeting of the PI3K/Akt pathway and the androgen-receptor axis in the clinic using AZD5363 and ENZ, respectively.
Targeting of the Akt and androgen receptor pathways with AZD5363 and enzalutamide, respectively, significantly delayed the development of enzalutamide-resistant prostate cancer through increased apoptosis and cell cycle arrest. This preclinical synergy provides a strong rationale for clinical evaluation of this combination.
Keywords: Akt inhibitor, Apoptosis, Enzalutamide, Prostate cancer.
While new androgen receptor (AR)-targeting therapies such as abiraterone and enzalutamide (ENZ) highlight the success of targeting the AR axis in castrate-resistant prostate cancer (CRPC), these therapies are not curative and resistance inevitably develops. Therefore, there remains a need for rationale therapeutic strategies to delay the development of resistance.
The Akt pathway is upregulated in CRPC and predicts a poorer prognosis  . Akt signalling is involved in many oncogenic cellular processes, including cell growth and survival. Preclinical research demonstrates that the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/mechanistic target of rapamycin (serine/threonine kinase) (mTOR) and AR pathways interact reciprocally both in settings of prostate cancer (PCa) resistance and carcinogenesis and . Furthermore, these experiments provide a rationale explaining disappointing results of prior trials of Akt pathway inhibitors as monotherapy in advanced PCa and .
AZD5363 functions as an orally available adenosine triphosphate-competitive pan-Akt inhibitor  . In this study, we evaluated the role of Akt inhibition with AZD5363 to delay the development of ENZ-resistant PCa.
The Akt pathway is a key targetable pathway in models of ENZ resistance. We and others found the Akt pathway is activated following treatment with ENZ (Supplementary Fig. 1a)  . Therefore, using ENZ-resistant MR49C and MR49F cells derived through serial passage of LNCaP xenografts treated with ENZ  , we assessed the efficacy of AZD5363 in models of ENZ resistance. Consistent with other adenosine triphosphate-competitive, catalytic inhibitors of Akt, AZD5363 resulted in an accumulation of inactive p-Akt  , demonstrated by downstream decreases in p-S6 and 4e-BP-1 ( Fig. 1 A). A dose-dependent decrease in cell viability was observed in ENZ resistant cells ( Fig. 1 B). Mechanistically, this was caused by increased apoptosis and cell cycle arrest ( Fig. 1 C; Supplementary Fig. 1b and 1c).
In vivo, we selected the rapidly proliferating, prostate-specific antigen (PSA)-producing MR49F xenograft model  , as MR49F cells had a highly active Akt pathway (Supplementary Fig. 2a). We observed a significant response to AZD5363 treatment with evident tumour growth inhibition and delayed PSA rises ( Fig. 1 D and F). On a microarray of collected tumours, increased pAkt levels, an expected on-target effect of AZD5363, were observed. A decrease in Ki67 staining confirmed the antiproliferative action in vivo ( Fig. 1 E; Supplementary Fig. 2b). This improved cancer-specific and overall survival (Supplementary Fig. 3a) with no toxicity (Supplementary Fig. 3b). Tumour growth inhibition was seen with lower doses of AZD5363, but ENZ given concomitantly appeared to act as an agonist (Supplementary Fig. 3c–3f). Our unpublished data confirm that ENZ acts as an agonist in MR49C and MR49F cells due to the presence of the previously described F876L AR ligand-binding domain mutation  . The rising PSA level in MR49F xenografts that became resistant to AZD5363 ( Fig. 1 D) suggested the resistance to AZD5363 in this model was AR driven.
Therefore, we hypothesised that earlier targeting of Akt and AR may delay the onset of ENZ resistance. Using LNCaP, C4-2, and 22RV1 cells, protein signalling pathways were analysed. We found that combination AZD5363 and ENZ resulted in a greater induction of poly adenosine diphosphate ribose polymerase cleavage and caspase-3 activity ( Fig. 2 A and 2B; Supplementary Fig. 5b and 5d), increased subG1/G0 population (Supplementary Fig. 4a) with concomitant decrease of cyclin D1 ( Fig. 2 A), and, in most cases, a greater decrease in phosphorylation of Akt downstream effectors ( Fig. 2 A; Supplementary Fig. 4b). These effects were translated to cell viability, where a synergistic effect of AZD5363 with ENZ was observed ( Fig. 2 C). Results in 22RV1 cells also demonstrated synergy with the combination, despite the cells being generally resistant to monotherapy on the different assays (Supplementary Fig. 4b–4d).
The combination of AZD5363 and ENZ was assessed at different disease time points using the LNCaP xenograft model (Supplementary Fig. 5a). Combination therapy for CRPC resulted in significant regression of tumours without regrowth, as well as a significant and sustained decline in PSA level compared to either monotherapy ( Fig. 2 D; Supplementary Fig. 5b and 5c). Immunohistochemical staining for markers of proliferation and downstream signalling corroborated these results ( Fig. 2 E; Supplementary Fig. 6).
In a separate in vivo study of the combination AZD5363 plus ENZ given at time of castration, tumour volume dropped similarly and PSA levels dropped to a relatively greater extent compared to the prior study ( Fig. 2 F and 2G). The mean PSA nadir was lowered to a greater extent with combination therapy given at castration relative to vehicle (51-fold difference;p = 0.064) compared to AZD5363 plus ENZ given at time of CPRC relative to ENZ treatment (12-fold difference;p = 0.065) among mice with ≥4 wk of treatment. Drugs were well tolerated in both in vivo studies (Supplementary Fig. 6d–6f).
While ENZ improves survival and quality of life in CRPC patients  , resistance invariably develops and thus there is a need for strategies to delay resistance. Resistance to ENZ may emerge through multiple mechanisms that allow for continued AR pathway signalling, such as AR mutations, splice variants, and glucocorticoid receptor signalling. Our study suggests that early cotargeting of the Akt pathway in combination with potent AR antagonists can delay the onset of ENZ-resistance.
Our prior work has demonstrated that AZD5363 synergizes with bicalutamide  . Using the more potent AR antagonist ENZ, we show that combined targeting with AZD5363 can delay ENZ resistance through mechanisms of cell cycle arrest and induction of apoptosis. The benefit of earlier combination treatment observed in our study mirrors recently reported results of combination docetaxel and androgen deprivation in hormone-sensitive metastatic disease  . More research is needed to understand which targeted inhibitor of the PI3K/Akt/mTOR pathway is optimally paired with AR pathway inhibitors, but our results support the concept that upstream Akt inhibition may be more efficacious than targeting downstream mTOR by avoiding feedback loops that occur with mTOR inhibition alone.
Another question in the use of PI3K/Akt/mTOR inhibitors surrounds patient selection. Interestingly, the combination demonstrated synergy in the phosphatase and tensin homolog (PTEN) wild-type 22RV1 cells, suggesting that while the response to PI3K/Akt/mTOR inhibition is dependent on activation of the pathway (eg, by PTEN loss), the synergy of AR and Akt pathway inhibitors may not be.
In conclusion, our preclinical studies in resistant PCa models demonstrate an impressive response in delaying and treating ENZ-resistant disease using the novel Akt inhibitor AZD5363 with ENZ. These results provide a strong rationale for further evaluation of this combination in the clinic.
Author contributions:Amina Zoubeidi had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study concept and design:Zoubeidi, Gleave, Toren.
Acquisition of data:Toren, Kim, Cordonnier, Fazli.
Analysis and interpretation of data:Zoubeidi, Toren.
Drafting of the manuscript:Zoubeidi, Toren.
Critical revision of the manuscript for important intellectual content:Zoubeidi, Davies, Crafter, Toren.
Statistical analysis:Toren, Fazli.
Obtaining funding:Zoubeid, Gleave.
Administrative, technical, or material support:Gleave, Zoubeidi, Fazli.
Financial disclosures:Amina Zoubeidi certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: C. Crafter and B. Davies are employees of AstraZeneca. M. Gleave and A. Zoubeidi receive research funding from AstraZeneca. M. Gleave is a consultant for AstraZeneca.
Funding/Support and role of the sponsor:AstraZeneca, Prostate Cancer Canada, and the Canadian Institutes of Health Research sponsored this research and were involved in the design and conduct of the study and approval of the manuscript.
-  R.L. Bitting, A.J. Armstrong. Targeting the PI3K/Akt/mTOR pathway in castration-resistant prostate cancer. Endocr Relat Cancer. 2013;20:R83-R99 Crossref
-  B.S. Carver, C. Chapinski, J. Wongvipat, et al. Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer. Cancer Cell. 2011;19:575-586 Crossref
-  D.J. Mulholland, L.M. Tran, Y. Li, et al. Cell autonomous role of PTEN in regulating castration-resistant prostate cancer growth. Cancer Cell. 2011;19:792-804 Crossref
-  K.G. Chee, J. Longmate, D.I. Quinn, et al. The AKT inhibitor perifosine in biochemically recurrent prostate cancer: a phase II California/Pittsburgh cancer consortium trial. Clin Genitourin Cancer. 2007;5:433-437 Crossref
-  B.R. Davies, H. Greenwood, P. Dudley, et al. Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background. Mol Cancer Ther. 2012;11:873-887 Crossref
-  H. Kuruma, H. Matsumoto, M. Shiota, et al. A novel antiandrogen, compound 30, suppresses castration-resistant and MDV3100-resistant prostate cancer growth in vitro and in vivo. Mol Cancer Ther. 2013;12:567-576 Crossref
-  M. Korpal, J.M. Korn, X. Gao, et al. An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide). Cancer Discov. 2013;3:1030-1043 Crossref
-  H.I. Scher, K. Fizazi, F. Saad, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2012;367:1187-1197
-  C. Thomas, F. Lamoureux, C. Crafter, et al. Synergistic targeting of PI3K/AKT pathway and androgen receptor axis significantly delays castration-resistant prostate cancer progression in vivo. Mol Cancer Ther. 2013;12:2342-2355 Crossref
-  C. Sweeney, Y. Chen, M. Carducci, et al. Impact on overall survival (OS) with chemohormonal therapy versus hormonal therapy for hormone-sensitive newly metastatic prostate cancer (mPrCa): an ECOG-led phase III randomized trial [abstract LBA2]. J Clin Oncol. 2014;:32
© 2014 European Association of Urology, Published by Elsevier B.V.