Evaluation of in vivo responses of sorafenib therapy in a preclinical mouse model of PTEN-deficient of prostate cancer
© Yamamoto et al.; licensee BioMed Central. 2015
Received: 7 January 2015
Accepted: 28 April 2015
Published: 8 May 2015
Despite recent advances in the treatment for advanced prostate cancer, outcomes remain poor. This lack of efficacy has prompted the development of alternative treatment strategies. In the present study we investigate the effects of the multikinase inhibitor sorafenib in a genetically engineered mouse model of prostate cancer and explore the rational combination with the mTOR inhibitor everolimus.
Conditional prostate specific PTEN-deficient knockout mice were utilized to determine the pharmacodynamic and chemopreventive effects of sorafenib. This mouse model was also used to examine the therapeutic efficacy of sorafenib alone or in combination with everolimus. Preclinical efficacy was assessed by comparing the reduction of tumor burden, proliferation, angiogenesis and the induction of apoptosis. Molecular responses were assessed by immunohistochemical, TUNEL and western blot assays.
Pharmacodynamic analysis revealed that a single dose of sorafenib decreased activation of the PI3K/AKT/mTOR signaling axis at doses of 30–60 mg/kg, but activated JAK/STAT3 signaling. Levels of cleaved casapase-3 increased in a dose dependent manner. Chemoprevention studies showed that chronic sorafenib administration was capable of inhibiting tumor progression through the reduction of cancer cell proliferation, angiogenesis and the induction of apoptosis. In intervention models of established castration-naïve and castration-resistant prostate cancer, treatment with sorafenib provided modest but statistically insignificant reduction in tumor burden. However, sorafenib significantly inhibited cancer cell proliferation and MVD but had minimal effects on the induction of apoptosis. Interestingly, the administration of sorafenib increased the expression levels of the androgen receptor, p-GSK3β and p-ERK1/2 in castration-resistant prostate cancers. In both intervention models, combination therapy demonstrated a clear tendency of enhanced antitumor effects over monotherapy. Notably, the treatment combination of sorafenib and everolimus overcame therapeutic escape from single agent therapy in castration-resistant prostate cancers.
In summary, we provide insights into the molecular responses of sorafenib therapy in a clinically relevant model of prostate cancer and present preclinical evidence for the development of targeted treatment strategies based on the use of multikinase inhibitors in combination with mTOR inhibitors for the treatment of advanced prostate cancer.
Strides have been made for the treatment of localized prostate cancer, including surgery and radiotherapy, however, androgen deprivation therapy (ADT) remains the standard treatment of advanced prostate cancer. Even though tumors initially respond to ADT, most patients invariably develop lethal castration-resistant prostate cancer (CRPC) within only a few years after the initiation of ADT [1-3]. Several new agents such as docetaxel, cabazitaxel, abiraterone acetate, enzalutamide and sipuleucel-T have shown some clinical improvements for CRPC patients, however, responses are modest and the median increase in survival remains poor [4-8]. Thus, novel treatment strategies that could prevent disease progression in CRPC are needed. Recently, a number of small molecules targeting kinases have been developed and have been or are undergoing clinical evaluation. Kinases have the potential to be effective targets for anticancer therapy since these modulate a number of signal transduction cascades. Moreover, many of these kinases play roles in modulating transformed malignant cells as well as non-malignant cells in the tumor microenvironment.
Sorafenib is an oral multi-targeted kinase inhibitor that has demonstrated the ability to suppress Raf kinases as well as a number of receptor tyrosine kinases (RTK) implicated in tumor progression and angiogenesis . Sorafenib has been shown to act directly on human prostate cancer cells decreasing cellular proliferation and inducing apoptosis through the downregulation of AKT and androgen receptor pathways . Moreover sorafenib has been shown to inhibit the activity of full length AR and AR lacking the AR-ligand binding domain in CRPC cells . However, neither sorafenib nor sunitinib, another multi-targeted RTK inhibitor, have lived up to their promise as single agent therapy having shown only modest clinical benefits in human CRPC [12-17]. This lack of efficacy suggests that other pathways are involved in the survival and growth of tumors. In addition, treatment with sorafenib can lead to the activation of c-Met and mTOR in some cell types .
The PI3K/AKT/mTOR pathway plays a crucial role in the development of prostate cancer and transformation to CRPC. In metastatic prostate cancer, alterations of the PI3K/AKT/mTOR pathway have been reported in 100% of cases . These alterations occur as a result of AKT activation due to loss of function of the tumor suppressor phosphatase and tensin homolog (PTEN) . As a result, a great deal of interest has been focused in developing therapeutic agents targeting the PI3K/AKT/mTOR signaling axis. Due to their promising results in preclinical models, several rapalogs, including everolimus, have been tested in the clinical setting but have met disappointing outcomes [17,21-23]. Recent studies have shown that in certain cancer cell types, combining sorafenib with mTOR inhibitors can overcome therapeutic escape [18,24-29].
In this study, we utilize a genetically engineered mouse model of PTEN-deficient prostate cancer to characterize the effects of sorafenib on castration-naïve and castration-resistant prostate tumors. We also utilize this mouse model to explore the efficacy of combination therapy with sorafenib and the mTOR inhibitor everolimus.
Material and methods
Reagents and antibodies
Sorafenib tysolate and everolimus were purchased form L.C. Laboratories (Woburn, MA). For all in vivo studies, sorafenib and everolimus were prepared as previously described [9,30]. Antibodies for western blot and immunohistochemical analysis were purchased as follows: β-catenin (#8480), Bcl-2 (#2870), BIM (#2933), cleaved caspase-3 (#9661), ERK (#9102), GAPDH (#2118), mTOR (#2983), p-β-catenin (#9561), p-ERK (#4370), p-mTOR (#2971), p-S6 (#2211), p-STAT3-pY705 (#9145), p-STAT3-pY727 (#9134), S6 (#2217) and STAT3 (#4904) antibodies from Cell Signaling Technology (Danvers, MA); AR (#RB-9030), PCNA (#RB-9055) and Ki67 (#RB-9043) antibodies from Thermo Scientific (Waltham, MA); cyclin B1 (sc-752), cyclin D1 (sc-753) antibodies from Santa Cruz Biotechnology (Dallas, TX); CD31 (#550300) from BD Biosciences (San Jose, CA).
Homozygous PTEN-conditional knockout (PSA Cre /PTEN Flox/Flox ) mice were used for this study. The characterization of this animal model has been described previously . Briefly, we used the PSA-Cre promoter to mediate Cre-lox recombination of exons 4 and 5 (exon 5 encodes the crucial phosphatase core motif). This results in the prostate-specific inactivation of PTEN in the dorsolateral and ventral lobes of the prostate. In homozygous mutants, PSA-Cre driven inactivation of PTEN leads to the development of mouse low grade prostatic intraepithelial neoplasia (lgPIN) at 8 weeks of age which progresses to mouse high grade PIN (hgPIN, a precursor to invasive adenocarcinoma) by 15 weeks of age. To induce CRPC, homozygous PTEN-conditional knockout mice were anesthetized and surgically castrated at 10–12 weeks of age. All experiments were approved by the Institutional Review Committee at Kinki University Faculty of Medicine. Mice were maintained in accordance with institutional guidelines and procedures were carried out in compliance with the standards for use of laboratory animals.
Pharmacodynamics effects of sorafenib were performed on 20-week-old PTEN-mutant mice harboring castration-naïve prostate cancer (CNPC). Sorafenib was administered as a single dose and mice were sacrificed after 24 h. Tumor samples were dissected and processed for western blot analysis.
Drug chemoprevention studies
Chemoprevention studies were performed on six-week-old PTEN-mutant mice. Sorafenib was administered orally by gavage (30 mg/kg, 3X/week) beginning at six weeks of age and was continued until the animals were 15 weeks or 20 weeks of age, at which time the experiment was terminated. The animals were sacrificed and the genitourinary tracts (GUT) were collected en bloc, weighed, imaged, and processed for histopathological, immunohistochemical (IHC), and western blot analysis.
Drug intervention studies
Drug efficacy was determined by performing drug intervention studies on 16-week-old PTEN-mutant mice harboring CNPC or CRPC. To induce CRPC, mice were surgically castrated at 10 weeks of age as previously described . Mice were randomized and assigned to treatment groups consisting of control (equal volume of vehicle, 5X/week), sorafenib (gavage, 30 mg/kg, 5X/week), everolimus (oral gavage, 10 mg/kg, 3X/week) or combination sorafenib (gavage, 30 mg/kg, 5X/week) and everolimus (gavage, 10 mg/kg, 3X/week) for 4 weeks. Mice were sacrificed and the GUTs were removed en bloc, weighed, imaged and processed for histopathological, IHC, TUNEL and western blot analysis.
Tumor burden and efficacy determination
Tumor burden was determined by fresh GUT weight or prostate surface area. The surface area of prostate, containing tumor tissue was determined using gross images of fresh GUTs as previously described . Digital images were captured using a Nikon Coolpix 995 digital camera attached to an Olympus SX61 stereomicroscope. The images were spatially calibrated and area measurements were obtained using ImageJ image analysis software . Drug antitumor efficacy was determined by differences in GUTs weight or prostate surface area.
Histology and immunohistochemical analysis
GUTs were fixed overnight in 10% neutral buffered formalin. Tissues were processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin using standard methods. IHC analysis was performed according to the standard protocols described previously . Assessment of staining was performed visually or using ImageJ analysis software with the Landini color deconvolution plugin V1.5 .
Distribution analysis of tumor stroma and epithelial cells compartments was performed on photomicrographs of H&E stained cross sections of the GUT taken at 40X magnification and digitally stitched with Photoshop CS5 Extended (Adobe Systems Inc. San Jose CA). Images were spatially calibrated and digital image masks corresponding to dorsolateral and ventral prostate lobes were painted on. In addition, separate masks corresponding to stroma, normal acini, lgPIN and hgPIN within the dorsolateral and ventral prostates were also generated (Additional file 1: Figure S1). Area measurements corresponding to each individual mask were recorded by the software and exported to a spreadsheet for further statistical analysis.
Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling assay
Terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling (TUNEL) assays were determined using the In Situ Cell Death Detection Kit (Roche Diagnostics Corporation, Indianapolis, IN) according to methods previously described .
Western blot analysis
Protein extraction and immunoblotting were performed as previously described . Semi-quantitative densitometric analyses were assessed using ImageJ analysis software. For all densitometric analyses, protein levels were normalized to GAPDH or total protein.
Data were reported as mean values ± standard error and were statistically analyzed using the Student’s t-test for paired analysis and one-way ANOVA for multiple comparisons. P < 0.05 was considered statistically significant. Statistical analysis was carried out using Sigmaplot v.13.0 (Systat Software, Inc. San Jose, CA).
Pharmacodynamic effects of sorafenib
Sorafenib suppresses PTEN-deficient tumor progression
To further assess the effects on tumor progression after treatment with sorafenib, we performed histological analysis of the tissue distribution patterns of stromal and glandular tissue within the prostate tumors. This analysis measures differences in tumor development and progression. Control mice showed an increase in the prostate area, consisting of the dorsolateral and ventral prostates between 15 and 20 weeks (Figure 2C). In addition, increases in the stromal and epithelial compartments were also observed. Significant differences between the absolute area measurements of normal and cancerous (hgPIN) glands were observed, but these did not reflect changes in the relative proportions of transformed glands (Figure 2D-E). Notably, mice treated with sorafenib exhibited decreases in the absolute area measurements of the whole prostate, epithelial and stromal compartments, and normal and cancerous glands (Figure 2D). Moreover, marked differences were observed in the relative proportions of the distribution of PIN and cancerous glands in the prostates of control and sorafenib-treated mice at 20 weeks.
Therapeutic intervention with sorafenib reduces PCa growth and reverses resistance to everolimus in CRPC
IHC analysis of Ki67 expression revealed significant suppression of tumor proliferation rates in all mice receiving drug treatments in the CNPC intervention model. Tumor cell proliferation was more strongly inhibited in mice receiving sorafenib compared to everolimus; still, the combination of the two drugs did not augment the antiproliferative effect (Figure 4D). In the CRPC intervention model, only modest reductions in tumor proliferation rates were observed in mice receiving monotherapy with either sorafenib or everolimus, however, combined therapy synergized to enhance the inhibition of tumor cell proliferation (Figure 4D). The apoptotic rate of CNPC tumors treated with combination therapy was significantly greater than that after monotherapy with either sorafenib or everolimus (Figure 4E). In the CRPC model, combination therapy did not result in improved apoptotic rates compared to monotherapy (Figure 4E).
Molecular characterization of major signal pathways in response to combination therapy with sorafenib and everolimus
As expected, levels of AKT phosphorylation increased in response to treatment with everolimus. However, responses to sorafenib varied between individual animals in both CNPC and CRPC intervention models. Interestingly, combination therapy reduced p-AKT levels in the CNPC treatment group, but levels rose in the CRPC group. Notably, phosphorylation of GSK3β in CRPC decreased mice despite elevated levels of phosphorylated AKT suggesting that the combination of so sorafenib and everolimus might cooperate to suppress GSK3 β activity in CRPC. For the most part, other AKT substrates showed similar tendencies of phosphorylation as p-AKT. Treatment with sorafenib alone reduced the levels of Erk1/2 phosphorylation and the effect was further enhanced by the co-administration of everolimus in CNPC (Figure 6A,B). In sharp contrast, mice with CRPC showed a ~2-fold increase in the levels of Erk1/2 phosphorylation after treatment with sorafenib. Relative levels of p-Erk1/2 did not vary with the administration of everolimus. In fact, adding everolimus to sorafenib contributed to reducing the upsurge of Erk1/2 phosphorylation (Figure 6A,C).
We examined the activation of the JAK/STAT3 pathway by analyzing the transcription activation of STAT3 though its phosphorylation at tyrosine 705 (pY705) and serine 727 (pY727) residues. In the CNPC intervention model, levels of constitutively active STAT3-pY705 decreased in response to treatment with sorafenib or everolimus, and combination therapy with both agents markedly reduced transcriptional activity. Overall, the relative levels of STAT3-pY727 phosphorylation were unchanged with monotherapy but were notably reduced with combination therapy (Figure 6A,B). In the CRPC intervention model, treatment with sorafenib did not affect phosphorylation of STAT3-pY705, however, treatment with everolimus alone or in combination with sorafenib tended to increase the expression levels of STAT3-pY705 (Figure 6A,C). Levels of STAT3-pY727 increased in response to treatment with sorafenib. However, this finding was not unexpected since phosphorylation of the serine residue is regulated by the MAPK pathway and the expression pattern of p-STAT3-pY727 paralleled that of p-Erk1/2 (Figure 6A,C).
Prostate tumors are complex structures made up of normal and aberrant epithelial cells that are surrounded by a stromal component composed of fibroblast, endothelial and inflammatory cell populations. This heterogeneous cell mixture provides the structural and metabolic support that allows cancer cells to survive and progress into aggressive phenotypes. The tumor’s stromal vascular network plays a particularly important role since it co-evolves along neoplastic and structural cells. This vascular network, composed of neoangiogenic microvessel, functions in the transport of oxygen, essential nutrients and growth factors to all cell populations within the tumor. In addition, these vascular networks facilitate the physical recruitment of inflammatory cells and the dissemination of metastatic cancer cells. As a result, tumor vasculature has become an important therapeutic target for molecular targeting agents . In this study, we have used a genetically engineered mouse model of PTEN-deficient prostate cancer to demonstrate that targeting both dysplastic epithelial cells and the stromal vascular network with sorafenib suppresses tumor growth. Furthermore, we show differences in sorafenib-induced patterns of molecular response between CNPC and CRPC. We also provide preclinical evidence for enhanced antitumor activity of sorafenib when co-administered with everolimus in CRPC.
The mouse model used in this study uses the human PSA promoter to drive the conditional inactivation of PTEN in the luminal cells of the adult prostate gland . In this manner, normal PTEN expression and function is maintained in other tissue. Inactivation of PTEN results in the stage-specific development of prostate cancer that recapitulates many of the features associated with the human disease. Tumors in these mice arise from normal tissues, but follow a multistage process of disease progression. In essence, this provides an excellent window of opportunity to evaluate the chemopreventive effects of potential anticancer agents by evaluating differences in the rates of disease progression. In the present study, pharmacological administration of sorafenib did not alter the onset of cancer, nevertheless, it did suppress tumor growth and progression. The antitumor effects of sorafenib correlated to the inhibition of cell proliferation and the induction of apoptosis in epithelial cancer cells as well as the reduction of MVD and reactive stroma in non-epithelial tumor cells.
The therapeutic effects of sorafenib were also investigated on established CNPC and CRPC. In both intervention models, treatment with sorafenib significantly inhibited cancer cell proliferation and MVD. However, the administration of sorafenib yielded only modest, statistically insignificant therapeutic responses. Interestingly, it was only in the CNPC intervention model that sorafenib induced apoptosis. It is also important to note that sorafenib inhibited p-Erk1/2 in CNPC. Of note, the treatment combination of sorafenib and everolimus inhibited phosphorylation of GSK3β in CRPC mice despite elevated levels of phosphorylated AKT. Nevertheless, phosphorylation of β-catenin increased suggesting that the combination of so sorafenib and everolimus might cooperate to suppress GSK3b activity in CRPC. However, sorafenib elicited the upregulation of phosphorylated Erk1/2 and STAT3-pY727 in CRPC. The activation of MAPK and STAT3 signal transduction pathways has been associated with the abrogation of apoptosis [35,36]. Typically, activation of STAT3 occurs by cytokine mediated JAK phosphorylation of tyrosine 705, however, a second phosphorylation site exists at serine 727 and is phosphorylated through MEK and its transient or constitutive activation in cancer cells has been associated with survival [37,38]. This effect could very well explain why we did not see the induction of apoptosis in CRPC mice treated with sorafenib.
Previous reports have suggested that sorafenib could inhibit prostate cancer cell survival by decreasing proliferation and inducing apoptosis through the downregulating of AR. However, in our study, treatment with sorafenib failed to downregulate AR expression in vivo. In fact, treatment with sorafenib increased AR expression in CRPC. This effect could be attributed to the increased levels of MAPK and STAT3 pathway activation resulting from sorafenib administration especially since both MAPK and STAT3 signal pathways are associated with the ligand independent activation of AR in CRPC [39,40]. Still, the clinical efficacy of sorafenib monotherapy for advance prostate cancer has been limited despite favorable outcomes in other cancer types such as kidney and liver [41,42]. We believe that the findings from our study may shed some light for the shortcomings of sorafenib monotherapy in treating human CRPC.
Recent reports have shown that the treatment combination of sorafenib and mTOR inhibitors result in a greater antitumor effect. [24,26-28]. Therefore, we hypothesized that by using the combination of sorafenib and everolimus, we could target the tumor microenvironment as well as direct cancer cell survival and compensatory pathways that could possibly restore treatment responses of CRPC. In both CNPC and CRPC intervention models, combination therapy demonstrated a clear tendency of improved tumor suppression. More importantly, the treatment combination of sorafenib and everolimus overcame therapeutic escape from single agent therapy in CRPC.
In summary, we have utilized a genetically engineered mouse model of prostate cancer to demonstrate differential effects of treatment with sorafenib in PTEN-deficient CNCP and CRPC and provide mechanistic insights into the molecular responses. Finally, we provide preclinical evidence for the development of targeted treatment strategies based on the use of multikinase inhibitors, such as sorafenib, in combination with mTOR inhibitors for the treatment of advanced prostate cancer.
Funding for this study was in part provided by a grant from the Japan Society for the Promotion of Science, Grant-in-Aid for Scientific Research (C) number 22591781 to MADV.
- Petrylak DP. New paradigms for advanced prostate cancer. Rev Urol. 2007;9 Suppl 2:S3–S12.PubMed CentralPubMedGoogle Scholar
- Berry WR. The evolving role of chemotherapy in androgen-independent (hormone-refractory) prostate cancer. Urology. 2005;65(Suppl6):2–7.View ArticlePubMedGoogle Scholar
- Tran C, Ouk S, Clegg NJ, Chen Y, Watson PA, Tran C, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. 2009;324:787–90.View ArticlePubMed CentralPubMedGoogle Scholar
- Berthold DR, Pond GR, Soban F, de Wit R, Eisenberger M, Tannock IF. Docetaxel plus prednisone or mitoxantrone plus prednisone for advanced prostate cancer: updated survival in the TAX 327 study. J Clin Oncol. 2008;26:242–5.View ArticlePubMedGoogle Scholar
- De Bono JS, Oudard S, Ozguroglu M, Hansen S, Machiels JP, Kocak I, et al. TROPIC Investigators: Prednisone plus cabazitaxel or mitoxantrone for metastatic castration-resistant prostate cancer progression after docetaxel treatment: a randomised open-label trial. Lancet. 2010;376:1147–54.View ArticlePubMedGoogle Scholar
- De Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, et al. COU-AA-301 Investigators: Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011;364:1995–2005.View ArticlePubMed CentralPubMedGoogle Scholar
- Scher HI, Fizazi K, Saad F, Taplin ME, Sternberg CN, Miller K, et al. AFFIRM Investigators: Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med. 2011;367:1187–97.Google Scholar
- Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, et al. IMPACT Study Investigators: Sipuleucel-T immunotherapy for castration-resistant prostate cancer. N Engl J Med. 2010;363:411–22.View ArticlePubMedGoogle Scholar
- Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, et al. BAY 43–9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res. 2004;64:7099–109.View ArticlePubMedGoogle Scholar
- Oh SJ, Erb HH, Hobisch A, Santer FR, Culig Z. Sorafenib decreases proliferation and induces apoptosis of prostate cancer cells by inhibition of the androgen receptor and Akt signaling pathways. Endocr Relat Cancer. 2012;9:305–19.View ArticleGoogle Scholar
- Zengerling F, Streicher W, Schrader AJ, Schrader M, Nitzsche B, Cronauer MV, et al. Effects of sorafenib on C-terminally truncated androgen receptor variants in human prostate cancer cells. Int J Mol Sci. 2012;13:11530–42.View ArticlePubMed CentralPubMedGoogle Scholar
- Chi KN, Ellard SL, Hotte SJ, Czaykowski P, Moore M, Ruether JD, et al. A phase II study of sorafenib in patients with chemo-naive castration-resistant prostate cancer. Ann Oncol. 2008;19:746–51.View ArticlePubMedGoogle Scholar
- Aragon-Ching JB, Jain L, Gulley JL, Arlen PM, Wright JJ, Steinberg SM, et al. Final analysis of a phase II trial using sorafenib for metastatic castration-resistant prostate cancer. BJU Int. 2009;103:1636–40.View ArticlePubMed CentralPubMedGoogle Scholar
- Michaelson MD, Oudard S, Ou YC, Sengeløv L, Saad F, Houede N, et al. Randomized, placebo-controlled, phase III trial of sunitinib plus prednisone versus prednisone alone in progressive, metastatic, castration-resistant prostate cancer. J Clin Oncol. 2014;32:76–82.View ArticlePubMedGoogle Scholar
- Amato RJ, Jac J, Mohammad T, Saxena S. Pilot study of rapamycin in patients with hormone-refractory prostate cancer. Clin Genitourin Cancer. 2008;6:97–102.View ArticlePubMedGoogle Scholar
- Sarker D, Reid AH, Yap TA, de Bono JS. Targeting the PI3K/AKT pathway for the treatment of prostate cancer. Clin Cancer Res. 2009;15:4799–805.View ArticlePubMedGoogle Scholar
- Templeton AJ, Dutoit V, Cathomas R, Rothermundt C, Bärtschi D, Dröge C, et al. Swiss Group for Clinical Cancer Research (SAKK): Phase 2 trial of single-agent everolimus in chemotherapy-naive patients with castration-resistant prostate cancer (SAKK 08/08). Eur Urol. 2013;64:150–8.View ArticlePubMedGoogle Scholar
- Huynh H, Ngo VC, Koong HN, Poon D, Choo SP, Thng CH, et al. Sorafenib and rapamycin induce growth suppression in mouse models of hepatocellular carcinoma. J Cell Mol Med. 2009;13:2673–83.View ArticlePubMedGoogle Scholar
- Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. Integrative genomic profiling of human prostate cancer. Cancer Cell. 2010;18:11–22.View ArticlePubMed CentralPubMedGoogle Scholar
- Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998;273:13375–8.View ArticlePubMedGoogle Scholar
- Bitting RL, Armstrong AJ. Targeting the PI3K/Akt/mTOR pathway in castration-resistant prostate cancer. Endocr Relat Cancer. 2013;20:R83–99.View ArticlePubMedGoogle Scholar
- Armstrong AJ, Netto GJ, Rudek MA, Halabi S, Wood DP, Creel PA, et al. A pharmacodynamic study of rapamycin in men with intermediate- to high-risk localized prostate cancer. Clin Cancer Res. 2010;16:3057–66.View ArticlePubMed CentralPubMedGoogle Scholar
- Nakabayashi M, Werner L, Courtney KD, Buckle G, Oh WK, Bubley GJ, et al. Phase II trial of RAD001 and bicalutamide for castration-resistant prostate cancer. BJU Int. 2012;110:1729–35.View ArticlePubMedGoogle Scholar
- Newell P, Toffanin S, Villanueva A, Chiang DY, Minguez B, Cabellos L, et al. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol. 2009;51:725–33.View ArticlePubMed CentralPubMedGoogle Scholar
- Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, et al. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and PI3K/AKT/mTOR pathways. Anticancer Res. 2010;30:4951–8.PubMed CentralPubMedGoogle Scholar
- Piguet AC, Saar B, Hlushchuk R, St-Pierre MV, McSheehy PM, Radojevic V, et al. Everolimus augments the effects of sorafenib in a syngeneic orthotopic model of hepatocellular carcinoma. Mol Cancer Ther. 2011;10:1007–17.View ArticlePubMedGoogle Scholar
- Mariniello B, Rosato A, Zuccolotto G, Rubin B, Cicala MV, Finco I, et al. Combination of sorafenib and everolimus impacts therapeutically on adrenocortical tumor models. Endocr Relat Cancer. 2012;19:527–39.View ArticlePubMedGoogle Scholar
- Pawaskar DK, Straubinger RM, Fetterly GJ, Hylander BH, Repasky EA, Ma WW, et al. Synergistic interactions between sorafenib and everolimus in pancreatic cancer xenografts in mice. Cancer Chemother Pharmacol. 2013;71:1231–40.View ArticlePubMed CentralPubMedGoogle Scholar
- Pignochino Y, Dell’Aglio C, Basiricò M, Capozzi F, Soster M, Marchiò S, et al. The Combination of Sorafenib and Everolimus Abrogates mTORC1 and mTORC2 upregulation in osteosarcoma preclinical models. Clin Cancer Res. 2013;19:2117–31.View ArticlePubMedGoogle Scholar
- De Velasco MA, Tanaka M, Yamamoto Y, Hatanaka Y, Koike H, Nishio K, et al. Androgen deprivation induces phenotypic plasticity and promotes resistance to molecular targeted therapy in a PTEN-deficient mouse model of prostate cancer. Carcinogenesis. 2014;35:2142–53.View ArticlePubMed CentralPubMedGoogle Scholar
- Schneider CA, Rasband WS, Eliceiri KW. NIH Image to ImageJ: 25 years of image analysis. Nature Methods. 2012;9:671–5.View ArticlePubMedGoogle Scholar
- Tomioka A, Tanaka M, De Velasco MA, Anai S, Takada S, Kushibiki T, et al. Delivery of PTEN via a novel gene microcapsule sensitizes prostate cancer cells to irradiation. Mol Cancer Ther. 2008;7:1864–70.View ArticlePubMedGoogle Scholar
- Ruifrok AC, Johnston DA. Quantification of histochemical staining by color deconvolution. Anal Quant Cytol Histol. 2001;23(4):291–9.PubMedGoogle Scholar
- Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med. 2011;17:1359–70.View ArticlePubMedGoogle Scholar
- Lu Z, Xu S. ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life. 2006;58:621–31.View ArticlePubMedGoogle Scholar
- Grandis JR, Drenning SD, Zeng Q, Watkins SC, Melhem MF, Endo S, et al. Constitutive activation of Stat3 signaling abrogates apoptosis in squamous cell carcinogenesis in vivo. Proc Natl Acad Sci U S A. 2000;97:4227–32.View ArticlePubMed CentralPubMedGoogle Scholar
- Sakaguchi M, Oka M, Iwasaki T, Fukami Y, Nishigori C. Role and regulation of STAT3 phosphorylation at Ser727 in melanocytes and melanoma cells. J Invest Dermatol. 2012;132:1877–85.View ArticlePubMedGoogle Scholar
- Yu H, Lee H, Herrmann A, Buettner R, Jove R. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat Rev Cancer. 2014;14:736–46.View ArticlePubMedGoogle Scholar
- Abreu-Martin MT, Chari A, Palladino AA, Craft NA, Sawyers CL. Mitogen-activated protein kinase kinase kinase 1 activates androgen receptor-dependent transcription and apoptosis in prostate cancer. Mol Cell Biol. 1999;19:5143–54.PubMed CentralPubMedGoogle Scholar
- Yang L, Wang L, Lin HK, Kan PY, Xie S, Tsai MY, et al. Interleukin-6 differentially regulates androgen receptor transactivation via PI3K-Akt, STAT3, and MAPK, three distinct signal pathways in prostate cancer cells. Biochem Biophys Res Commun. 2003;305:462–9.View ArticlePubMedGoogle Scholar
- Escudier B, Eisen T, Stadler WM, Szczylik C, Oudard S, Siebels M, et al. TARGET Study Group: Sorafenib in advanced clear-cell renal-cell carcinoma. N Engl J Med. 2007;356:125–34.View ArticlePubMedGoogle Scholar
- Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. SHARP Investigators Study Group: Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008;359:378–90.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.