The mammalian target of rapamycin (mTOR) has proven to be a valid therapeutic target in a number of human cancers, and it is a candidate for clinical trials in human breast cancer. We report on a biomarker-based translational medicine approach to assess the efficacy and mechanism of action for the mTOR inhibitor temsirolimus (CCI-779) in a mammary carcinoma OncoMouse model [polyomavirus middle T antigen (PyMT)]. The mTOR signaling pathway biomarkers were assessed using a reverse-phase protein array. Pharmacokinetics studies were conducted in both the tumor and plasma compartments. Pharmacodynamic biomarkers for compound-target engagement of tumor phospho-S6 proteins were assayed by Western blot. Temsirolimus (intravenously once a week for 2 weeks) was administered in both early and advanced stages of tumors. Biomarkers for temsirolimus effects on tumor progression were assessed by three-dimensional ultrasound imaging in combination with immunohistochemistry to assess vascular density (Texas red-dextran and CD31 immunostaining) and macrophage burden (F4/80 antigen). Tumor growth was significantly arrested in temsirolimus (25 ± 14% from 8 to 10 weeks, p < 0.05, and 26 ± 17% from 11 to 13 weeks, p < 0.01), compared with 493 ± 160 and 376 ± 50% increases, respectively, in vehicle-treated groups. Temsirolimus reduced tumor vascular density, 36 to 48 and 58 to 60%, p < 0.05, by the Texas red-dextran method or CD31-positive vessel count, respectively. Temsirolimus reduced tumor macrophage burden by 46% at 13 weeks (p < 0.05). Temsirolimus inhibited (p < 0.05) the phosphoproteins S6 pS235/236 and S6 pS240/244 up to 81 and 87%, respectively. We conclude that the multimodal biomarkers of temsirolimus efficacy and mechanism of action (phosphoproteins) strongly suggest that it might translate to therapeutic efficacy in human tumors that bear congruency to features present in the mammary carcinoma of PyMT tumors.
The mammalian target of rapamycin (mTOR) is a valid therapeutic target in cancers (Abraham and Eng, 2008). mTOR is a downstream mediator in the phosphatidylinositol-3 kinase (PI3K)/Akt (protein kinase B) signaling pathway, which receives input from multiple signals (including growth factors and nutrients) to regulate protein synthesis through two main pathways, ribosomal S6 kinase (S6K) 1 and 2, and eukaryotic initiation factor 4E-binding protein 1, that play fundamental roles in ribosome biogenesis and cap-dependent translation, respectively (Brown et al., 1995; Brown and Schreiber, 1996). Rapamycin, a bacterially derived natural product, has been shown to be an immunosuppressant and anticancer agent (Sehgal et al., 1994; Huang et al., 2003). Both immunosuppressive and anticancer properties of rapamycin are attributed, at least in part, to its mTOR signaling pathway inhibition by controlling mRNA translation and cell proliferation. CCI-779 (temsirolimus), a synthetic analog of rapamycin, is an inhibitor of mTOR that is marketed for treatment of human renal carcinoma and has shown activity against a wide range of cancers in preclinical models (Geoerger et al., 2001; Neshat et al., 2001; Podsypanina et al., 2001; Yu et al., 2001; Frost et al., 2004; Teachey et al., 2006; Fung et al., 2009), including those of breast cancer xenografts (Yu et al., 2001; Fung et al., 2009). It is noteworthy that although temsirolimus demonstrated positive outcome in breast cancer xenografts, it was more effective for the therapeutics in human prostate cancer xenografts (Fung et al., 2009).
Mammary gland-specific expression of polyomavirus middle T antigen (PyMT) under the control of the mouse mammary tumor virus (MMTV) promoter in transgenic mice (MMTV-PyMT; cited as PyMT mice here) results in the transformation of mammary gland epithelium, leading to the development of multifocal mammary adenocarcinomas and metastasis to lymph nodes and the lung (Guy et al., 1992; Lin et al., 2003). Tumor formation and progression in these mice is characterized by four stages: hyperplasia, adenoma/mammary intraepithelial neoplasia, and early and late carcinoma (Lin et al., 2003). The congruency of this model to some forms of human breast cancer is exemplified by its gradual loss of steroid hormone receptors (estrogen and progesterone) and β1-integrin that is associated with overexpression of ErbB2 (HER2) and cyclin D1 in late-stage metastatic cancer (Maglione et al., 2001; Lin et al., 2003). The PyMT mouse model of breast cancer is further typified by short latency, high penetrance, and a high incidence of lung metastasis in the late stage (Lin et al., 2003). Furthermore, the immune-competent feature of PyMT mice provides advantages for tumor-associated inflammation relative to tumor xenografts. Taken together, these studies suggest that PyMT mice could serve as a useful preclinical model for evaluating the efficacy and mechanism of action of agents against tumor survival pathways such as the mTOR.
Because the role of the mTOR signaling pathway in PyMT-induced mammary tumorigenesis remains unclear, we embarked on an investigation of the mTOR pathway in this model using a multimodal biomarkers approach. We aimed to validate this model by using biomarkers for mTOR signaling pathway activation and probe the efficacy of CCI-779 to modify tumor growth kinetics along with several key tumor microenvironment and growth-promoting elements, including vasculogenesis and inflammation, that might govern tumor progression. The studies were conducted after pilot experiments that defined an optimal drug exposure along with pharmacokinetic and pharmacodynamic assessment. Tumor progression and regression were monitored using noninvasive ultrasound imaging. Tumor vasculogenesis and macrophages were evaluated by quantitative immunohistochemistry (IHC). These biomarker-based studies provide new insights into the mechanism of action and efficacy of rapamycin-related drugs toward PyMT-driven mammary tumors.
Materials and Methods
FVB/N-Tg (MMTV-PyMT)634Mul/J (PyMT) transgenic mice were provided by The Jackson Laboratory (Bar Harbor, ME). Heterozygous female PyMT mice were used for the present study. Genotype was determined by polymerase chain reaction using primers 5′-CAAATGTTGCTTGTCTGGTG-3′ and 5′-GTCAGTCGAGTGCACAGTTT-3′ as forward and reverse primers, respectively, for wild-type gene (200 base pairs) and 5′-GGAAGCAAGTACTTCACAAGGG-3′ and 5′-GGAAAGTCACTAGGAGCAGGG-3′ for PyMT transgenic gene (556 base pairs). Mice were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996). The Institutional Animal Care and Use Committees approved all procedures described in this study.
After pilot study on tumor progression in PyMT mice, using validated ultrasound technology (Zhao et al., 2010), both early (8–10 weeks of age) and advanced (11–13 weeks) tumors in mice were selected for the present study. Pharmacokinetic (PK) studies of CCI-779 and its metabolite rapamycin were conducted in PyMT mice after a single dose of 25 mg/kg i.v. CCI-779. Pharmacodymanic biomarkers, phospho-S6 cluster proteins, were measured by Western blot analysis. Ultrasound was used for noninvasive monitoring of tumor growth (volume) before and after two treatments (once a week) of CCI-779. This study was guided by pilot, dose-dependent (vehicle, 5 and 25 mg/kg i.v. CCI-779, once weekly) and time-dependent (treatment for 1–4 weeks) pharmacological studies in PyMT mice. The doses of CCI-779 at 5 and 25 mg/kg were selected according to previous studies in xenograft models (Yu et al., 2001 and Frost et al., 2004), of which 5 mg/kg was expected to have the least and 25 mg/kg the maximum efficacy without adverse effects. Tumor vascular density was evaluated by two methods: IHC using a fluorescence agent, Texas red-dextran, and an endothelium marker, CD31. Tumor macrophage burden was evaluated by IHC using an F4/80 antibody (for a pan-specific macrophage marker) as described previously (Lin et al., 2003).
Assessment of Tumor Volume by Ultrasound Imaging.
In each mouse, the largest (single) tumor was measured by ultrasound (Vevo 770 high-resolution in vivo micro-imaging system; VisualSonics Inc., Toronto, Canada) according to the manufacturer's specification. Tumor volume was acquired through three-dimensional reconstruction of two-dimensional tumor panels uniformly spaced at defined intervals using the software provided by the manufacturer (Vevo 770 Analytic Software; VisualSonics, Inc.) (Zhao et al., 2010). The correlation between three-dimensional volume and the total tumor burden (by weight at postmortem) was validated in the same mouse.
In Vivo Vessel Labeling and Histological Analysis.
Texas red-conjugated dextran (molecular weight 70,000; Molecular Probes, Carlsbad, CA) was used for in vivo labeling of tumor vasculature as described previously (Lin et al., 2006), using 6.2 mg/ml in phosphate-buffered saline; 21 μg/g of mouse body weight, intravenously injected 5 min before euthanasia. Tumors were isolated and preserved by either freezing or being formalin-fixed and paraffin-embedded, then sectioned for histological assessment of vascular density. For quantitative analysis of vessel distribution in the tumor, the images of the entire midline section of the largest tumor were captured with a Zeiss Axio Imager equipped with an automatic motorized stage (Carl Zeiss, Inc., Thornwood, NY). Texas red-dextran signals were analyzed using the Axio Imager data analysis program (Carl Zeiss, Inc.). The numbers of animals used for the studies are indicated in each figure legend.
Rat monoclonal (CI:A3–1) anti-F4/80 antibody against a pan macrophage marker, BM8 (Abcam Inc., Cambridge, MA) was used to detect tumor macrophages as described previously (Lin et al., 2006), and rat anti-mouse CD31 antibody (BD Biosciences, San Jose, CA) was used to assess the total tumor vasculature. Standard procedure was used for the IHC analysis except that frozen sections were required for CD31 immunostaining using tissues fixed in 5% formalin in 20% sucrose/phosphate-buffered saline solution at 4°C for 24 h. Because of tumor heterogeneity, the photomicrographs of the immunoreactive signals from the entire tumor area were captured using the Zeiss Axio Imager equipped with an automatic motorized stage, and the areas were quantified (as the percentage of the total tumor area) by using the Axio Imager data analysis program. At least three sections from each animal (n = 6) were used to study the effects of CCI-779.
Drug Dosing and PK Analysis.
CCI-779 (Pfizer, Collegeville, PA) was dissolved in a vehicle containing 2% Tween 80, 4% ethanol, and 5% polyethylene glycol 400 in water (as a stock solution for compound preparation) and mixed with 5% dextrose containing 0.25% lactic acid. Vehicle or 5 or 25 mg/kg CCI-779 was dosed intravenously via the tail vein once a week for 2 weeks.
PK study (n = 3) was performed in 11-week-old PyMT mice after a single dose of 25 mg/kg i.v. CCI-779. In our initial PK study, both plasma and tumor samples were collected at time 0, 1, 3, and 7 days after a single dosing. PK study at early time points was also conducted for both plasma and tumor drug concentrations for CCI-779 and its metabolite, rapamycin, at 0, 5, 15, and 30 min and 1, 6, 24 h after a single dose of 25 mg/kg i.v. Drug concentrations in plasma and tumor were determined by liquid chromatography/tandem mass spectrometry. Tumors were homogenized in 1:10 volume of water. The limitation of quantification for mass spectrometry is 1 ng/ml drug using a 50 μl of plasma and/or tumor. The specific extraction and reconstitution procedure included: 1) 50 μl of plasma sample or plasma for standard was loaded in each well of a 96-deep wall plate; 2) 50 μl of water was added to each sample and standard; 3) 10 μl of internal standard (either CCI-779 or rapamycin) was added (for a standard curve at 1–2500 ng/ml) and votexed for 5 min; 4) 400 μl of acetonitrile was added and votexed for 5 min; 5) for separation, samples were centrifuged at 3400 rpm for 10 min at 4°C; 6) 350 μl of supernatant was transferred into a 3M Empore filter plate (3M, St. Paul, MN) using the Tomtec Quadra 96 (Tomtec, Hamden, CA) and dried under nitrogen at 30°C; 7) samples were reconstituted with 200 μl of water/methanol (1:1); and 8) 10 μl of the reconstituted solution was injected into a liquid chromatographer/mass spectrometer for analysis. Chromatographic separation was performed using an Agilent 1200 LC system (Agilent Technologies, Santa Clara, CA), which was equipped with an ESI FlouroSep-RP phenyl HS 50 × 2.1 mm, 5-μ column. The mobile phase consisted of 10 mM ammonium acetate in water (solvent A) and 10 mM ammonium acetate in acetonitrile (solvent B). A gradient with a flow rate of 0.5 ml/min was initiated at 95% solvent A, increased to 95% solvent B at 4.5 min, and changed back to 95% solvent A for another 1.5 min. The mass spectrometry (using Sciex API-4000-Einstein Model ESI+; Applied Biosystems, Foster City, CA) conditions include multiple reaction monitoring, turbo ion-spray voltages at 5000 kV, and temperature at 500°C.
Western Blot Analysis.
Antibodies targeting total S6 and phospho-S6, pS235/236, and pS240/244 proteins and anti-β-actin were purchased from Cell Signaling Technology (Danvers, MA). Tumor samples were collected from PyMT mice treated with CCI-779 or vehicle (n = 3) and immediately frozen in liquid nitrogen. Western blots were conducted on pulverized tumors after porcelain mortar and pestle grinding under liquid nitrogen. Western blotting was used to detect the phosphoproteins and nonphosphoproteins, and the specific band intensity was quantified as described previously (Wang et al., 2003).
mTOR Pathway Analysis.
To validate the activation of mTOR signaling pathway in PyMT mice, the proteins and phosphoproteins, Akt, Akt pS473, Akt pT308, mTOR pS2488, p70S6K, P70S6K pT389, S6, S6 pS235/236, and S6 pS240/244 were analyzed in tumors isolated from 13-week-old mice (n = 5). Tumors were rapidly dissected out and immediately frozen in liquid nitrogen. The pulverized tissues were used for reverse protein array analysis by Bay Point Biosystems Inc. (Houston, TX) as described previously (Grote et al., 2008).
All data are presented as mean ± S.E. Statistical comparisons were made by analysis of variance (with mean differences determined using Fisher's protected least-squares difference) test. Values were considered to be significant when p < 0.05 for CCI-779-treated groups compared with vehicle-treated groups as indicated in figure legends.
mTOR Signaling Pathway Analysis.
Figure 1 illustrates the PI3K/Akt/mTOR signaling pathway (Abraham and Eng, 2008; Le Tourneau et al., 2008) and the histogram data of the selected proteins and phosphoproteins of the mTOR signaling pathway detected by a reverse-phase protein array in the tumors of 13-week old PyMT mice (n = 5). The following proteins/phosphoproteins were evaluated in this study: Akt, Akt pS473, Akt pT308, mTOR pS2448, P70S6K, P70S6K pT389, S6, S6 pS235/236 and S6 pS240/244. Although various levels of expression were observed, every member evaluated within this pathway was detected within the acceptable sensitivity of the methodology applied for the PyMT tumors. The maximum inhibition of S6 pS235/236 and S6 pS240/244 phosphoproteins reached 81 and 87%, respectively, at 1 to 6 h in the tumors after CCI-779 dosing (Fig. 1).
Pharmacokinetics of CCI-779 in PyMT Mice.
PK study was conducted for both CCI-779 and its metabolite, rapamycin, in both plasma and tumors after a single dose of 25 mg/kg i.v. in PyMT mice. Plasma rapamycin levels reached peak immediately after dosing (27,500 ng/ml or 30,085 nM at 5 min) and remained elevated throughout 24 h (690 ng/ml or 755 nM at 24 h; Fig. 2A, top), whereas plasma CCI-779 levels reached peak (10,556 ng/ml or 11,548 nM at 5 min) and significantly diminished (1.7 ng/ml or 2 nM at 24 h). In contrast, concentrations for both compounds in tumors remained substantially higher (47 ng/ml or 51 nM for CCI-779 and 123 ng/ml or 135 nM for rapamycin in the tumors 24 h after dosing; Fig. 2A, top) and could still be detected 7 days after dosing (Fig. 2A, bottom; no data were available for rapamycin up to 7 days). As the result, the ratio of tumor/plasma drug was significantly higher for CCI-779 than rapamycin. Our additional studies showed a similar level of drug exposure in tumors for CCI-779 and rapamycin at 7 days after treatment of CCI-779, 25 mg/kg/week i.v. for 2 weeks (data not shown). A rapid decrease of CCI-779 in plasma in first 24 h (Fig. 1, top) may reflect the biodistribution of this compound and its quick conversion to metabolites (as shown for the sustained plasma levels of rapamycin), whereas a sustained level for a longer time (up to 7 days; Fig. 1, bottom) may reflect the long half-life of the compound and/or continued release from previously retained compartment such as tumors.
Effect of CCI-779 on S6 and Phospho-S6 Protein Expression by Western Blot.
As illustrated in Fig. 3, total S6 protein levels were maintained at a similar level up to 24 h after CCI-779 administration, yet both phospho-S6 S235/236 and S6 S240/244 proteins rapidly declined (starting at 15 min) and reached statistically significance at 60 min after dosing (Fig. 3).
CCI-779 Effect on Tumor Growth.
Significant inhibition of tumor growth progression was observed for both 5 and 25 mg/kg treatment regimens after 1 week and was sustained through the entire course of the study (Fig. 4A). The correlation between the ultrasound assessment and direct tumor weight was determined in 27 animals collected from three experiments. Correlation was established, with coefficient of r2 = 0.80 (Fig. 4B). It was also noted that significant correlation was also established in the subgroup with total tumor burden <2 g (r2 = 0.88, n = 18 for tumor weight <2 g).
Effects of CCI-779 on Imaging Biomarkers for Tumor Growth in Early and Advanced Tumors.
As shown in Fig. 5, CCI-779, 25 mg/kg/week i.v. for 2 weeks, significantly reduced tumor volume (n = 6), with 25 ± 14% increases in animals from weeks 8 to 10 (p < 0.05) and 26 ± 17% decreases from weeks 11 to 13 (p < 0.01) compared with 493 ± 160 and 376 ± 50% increases, respectively, in vehicle-treated groups (Fig. 5).
Effects of CCI-779 on Biomarkers for Tumor Microvasculature in Early and Advanced Tumors.
As shown in Fig. 6, mice (n = 6) treated with CCI-779, 25 mg/kg/week i.v. for 2 weeks, significantly reduced tumor vascular density, with 36 ± 6 and 58 ± 9% decreases (p < 0.05) at 10 and 13 weeks, respectively, based on Texas red-dextran perfusion areas in CCI-779 treated animals over vehicles or 48 ± 5 and 60 ± 17% decreases (p < 0.01) at 10 and 13 weeks, respectively, based on CD31-positive immunostaining vascular area.
Effects of CCI-779 on Biomarkers for Macrophage Infiltration in Early and Advanced Tumors.
Representative immunohistochemical images in mice at 10 and 13 weeks after vehicle or 25 mg/kg/week CCI-779 (intravenously once each week) are illustrated in Fig. 7A. Significant reduction in macrophage burden was observed in tumors from mice treated with CCI-779, 46 ± 19% decrease at 13 weeks compared with vehicle treatment (p < 0.05; n = 6; Fig. 7B). A more moderate reduction in macrophage burden was observed in the early tumors (8–10 weeks; p not significant). There was a significant increase in macrophage burden along with the tumor progression (57.9% increase from 10 to 13 weeks; p < 0.05; n = 7–9), as reflected the change of macrophage staining from 2.4 ± 0.4 and 3.8 ± 0.5% for the early (10 weeks) and late (13 weeks) tumors, respectively, in the vehicle-treated mice. However, there were no changes in macrophage burden in the CCI-779-treated groups, with 1.9 ± 0.3 to 2.0 ± 0.4%, respectively, for the early and late stages of tumors (Fig. 7B).
The studies presented in this article have been designed to address important aspects of preclinical drug transition into clinical development by seeking a more congruent animal model of breast cancer combined with biomarkers that allow more rigorous prediction of efficacy and confirmation of engagement of the compound with its signaling pathway elements by using multimodal biomarker imaging and pharmacological and biochemical approaches. Our data demonstrated for the first time that the potent and selective mTOR inhibitor CCI-779 is highly efficacious in arresting tumor progression in the PyMT mouse model of mammary carcinoma. The relative PK profiles of CCI-779 and rapamycin observed in the present study are in agreement with those observed in patients with breast and/or renal cancers (Raymond et al., 2004; Boni et al., 2005). The antitumor efficacy of CCI-779 was associated with significantly suppressed tumor vascular density and macrophage burden (Figs. 6 and 7), two key microenvironmental elements associated with tumor progression (Leek and Harris, 2002; Furuya and Yonemitsu, 2008; Li et al., 2008; De Palma and Lewis, 2011). Our present study not only confirmed a previous report for the antitumor activity of rapamycin in a transgenic mouse model of ErbB2-dependent human breast cancer (Liu et al., 2005), but also provided novel information on macrophage infiltration and PK/pharmacodynamic correlation of CCI-779.
The precise mechanisms of CCI-779 antitumor progression efficacy in PyMT mice remain to be elucidated. Our current knowledge of mTOR signaling pathway in tumorgenesis suggests several potential mechanisms of CCI-779 in blocking tumor progression. First, CCI-779 and rapamycin are known to be potent inhibitors of the mTOR complex 1, which is known to play a role in controlling cell autonomous growth, in response to diverse extracellular signals, including growth factors and nutrients (Chiang and Abraham, 2007; Le Tourneau et al., 2008; Guertin and Sabatini, 2009). The expression and activation state of several key components of the mTOR signaling pathway was demonstrated in the mammary tumors of PyMT mice (Fig. 1). Thus, one potential mechanism of CCI-779 in suppressing tumor growth in this PyMT model might be associated with its down-regulation of the PI3K/Akt/mTOR pathway. Data provided in this article demonstrated inhibition by CCI-779 of both phospho-S6 proteins in the tumors (Fig. 3), which is also in agreement with a previous report that demonstrated a direct role of CCI-779 in the repression of global protein synthesis (Shor et al., 2008).
Second, mTOR is also known to be downstream of the VEGF receptor signaling pathway, and mTOR inhibitors such as CCI-779 and rapamycin were shown to be antiangiogenic (Heng and Bukowski, 2008; Li et al., 2008). Rapamycin and related mTOR inhibitors inhibit endothelial cell VEGF expression and VEGF-induced endothelial cell proliferation (Dormond et al., 2007). CCI-779 has been reported to have antiangiogenic activity in a Matrigel assay in vivo (Del Bufalo et al., 2006) and also in a mouse xenograft model (Wan et al., 2006). In agreement with these previous findings, our present study provides more direct evidence that treatment of PyMT mice with CCI-779 significantly reduced tumor vascular density (Fig. 6). It also should be pointed out that the antiangiogenic effects of rapamycin may represent a downstream consequence of mTOR inhibition on the tumor cells (production of proangiogenic cytokines) rather than on the tumor vasculature itself in a xenograft model (Amornphimoltham et al., 2008).
Third, the antitumor effects of CCI-779 in PyMT mice might have been, at least in part, associated with the reduction in tumor macrophage burden. In particular, macrophages infiltrated in tumors have been recently demonstrated to play a critical role in tumor chemoresistance (De Palma and Lewis, 2011). A previous study demonstrated that infiltrated macrophages in primary mammary tumors play a causal role in promoting tumor angiogenesis and the progression to malignancy in PyMT mice (Lin et al., 2006). In this regard, VEGF produced by macrophages has been suggested to play a key role in this process (Lin et al., 2007). In the present work, we have documented the increase in the number of macrophages in the primary mammary tumors along with tumor progression in PyMT mice (from early to late stages; Fig. 7). CCI-779 treatment markedly suppressed macrophage content, especially in the advanced tumors (from 11 to 13 weeks) (Fig. 7). In contrast to the late time point, the effect of CCI-779 on macrophage accumulation in tumors at 10 weeks was much less. The reason associated with this differential effect is not known. It might be involved in the different tumor microenvironment and/or the subpopulations of macrophages. Because CCI-779 suppressed tumor growth in both early and late stages, it is likely that macrophages in the early stage might be more resistant to CCI-779. At least two subtypes of macrophages (M1 and M2 types) have been characterized in tumors (Leek and Harris, 2002; Allavena et al., 2008). However, their potential differential distribution and response in the tumors to CCI-779 treatment remain to be investigated.
Taken together, this work illustrates the utility of biomarkers that report on the mechanism of action (mTOR in respect to survival pathway) and tumor environment growth-promoting elements (vasculogenesis and macrophage burden) in tandem with the tumor growth arrest efficacy of a compound in early and late tumor stages. The data from this PyMT model with immune competency may provide a better value for drug discovery and translational research of human disease.
Participated in research design: Wang, Zhan, Zhao, Alvarez, Zhou, Abraham, and Feuerstein.
Conducted experiments: Wang, Zhan, and Zhao.
Performed data analysis: Wang, Zhan, Zhao, and Chaudhary.
Wrote or contributed to the writing of the manuscript: Wang, Zhan, Zhao, Alvarez, Zhou, Abraham, and Feuerstein.
We thank Salvatore Alesci, Daniel Johnston, Jay Gibbons, and Hans Peter Gerber (Pfizer Inc.) for helpful comments and discussions; and the staff at BioResources at the Pfizer Inc., Collegeville facility for excellent technical support with animal care and husbandry.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- mammalian target of rapamycin
- polyomavirus middle T antigen
- phosphatidylinositol-3 kinase
- ribosomal S6 kinase
- mouse mammary tumor virus
- vascular endothelial growth factor
- Received June 23, 2011.
- Accepted August 10, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics