To examine the in vitro and in vivo efficacy of the dual PI3K/mTOR inhibitor NVP-BEZ235 in treatment of PIK3CA wild-type colorectal cancer (CRC).
PIK3CA mutant and wild-type human CRC cell lines were treated in vitro with NVP-BEZ235, and the resulting effects on proliferation, apoptosis, and signaling were assessed. Colonic tumors from a genetically engineered mouse (GEM) model for sporadic wild-type PIK3CA CRC were treated in vivo with NVP-BEZ235. The resulting effects on macroscopic tumor growth/regression, proliferation, apoptosis, angiogenesis, and signaling were examined.
In vitro treatment of CRC cell lines with NVP-BEZ235 resulted in transient PI3K blockade, sustained decreases in mTORC1/mTORC2 signaling, and a corresponding decrease in cell viability (median IC50 = 9.0–14.3 nM). Similar effects were seen in paired isogenic CRC cell lines that differed only in the presence or absence of an activating PIK3CA mutant allele. In vivo treatment of colonic tumor-bearing mice with NVP-BEZ235 resulted in transient PI3K inhibition and sustained blockade of mTORC1/mTORC2 signaling. Longitudinal tumor surveillance by optical colonoscopy demonstrated a 97% increase in tumor size in control mice (p = 0.01) vs. a 43% decrease (p = 0.008) in treated mice. Ex vivo analysis of the NVP-BEZ235-treated tumors demonstrated a 56% decrease in proliferation (p = 0.003), no effects on apoptosis, and a 75% reduction in angiogenesis (p = 0.013).
Citation: Roper J, Richardson MP, Wang WV, Richard LG, Chen W, Coffee EM, et al. (2011) The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Induces Tumor Regression in a Genetically Engineered Mouse Model of PIK3CA Wild-Type Colorectal Cancer. PLoS ONE 6(9): e25132. https://doi.org/10.1371/journal.pone.0025132
Editor: Alfons Navarro, University of Barcelona, Spain
Received: June 14, 2011; Accepted: August 25, 2011; Published: September 26, 2011
Copyright: © 2011 Roper et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by grants from the National Cancer Institute (2U01CA084301) and the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)(5K08DK7803325, R03DK088014, and T32-DK07542). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
In 2011, colorectal cancer (CRC) will continue to be the third most common cause of cancer-related mortality in the U.S . Despite the growing arsenal of chemotherapeutic agents, the median survival for patients with metastatic CRC is still less than 20 months, which underscores the urgent need for the development of novel therapeutic approaches .
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cellular proliferation and apoptosis. mTOR binds regulatory associated protein of mTOR (Raptor) and mammalian LST8/G-protein β-subunit like protein (mLST8/GβL) to form the mTOR complex 1 (mTORC1), which promotes translation through phosphorylation of p70 S6 kinase (S6K), S6 ribosomal protein (S6), and eukaryotic initiation factor 4E binding protein 1(4E-BP1). Alternatively, mTOR can bind rapamycin-insensitive companion of mTOR (Rictor), mLST8/GβL, and mammalian stress-activated protein kinase interacting protein 1 (mSIN1) to form mTOR complex 2 (mTORC2) , .
The upstream phosphatidylinositol 3-kinase (PI3K) signaling pathway can activate mTOR. Class IA PI3Ks are activated by growth factor receptor tyrosine kinases (RTKs) and are composed of a heterodimer consisting of a p110α/p110β catalytic and a p85 regulatory subunit . The PIK3CA (phosphatidylinositol 3-kinase, catalytic, α-polypeptide) gene that encodes p110α is frequently mutated in many human cancers, including CRC . Point mutations in PIK3CA cluster at two hotspots: E545K in the helical domain (exon 9) and H1047R in the catalytic kinase domain (exon 20). These mutations increase p110α activity and promote CRC cell growth, invasion, and migration in vitro via activation of the PI3K pathway . Mutations in the helical and catalytic domains of PIK3CA confer essentially identical phenotypes in human CRC cell lines . AKT is a critical downstream effector of the PI3K pathway and promotes cell growth and survival via a number of mechanisms, including phosphorylation of TSC2, which results in mTORC1 activation . Full activation of AKT is achieved after phosphorylation at Thr308 and Ser473 by PDK1 and mTORC2, respectively , –.
Because of its central role in carcinogenesis, mTORC1 blockade is an attractive therapeutic strategy for CRC. Treatment of Apc Δ716 mice with the mTORC1 inhibitor everolimus inhibits cellular proliferation and tumor angiogenesis, resulting in a decrease in both number and size of intestinal tumors . We have recently reported that treatment of a genetically engineered mouse (GEM) model for sporadic CRC with the mTORC1 inhibitor rapamycin results in an 80% reduction in individual tumor growth, as observed by longitudinal colonoscopy surveillance . However, the clinical efficacy of mTORC1 blockade may be attenuated by the concomitant loss of an mTORC1-dependent negative feedback loop on PI3K signaling (reflected by increased AKT phosphorylation at Thr308), and continued mTORC2-mediated activation of AKT through phosphorylation at Ser473 –. Indeed, a Phase I clinical trial examining the efficacy of the mTORC1 inhibitor everolimus in advanced solid tumors demonstrated modest benefit in only one of 16 colorectal cancer patients and overall increased phosphorylation of AKT at Ser473 . Taken together, it appears that therapeutic strategies in which PI3K and mTOR are concurrently inhibited may be most efficacious.
NVP-BEZ235 (Novartis) is a dual pan-class I PI3K and mTOR kinase inhibitor that has been demonstrated to reduce tumor growth in a number of different xenograft and several genetically engineered mouse (GEM) models and is currently in clinical trials –. There has been suggestion that use of such agents may be limited to tumors with activating mutations in PIK3CA , . As activating PIK3CA mutations are seen in only 17% of CRC, this would imply such agents may be targeted towards only a small proportion of patients . Because NVP-BEZ235 inhibits the wild-type and mutant forms of PIK3CA with comparable efficacy , we hypothesized that NVP-BEZ235 may have significant efficacy in the treatment of PIK3CA wild-type CRC.
In this manuscript, we describe results from in vitro treatment studies demonstrating comparable efficacy of NVP-BEZ235 against both PIK3CA mutant and wild-type human CRC cell lines. We also describe results from in vivo treatment studies demonstrating significant efficacy in a GEM model for sporadic wild-type PIK3CA CRC. Taken together, our findings provide a compelling preclinical rationale for clinical trials to examine the use of NVP-BEZ235 in treatment of PIK3CA wild-type CRC patients.
Materials and Methods
In vitro treatment of human CRC cell lines
HCT116 (PIK3CA mutant; kinase domain at H1047R), DLD-1 (PIK3CA mutant; helical domain at E545K), and SW480 (PIK3CA wild-type) human CRC cell lines (ATCC) and isogenic DLD-1 PIK3CA mutant and wild-type cells (obtained from B. Vogelstein) were maintained in DMEM (Invitrogen) with 10% FBS and 1× Penicillin/Streptomycin (Invitrogen). Cells were plated at different initial densities (HCT116: 3,000 cells/well, DLD-1: 5,500 cells/well, SW480: 4,500 cells/well, DLD-1 PIK3CA mutant: 7,000 cells/well, and DLD-1 PIK3CA wild-type: 9,000 cells/well) to account for differential growth kinetics. After 16 hours, cells were incubated with increasing concentrations of NVP-BEZ235 (Novartis), and drug-containing growth medium was changed every 24 hours. Cell viability was assessed 16 hours after the initial plating and 48 hours after initiation of drug treatment using the colorimetric MTS assay CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega), as per the manufacturer's instructions. Cell viability after drug treatment was normalized to that of untreated cells also grown for 48 hours. IC50 values were calculated using 4 parameter nonlinear regression in GraphPad Prism 5 (GraphPad Software). For western blot analysis, cells were plated with 0 nM or maximal inhibitory dose (500 nM) NVP-BEZ235 for 2, 6, 24, or 48 hours.
Sequencing of colonic tumors from a GEM model for sporadic CRC
C57BL/6J Apc conditional knockout mice (Apc CKO) were treated with Adeno-Cre, as previously described . Following necropsy, 10 tumor specimens were collected in 1 ml RNA Later (Invitrogen, Inc), stored overnight in 4°C, then removed from RNA Later and archived in −80°C. RNA was extracted from specimens using RNeasy (Qiagen, Inc.), and cDNA was generated with reverse transcriptase (Omniscript RT, Qiagen, Inc). Primers designed by the study authors were used to create 553 bp and 477 bp amplicons (PCR performed using Platinum PCR SuperMix High Fidelity, Invitrogen, Inc) spanning codons 532–554 of exon 9 (helical domain; 9F: 5′ GCAGTGTGGTGAAGTTTCCA 3′, 9R: 5′ TGGCCAATCCTTTGATTTGT 3′) and c1011–1062 of exon 20 (kinase domain; 20F: 5′ ACTGCGTGGCAACCTTTATC 3′, 20R: 5′ TGATGGTGTGGAAGATCCAA 3′) of the Pik3ca gene, respectively, which include mutation hotspot regions. Sanger sequencing of the amplicons was performed at the BioPolymers Facility at Harvard Medical School, and results analyzed with Sequencher 4.10.1 (Gene Codes, Inc).
In vivo treatment of a GEM model for sporadic CRC
Apc CKO mice were treated with Adeno-Cre and followed by optical colonoscopy, as previously described . As a colonoscopic metric for tumor size, the Tumor Size Index (TSI) was calculated as (tumor area/colonic lumen area)×100 (%). Tumor-bearing mice were randomly assigned to treatment with either control vehicle alone (n = 8) or 45 mg/kg body weight NVP-BEZ235 in 10% 1-methyl-2-pyrrolidone/90% PEG 300 (n = 8) by daily oral gavage for 28 days. The treatment dose was chosen based on literature indicating that 40–50 mg/kg body weight NVP-BEZ235 effectively treats murine tumor models without adverse effects , , , , , . Based on pharmacokinetic studies demonstrating maximal tissue concentration one hour after NVP-BEZ235 administration, tumor-bearing mice were sacrificed one hour after final treatment dose . Colonic tumor volume was assessed using calipers (width×length×height) and tumors were harvested for both western blot analysis and immunohistochemistry.
Western blot analysis
Concentrations of whole cell or tumor lysates were determined by Bio-Rad Protein Assay (Bio-Rad). 10 µg and 25 µg protein lysate for whole cell and tumor, respectively, was separated on 10% SDS/PAGE gel, transferred to nitrocellulose membrane, blocked in 1% BSA for one hour, incubated at room temperature for two hours with primary antibody and one hour with secondary antibody. Detection was performed using the Amersham™ ECL™ Western Blot Detection Reagents (GE Healthcare). p-AKT Thr308 (1∶1000 dilution), p-AKT Ser473 (1∶2000 dilution), total AKT (1∶1000 dilution), p-S6 Ser240/244 (1∶3000 dilution), p-S6 Ser235/236 (1∶1000 dilution), S6 (1∶1000 dilution), cleaved caspase 3 (1∶1000 dilution), and cleaved PARP (1∶1000 dilution) were obtained from Cell Signaling Technologies (Beverly, MA). β-actin (1∶5000 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Peroxidase AffiniPure Donkey Anti- Rabbit Ig secondary antibody (1∶10,000 dilution) was obtained from Jackson ImmunoResearch (West Grove, PA) .
Five µm paraffin-embedded tissue sections were deparaffinized in xylene followed by alcohol rehydration. Antigen retrieval was performed in 1× citrate buffer (pH 6.0) (Zymed) using a Medical Decloaking Chamber (Biocare Medical). Slides were blocked at room temperature with Peroxidase Blocking Reagent (DAKO), normal donkey/rabbit serum, and Avidin/Biotin Blocking Kit (Vector Laboratories). Slides were incubated overnight at 4°C with primary antibody and 30 minutes at room temperature with secondary antibody. The Vectastain ABC kit (Vector Laboratories) was used for detection per manufacturer's instructions. Slides were stained with the Liquid DAB+Substrate Chromogen System (Dako) per manufacturer's instructions and counterstained with Mayer's hematoxylin solution and Scott's Bluing solution. p-AKT Ser473 (1∶50 dilution), p-S6 Ser240/244 (1∶50 dilution), p-S6 Ser235/236 (1∶100 dilution), were obtained from Cell Signaling Technologies (Beverly, MA). CD31 (1∶100 dilution) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). KI-67 (1∶100 dilution) was obtained from US Biological (Swampscott, MA). TUNEL assay (Apoptag) was purchased from Millipore (Billerica, MA) . The KI-67 proliferation index was calculated as the mean number of KI-67 positive cells/total number of glandular cells per high power field (mean of 16 high power fields)×100, and microvessel density (MVD) was calculated as number of CD31 positive cells per high power field (mean of 16 high power fields). TUNEL positivity index was calculated as mean number of TUNEL positive cells/total number of glandular cells per high power field (mean of 16 high power fields)×100. Measurements were performed by three blinded, independent observers in four control and four treated tumors.
Comparisons of final tumor volume, KI-67, MVD, and apoptotic cells between control and NVP-BEZ235-treated cohorts were calculated using the two-tailed Independent-Samples T Test. Pre- and post-treatment TSI values were compared with the Wilcoxon signed-rank test. P<0.05 was considered significant for all analyses. All analyses were calculated using SPSS 18.0 for Windows (IBM, Inc).
In vitro NVP-BEZ235 treatment of human CRC cell lines decreases cellular proliferation but has no effect on apoptosis
To examine the effects of in vitro NVP-BEZ235 treatment on cellular viability, three human CRC cell lines (HCT116, DLD-1, and SW480) were treated with increasing amounts of NVP-BEZ235 for 48 hours, and cellular viability was assessed by a colorimetric MTS assay. These studies revealed a similar dose-dependent decrease in viability after NVP-BEZ235 treatment (mean IC50 of three separate experiments = 14.3±6.4, 9.0±1.5, and 12.0±1.6 nM for HCT116, DLD-1, and SW480 cell lines, respectively; p = 0.74) for all three cell lines (Figure 1A). To determine if the observed decrease in cellular viability after NVP-BEZ235 treatment resulted from an induction of apoptosis, western blot analysis for cleaved caspase-3 and cleaved PARP was performed, revealing no increase in these apoptotic markers with NVP-BEZ235 treatment (Figure 1B). Taken together, these studies suggest that in vitro treatment with NVP-BEZ235 results in an equivalent decrease in cellular proliferation in two CRC cell lines harboring distinct PIK3CA mutations (HCT116 and DLD-1) as well as in a PIK3CA wild-type colorectal cancer cell (SW480), with no effect on apoptosis.
(A) Cell viability of HCT116, DLD-1, and SW480 CRC cell lines was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of three independent experiments. (B) Western blot analysis for p-AKTThr308, p-AKTSer473, p-S6Ser240/244, p-S6Ser235/236, cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.
In vitro NVP-BEZ235 treatment of human CRC cell lines results in sustained mTORC1 and mTORC2 inhibition, but transient PI3K blockade
To examine the effects of in vitro NVP-BEZ235 treatment on PI3K (p-AKTThr308), mTORC1 (p-S6Ser235/236 and p-S6Ser240/244), and mTORC2 (p-AKTSer473) mTOR signaling, western blot analysis was performed. After two hours of NVP-BEZ235 treatment, levels of p-AKTThr308, p-AKTSer473, p-S6Ser235/236, and p-S6Ser240/244 were all significantly decreased. Whereas a sustained decrease was observed in the levels of p-AKTSer473, p-S6Ser235/236, and p-S6Ser240/244, full inhibition of p-AKTThr308 was lost in as little as six hours (Figure 1B). Taken together, these results suggest that in vitro NVP-BEZ235 treatment results in sustained inhibition of mTORC1 and mTORC2 signaling, but that PI3K blockade is transient.
The efficacy of in vitro NVP-BEZ235 treatment of human CRC cell lines does not depend on PIK3CA mutational status
The comparable efficacy of NVP-BEZ235 treatment in PIK3CA mutant (HCT116 and DLD-1) and PIK3CA wild type (SW480) human CRC cell lines suggests that its clinical efficacy might not be limited to those patients whose tumors contain activating PIK3CA mutations. To further examine this possibility, we assessed the effect of NVP-BEZ235 on cellular proliferation and intracellular cell signaling in paired PIK3CA mutant and wild-type cell lines. The PIK3CA mutant cell line was derived from DLD-1 through disruption of the wild-type PIK3CA allele by targeted homologous recombination, whereas the PIK3CA wild-type cell line was derived through disruption of the mutant PIK3CA allele (a kind gift from B. Vogelstein) . As such, the genetic composition of these cells differs only at the PIK3CA locus, making these otherwise perfect isogenic controls. We found similar IC50's for NVP-BEZ235 in both PIK3CA mutant and wild-type DLD-1 cells (mean IC50 of three separate experiments = 15.1±6.0 and 12.1±4.3 nM for DLD-1 PIK3CA mutant and wild-type cell lines, respectively; p = 0.82, Figure 2A). Western blot analysis of p-AKT and p-S6 revealed sustained inhibition of p-AKTSer473, p-S6Ser235/236, and p-S6Ser240/244; however, the inhibition of p-AKTThr308 was transient in both cell lines (Figure 2B). Furthermore, NVP-BEZ235 treatment did not increase levels of cleaved caspase-3 and cleaved PARP in either cell line (Figure 2B). Overall, these findings suggest that PIK3CA mutational status does not predict the efficacy of NVP-BEZ235 treatment in human CRC cell lines.
(A) Cell viability of mutant and wild-type isogenic PIK3CA cells was assessed by MTS assay after treatment with increasing concentrations (0–500 nM) of NVP-BEZ235 for 48 hours. Results shown are the mean of four independent experiments. (B) Western blot analysis for p-AKTThr308, p-AKTSer473, p-S6Ser240/244, p-S6Ser235/236, cleaved caspase 3, and cleaved PARP was performed after 2, 6, 24, and 48 hours incubation with (−) 0 or (+) 500 nM NVP-BEZ235.
In vivo NVP-BEZ235 treatment induces tumor regression in a GEM model for sporadic PIK3CA wild-type CRC
To examine the effects of in vivo NVP-BEZ235 treatment on colonic tumor growth, we used a novel GEM model for sporadic CRC that we have recently described . Adenovirus expressing Cre recombinase (Adeno-Cre) was used to induce colonic tumors in floxed Apc mice. Tumors from 10 mice were analyzed for the presence of activating mutations by direct sequencing of exons 9 and 20 of the Pik3ca gene. No mutations were identified, which suggests that these mice are representative of PIK3CA wild-type CRC. Optical colonoscopy was used to randomize treatment of comparably sized colonic tumors with control drug vehicle or 45 mg/kg NVP-BEZ235 by daily oral gavage for 28 days. We noted no toxicity or side effects during this drug treatment regimen. Subsequent longitudinal growth or regression of individual colonic tumors was determined by optical colonoscopy, as previously described . For each tumor, a relative Tumor Size Index (TSI) metric was calculated as tumor size (T) normalized to colonic luminal area (L) (Figure 3A).
Mice with colonic tumors were randomized to treatment with control diluent (N = 8) or 45 mg/kg NVP-BEZ235 (N = 8) by daily oral gavage for 28 days. Resulting colonic tumor growth or regression was serially examined by optical colonoscopy. (A) Tumor Size Index (TSI) was calculated as Tumor Area (T) divided by Lumen Area (L) ×100. (B) Representative tumor colonoscopy still images during the 28 day treatment period. (C) Tumor volume of control and NVP-BEZ235-treated tumors at necropsy (mean 65 mm3 vs. 5 mm3; p = 0.01). (D) Final TSI vs. Tumor Volume (R2 = 0.89, P<0.0001). Change in mean TSI for (E) control (32% pre-treatment vs. 57% post-treatment, P = 0.01) and (F) treated (32% vs. 20%, P = 0.02) cohorts.
A representative time course of colonoscopy images for control (n = 8) and NVP-BEZ235-treated tumors (n = 8) is shown in Figure 3B. Tumor volume at necropsy was significantly larger in control vs. treated groups (65 mm3 vs. 5 mm3; p = 0.01; Figure 3C). In accordance with our previous analyses , the final TSI in both treatment groups positively correlated with tumor volume at necropsy (R2 = 0.89, p = 0.0001; Figure 3D). The mean TSI in the control group significantly increased over the treatment period (32% pre-treatment vs. 57% post-treatment, p = 0.01; Figure 3E), whereas that in the NVP-BEZ235 cohort significantly decreased (36% vs. 20%, p = 0.008; Figure 3F). Every tumor in the control group increased in size; treated tumors all decreased in size. Taken together, these results demonstrate that in vivo treatment with NVP-BEZ235 results in significant regression in PIK3CA wild-type colonic tumors.
In vivo NVP-BEZ235 treatment of a GEM model for sporadic CRC results in sustained mTORC1 and mTORC2 inhibition, but transient PI3K blockade
To examine the effects of in vivo NVP-BEZ235 treatment on PI3K and mTOR signaling, western blot analysis was performed in colonic tumors that were harvested one hour after final drug dosing on days 5 and 28 of treatment. Western blot analysis for levels of p-AKTThr308, p-S6Ser240/244, and p-AKTSer473 was performed as surrogates for activation of the PI3K, mTORC1, and mTORC2 pathways, respectively. A sustained decrease in levels of p-AKTSer473 and p-S6Ser240/244 was observed in the tumors of mice treated with NVP-BEZ235, as compared to control diluent (Figure 4A and 4B). These findings were confirmed by tumor immunohistochemistry (Figure 4C). As with our in vitro studies, an initial decrease in levels of p-AKTThr308 was observed at five days after treatment, but normalized by 28 days (Figures 4A and 4B). Taken together, these results suggest that in vivo NVP-BEZ235 treatment results in sustained inhibition mTORC1 and mTORC2, but transient PI3K blockade.
Mice with colonic tumors were randomized to treatment with control diluent or 45 mg/kg NVP-BEZ235 by daily oral gavage for five days. Western blot analysis of p-AKTThr308, p-AKTSer473, and p-S6Ser240/244 was performed for tumors treated with (−) control diluent or (+) NVP-BEZ235 for (A) five and (B) 28 days. (C) Immunohistochemistry of p-AKTSer473, p-S6Ser240/244, and p-S6Ser235/236 was performed for tumors treated with control diluent or NVP-BEZ235 for 28 days.
In vivo NVP-BEZ235 treatment of a GEM model for sporadic CRC inhibits proliferation, has no effect on apoptosis, and blocks tumor angiogenesis
To examine the effects of in vivo NVP-BEZ235 treatment on cellular proliferation, colonic tumors were examined by immunohistochemistry for the proliferation marker KI-67. This analysis revealed that proliferation decreased by 56% after 28 days of NVP-BEZ235 treatment (p = 0.003, Figure 5A). To assess the effect of NVP-BEZ235 treatment on cellular apoptosis, colonic tumors were examined by TUNEL assay. No differences were seen between tumors treated with control diluent and NVP-BEZ235 (p = 0.9, Figure 5B). As the PI3K and mTOR pathways play a significant role in tumor angiogenesis , , we examined the effects of NVP-BEZ235 treatment on microvessel density (MVD) through immunohistochemistry for the endothelial marker CD31. This analysis revealed that MVD decreased by 75% after 28 days of NVP-BEZ235 treatment (p = 0.013, Figure 5C). Taken together, these results suggest that in vivo treatment with NVP-BEZ235 results in a significant decrease in tumor proliferation, does not induce cellular apoptosis, and inhibits tumor angiogenesis.
(A) Representative immunohistochemistry for the proliferation marker KI-67 after treatment with control diluent or NVP-BEZ2355 for 28 days (p = 0.003). KI-67 proliferation index was calculated as average number of positive tumor cells per high power field. (B) TUNEL immunohistochemistry was performed in tumors treated with control diluent or NVP-BEZ235 for 28 days (p = 0.9). TUNEL staining was quantified as percent positive cells per high powered field. (C) Representative immunohistochemistry for the endothelial marker PECAM after treatment with control diluent or 45 mg/kg NVP-BEZ2355 (p = 0.013). Microvessel density was calculated as average number of positive vessels per high power field.
Because of their central role in the initiation and progression of CRC, blockade of the mTOR and PI3K signaling pathways has emerged as a compelling target for the development of novel CRC therapeutics , –. We and others have demonstrated the efficacy of mTORC1 pathway inhibition in preclinical CRC models , . However, an early clinical trial examining mTORC1 inhibitors in human CRC patients demonstrated modest results, perhaps due to loss of an mTORC1-dependent feedback loop that limits PI3K activation and/or continued mTORC2-mediated activation of AKT , . Taken together, it appears dual PI3K/mTOR inhibitors, such as NVP-BEZ235, are required for maximal therapeutic efficacy.
Some studies have suggested that PIK3CA mutant cancers are “oncogenically addicted” to PI3K signaling, leading to increased sensitivity to PI3K inhibitor therapy , , . However, one group has reported no association between PIK3CA mutational status and response to PI3K inhibitors . Nonetheless, another group has reported exclusively targeting PIK3CA mutant patients for PI3K/mTOR therapy . As PIK3CA mutations are seen in only 17% of human CRC, this approach would significantly limit the clinical impact of such agents . Because NVP-BEZ235 equally inhibits the mutant and wild-type forms of PIK3CA, we hypothesized that NVP-BEZ235 would have significant efficacy in human wild-type PIK3CA CRC . In support of this, we found comparable sensitivity to in vitro NVP-BEZ235 treatment in PIK3CA mutant (HCT116, kinase domain mutation; DLD-1, helical domain mutation) and PIK3CA wild-type (SW480) CRC cell lines, as well as in matched PIK3CA mutant and wild-type isogenic cell lines. Direct sequencing of the hot spot regions at exons 9 and 20 of Pik3ca in the colonic tumors validated our GEM model as a surrogate for PIK3CA wild-type CRC, which we used in our in vivo NVP-BEZ235 treatment studies. Taken together, these findings suggest that NVP-BEZ235 treatment may have clinical benefit in the 83% of CRC patients with wild-type PIK3CA. To further assess this feature, we sought to examine whether NVP-BEZ235 would be effective in a GEM model for sporadic PIK3CA wild-type CRC.
Traditional in vivo target validation approaches rely on xenograft platforms. Unfortunately, these models are not truly predictive of response in human patients, because they derive from high passage tumor cell lines grown in vitro. These cell lines are implanted into ectopic sites that do not resemble the colonic microenvironment, and therefore fail to recapitulate the heterogeneous nature of cancer and its supporting stroma . Although GEM cancer models address these shortcomings, most GEM models employ germ-line or tissue-wide modification of genes known to be mutated in human CRC. In addition, many CRC GEM models mainly present with small intestinal tumors . To accurately model human sporadic CRC, we have recently described a procedure in which Adeno-Cre is administered to floxed mice to somatically inactivate the Apc gene in a stochastic fashion and restrict tumor formation to the distal colon. The reproducible anatomical location of these tumors permits the use of optical colonoscopy to examine individual tumors in a longitudinal fashion .
To examine the efficacy of in vivo NVP-BEZ235 treatment, we used our GEM model for sporadic CRC. In our studies, there was strong correlation between the final tumor sizes assessed by colonoscopy versus necropsy (R2 = 0.89), thus validating our colonoscopy-based tumor sizing protocol. In accordance with our in vitro NVP-BEZ235 treatment of human CRC cell lines, colonic tumors from the GEM model decreased by 43% in size after in vivo treatment with NVP-BEZ235, whereas control tumors increased in size by 97% (p<0.0001). Similar findings have been reported with another dual PI3K/mTOR inhibitor, PKI-587, in a xenograft model of CRC . Taken together, these results suggest that NVP-BEZ235 would be an effective treatment in human CRC patients.
We studied the effect of NVP-BEZ235 treatment on the PI3K, mTORC1, and mTORC2 signaling pathways. Both in vitro and in vivo studies demonstrated sustained inhibition of mTORC1 and mTORC2 (reflected in decreased phosphorylation of S6 and AKTSer473, respectively), but transient blockade of PI3K activation by PDK1 (assessed by levels of p-AKTThr308). These findings are consistent with a report demonstrating the parallel roles of mTORC1 and mTORC2 in CRC carcinogenesis . Findings from other cancer models suggest that NVP-BEZ235 prevents mTORC2-dependent AKTSer473 phosphorylation, and that PI3K inhibition with NVP-BEZ235 may be short-lived due to loss of feedback inhibition of PI3K activity by the mTORC1 target S6 kinase , , . Therefore, we speculate that the addition of a selective PI3K inhibitor with efficacy against wild-type and mutant PIK3CA may augment the effectiveness of NVP-BEZ235 therapy. Taken together, our findings suggest that the efficacy of NVP-BEZ235 may derive predominantly from inhibition of mTORC1 and mTORC2 signaling.
We also examined possible mechanisms by which NVP-BEZ235 might induce tumor regression. In vivo NVP-BEZ235 treatment of colonic tumors resulted in a 56% decrease in cellular proliferation, but the absence of an induction in apoptosis. Although induction of apoptosis by NVP-BEZ235 treatment has been reported in lung, breast, renal cell carcinoma, and ovarian models , , , the absence of apoptosis has been found after treatment of glioma and sarcoma models , . Furthermore, in vivo NVP-BEZ235 treatment of colonic tumors resulted in a 75% decrease in microvessel density after NVP-BEZ235 treatment. Similar findings have been described in pancreatic, glioma, and neuroendocrine tumor systems , , . It is interesting that though we observed a decrease in macroscopic tumor size after in vivo NVP-BEZ235 treatment, we did not see a corresponding induction of apoptosis. This finding could be explained by an increase in constitutive cell turnover resulting from a drug-induced inhibition of angiogenesis coupled to a simultaneous drug-induced decrease in cellular proliferation. Because this process occurred slowly over the 28 day time course, frank necrosis would not be seen. Taken together, these findings support the further investigation of combination therapy with NVP-BEZ235, cytotoxic, and/or anti-angiogenic agents in CRC patients.
In summary, we present evidence for the therapeutic efficacy of NVP-BEZ235 in PIK3CA wild-type CRC. Furthermore, our results suggest that the efficacy of NVP-BEZ235 treatment may be augmented in if used in combination with additional PI3K inhibition, cytotoxic, and/or anti-angiogenic agents. Taken together, our findings provide compelling evidence for the further exploration of NVP-BEZ235-based therapies in clinical trials for CRC.
We would like to thank Alain Charest and Raju Kucherlapati for critical review of this manuscript.
Conceived and designed the experiments: JR MR KH. Performed the experiments: JR MR MS EC LL WW LR WC. Analyzed the data: JR MR KH. Contributed reagents/materials/analysis tools: JR MR MS EC LL WW RB EM KH PC. Wrote the paper: JR MR KH.
- 1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60: 277–300.
- 2. Wolpin BM, Mayer RJ (2008) Systemic treatment of colorectal cancer. Gastroenterology 134: 1296–1310.
- 3. Efeyan A, Sabatini DM (2010) mTOR and cancer: many loops in one pathway. Current Opinion in Cell Biology 22: 169–176.
- 4. Zoncu R, Efeyan A, Sabatini DM (2011) mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 12: 21–35.
- 5. Engelman JA, Luo J, Cantley LC (2006) The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet 7: 606–619.
- 6. Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, et al. (2004) High frequency of mutations of the PIK3CA gene in human cancers. Science 304: 554.
- 7. Samuels Y, Diaz LA, Schmidt-Kittler O, Cummins JM, Delong L, et al. (2005) Mutant PIK3CA promotes cell growth and invasion of human cancer cells. Cancer Cell 7: 561–573.
- 8. Alessi DR, James SR, Downes CP, Holmes AB, Gaffney PR, et al. (1997) Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7: 261–269.
- 9. Currie RA, Walker KS, Gray A, Deak M, Casamayor A, et al. (1999) Role of phosphatidylinositol 3,4,5-trisphosphate in regulating the activity and localization of 3-phosphoinositide-dependent protein kinase-1. Biochem J 337(Pt 3): 575–583.
- 10. Fujishita T, Aoki M, Taketo MM (2009) The role of mTORC1 pathway in intestinal tumorigenesis. Cell Cycle 8: 3684–3687.
- 11. Hung KE, Maricevich MA, Richard LG, Chen WY, Richardson MP, et al. (2010) Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment. Proc Natl Acad Sci USA 107: 1565–1570.
- 12. Fujishita T, Aoki K, Lane HA, Aoki M, Taketo MM (2008) Inhibition of the mTORC1 pathway suppresses intestinal polyp formation and reduces mortality in ApcDelta716 mice. Proc Natl Acad Sci USA 105: 13544–13549.
- 13. Tabernero J, Rojo F, Calvo E, Burris H, Judson I, et al. (2008) Dose- and schedule-dependent inhibition of the mammalian target of rapamycin pathway with everolimus: a phase I tumor pharmacodynamic study in patients with advanced solid tumors. J Clin Oncol 26: 1603–1610.
- 14. Sturgill TW, Hall MN (2009) Activating mutations in TOR are in similar structures as oncogenic mutations in PI3KCalpha. ACS Chem Biol 4: 999–1015.
- 15. Cao P, Maira S-M, García-Echeverría C, Hedley DW (2009) Activity of a novel, dual PI3-kinase/mTor inhibitor NVP-BEZ235 against primary human pancreatic cancers grown as orthotopic xenografts. Br J Cancer 100: 1267–1276.
- 16. Chiarini F, Grimaldi C, Ricci F, Tazzari PL, Evangelisti C, et al. (2010) Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against T-cell acute lymphoblastic leukemia. Cancer Res 70: 8097–8107.
- 17. McMillin DW, Ooi M, Delmore J, Negri J, Hayden P, et al. (2009) Antimyeloma activity of the orally bioavailable dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235. Cancer Res 69: 5835–5842.
- 18. Xu C-X, Zhao L, Yue P, Fang G, Tao H, et al. (2011) Augmentation of NVP-BEZ235's anticancer activity against human lung cancer cells by blockage of autophagy. Cancer Biol Ther 12: [Epub ahead of print].
- 19. Zitzmann K, Rüden J von, Brand S, Göke B, Lichtl J, et al. (2010) Compensatory activation of Akt in response to mTOR and Raf inhibitors - a rationale for dual-targeted therapy approaches in neuroendocrine tumor disease. Cancer Lett 295: 100–109.
- 20. Sunayama J, Matsuda K-I, Sato A, Tachibana K, Suzuki K, et al. (2010) Crosstalk Between the PI3K/mTOR and MEK/ERK Pathways Involved in the Maintenance of Self-Renewal and Tumorigenicity of Glioblastoma Stem-Like Cells. Stem Cells 28: 1930–9.
- 21. Faber AC, Li D, Song Y, Liang M-C, Yeap BY, et al. (2009) Differential induction of apoptosis in HER2 and EGFR addicted cancers following PI3K inhibition. Proc Natl Acad Sci USA 106: 19503–19508.
- 22. Sunayama J, Sato A, Matsuda K-I, Tachibana K, Suzuki K, et al. (2010) Dual blocking of mTor and PI3K elicits a prodifferentiation effect on glioblastoma stem-like cells. Neuro Oncol 12: 1205–19.
- 23. Bhatt AP, Bhende PM, Sin S-H, Roy D, Dittmer DP, et al. (2010) Dual inhibition of PI3K and mTOR inhibits autocrine and paracrine proliferative loops in PI3K/Akt/mTOR-addicted lymphomas. Blood 115: 4455–4463.
- 24. Konstantinidou G, Bey EA, Rabellino A, Schuster K, Maira MS, et al. (2009) Dual phosphoinositide 3-kinase/mammalian target of rapamycin blockade is an effective radiosensitizing strategy for the treatment of non-small cell lung cancer harboring K-RAS mutations. Cancer Res 69: 7644–7652.
- 25. Chapuis N, Tamburini J, Green AS, Vignon C, Bardet V, et al. (2010) Dual inhibition of PI3K and mTORC1/2 signalling by NVP-BEZ235 as a new therapeutic strategy for acute myeloid leukemia. Clin Cancer Res 16: 5424–35.
- 26. Santiskulvong C, Konecny GE, Fekete M, Chen K-YM, Karam A, et al. (2011) Dual Targeting of Phosphoinositide 3-Kinase and Mammalian Target of Rapamycin Using NVP-BEZ235 as a Novel Therapeutic Approach in Human Ovarian Carcinoma. Clin Cancer Res 17: 2373–84.
- 27. Roccaro AM, Sacco A, Husu EN, Pitsillides C, Vesole S, et al. (2010) Dual targeting of the PI3K/Akt/mTOR pathway as an antitumor strategy in Waldenstrom macroglobulinemia. Blood 115: 559–569.
- 28. Engelman JA, Chen L, Tan X, Crosby K, Guimaraes AR, et al. (2008) Effective use of PI3K and MEK inhibitors to treat mutant Kras G12D and PIK3CA H1047R murine lung cancers. Nat Med 14: 1351–1356.
- 29. Schnell CR, Stauffer F, Allegrini PR, O'Reilly T, McSheehy PMJ, et al. (2008) Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical imaging. Cancer Res 68: 6598–6607.
- 30. Pollizzi K, Malinowska-Kolodziej I, Stumm M, Lane H, Kwiatkowski D (2009) Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis. Mol Cancer 8: 38.
- 31. Masuda M, Shimomura M, Kobayashi K, Kojima S, Nakatsura T (2011) Growth inhibition by NVP-BEZ235, a dual PI3K/mTOR inhibitor, in hepatocellular carcinoma cell lines. Oncol Rep. [Epub ahead of print].
- 32. Maira S-M, Stauffer F, Brueggen J, Furet P, Schnell C, et al. (2008) Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther 7: 1851–1863.
- 33. Kong D, Dan S, Yamazaki K, Yamori T (2010) Inhibition profiles of phosphatidylinositol 3-kinase inhibitors against PI3K superfamily and human cancer cell line panel JFCR39. Eur J Cancer 46: 1111–1121.
- 34. Buonamici S, Williams J, Morrissey M, Wang A, Guo R, et al. (2010) Interfering with resistance to smoothened antagonists by inhibition of the PI3K pathway in medulloblastoma. Sci Transl Med 2: 51ra70.
- 35. Lee M, Theodoropoulou M, Graw J, Roncaroli F, Zatelli MC, et al. (2011) Levels of p27 Sensitize to Dual PI3K/mTOR Inhibition. Mol Cancer Ther. [Epub ahead of print] doi: 10.1158/1535-7163.MCT-11-0188.
- 36. Manara MC, Nicoletti G, Zambelli D, Ventura S, Guerzoni C, et al. (2010) NVP-BEZ235 as a new therapeutic option for sarcomas. Clin Cancer Res 16: 530–540.
- 37. Martin SK, Fitter S, Bong LF, Drew JJ, Gronthos S, et al. (2010) NVP-BEZ235, a dual pan class I PI3 kinase and mTOR inhibitor, promotes osteogenic differentiation in human mesenchymal stromal cells. J Bone Miner Res 25: 2126–2137.
- 38. Serra V, Markman B, Scaltriti M, Eichhorn PJA, Valero V, et al. (2008) NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations. Cancer Res 68: 8022–8030.
- 39. Liu T-J, Koul D, LaFortune T, Tiao N, Shen RJ, et al. (2009) NVP-BEZ235, a novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, elicits multifaceted antitumor activities in human gliomas. Mol Cancer Ther 8: 2204–2210.
- 40. Eichhorn PJA, Gili M, Scaltriti M, Serra V, Guzman M, et al. (2008) Phosphatidylinositol 3-kinase hyperactivation results in lapatinib resistance that is reversed by the mTOR/phosphatidylinositol 3-kinase inhibitor NVP-BEZ235. Cancer Res 68: 9221–9230.
- 41. Brachmann SM, Hofmann I, Schnell C, Fritsch C, Wee S, et al. (2009) Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells. Proc Natl Acad Sci USA 106: 22299–22304.
- 42. Marone R, Erhart D, Mertz AC, Bohnacker T, Schnell C, et al. (2009) Targeting melanoma with dual phosphoinositide 3-kinase/mammalian target of rapamycin inhibitors. Mol Cancer Res 7: 601–613.
- 43. Roulin D, Waselle L, Dormond-Meuwly A, Dufour M, Demartines N, et al. (2011) Targeting renal cell carcinoma with NVP-BEZ235, a dual PI3K/mTOR inhibitor, in combination with sorafenib. Mol Cancer 10: 90.
- 44. Xu C-X, Li Y, Yue P, Owonikoko TK, Ramalingam SS, et al. (2011) The Combination of RAD001 and NVP-BEZ235 Exerts Synergistic Anticancer Activity against Non-Small Cell Lung Cancer In Vitro and In Vivo. PLoS ONE 6: e20899.
- 45. Herrera VA, Zeindl-Eberhart E, Jung A, Huber RM, Bergner A (2011) The dual PI3K/mTOR inhibitor BEZ235 is effective in lung cancer cell lines. Anticancer Res 31: 849–854.
- 46. Bhende PM, Park SI, Lim MS, Dittmer DP, Damania B (2010) The dual PI3K/mTOR inhibitor, NVP-BEZ235, is efficacious against follicular lymphoma. Leukemia 24: 1781–1784.
- 47. Cho DC, Cohen MB, Panka DJ, Collins M, Ghebremichael M, et al. (2010) The efficacy of the novel dual PI3-kinase/mTOR inhibitor NVP-BEZ235 compared with rapamycin in renal cell carcinoma. Clin Cancer Res 16: 3628–3638.
- 48. Baumann P, Mandl-Weber S, Oduncu F, Schmidmaier R (2009) The novel orally bioavailable inhibitor of phosphoinositol-3-kinase and mammalian target of rapamycin, NVP-BEZ235, inhibits growth and proliferation in multiple myeloma. Exp Cell Res 315: 485–497.
- 49. Brünner-Kubath C, Shabbir W, Saferding V, Wagner R, Singer CF, et al. (2011) The PI3 kinase/mTOR blocker NVP-BEZ235 overrides resistance against irreversible ErbB inhibitors in breast cancer cells. Breast Cancer Res Treat 129: 387–400.
- 50. Dubrovska A, Kim S, Salamone RJ, Walker JR, Maira S-M, et al. (2009) The role of PTEN/Akt/PI3K signaling in the maintenance and viability of prostate cancer stem-like cell populations. Proc Natl Acad Sci USA 106: 268–273.
- 51. Di Nicolantonio F, Arena S, Tabernero J, Grosso S, Molinari F, et al. (2010) Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus. J Clin Invest 120: 2858–2866.
- 52. Tanaka H, Yoshida M, Tanimura H, Fujii T, Sakata K, et al. (2011) The Selective Class I PI3K Inhibitor CH5132799 Targets Human Cancers Harboring Oncogenic PIK3CA Mutations. Clinical Cancer Research 17: 3272–3281.
- 53. Baba Y, Nosho K, Shima K, Hayashi M, Meyerhardt JA, et al. (2011) Phosphorylated AKT expression is associated with PIK3CA mutation, low stage, and favorable outcome in 717 colorectal cancers. Cancer 117: 1399–1408.
- 54. Chang Q, Chen E, Hedley DW (2009) Effects of combined inhibition of MEK and mTOR on downstream signaling and tumor growth in pancreatic cancer xenograft models. Cancer Biol Ther 8: 1893–1901.
- 55. Lang SA, Gaumann A, Koehl GE, Seidel U, Bataille F, et al. (2007) Mammalian target of rapamycin is activated in human gastric cancer and serves as a target for therapy in an experimental model. Int J Cancer 120: 1803–1810.
- 56. Guertin DA, Sabatini DM (2007) Defining the role of mTOR in cancer. Cancer Cell 12: 9–22.
- 57. Bunney TD, Katan M (2010) Phosphoinositide signalling in cancer: beyond PI3K and PTEN. Nat Rev Cancer 10: 342–352.
- 58. Liu P, Cheng H, Roberts TM, Zhao JJ (2009) Targeting the phosphoinositide 3-kinase pathway in cancer. Nat Rev Drug Discov 8: 627–644.
- 59. Chalhoub N, Baker SJ (2009) PTEN and the PI3-kinase pathway in cancer. Annu Rev Pathol 4: 127–150.
- 60. Bhaskar PT, Hay N (2007) The Two TORCs and Akt. Developmental Cell 12: 487–502.
- 61. Ihle NT, Lemos R, Wipf P, Yacoub A, Mitchell C, et al. (2009) Mutations in the Phosphatidylinositol-3-Kinase Pathway Predict for Antitumor Activity of the Inhibitor PX-866 whereas Oncogenic Ras Is a Dominant Predictor for Resistance. Cancer Research 69: 143–150.
- 62. Guo X-N, Rajput A, Rose R, Hauser J, Beko A, et al. (2007) Mutant PIK3CA-bearing colon cancer cells display increased metastasis in an orthotopic model. Cancer Res 67: 5851–5858.
- 63. Martin-Fernandez C, Bales J, Hodgkinson C, Welman A, Welham MJ, et al. (2009) Blocking phosphoinositide 3-kinase activity in colorectal cancer cells reduces proliferation but does not increase apoptosis alone or in combination with cytotoxic drugs. Mol Cancer Res 7: 955–965.
- 64. Janku F, Tsimberidou AM, Garrido-Laguna I, Wang X, Luthra R, et al. (2011) PIK3CA Mutations in Patients with Advanced Cancers Treated with PI3K/AKT/mTOR Axis Inhibitors. Mol Cancer Ther 10: 558–565.
- 65. Sharpless NE, Depinho RA (2006) The mighty mouse: genetically engineered mouse models in cancer drug development. Nat Rev Drug Discov 5: 741–754.
- 66. Taketo MM, Edelmann W (2009) Mouse models of colon cancer. Gastroenterology 136: 780–798.
- 67. Mallon RG, Feldberg LR, Lucas J, Chaudhary I, Dehnhardt C, et al. (2011) Antitumor Efficacy of PKI-587, a Highly Potent Dual PI3K/mTOR Kinase Inhibitor. Clin Cancer Res 17: 3193–203.
- 68. Gulhati P, Bowen KA, Liu J, Stevens PD, Rychahou PG, et al. (2011) mTORC1 and mTORC2 regulate EMT, motility and metastasis of colorectal cancer via RhoA and Rac1 signaling pathways. Cancer Res 71(9): 3246–56.
- 69. Breuleux M, Klopfenstein M, Stephan C, Doughty CA, Barys L, et al. (2009) Increased AKT S473 phosphorylation after mTORC1 inhibition is rictor dependent and does not predict tumor cell response to PI3K/mTOR inhibition. Mol Cancer Ther 8: 742–753.