Antitumor activities of ATP-competitive inhibitors of mTOR in colon cancer cells
© Blaser et al; licensee BioMed Central Ltd. 2012
Received: 20 October 2011
Accepted: 8 March 2012
Published: 8 March 2012
The mammalian target of rapamycin (mTOR) is frequently activated in colon cancers due to mutations in the phosphatidylinositol 3-kinase (PI3K) pathway. Targeting mTOR with allosteric inhibitors of mTOR such as rapamycin reduces colon cancer progression in several experimental models. Recently, a new class of mTOR inhibitors that act as ATP-competitive inhibitors of mTOR, has been developed. The effectiveness of these drugs in colon cancer cells has however not been fully characterized.
LS174T, SW480 and DLD-1 colon cancer cell lines were treated with PP242 an ATP-competitive inhibitor of mTOR, NVP-BEZ235, a dual PI3K/mTOR inhibitor or rapamycin. Tumor cell growth, proliferation and survival were assessed by MTS assay, 5-bromo-2'-deoxyuridine (BrDU) incorporation or by quantification of DNA fragmentation respectively. In vivo, the anticancer activity of mTOR inhibitors was evaluated on nude mice bearing colon cancer xenografts.
PP242 and NVP-BEZ235 reduced the growth, proliferation and survival of LS174T and DLD-1 colon cancer cells more efficiently than rapamycin. Similarly, PP242 and NVP-BEZ235 also decreased significantly the proliferation and survival of SW480 cells which were resistant to the effects of rapamycin. In vivo, PP242 and NVP-BEZ235 reduced the growth of xenografts generated from LS174T and SW480 cells. Finally, we also observed that the efficacy of ATP-competitive inhibitors of mTOR was enhanced by U0126, a MEK inhibitor.
Taken together, these results show that ATP-competitive inhibitors of mTOR are effective in blocking colon cancer cell growth in vitro and in vivo and thus represent a therapeutic option in colon cancer either alone or in combination with MEK inhibitors.
KeywordsColon cancer mTOR Rapamycin NVP-BEZ235 PP242 Proliferation Xenograft
Colorectal cancer (CRC) is one of the leading cause of cancer-related deaths worldwide . Over the last decade, new therapeutic options for the treatment of CRC have been developed including targeted therapies. For example, drugs that block the vascular endothelial growth factor or the epidermal growth factor receptor have shown clinical activities and have been approved for the treatment of CRC . However, despite these new treatments, the prognosis of CRC remains poor and new therapeutic strategies still need to be explored.
The mammalian target of rapamycin (mTOR) is a serine/threonine kinase, present in two functionally distinct complexes mTORC1 and mTORC2. While mTORC1 is composed of mTOR, mLST8, raptor, deptor and PRAS40, mTORC2 consists of mTOR, rictor protor, mLST8, deptor and sin1 [3, 4]. mTORC1 regulates cell growth by controlling mRNA translation initiation and progression by phosphorylating two well characterized downstream effectors: S6K1 and 4E-BP1 . In addition, mTORC1 also regulates ribosome biogenesis, autophagy and lipid biosynthesis. mTORC2 is involved in cell survival and proliferation by phosphorylating members of the AGC kinase family including Akt, protein kinase C and serum-and glucocorticoid-regulated kinase [6–8]. Of note, whereas mTORC1 is sensitive to acute exposure to rapamycin, mTORC2 is not. However in a subset of cells, prolonged exposure to rapamycin also inhibits mTORC2 .
Emerging data have shown that mTOR is implicated in the progression of CRC and represents a promising target in the treatment of CRC. Indeed, components of mTOR signaling pathway are frequently activated or over-expressed in CRC [10, 11]. For example, genetic aberrations of the catalytic subunit of the phosphatidylinositol 3-kinase (PI3K), an upstream effector of mTORC1 and mTORC2, are frequent in colon cancer [12, 13].Moreover, the inhibition of mTOR signals by allosteric inhibitors such as rapamycin or small interfering RNA has been shown to reduce colon cancer growth in different experimental settings [10, 11, 14, 15]. Recently, a new class of mTOR inhibitors have been developed that target the kinase domain of mTOR and referred as ATP-competitive inhibitors of mTOR [16, 17]. In contrast to rapamycin which targets only certain functions of mTORC1, ATP-competitive inhibitors of mTOR inhibit both mTORC1 and mTORC2. Furthermore, a subset of these inhibitors also blocks PI3K in addition to inhibit mTORC1 and mTORC2 . In this study, we have determined the anticancer activity of PP242 , a kinase inhibitor of mTOR and NVP-BEZ235 , a dual PI3K/mTOR inhibitor, in colon cancer cells, both in vitro and in vivo.
Cell lines, antibodies and reagents
The human colon cancer cell lines LS174T, DLD-1, SW480, SW620, HT29, Caco-2, and HCT-116 were maintained in Dulbecco's modified eagle's medium supplemented with 10% fetal calf serum. Antibodies directed against phospho-Akt (Ser473), Akt, phospho-S6 ribosomal protein (Ser235/236), S6 ribosomal protein and cleaved caspase-3 were from Cell signaling technology (Danvers, MA, USA). Rapamycin, U0126 and NVP-BEZ235 were from LC laboratories (Woburn, MA, USA). PP242 was from Chemdea (Ridgewood, NJ, USA). For in vitro experiments, all inhibitors were dissolved in dimethyl sulfoxide (DMSO).
Western blot analysis
Western blot were performed as previously described .
MTS proliferation assay
LS174T, SW480, DLD-1, Caco-2, HCT-116, SW620 and HT-29 cells were plated on 96-well plates (Costar) at 10'000 cells per well and cultured in DMEM 10% FBS. Twelve hours later, cells were treated with rapamycin (10 nM), NVP-BEZ235 (100 nM), PP242 (100 nM) or DMSO as a control. Cellular proliferation was monitored after 48 hours of treatment with the CellTiter 96® Aqueous One Solution (Promega Corporation) colorimetric assay by following the manufacturer's instructions.
BrDU incorporation assay
BrDU incorporation assay was performed as previously described .
Cell survival studies
LS174T, SW480, DLD-1 cells were plated in 96-well plates at 30,000 cells per well. Twelve hours later, cells were treated with rapamycin (10 nM), NVP-BEZ235 (100 nM), PP242 (100 nM), either alone or in combination with U0126 (10 μM) for 48 hours. Subsequently cells were harvested and apoptosis was determined using the Cell Death Detection ELISA plus kit (Roche) and following the manufacturer's instructions. Results are represented as the mean enrichment factor (absorbance of the treated cells/absorbance of the control cells).
In addition, cell apoptosis was also quantified using flow cytometry. LS174T, SW480 and DLD-1 cells were plated in 6-well plates at 300 000 cells per well and treated as above. After 48 hours of treatment cells were collected and fixed in 70% ethanol for 24 hours. Cells were subsequently resuspended in phosphate buffered saline (PBS) containing 20 μg/ml propidium iodide and 200 μg/ml RNAse and incubated for 30 minutes at 37°C. The percentages of sub-G1 population were determined by flow cytometry.
Animal experiments were approved by the ethics committee of the cantonal veterinary office of Canton Vaud (Authorization 2047) and conducted in accordance with the regulations of the Service of Consumables and Veterinary Affairs-Division of Animal Protection (SCAV-EXPANIM). Female nude mice aged 8 weeks were purchased from Charles River (Charles River Laboratories, St. Germain sur l'Arbresle, France). One million LS174T or SW480 cells were injected subcutaneously into the flank of nude mice. Once the tumor xenografts reached 25 mm3, mice were randomized into different groups (n = 5 in each group). Mice were treated with rapamycin (1.5 mg/kg/d, i.p.), NVP-BEZ235 (30 mg/kg/d, p.o.), PP242 (60 mg/kg/d, p.o.) either alone or in combination with U0126 (40 μmol/kg/d, i.p.). All mice received both p.o. and i.p. doses of vehicle to control for morbidity associated with treatment. NVP-BEZ235 was solubilized in one volume of N-methylpyrrolidone and further diluted in nine volumes of PEG 300. PP242 was dissolved in PEG 300. Stock solutions of rapamycin and U0126 were prepared in DMSO and further diluted in PBS before injection. Tumor volumes were measured using caliper measurements every day and calculated with the formula V = π/(6a2b) where a is the short axis and b the long axis of the tumor. Animals were sacrificed after 20 days of treatment and the tumors were excised and processed for further analysis.
Tumor xenografts were carefully removed and rapidly frozen in OCT compound (Tissue-Teck) on dry ice. Eight μm transverse sections were cut on a cryostat (CM 1850, Leica), and processed for immunolabeling with an anti-Ki-67 (Novocastra) as previously described . Ki-67 positivity was quantified and expressed as % of cells positive for Ki-67/total number of cells (300 cells counted per tumor; five tumors in each group).
Data were analyzed by Student's t-test or one way ANOVA. Values of P < 0.05 were considered statistically significant.
Concentration-dependent effects of ATP-competitive inhibitors of mTOR on mTORC1 and mTORC2 activity in colon cancer cells
Effect of ATP-competitive inhibitors of mTOR compared to rapamycin on colon cancer cell proliferation and survival
To next investigate whether the effects induced by mTOR inhibitors on colon cancer cell growth result from a reduction of cell proliferation, we performed 5-bromo-2'-deoxyuridine (BrDU) incorporation assay. NVP-BEZ235 and PP242 significantly decreased BrDU incorporation in colon cancer cell lines. Similarly to what we observed on cell growth, rapamycin decreased BrDU incorporation in LS174T and DLD-1 cells but not in SW480 cells (Figure 2B). Finally, we also investigated whether mTOR inhibitors induce apoptosis of colon cancer cells by using a cell death detection ELISA. We observed that NVP-BEZ235 and PP242 increased colon cancer cell apoptosis in all cell lines tested. The effect of NVP-BEZ235 was significantly stronger than PP242. In contrast, rapamycin failed to induce colon cancer cell apoptosis in LS174T and SW480 cells and significantly reduced apoptosis in DLD-1 cells (Figure 2C). Similar results were obtained by quantifying the apoptotic population of colon cancer cells following treatments using propidium iodide staining and flow cytometry analysis (Additional File 2). Taken together, these results show that ATP-competitive inhibitors of mTOR reduce colon cancer cell proliferation and survival.
ATP-competitive inhibitors of mTOR reduce the growth of colon cancer xenografts
Effect of ATP-competitive inhibitors of mTOR in combination with U0126 on colon cancer cell growth
mTOR represents a promising target in colon cancer. Indeed, components of mTOR signaling pathways are frequently over-expressed and activated in human samples of colon cancer [10, 11]. In addition, in experimental settings, the inhibition of mTOR components using siRNA or shRNA results in a marked reduction of colon cancer cell growth in vitro and tumor xenograft growth in vivo [10, 11, 14]. Furthermore, in a transgenic mouse model in which the adenomatous polyposis coli tumor suppressor gene has been mutated, the inhibition of mTORC1 by the rapamycin analog everolimus, decreased the formation of intestinal polyps and reduced mortality of these mice .
Initial studies used rapalogs to target mTOR. However, recent findings have demonstrated that targeting mTOR signaling pathway with rapalogs might not be optimal . In fact, rapalogs block only certain functions of mTORC1 and have no effects on mTORC2. Moreover, the inhibition of mTORC1 by rapalogs also results in the activation of proliferative and survival signals such as the PI3K/Akt and MEK/MAPK signaling pathways through the removal of a negative feedback loop . To overcome these limitations, a new class of mTOR inhibitors has been developed that block the kinase domain of mTOR and therefore inhibit both mTORC1 and mTORC2 [16, 29]. In this study, we found that two such inhibitors, PP242, a specific inhibitor of mTOR and NVP-BEZ235, a dual PI3K/mTOR inhibitor, effectively reduced colon cancer cell proliferation and survival and the growth of colon cancer tumor xenografts. Consistent with our findings, a recent study also demonstrated the efficacy of NVP-BEZ235 in a genetically engineered mouse model of CRC . Therefore our results provide rationale for the clinical evaluation of ATP-competitive inhibitors of mTOR in colon cancer patients.
We initially hypothesized that ATP-competitive inhibitors of mTOR would produce anticancer activity only in cells harboring PI3KCA mutations. To support this hypothesis it was previously reported that NVP-BEZ235 was effective in PI3K but not in KRAS mutated breast cancer cells and similar findings were reported in a murine model of lung cancer [31, 32]. However, we observed here that ATP-competitive inhibitors of mTOR exhibited anticancer effects on both PI3KCA mutated as well as on PI3KCA wild type colon cancer cells. Consistent with our findings, NVP-BEZ235 is effective in a mouse model of sporadic PI3KCA wild type CRC suggesting that the antitumor activity of ATP-competitive inhibitors of mTOR is not restricted to PI3KCA mutated colon cancer cells .
The anticancer efficacy of NVP-BEZ235 and PP242 was both in vitro and in vivo superior to rapamycin. It is however worth noting that despite blocking mTORC1 activity in vivo, the doses of rapamycin that we used (1.5 mg/kg/day) were lower than those reported by other groups (5 mg/kg/day and 20 mg/kg/day) [33, 34]. Therefore a comparison between ATP-competitive inhibitors of mTOR and higher concentrations of rapamycin is needed to conclude that ATP-competitive inhibitors of mTOR are more efficient than rapamycin. Nevertheless, similar to what we found, it was reported in renal cell carcinoma, that the anticancer efficacy of NVP-BEZ235 was superior to rapamycin used at 3.5 mg/kg/day .
Our findings also suggest that ATP-competitive inhibitors of mTOR display a broader anticancer activity than rapalogs. We found that while rapamycin had no effect on SW480 colon cancer cells, PP242 and NVP-BEZ235 reduced SW480 cell proliferation and survival as well as the growth of SW480 xenografts. Similarly, it was reported that blocking mTORC1 by rapamycin or by the use of raptor siRNA had no effect on the proliferation of SW480 cells. In contrast, targeting mTORC2 with rictor siRNA efficiently reduced SW480 cell proliferation . Therefore, by blocking mTORC2 in addition to mTORC1, the anticancer activity of ATP-competitive inhibitors of mTOR appear to be broader than rapamycin.
Emerging evidence has shown that blocking mTORC1 results in the removal of a negative feedback loop resulting in the activation of the PI3K/Akt and MEK/MAPK signaling pathways that counteract the anticancer efficacy of mTOR inhibitors . In our study, we observed that ATP-competitive inhibitors of mTOR increased MAPK phosphorylation in LS174T cells (Figure 4a). Similar effects were reported in other cell types including renal cancer cells, Waldenstrom macroglobulinemia cells, sarcoma cells and endothelial cells [35–38]. We further observed that targeting MAPK with a MEK inhibitor in combination with mTOR inhibitors resulted in synergistic inhibition of LS174T and SW480 colon cancer cell growth (Figure 4b-d). Noteworthy, we found that ATP-competitive inhibitors of mTOR did not increase MAPK phosphorylation in SW480 suggesting that MEK inhibitors would potentiate the anticancer efficacy of mTOR inhibitors regardless of whether mTOR inhibitors increase MAPK phosphorylation.
Overall, our study shows that ATP-competitive inhibitors of mTOR efficiently reduced the growth of colon cancer cells both in vitro and in vivo. In addition, it also shows that the anticancer efficacy of ATP-competitive inhibitors of mTOR is potentiated by the simultaneous pharmacological blockade of the MEK/MAPK signaling pathway in colon cancer cells. Therefore, ATP-competitive inhibitors represent promising agents in the treatment of CRC that warrant to be tested in clinical trials.
Mammalian target of rapamycin
Catalytic subunit of phosphatidylinositol 3-kinase
Phosphate buffered saline.
This work was supported by a research grant of the Swiss National Science Foundation (SCORE 32323B-123821 to OD).
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