PP242

The Dual mTORC1 and mTORC2 Inhibitor PP242 Shows Strong Antitumor Activity in a Pheochromocytoma PC12 Cell Tumor Model

Xiaohua Zhang, Xianjin Wang, Liang Qin, Tianyuan Xu, Zhaowei Zhu, Shan Zhong, Minguang Zhang, and Zhoujun Shen

OBJECTIVE

To assess the activity of mTOR and downstream effector proteins in the mTOR pathway after treatment with a dual mTOR complex 1 and 2 (mTORC1/2) inhibitor (PP242) compared with that of mTOR complex 1 (mTORC1) inhibitor (rapamycin) using a xenograft tumor model.

METHODS

Pheochromocytoma PC12 cell were xenografted into nude mice. Animals were treated with PP242 and rapamycin. Mean tumor volume was compared across groups. Terminal deoxy- nucleotidyl transferaseemediated dUTP nick-end labeling staining was used to detect apoptosis. Immunoblot analysis was performed to assess mTORC1/2 activity using p-Akt, p-S6, and p-4E- BP1. The expression of the antiapoptotic protein Bcl-2, pro-apoptotic protein Bax, and the mediator of angiogenesis vascular endothelial growth factor were also investigated.

RESULTS

The mean tumor volume of PP242 was significantly lower than in other groups. The terminal deoxynucleotidyl transferaseemediated dUTP nick-end labeling results showed that PP242 markedly increased cell apoptosis compared with other groups. Immunoblot analysis of tumor lysates treated with PP242 demonstrated inhibition of activated p-Akt. We also observed that only PP242, but not rapamycin, significantly reduced Bcl-2 expression and markedly increased Bax expression. Rapamycin decreased vascular endothelial growth factor expression, but not nearly as striking as seen in the PP242 group.

CONCLUSION

Our study showed that PP242 showed strong antitumor activity in a pheochromocytoma PC12 cell tumor model. Based on our study, dual mTORC1/2 kinase inhibitors warrant further investigation as a potential treatment for malignant pheochromocytomas or paragangliomas. UROLOGY 85: heochromocytomas (PCCs) and paragangliomas (PGLs) are rare neuroendocrine tumors that develop in the sympathetic and parasympathetic nervous systems. They can occur sporadically or as a part of familial syndrome, and can be benign or malignant tumors. The majority of PCCs are benign, and around 10% of PCCs and up to 40% of PGLs are malignant.1 To date, malignancy is still defined as the criterion that distant metastasis is developed. Surgical resection presents the treatment of choice when technically feasible. Pa- tients affected with malignant forms of the disease have a variable but mostly poor clinical outcome resulting in a 5-year cancer-specific survival of <50%.2 There are a lot of therapeutic modalities in the management of malignant PCC or PGL, such as chemotherapy and radionuclide therapy with either 131 I-metaiodobenzylguanidine or radiolabeled somatostatin analogs. These treatments may improve symptoms and hormonal markers, but the out- comes for the control of tumor bulk are less favorable and frequently short lived.3 Undoubtedly, difficulties in treat- ing patients with malignant PCC or PGL disease remain. Molecular targeted therapies are nowadays considered as the most promising strategies for the treatment of pa- tients with malignant PCC or PGL, which include tar- geting of the vascular endothelial growth factor (VEGF) pathway using either humanized monoclonal anti-VEGF antibodies (bevacizumab) or small tyrosine kinase inhibitors such as sorafenib or sunitinib, as well as mTOR inhibitors such as everolimus (RAD001).2,4,5 Among these strategies, target of mTOR is thought to be highly promising choice in future because hyperactivity of mTOR pathway is a well-established factor leading to increased angiogenesis in malignant PCC or PGL, which has been shown to be associated with the development of metastases, poor prognosis, and reduced survival.2,4-6 Numerous studies have shown that rapamycin and rapalogs (everolimus and temsirolimus), the first genera- tion mTOR inhibitor, can inhibit the proliferation of cancer cell lines and have got some success in caner treatment.7-9 Unfortunately, their overall efficacy as cancer therapeutics has been limited.10,11 It is increas- ingly recognized that the mechanism of action of rapa- mycin as a partial mTOR inhibitor is not sufficient for achieving broad and robust antitumor effect, at least when these agents are used in a monotherapy setting. The major drawbacks of first generation mTOR inhibitor are following: (1) S6K is exquisitely inhibited, yet the control of 4E binding protein 1 (4E-BP1) and messenger ribo- nucleic acid translation is far less sensitive, (2) mTOR complex 2 (mTORC2) activity is not acutely blocked, and (3) there is a feedback loop between mTOR complex 1 (mTORC1) and Akt.12,13 Recent studies in cancer biology indicate that mTORC2 is emerging as a promising therapeutic target because its activity is essential for the transformation and vitality of a number of cancer cell types.14 Therefore, mTOR adenosine triphosphateecompetitive inhibitors (such as PP242), the second generation mTOR inhibitor, have been developed recently that are able to completely suppress both mTORC1 and mTORC2 complexe mediated signaling.15-18 Importantly, they have shown marked improvement of antitumor activity in vivo and in vitro, and the effectiveness of these drugs in cancer treatment is currently being tested in clinical trials.19-22Growing evidence has shown that the PI3K/Akt/ mTOR pathway plays an important role in the patho- genesis of malignant PCC and PGL. Investigational mTOR kinase inhibitors may provide a novel therapeutic approach for these tumors. Therefore, this study was designed to evaluate the antitumor activity of a dual mTORC1/2 inhibitor PP242 compared with mTORC1 inhibitor rapamycin in a pheochromocytoma PC12 cell tumor model. Using a xenograft tumor model, we assessed the activity and expression of downstream proteins in the mTOR pathway after treatment with PP242 or rapamycin. We also focused on its mechanism of antitumor activity. METHODS Cell Line The pheochromocytoma PC12 cell line was obtained from the Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China) and were cultured in Dulbecco’s Modified Eagle Media medium (Gibco BRL Co Ltd, Life Technologies, Grand Island, NY) supplemented with 10% inactivated horse serum (Gibco) under an atmosphere of 5% CO2 at 37◦C. Xenograft Animal Tumor Model All studies were approved by the Shanghai Jiaotong University School of Medicine Institutional Animal Care and Use Com- mittee and conducted in accordance with Institutional Animal Care and Use Committee guidelines. Four-week-old male BALB/c nude mice, weighing 16-20 g, were obtained from the Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai, China). PC12 cell suspension (1 × 107/ 0.1 mL) was injected subcutaneously into the left flanks of mice. When mean palpable tumor volume was approximately 30 mm3 (arose within 7-10 days) calculated using external calipers and the standard formula for volume V ¼ 1/2 × (width2 × length), mice were randomized into 3 groups. Each treatment group consisted of 5 mice. Xenograft Tumor Model Treatment Protocols Treatment was performed for 2 weeks. PP242 and rapamycin were administered once a day. Mice were randomized into the following treatment groups: (1) control, treated by oral gavage with vehicle, 100 mL normal saline; (2) PP242, 10 mg/kg PP242 in 100 mL by oral gavage; (3) rapamycin, 1 mg/kg rapamycin in 100 mL by oral gavage. PP242 or rapamycin was suspended in water containing 5% 1-methyl-2-pyrrolidinone and 15% poly- vinylpyrrolidone as manufacturer instructions as previously reported.20 Assessment of Xenograft Tumor Treatment Response and Obtaining Specimen of Tumor Tumor size was measured by caliper of the 2 perpendicular di- ameters every other day, and the volume of the tumor was calculated with the formula V ¼ 1/2 × (width2 × length) as described previously. At the end of the experiment on day 15 after injection, mice were sacrificed, and tumors were rapidly removed, weighed, and then extracted for protein analysis or were formalin fixed and paraffin embedded. TUNEL Method for Detection of Apoptosis in Tumor Tissues Terminal deoxynucleotidyl transferaseemediated dUTP nick- end labeling (TUNEL) staining using an apoptosis detection kit (KeyGen Biotech, Nanjing, China) was carried out by following the manufacturer’s instructions. Briefly, paraffin- embedded samples of tumor tissue were cut in 5-mm sections and mounted on glass slides. The slides were incubated over- night at 56◦C, deparaffinized in xylene, and then hydrated in pure ethanol, 95% and 70% for 3 minutes. Then, they were treated once with phosphate-buffered saline (PBS) for 5 mi- nutes. The slides were then incubated in proteinase K for 15 minutes at room temperature, and then washed twice for 2 minutes with dH2O. The slides were immersed in 3% H2O2 for 5 minutes, washed twice for 5 minutes with PBS. The tissue slides were treated with equilibration buffer for 10 minutes. Then, the slides were incubated with TdT enzyme at 37◦C for 1 hour in a humidified chamber. Thereafter, stop or wash buffer was applied for 10 minutes, and the slides were washed with PBS 3 times for 5 minutes in each wash. The slides were then incubated for half an hour in a humidified chamber with anti- digoxigenin conjugate and washed 4 times for 5 minutes with PBS. Peroxidase substrate was applied for 6 minutes at room temperature. The slides were then washed with dH2O 3 times for 1 minute and once for 5 minutes. At last, the slides were dehydrated, transparated with xylene, and mounted with per- manent mounting medium. Normal female rodent mammary gland tissue sections were used as positive control. Five randomly selected high-power lens visual fields were evaluated in each section, and the apoptotic cells were identified based on intense brown nuclear staining. The percent of apoptotic cells was calculated, and the data were expressed as the average of apoptotic cell numbers per sample. Figure 1. Dual mTORC1/2 inhibitor PP242 showing strong antitumor activity in a xenotransplant animal model. (A) Photo- graphs show the tumors resected from representative mice at day 15. (B) Tumor growth curves of PC12 xenografts in nude mice treated with saline, rapamycin, or PP242 daily. Each group comprised 5 mice. Each data point means estimated volume of the tumors. The error bars are standard deviations of the mean. Immunoblot Analyses For tumor immunoblotting studies, tumors were rapidly harvested into radio-immunoprecipitation assay buffer. Tumors were extracted by homogenization in radio-immunoprecipitation assay buffer followed by centrifugation at 4◦C for 10 minutes at 13,000×g to remove insoluble material. Protein concentration was determined using bicinchonininc acid assay kit (KeyGen Biotech, Nanjing, China). To determine the total levels of spe- cific proteins, equal amounts of protein from lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis then transferred to polyvinylidene difluoride membranes and analyzed by immunoblotting with specific primary antibodies against Akt, p-Akt (S473), S6, p-S6 (S235/236), 4E-BP1, and p-4E-BP1 (T37; all from Cell Signaling Technology, Beverly) and Bcl-2 (Santa Cruz Biotechnology), Bax (Santa Cruz Biotechnology), and VEGF (Abcam, England) at dilutions specified by the manufacturer. Subsequently, the membranes were washed 3 times in tris buffered saline tween 20 (0.1% by volume) and incubated with the corresponding horseradish peroxidaseeconjugated sec- ondary antibodies at room temperature for 1 hour. After washing 3 times with tris buffered saline tween 20, the bound secondary antibody was detected using an enhanced chemiluminescence system (NENTM Life Science Products, Inc). Statistical Analyses All the quantitative data were expressed as the mean standard deviation. Tumor volume was compared between groups using analysis of variance. The Student t test was used to compare quantitative data. The statistical analyses were performed using SPSS 17.0 statistical software (SPSS, Chicago, IL). Values of P <.05 were considered to be significant. RESULTS Dual mTORC1/2 Inhibitor PP242 Showing Strong Antitumor Activity in a Xenotransplant Animal Model Mice were treated as described in the Methods section for 2 weeks and tumor volumes were measured throughout that period. At the end of the treatment, the mean tumor volume in the PP242 group was smallest (854 152 mm3), rapamycin group (1501 88 mm3) followed, and the average tumor volume in the control group was largest (2139 120 mm3; Fig. 1A). The difference in mean tumor volume was statistically significant when comparing tumor volume in the PP242 group with mean tumor volumes in another 2 groups at the completion of treatment (P <.005). As shown in the tumor growth curve, compared with vehicle group, oral administration of rapamycin only slightly inhibited the tumor growth. However, the PP242 therapy dramatically inhibited tumor growth than rapamycin (Fig. 1B). Taken together, the PP242 treatment group had the largest treatment effect with smallest tumor volume at the end of treatment. There was some antitumor activity in the rapamycin group but not nearly as striking as seen in the PP242 group. Effect of Dual mTORC1/2 Inhibitor PP242 or mTORC1 Inhibitor Rapamycin on the Downstream mTOR Effector Pathway in Xenotransplant Tumor Tissues We assessed the effect of dual mTORC1/2 inhibitor PP242 or mTORC1 inhibitor rapamycin on the down- stream mTOR effector pathway in xenotransplant tumor tissues, which demonstrated the superior blockade by mTORC1/2 inhibitor on downstream effectors of the mTOR pathway. Representative immunoblotting ana- lyses were performed on excised tumors taken at 2 hours after final treatment. We found that S6 protein phos- phorylation was strongly decreased in tumor lysates from both rapamycin- and PP242-treated samples (Fig. 2). In contrast, only mTORC1/2 inhibitor PP242, but not mTORC1 inhibitor rapamycin, strongly decreased phos- phorylation of 4E-BP1 (Fig. 2). We also observed that only dual mTORC1/2 inhibitor PP242 resulted in strong downregulation of Akt activation, shown by the striking reduction in p-Akt at Ser 473, which was not observed in rapamycin-treated tumors (Fig. 2). Taken together, these data provide evidence for the importance of inhibiting mTORC2 and blockade of the Akt upregulatory loop in tumor control. Figure 2. Effect of dual mTORC1/2 inhibitor PP242 or mTORC1 inhibitor rapamycin on the downstream mTOR effector pathway in xenotransplant tumor tissues. (A) Tumor lysates were subjected to immunoblotting for levels of Akt, phospho-Akt (S473), S6, phospho-S6 (S235/236), 4E-BP1, phospho-4E-BP1 (T37), and b-actin. We ran Western blotting on implants from each animal in our study. One of the representative graphs of experimental results was showed. (B) We performed the densitometry of these blots and presented the data as a graph. Effect of Dual mTORC1/2 Inhibitor PP242 or mTORC1 Inhibitor Rapamycin on Apoptosis in Xenograft The TUNEL results of each group are shown in Figure 3. PP242 markedly increased cell apoptosis (17.6 0.9%) compared with the control group (1.8 0.8%). However, rapamycin decreased the proportion of apoptotic cells significantly (9 1%; P <.05) compared with the control group, not nearly as striking as seen in the PP242 group.The antiapoptotic effect of rapamycin is lower than that of PP242 according to comparison between the rapamy- cin group and the PP242 group (P <.05). Effect of PP242 on Antiapoptotic and Proapoptotic Proteins in Xenotransplant Tumor Tissues The expressions of the antiapoptotic protein Bcl-2 and proapoptotic Bax were investigated in xenotransplant tumor tissues after PP242 or rapamycin treatment. As shown in Figure 4, only dual mTORC1/2 inhibitor PP242, but not mTORC1 inhibitor rapamycin, signifi- cantly reduced Bcl-2 expression, indicating that it may induce its apoptotic effect through inhibition of anti- apoptotic protein expression. Furthermore, as shown in Figure 4, proapoptotic Bax expression was markedly increased, implying that PP242 contributes to the acti- vation of this proapoptotic factor. Effect of Dual mTORC1/2 Inhibitor PP242 on Angiogenesis in Xenograft Angiogenesis is an essential process for growth of tumor and metastasis of solid malignancy. One of the most potent endothelial mitogens and mediators of angiogen- esis is VEGF. The induction of VEGF in cancer cells can be mediated through activation of PI3K/Akt/mTOR signaling pathway. To make sure whether the function of PP242 or rapamycin has an effect on VEGF expression, expression of VEGF protein was determined by Western blotting in xenotransplant tumor tissues after PP242 or rapamycin treatment. As shown in Figure 4, mTORC1 inhibitor rapamycin decreased VEGF expression, but not nearly as striking as seen in the PP242 group. COMMENT PCC and PGL are rare neuroendocrine tumors that develop in the sympathetic and parasympathetic nervous systems. The majority of PCC or PGL tumors are benign and, when feasible, treated by surgical resection.4-6 Chemotherapy and radiotherapy become the main treatment options for those malignant tumors. However, these methods are not an effective therapy for malignant PCC or PGL.2,4-6 Thus, the search of a potential alter- native therapy for malignant PCC or PGL is urgent and extremely important. Figure 3. Effect of dual mTORC1/2 inhibitor PP242 or mTORC1 inhibitor rapamycin on apoptosis in xenograft. PC12 xeno- grafts in nude mice treated with saline, rapamycin, or PP242 were stained with TUNEL apoptosis detection kit, and the percent of apoptotic cells was calculated under a microscope (×100). P <.05 for comparison of PP242 vs control or rapa- mycin therapy. TUNEL, terminal deoxynucleotidyl transferaseemediated dUTP nick-end labeling. Figure 4. Effect of PP242 on the expresion of Bcl-2, Bax, and vascular endothelial growth factor in xenotransplant tumor tissues. (A) Tumor lysates were subjected to immunoblotting for levels of Bcl-2, Bax, and vascular endothelial growth factor. We ran Western blotting on implants from each animal in our study. One of the representative graphs of experimental results was showed. (B) We performed the densitometry of these blots and presented the data as a graph. VEGF, vascular endo- thelial growth factor. Based on the findings that hyperactivity of mTOR pathway is a well-established factor leading to increased angiogenesis in malignant PCC or PGL, which has been shown to be associated with the development of metas- tases, poor prognosis, and reduced survival,2,4-6 target of mTOR is thought to be a highly promising choice in future. mTOR is a serine/threonine kinase at the nexus between oncogenic phosphoinositide 3-kinase (PI3K)/ Akt signaling and critical downstream pathways that plays a pivotal role in cell metabolism, growth, prolifer- ation, and survival.23,24 Based on their sensitivity to rapamycin treatment, mTOR kinase have 2 distinct multiprotein complexes: one is mTORC1, a complex composed of mTOR, Raptor, Deptor, and PRAS40, and another is mTORC2, which contains mTOR, Rictor, mLST8, and Protor.25 mTORC1, or the rapamycin- sensitive mTOR complex, phosphorylates downstream targets including p70S6K (S6K) and 4E-BP1 and is important for protein translation and cell growth. mTORC2, or the rapamycin-insensitive mTOR complex, phosphorylates Akt at Ser 473 and increases its enzymatic activity.26,27 In the past 2 decades, numerous studies have shown that rapamycin and rapalogs (everolimus and temsirolimus), the first generation mTOR inhibitor, can inhibit the prolifer- ation of cancer cell lines and have achieved some success in cancer treatment.7-9 Nevertheless, their overall efficacy as cancer therapeutics has been limited to a few rare cancers.10,11 Fortunately, recent studies in cancer biology indicate that mTORC2 is emerging as a promising thera- peutic target because its activity is essential for the trans- formation and vitality of a number of cancer cell types.14 Therefore, mTOR adenosine triphosphateecompetitive inhibitors (such as PP242), the second generation mTOR inhibitor, have been developed recently that are able to completely suppress both mTORC1 and mTORC2 complexemediated signaling.15-18 In the present study, we evaluated the antitumor ac- tivity of a dual mTORC1 and mTORC2 inhibitor PP242 compared with mTORC1 inhibitor rapamycin in a pheochromocytoma PC12 cell tumor model. Our results demonstrated that the PP242 therapy dramatically inhibited tumor growth. Also, we found that there was some antitumor activity in the rapamycin group but not nearly as striking as seen in the PP242 group. Immuno- blot analysis of tumors from mice in the different treat- ment groups demonstrated that dual mTORC1/2 inhibitor PP242 strongly reduces activation of effector proteins in the mTOR pathway, particularly 4E-BP1. Further, this abolished any evidence for the upregulation of Akt activity, which likely is the mediator of rapamycin treatment efficacy concerns. Importantly, we also demonstrated that reliance solely on S6 phosphorylation as a surrogate measure of mTOR activity is unreliable. Use of S6 phosphorylation to measure mTORC1 activity is standard practice in many preclinical and clinical studies. TUNEL analysis demonstrated that PP242 markedly increased cell apoptosis compared with the control group. However, rapamycin decreased the proportion of apoptotic cells significantly compared with the control group, not nearly as striking as seen in the PP242 group. We also observed that only PP242, but not rapamycin, significantly reduced antiapoptotic protein Bcl-2 expression and markedly increased proapoptotic Bax expression by Western blotting, indicating that it may induce its apoptotic effect through inhibition of anti- apoptotic protein and upregulation of the proapoptotic expression. Angiogenesis is an essential process for growth of tumor and metastasis of solid malignancy. One of the most potent endothelial mitogens and mediators of angiogen- esis is VEGF. The induction of VEGF in cancer cells can be mediated through activation of PI3K/Akt/mTOR signaling pathway.1 PCC and PGL are very highly vas- cularized tumors and, therefore, potentially strongly dependent on angiogenesis-mediated growth and sur- vival.28 Targeting the VEGF pathway is the most commonly used antiangiogenic strategy in cancer and appeared as the best candidate for inhibiting angiogenesis in PCC or PGL.29 To make sure whether the PP242 has an effect on VEGF expression, expression of VEGF pro- tein was determined by Western blotting the tumors from mice in the different treatment groups. Our study demonstrated that PP242 significantly downregulated VEGF protein level in xenotransplant tumor tissues. Growing evidence has shown that the PI3K/Akt/ mTOR pathway plays an important role in the patho- genesis of malignant PCC or PGL.3,29,30 Those findings, combined with those of our present study, indicate that PP242 shows its strong antitumor activity, at least in part, through inhibition of the PI3K/Akt/mTOR pathway in the treatment of malignant PCC or PGL. However, we would like to emphasize the limitations of this study. It is known that PC12 cells, although well established and extensively used, do not accurately reflect the pathogen- esis of malignant cells. CONCLUSION The present study found that PP242 showed strong antitumor activity in a pheochromocytoma PC12 cell tumor model. Our present results suggest that PP242 may be further investigated in the treatment of malignant PCC or PGL in clinical trials.

References

1. Favier J, Igaz P, Burnichon N, et al. Rationale for anti-angiogenic therapy in pheochromocytoma and paraganglioma. Endocr Pathol. 2012;23:34-42.
2. Lowery AJ, Walsh S, McDermott EW, et al. Molecular and thera- peutic advances in the diagnosis and management of malignant pheochromocytomas and paragangliomas. Oncologist. 2013;18: 391-407.
3. Druce MR, Kaltsas GA, Fraenkel M, et al. Novel and evolving therapies in the treatment of malignant phaeochromocytoma: experience with the mTOR inhibitor everolimus (RAD001). Horm Metab Res. 2009;41:697-702.
4. Jimenez C, Rohren E, Habra MA, et al. Current and future treat- ments for malignant pheochromocytoma and sympathetic para- ganglioma. Curr Oncol Rep. 2013;15:356-371.
5. Buzzoni R, Pusceddu S, Damato A, et al. Malignant pheochromo- cytoma and paraganglioma: future considerations for therapy. Q J Nucl Med Mol Imaging. 2013;57:153-160.
6. Parenti G, Zampetti B, Rapizzi E, et al. Updated and new perspec- tives on diagnosis, prognosis, and therapy of malignant pheochro- mocytoma/paraganglioma. J Oncol. 2012;2012:872713.
7. Benjamin D, Colombi M, Moroni C, Hall MN. Rapamycin passes the torch: a new generation of mTOR inhibitors. Nat Rev Drug Discov. 2011;10:868-880.
8. Okazaki H, Matsunaga N, Fujioka T, et al. Circadian regulation of mTOR by the ubiquitin pathway in renal cell carcinoma. Cancer Res. 2014;74:543-551.
9. Pezzani R, Rubin B, Redaelli M, et al. The antiproliferative effects of ouabain and everolimus on adrenocortical tumor cells. Endocr J. 2014;61:41-53.
10. Houghton PJ. Everolimus. Clin Cancer Res. 2010;16:1368-1372.
11. Guertin DA, Sabatini DM. The pharmacology of mTOR inhibi- tion. Sci Signal. 2009;2:pe24.
12. Thoreen CC, Sabatini DM. Rapamycin inhibits mTORC1, but not completely. Autophagy. 2009;5:725-726.
13. Carew JS, Kelly KR, Nawrocki ST. Mechanisms of mTOR inhibitor resistance in cancer therapy. Targeted Oncol. 2011;6:17-27.
14. Sparks CA, Guertin DA. Targeting mTOR: prospects for mTOR complex 2 inhibitors in cancer therapy. Oncogene. 2010;29:3733- 3744.
15. Feldman ME, Apsel B, Uotila A, et al. Active-site inhibitors of mTOR target rapamycin-resistant outputs of mTORC1 and mTORC2. PLoS Biol. 2009;7:e38.
16. Yu K, Shi C, Toral-Barza L, et al. Beyond rapalog therapy: pre- clinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2. Cancer Res. 2010;70:621-631.
17. Thoreen CC, Kang SA, Chang JW, et al. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin- resistant functions of mTORC1. J Biol Chem. 2009;284:8023-8032.
18. Janes MR, Limon JJ, So L, et al. Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor. Nat Med. 2010; 16:205-213.
19. Zhang YJ, Duan Y, Zheng XF. Targeting the mTOR kinase domain: the second generation of mTOR inhibitors. Drug Discov Today. 2011;16:325-331.
20. Li H, Lin J, Wang X, et al. Targeting of mTORC2 prevents cell migration and promotes apoptosis in breast cancer. Breast Cancer Res Treat. 2012;134:1057-1066.
21. Altman JK, Szilard A, Goussetis DJ, et al. Autophagy is a survival mechanism of acute myelogenous leukemia precursors during dual mTORC2/mTORC1 targeting. Clin Cancer Res. 2014;20:2400-2409.
22. Jordan NJ, Dutkowski CM, Barrow D, et al. Impact of dual mTORC1/2 mTOR kinase inhibitor AZD8055 on acquired endo- crine resistance in breast cancer in vitro. Breast Cancer Res. 2014;16: R12.
23. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cel Biol. 2011;12:21-35.
24. Guertin DA, Sabatini DM. An expanding role for mTOR in cancer.
Trends Molecular Medicine. 2005;11:353-361.
25. Betz C, Hall MN. Where is mTOR and what is it doing there? J Cell
Biol. 2013;203:563-574.
26. Sun SY. mTOR kinase inhibitors as potential cancer therapeutic drugs. Cancer Lett. 2013;340:1-8.
27. Laplante M, Sabatini DM. mTOR signaling at a glance. J Cel Sci. 2009;122:3589-3594.
28. Feng F, Zhu Y, Wang X, et al. Predictive factors for malignant pheochromocytoma: analysis of 136 patients. J Urol. 2011;185: 1583-1590.
29. Saito Y, Tanaka Y, Aita Y, et al. Sunitinib induces apoptosis in pheochromocytoma tumor cells by inhibiting VEGFR2/Akt/ mTOR/S6K1 pathways through modulation of Bcl-2 and BAD. Am J Physiol Endocrinol Metab. 2012;302:E615-625.
30. Motylewska E, Lawnicka H, Kowalewicz-Kulbat M, et al. Interferon alpha and rapamycin inhibit the growth of pheochromocytoma PC12 line in vitro. Endokrynol Pol. 2013;64:368-374.