Effect Of NVP-BEZ235 Research Articles

The synergistic antitumor effect of IL-6 neutralization with NVP-BEZ235 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) still ranks among the top cancers worldwide with high incidence and mortality. Due to abnormal activation of the PI3K/AKT/mTOR signalling pathway in HCC, targeting this pathway represents a potential therapeutic strategy. NVP-BEZ235 is a novel dual-targeted ATP-competitive PI3K/mTOR inhibitor that has shown effective antitumor effects. In this study, we found that interleukin-6 (IL-6) was significantly increased after exposure to NVP-BEZ235, and we proposed a treatment in which an anti-IL-6 antibody was combined with NVP-BEZ235 for HCC. In vitro results revealed that targeted inhibition of IL-6 potentiated the antitumor effects of NVP-BEZ235 in HCC cells. The mechanism might be attributed to their synergistic inhibitory activity on the PI3K/AKT/mTOR signalling pathway. Furthermore, an in vivo study demonstrated that combined administration of NVP-BEZ235 and anti-IL-6 Ab reduced HCC tumour load more effectively than either NVP-BEZ235 or anti-IL-6 Ab treatment alone. These findings add guidance value to the analysis of HCC and provide a reference for clinical treatment.

Abstract 3908: The combination of NVP-BEZ235 & endostatin exerts synergistic anticancer activity against triple-negative breast cancer in vitro and in vivo

Abstract Background: Triple-negative breast cancer(TNBC) take up 15% of invasive breast cancer. TNBC has no effective molecular targets & unfavourable prognosis. We investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, a combine with or not an angiogenesis inhibitor Endostatin in the orthotopic TNBC model & explored its anti-tumor & anti-angiogenesis mechanisms. Material & Methods: Assess sensitivity of the cells to the drug by MTT assays.Orthotopic xenografts were performed for tumor growth and survival studies. Additionally, Immunohistochemistry for VEGF and MVD(CD34) was performed using the EnVision/HRP technique,Serum VEGF was detected by ELISA method. Results: BEZ235 effectively inhibited cell proliferation in vitro & provided additive effects when combine with Endostatin.Treatment with BEZ235 & Endostatin resulted in inhibition of tumor growth & prolongation of survival time of tumor-bearing mice in vivo.Immunohistochemical analysis revealed that intratumoral proliferation & angiogenesis was significantly suppressed. Furthermore, Changing of the number of Serum VEGF support the finding of angiogenesis inhibition too. Conclusion: Our findings suggest that BEZ235 exerts antitumor effects against TNBC & enhances effects of Endostatin through inhibition of cell proliferation & tumor angiogenesis. This approach may represent a promising combination targeted therapy for TNBC treatment. Note: This abstract was not presented at the meeting. Citation Format: Xiaoshan Wang, Xiao Zhang, Fanghua Li. The combination of NVP-BEZ235 & endostatin exerts synergistic anticancer activity against triple-negative breast cancer in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3908.

Abstract 2941: Combination of hemiasterlin derivative (R)(S)(S)-BF65 and PI3K/mTOR dual inhibitor NVP-BEZ235 as a new strategy to inhibit ovarian cancer growth and metastasis

Abstract Metastasis is a big issue to cancer therapy and is the major cause of therapeutic failure and cancer death. Therefore, inhibiting cancer metastasis is a key challenge in cancer treatment. Ovarian cancer is the most deadly cancer among women and combination treatment strategy is usually used to fight against this disease not only to reduce the toxicity and side effects, but also to enhance the anticancer effect. (R)(S)(S)-BF65 is a hemiasterlin derivative and potent anti-tubulin agent with IC50 values in the low nanomolar range against various cancer cells. Previous studies indicated that (R)(S)(S)-BF65 in combination with an allosteric Akt inhibitor MK-2206 synergistically inhibited the growth of SKOV3 human ovarian cancer cells. Using the MTT assay, we demonstrated that (R)(S)(S)-BF65 could also enhanced the growth inhibitory effect of NVP-BEZ235, a PI3K/mTOR dual inhibitor in SKOV3 cells. The IC50 was 11.6 μM for NVP-BEZ235 and 20.6 nM for (R)(S)(S)-BF65 after 48 h-treatment. With a molar ratio of 1:1000 or 1:500, the combination of (R)(S)(S)-BF65 and NVP-BEZ235 displayed an additive to mild synergistic cytotoxic effect with CI50 values of 0.96 and 0.91, respectively. Interestingly, the combination of (R)(S)(S)-BF65 and NVP-BEZ235, at concentrations much lower than those required for growth inhibition, significantly inhibited cell migration and invasion as demonstrated by wound healing migration, transwell migration and invasion assays. The combinatorial effect was much more effective than single treatments. Epithelial-mesenchymal transition (EMT) plays an important role in cancer metastasis. It has been reported that activation of the PI3K/Akt pathway leads to phosphorylation and inactivation of GSK3β, which may in turn induce EMT. Western blot analysis of EMT markers revealed that NVP-BEZ235 downregulated mesenchymal markers β-catenin and Slug, and upregulated the epithelial marker, E-cadherin. (R)(S)(S)-BF65 also downregulated mesenchymal markers β-catenin and Snail, but had no effect on E-cadherin. The combination of both led to downregulation of more mesenchymal markers including β-catenin, Slug, Snail and ZEB1, and upregulation of E-cadherin, which may at least in part explain why the combination was more effective than single treatments to suppress cell migration and invasion. Taken together, the combination of (R)(S)(S)-BF65 and NVP-BEZ235 may be a promising strategy to inhibit ovarian cancer growth and metastasis. Citation Format: Po-Chia Su, Tzu-En Huang, Lih-Ching Hsu. Combination of hemiasterlin derivative (R)(S)(S)-BF65 and PI3K/mTOR dual inhibitor NVP-BEZ235 as a new strategy to inhibit ovarian cancer growth and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2941.

Abstract 3468: Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells

Abstract Molecular target drugs are used in the treatment of a variety of malignant tumors, but this approach to developing novel therapies for oral squamous cell carcinoma (OSCC) has lagged behind the progress seen for other cancers. The PI3K/AKT/mTOR and RAS/MAPK pathways are important pathways for the growth and survival of cancer cells. These pathways also have been activated such as AKT and ERK overexpression in OSCC. In this study, we investigated the antitumor effect of PI3K/mTOR inhibitor, NVP-BEZ235 and MEK1/2 inhibitor, Trametinib, in human OSCC cells. First, we confirmed the inhibitory effect of NPV-BEZ235 and Trametinib in PI3K/AKT/mTOR and RAS/MAPK pathways by Western blotting. NPV-BEZ235 and Trametinib were reduced phosphorylation of AKT, p70S6K, and MEK1/2 in OSCC cells. Next, we examined the anti-proliferative effect of NVP-BEZ235 and Trametinib using 4 human OSCC cell lines by WST-8 assay. NVP-BEZ235 and Trametinib significantly inhibited the growth of all OSCC cells in a dose-dependent manner in vitro. Further, we investigated potential synergistic effects of inhibiting both the PI3K/AKT/mTOR and RAS/MAPK pathways, by treating with NVP-BEZ235 and Trametinib. The anti-proliferative effect of this combination was measured in OSCC cells by calculating the Combination Index. Combination treatment with NVP-BEZ235 and Trametinib showed a synergistic effect on the cell growth of all OSCC cells in vitro. Subsequently, we analyzed the effect of NBP-BEZ235 and Trametinib on the cell cycle of green fluorescent protein (GFP)-SAS cells by flow cytometry. Treated with NVP-BEZ235 showed no enhanced effect compared to control. Treatment of Trametinib resulted in the decrease in percentage of cells in G2 phase and increase in G1 phase. This indicated that the anti-proliferative effect of Trametinib was due to G1 arrest. Furthermore, treatment of Trametinib increased apoptosis cells in GFP-SAS cells. Next, we assessed the effect of NVP-BEZ235 and Trametinib on the in vivo growth of GFP-SAS cells. GFP-SAS tumor-bearing mice were treated with single, combination, or vehicle by oral daily. Treatment with NVP-BEZ235 and Trametinib significantly reduced the size of tumors compared with vehicle control. However, combination treatment showed no synergistic effect compared to single treatment. Finally, we investigated the gene expression changes with combination treatment using microarray analysis. The suppression of PI3K/AKT/mTOR and RAS/MAPK pathways caused overexpression of genes related to cell cycle and DNA-repair. These results suggest that activation of PI3K/AKT/mTOR and RAS/MAPK pathways support the growth of OSCC cells and NVP-BEZ235 and Trametinib may be a useful therapeutic strategy for OSCC. Citation Format: Norihiko Tokuzen, Koh-ichi Nakashiro. Antitumor effect by inhibition of PI3K/AKT/mTOR and RAS/MAPK pathways in human oral squamous cell carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3468.

Inhibitory effect of NVP-BEZ235 on gastric cancer in nude mice

Objective To investigate the effect of NVP-BEZ235 alone and combined with 5-fluorouracil (5-Fu) on NCI-N87 gastric cancer in nude mice. Methods human gastric cancer cell line NCI-N87 was used to prepare tumor bearing mice. Divided into control group (normal saline every 3 days 0.02 ml/g, every 3 days by intraperitoneal injection of saline 0.02 ml/g), group 5-Fu (normal saline every 3 days every 3 days by intraperitoneal injection of 0.02 ml/g, 5-Fu 0.025 mg/g), group NVP-BEZ235 (every 3 days by gavage once every 3 days NVP-BEZ235 0.04 mg/g, intraperitoneal injection of saline.0.02 ml/g), combination group (gavage once every 3 days NVP-BEZ235 0.04 mg/g, every 3 days by intraperitoneal injection of 5-Fu 0.025 mg/g). Recorded the volume and weight of tumor. Detection of apoptosis related proteins, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway related proteins and invasion related proteins by Western blotting. Results Compared with 5-Fu group (6.84±0.98), the relative volume of tumor in nude mice with NCI-N87 gastric cancer xenograft model was (8.47±1.44), and the difference was statistically significant (t=2.303, P=0.044)). Tumor weight (g): the combination group (0.83±0.16) was less than the 5-Fu group (1.20±0.32), the difference was statistically significant (t=2.524, P=0.038). Akt, p-Akt, the relative expression of mTOR in group NVP-BEZ235 (0.56±0.05, 0.23±0.04, 0.50±0.03) was significantly lower than the control group (0.96±0.11, 0.68±0.03, 0.64±0.12; t=9.739, 21.823, 3.366, P=0.000, 0.000, 0.006)), combination group (0.53±0.06, 0.20±0.03, 0.51±0.07) was significantly lower than that of group 5-Fu (0.93±0.10, 0.51±0.05, 0.67±0.04; t=8.234, 12.489, 4.628, P=0.000, 0.000, 0.002), there was significant difference between the four groups (F=46.457, 263.318, 6.678, P=0.000, 0.000, 0.002). The activation of apoptosis related protein cysteinyl aspartate-specific protease (Caspase)-3, B cell lymphoma/leukemia-2 associated X protein (bax) and B cell lymphoma/leukemia-2 (bcl-2), NVP-BEZ235 (1.28±0.06, 1.39±0.08, 0.59±0.04) was significantly higher than the control group (0.94±0.10, 0.99±0.11, 1.07±0.07; t=8.660, 8.077, 17.456, P=0.000, 0.000, 0.000) combined. The medicine group (1.57±0.09, 1.70±0.10, 0.37±0.07) was significantly higher than that of group 5-Fu (1.42±0.09, 1.50±0.05, 0.48±0.07; t=2.737, 4.472, 2.861, P=0.021, 0.003, 0.017), compared with a significant difference between the four groups (F=71.181, 81.393, 199.848, P=0.000, 0.000, 0.000). The relative expression of matrix metalloproteinase (MMP)-9 and vascular endothelial growth factor (VEGF), NVP-BEZ235 (0.44±0.04, 0.30±0.05) was significantly lower than the control group (0.59±0.06, 0.41±0.03; t=5.335, 5.550, P=0.000, 0.001), combination group (0.37±0.04, 0.24±0.07) was significantly lower than that of 5-Fu group (0.58±0.12, 0.36±0.04; t=4.144, 3.691, P=0.006, 0.005), compared with a significant difference between the four groups (F=15.164, 20.205, P=0.000, 0.000). Conclusion NVP-BEZ235 can down regulate the activation level of PI3K/Akt/mTOR pathway, promote apoptosis, inhibit tumor invasion and metastasis, and play a synergistic role with 5-Fu. Key words: Gastric cancer; NVP-BEZ235; Phosphatidylinositol 3 kinase/protein kinase B/mammalian target of rapamycin signaling pathway; Apoptosis; Invasion

Effects of NVP-BEZ235 on the proliferation, migration, apoptosis and autophagy in HT-29 human colorectal adenocarcinoma cells.

The phosphoinositide 3 kinase (PI3K)/Akt/mammalian target of the rapamycin (mTOR) pathway plays a significant role in colorectal adenocarcinoma. NVP-BEZ235 (dactolisib) is a novel dual inhibitor of PI3K/mTOR. The effects of NVP-BEZ235 in human colorectal adenocarcinoma are still unclear. In the present study, we aimed to explore the proliferation, migration, apoptosis and autophagy in HT-29 human colorectal adenocarcinoma cells. HT-29 human colorectal adenocarcinoma cells were treated with NVP-BEZ235 (0, 0.001, 0.01, 0.1, 1 and 3µM) for 24 and 48h, respectively. Cells were also treated with NVP-BEZ235 (0.1µM), DDP (100, 300 and 1,000µM), and NVP-BEZ235 (0.1µM) combined with DDP (100, 300 and 1,000µM) respectively, and cultured for 24h after treatment. MTT assay was utilized to evaluate the effects of NVP-BEZ235 alone or NVP-BEZ235 combined with cis-diamminedichloroplatinum (DDP) on proliferation of HT-29 cells. Cell wound-scratch assay was used detect cell migration. In addition, expression of microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B and LC3B) in HT-29 cells was detected by immunofluorescence at 48h after NVP-BEZ235 (1µM) treatment. Expression of proteins involved in cell cycle and proliferation (p-Akt, p-mTOR and cyclinD1), apoptosis (cleaved caspase-3), and autophagy (cleaved LC3B and Beclin-1) were detected by western blot analysis. NVP-BEZ235 inhibited the proliferation and migration of HT-29 human colorectal adenocarcinoma cells. NVP-BEZ235 decreased protein expression of p-Akt, p-mTOR and cyclinD1, and increased protein expression of cleaved caspase-3, cleaved LC3B and Beclin-1 as the concentrations and the incubation time of NVP-BEZ235 increased. In addition, NVP-BEZ235 and DDP had synergic effects in inhibiting cell proliferation and migration. The expression of protein involved in apoptosis (cleaved caspase-3) was higher in drug combination group compared to the NVP-BEZ235 single treatment group. NVP-BEZ235 inhibited the proliferation and migration, and induced apoptosis and autophagy of HT-29 human colorectal adenocarcinoma cells.

2806 PI3K/mTOR pathway inhibitors enhance radiosensitivity in radiation resistant oral squamous cell carcinomas by inducing cell cycle arrest

Background: Radiation therapy is often used to treat oral cancer but toxicity and local control remain problematic. PI3K and mTOR activation has been linked to decreased radiation responsiveness in oral cancer, so it limits efficacy of radiotherapy. In this study, we invertigated the NVPBEZ235, a dual PI3K/mTOR inhibitor in rediosensitization of OSCC cell lines and a radiation-resistant cell line. Additionally, we investigated further the effect of RAD001, NVP-BEZ235 on the primary OSCC tumors. Methods: We developed a radioresistant oral cancer cells (OML1-R), from the certain oral cancer patient in Taiwan by repeated exposure to step-wise fractionated ionizing radiation. We performed clonogenic survival assays on OSCC cell lines (SCC4, SCC25), OML1-R and exposed to increasing dose of radiation (0 to 4 Gy) in the presence of RAD001 and NVP-BEZ235. Analysis of target protein phosphorylation and cell cycle were performed. We also isolated four different primary cancer cells from tumor tissues of individual OSCC patients and examined the effects of radiation alone and in combination with NVP-BEZ235 by the clonogenic survival assay. Results: Combined treatment of the NVP-BEZ235 with radiation effectively inhibited phosphorylation of AKT, S6, 4EBP1 and eIF4E target proteins and reduced clonogenic survival in OSCC cell lines (SCC4 and SCC25) and OML1-R. Similarly, NVP-BEZ235 also sensitized all of the four primary OSCC cells to radiation. The treatment arrested OSCC cell lines, OML1-R and primary OSCC cells at G1 and G2/M phases of the cell cycle. NVPBEZ235 treatment reduced the protein levels of cyclin D1and CDK4 and also changed the protein levels of CDK inhibitors, p27 and p21 leading to G1 cell cycle delay. Besides, NVP-BEZ235 enhanced the cytotoxic effect of radiation on OSCC cells without inducing significant cell apoptotic and did not affect H2AX phosphorylation, as a marker of DNA damage. Conclusions: The present study identified radiosensitizing effect of NVPBEZ235 in oral cancer cells and primary tumors and the effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined, suggesting that BEZ235 may be as a potential therapeutic strategy for improving clinical outcome in OSCC. No conflict of interest.

Inhibition of PI3K Pathway Reduces Invasiveness and Epithelial-to-Mesenchymal Transition in Squamous Lung Cancer Cell Lines Harboring PIK3CA Gene Alterations

A prominent role in the pathogenesis of squamous cell carcinoma of the lung (SQCLC) has been attributed to the aberrant activation of the PI3K signaling pathway, due to amplification or mutations of the p110α subunit of class I phosphatidylinositol 3-kinase (PIK3CA) gene. The aim of our study was to determine whether different genetic alterations of PIK3CA affect the biologic properties of SQCLC and to evaluate the response to specific targeting agents in vitro and in vivo. The effects of NVP-BEZ235, NVP-BKM120, and NVP-BYL719 on two-dimensional/three-dimensional (2D/3D) cellular growth, epithelial-to-mesenchymal transition, and invasiveness were evaluated in E545K or H1047R PIK3CA-mutated SQCLC cells and in newly generated clones carrying PIK3CA alterations, as well as in a xenograft model. PIK3CA mutated/amplified cells showed increased growth rate and enhanced migration and invasiveness, associated with an increased activity of RhoA family proteins and the acquisition of a mesenchymal phenotype. PI3K inhibitors reverted this aggressive phenotype by reducing metalloproteinase production, RhoA activity, and the expression of mesenchymal markers, with the specific PI3K inhibitors NVP-BKM120 and NVP-BYL719 being more effective than the dual PI3K/mTOR inhibitor NVP-BEZ235. A xenograft model of SQCLC confirmed that PIK3CA mutation promotes the acquisition of a mesenchymal phenotype in vivo and proved the efficacy of its specific targeting drug NVP-BYL719 in reducing the growth and the expression of mesenchymal markers in xenotransplanted tumors. These data indicate that PIK3CA mutation/amplification may represent a good predictive feature for the clinical application of specific PI3K inhibitors in SQCLC patients.

Dual blockade of PI3K/AKT/mTOR (NVP-BEZ235) and Ras/Raf/MEK (AZD6244) pathways synergistically inhibit growth of primary endometrioid endometrial carcinoma cultures, whereas NVP-BEZ235 reduces tumor growth in the corresponding xenograft models

ObjectivesEndometrial carcinoma (EC) is the most common gynecological cancer in the Western World. Treatment options are limited for advanced and recurrent disease. Therefore, new treatment options are necessary. Inhibition of the PI3K/AKT/mTOR and/or the Ras/Raf/MEK pathways is suggested to be clinically relevant. However, the knowledge about the effect of combination targeted therapy in EC is limited. The aim of this study was to investigate the effect of these therapies on primary endometrioid EC cell cultures in vitro and in vivo. MethodsPrimary endometrioid EC cell cultures were incubated with Temsirolimus (mTORC1 inhibitor), NVP-BKM120 (pan-PI3K inhibitor), NVP-BEZ235 (pan-PI3K/mTOR inhibitor), or AZD6244 (MEK1/2 inhibitor) as single treatment. In vitro, the effect of NVP-BEZ235 with or without AZD6244 was determined for cell viability, cell cycle arrest, apoptosis induction, and cell signaling. In vivo, the effect of NVP-BEZ35 was investigated for 2 subcutaneous xenograft models of the corresponding primary cultures. ResultsNVP-BEZ235 was the most potent PI3K/AKT/mTOR pathway inhibitor. NVP-BEZ235 and AZD6244 reduced cell viability and induced cell cycle arrest and apoptosis, by reduction of p-AKT, p-S6, and p-ERK levels. Combination treatment showed a synergistic effect. In vivo, NVP-BEZ235 reduced tumor growth and inhibited p-S6 expression. The effects of the compounds were independent of the mutation profile of the cell cultures used. ConclusionsA synergistic antitumor effect was shown for NVP-BEZ235 and AZD6244 in primary endometrioid EC cells in vitro. In addition, NVP-BEZ235 induced reduction of tumor growth in vivo. Therefore, targeted therapies seem an interesting strategy to further evaluate in clinical trials.

Abstract 1253: Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells

Abstract The long-term goal of our research is to investigate the molecular carcinogenesis of esophageal squamous cell carcinoma and to develop strategies for esophageal cancer prevention. Previous studies in our laboratory demonstrated an association between activation of PI3K/AKT/mTOR pathways and the development of N-nitrosomethylbenzylamine (NMBA)-induced tumors in the rat esophagus. The objective of current study is to evaluate the effects of NVP-BEZ235, a dual PI3K/mTOR inhibitor, and MK2206, an AKT inhibitor, alone or in combination in esophageal cancer cell lines, KYSE-70, -150, -270 and -410. The cancer cell lines were treated with different doses of NVP-BEZ235 (1, 10, 50, 100, 1000 nMol/L) and MK2206 (0.5, 1, 3, 10, 20 uMol/L), or NVP-BEZ235 100 nMol/L plus MK2206 3 uMol/L. Western blot was performed to assess the activation of PI3K/AKT/mTOR pathways. Cell proliferation, apoptosis and cell cycle were analyzed by WST1 and flow cytometry. We found that the combination of NVP-BEZ235 and MK2206 significantly reduced the phosphorylation of p-AKTT308, p-AKTS473, p-mTORS2448, p-S6S235/236 and p-4EBP1T37/46, when compared to NVP-BEZ235 (1000 nMol/L) and MK2206 (20 uMol/L) alone (p<0.05). The cell proliferation rate was reduced by NVP-BEZ235 1000 nMol/L, MK2206 20 uMol/L, or NVP-BEZ235 plus MK2206 to 20% (p<0.01), 24% (p<0.01) and 17% (p<0.01), respectively. The NVP-BEZ235 1000 nMol/L and MK2206 10 uMol/L induced apoptosis in esophageal cancer cells to 30% and 20% (p<0.01), respectively. NVP-BEZ235 plus MK2206 induced apoptosis in esophageal cancer cells to 45% (p<0.01). The combination treatment increased the proportion of cells at G0/G1 phase compared to cells treated with NVP-BEZ235 or MK2206 alone. Our results indicate that co-targeting the PI3K/mTOR and AKT signaling pathways produces synergistic activity against esophageal carcinoma cell growth. This in-vitro study provides evidence to test the efficacy of NVP-BEZ235 in combination with MK2206 in inhibition of esophageal tumor development in vivo. (Supported by NIH/NCI R01 CA131073-01A1). Citation Format: Hao Yu, Ni Shi, Zui Pan, Tong Chen. Dual PI3K/mTOR inhibitor NVP-BEZ235 in combination with AKT inhibitor MK2206 in esophageal carcinoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1253. doi:10.1158/1538-7445.AM2014-1253

Effect of NVP-BEZ235, dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor, on radiosensitivity of oral cancer cell line through G2/M phase checkpoint regulation.

e13558 Background: Radioresistance is a major challenge in head and neck cancer radiotherapy (RT). PI3K/mTOR signal pathway has been considered an important regulatory mechanism of resistance to ra...

Abstract A63: NVP-BEZ235, a dual PI3K/mTOR inhibitor, elicits an alternate route for induction of cell death in PTEN null prostate cancer cells.

Abstract Dysregulation of the PI3K-AKT & mTOR pathway by (mutation and/or) deletion of tumor suppressor gene PTEN frequently occurs in human prostate cancer and is therefore considered to be attractive molecule as therapeutic targets. Here, we investigated the antitumor effect of NVP-BEZ235 in human prostate cancer cells dependent on PTEN genotype. In this setting, NVP-BEZ235 induced cell death in the PTEN-independent manner. However, NVP-BEZ235 selectively induced apoptotic cell death in prostate cancer cells presenting wild-type PTEN, DU145, but not in cells with deletion of PTEN, PC3. Treatment with NVP-BEZ235 resulted in autophagic cell death in PC3 cells with PTEN loss of function. Consistently, NVP-BEZ235 treatment did not result in the cleavage of caspase-3 and was link to active LC3-I/II as an indicator of autophagic cell death process, suggesting an alternate route for induction of cell death after exposure to NVP-BEZ235 in PTEN-null prostate cancer cells. These results suggest that PTEN/PI3K/Akt pathways are critical for prostate cancer survival and targeting PI3K signaling by NVP-BEZ235 may be beneficial in prostate cancer independent on PTEN genotype. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A63. Citation Format: Jeong Eun Kim, Seoung-Woo Hong, Jae-Sik Shin, Jai-Hee Moon, Ye-Seul Kim, Kyu-pyo Kim, Yong Sang Hong, Jae-Lyun Lee, Dong-Hoon Jin, Tae Won Kim. NVP-BEZ235, a dual PI3K/mTOR inhibitor, elicits an alternate route for induction of cell death in PTEN null prostate cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A63.

Increased activation of PI3K/AKT signaling pathway is associated with cholangiocarcinoma metastasis and PI3K/mTOR inhibition presents a possible therapeutic strategy

Phosphatidylinositol 3-kinase (PI3K) signaling plays a critical role in cholangiocarcinoma (CCA), as well as anti-cancer drug resistance and autophagy, the type II program cell death regulation. In this work, we aimed to: (1) determine the expression levels of several key components of PI3K signaling and (2) evaluate whether NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, could inhibit CCA cell growth. Immunohistochemistry for p85α, p110α, AKT, p-AKT (T308), mTOR, p-mTOR (S2448), GSK-3β, p-GSK-3β (S9), PTEN, and p-PTEN (S380, T382/383) was performed in 30 CCA patients. Western blotting was used to analyze PTEN and p-PTEN expression in the cell lines (KKU-OCA17, KKU-100, KKU-M055, KKU-M139, KKU-M156, KKU-M213, and KKU-M214). The effects of NVP-BEZ235 on CCA cells were evaluated using a growth inhibition assay, flow cytometer and migration assay. Increased activation of PI3K/AKT signaling was reproducibly observed in the CCA tissues. The expression of p85α, mTOR, and GSK-3β was significantly correlated with metastasis. Interestingly, PTEN suppression by loss of expression or inactivation by phosphorylation was observed in the majority of patients. Furthermore, NVP-BEZ235 effectively inhibited CCA cell growth and migration through reduced AKT and mTOR phosphorylation and significantly induced G1 arrest without apoptosis induction, although increase autophagy response was observed. In conclusion, the constitutive activation of PI3K/AKT pathway in CCA is mainly due to PTEN inactivation by either loss of expression or phosphorylation along with an increased expression in its pathway components heralding a poor prognosis for CCA patients. This work also indicates that inhibition of PI3K and mTOR activity by the inhibitor NVP-BEZ235 has anti-cancer activity against CCA cells which might be further tested for CCA treatment.

Effect of the combination of the dual mTOR/pI3K inhibitor NVP-BEZ235 with gemcitabine on growth inhibition in pancreatic cancer cells in vitro and in vivo.

e15070 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant tumours and is still associated with a very poor prognosis. Therefore new treatment strategies are needed. The PI3K/AKT and mTOR signaling pathways are frequently dysregulated in PDAC. Thus we investigated the effects of NVP-BEZ235, a novel dual PI3K/mTOR inhibitor, alone or in combination with gemcitabine first in vitro and after promising results also in vivo. Methods: We examined the effect of gemcitabine and NVP-BEZ235 (kindly provided by Novartis Pharma) on cell viability as single agents and in combination with sequential administrations in the four human pancreatic cancer cell lines MiaPaCa-2, Panc-1, AsPC-1 and BxPC-3. For in vivo experiments we used NOD SCID Mice, which were injected with BxPc3 into the right flank. Treatments consisted of Gemcitabine alone, NVP-BEZ235 alone, simultaneous application of both, first application of Gemcitabine followed by NVP-BEZ235 and NVP-BEZ235 followed by Gemcitabine. Results: Simultaneous incubation of gemcitabine and NVP-BEZ235 affected the PDAC cell lines significantly better than the single agent administration. But most effective was a sequential administration of gemcitabine followed by NVP-BEZ235. In vivo Gemcitabine and NVP-BEZ235 as single agents showed a slightly reduced tumor growth and the treatment in the sequence NVP-BEZ235 first, followed by Gemcitabine resulted in only a minimal reduction of tumor growth. The most effective results were obtained by simultaneous and even better in the sequence of Gemcitabine followed by NVP-BEZ235, respectively. Conclusions: The combination of gemcitabine with the dual PI3k/mTOR inhibitor NVP-BEZ235 enhanced the efficacy of PDAC treatment via down-regulation of the DDR related gene Survivin in vitro. This combination seems to be significantly more effective than single agent use in vitro and also in vivo. Furthermore we demonstrated that the sequence of administration of these agents could be a relevant issue. These promising results might offer a new and effective option for the treatment of pancreatic cancer in the future.

Abstract 2181: Dual targeting of phosphoinositide 3-kinase/mammalian target of rapamycin using NVP-BEZ235 suppressed tumor growth in a genetically engineered mouse model of serous ovarian cancer.

Abstract Introduction: The PI3K/Akt/mTOR pathway plays a central role in regulating cancer cell proliferation and survival, and activation of the PI3K/Akt/mTOR pathway is commonly seen in ovarian cancer. NVP-BEZ235 is a novel agent that targets two molecules in this pathway, PI3K and mTOR. Thus, we explored the effects of NVP-BEZ235 on proliferation in human ovarian cancer cell lines and inhibition of tumor growth in a genetically engineered mouse model of serous ovarian cancer. Methods: Four ovarian cancer cell lines (SKOV3, ES2, HEY, IGROV1) were treated with NVP-BEZ235 (NOVARTIS Pharmaceuticals) at different concentrations for up to 72 hours and proliferation was measured using the MTT assay. Cell cycle progression was evaluated by Cellometer. Apoptosis was determined by Annexin V-FITC assay. Phosphorylated (phos)-Akt, phos-S6 were evaluated by Western blot analysis. Adhesion was assessed by ELISA assay. The K18-gT121+/−;p53fl/fl;Brca1fl/fl (KpB) genetically engineered serous ovarian cancer mouse model was used for the in vivo studies. The KpB mice were subjected to a 60% calories-derived from fat in a high fat diet (HFD) versus 10% calories from fat in a low fat diet (LFD) to mimic diet-induced obesity and subsequently exposed to NVP-BEZ235 or placebo for 4 weeks. Results: NVP-BEZ235 inhibited proliferation in all four ovarian cancer cell lines, in both a dose and time dependent manner (IC50=30 nM for SKOV3, 42 nM for ES2, 7 nM for IGROV1, 30 nM for HEY, p=0.00001). Treatment with NVP-BEZ235 resulted in G1 cell cycle arrest but failed to induce apoptosis. Western blot analysis demonstrated that NVP-BEZ235 decreased phosphorylation of both Akt and S6 within 24 hours of exposure. Cellular adhesion was decreased by 34-51% in the ovarian cancer cell lines at a dose of 50 nM (p=0.00001). In the KpB mice fed a high fat diet (obese) and treated with NVP-BEZ235, tumor weight decreased by 77% (p=0.0015) when compared with control animals. Among KpB mice fed a low fat diet (non-obese), tumor weight decreased by 59% after treatment with NVP-BEZ235 (p=0.03). Conclusions: NVP-BEZ235 potently inhibited cell growth via G1 arrest and decreased cellular adhesion in human ovarian cancer cells. In vivo studies using the KpB mouse model found that treatment with NVP-BEZ235 inhibited tumor growth in both obese and non-obese mice, but was more efficacious in the obese mice. Given that alterations in the PI3K/Akt/mTOR parthway are common in both obeso- and onco-genesis disease processes, this may explain the enhanced effect of NVP-BEZ235 in the obese KpB mice. This work suggests that NVP-BEZ235 may be a novel chemotherapeutic agent for ovarian cancer treatment that is potentially more beneficial in the obese population. Citation Format: Yan Zhong, Chunxiao Zhou, Victoria Lin Bae-Jump. Dual targeting of phosphoinositide 3-kinase/mammalian target of rapamycin using NVP-BEZ235 suppressed tumor growth in a genetically engineered mouse model of serous ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2181. doi:10.1158/1538-7445.AM2013-2181

Abstract LB-359: Inhibition of DNA repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a novel radiosensitization strategy for glioblastoma.

Abstract Glioblastomas (GBM) are deadly brain tumors with extremely poor prognosis. The standard of care for GBM is tumor resection followed by treatment with ionizing radiation (IR) and the DNA alkylating agent temozolomide. As GBMs are highly radioresistant, it is crucial to identify effective radiosensitizers for improving therapeutic outcome. We find that NVP-BEZ235, a dual PI3K/mTOR inhibitor that is in clinical trials, potently inhibits both ATM and DNA-PKcs, the two major kinases that respond to IR-induced DNA double-strand breaks (DSBs). Consequently, this drug blocks non-homologous end joining (NHEJ) as well as homologous recombination (HR), the two major pathways of DSB repair. We find that low concentrations of NVP-BEZ235 (100 nM) can block DSB repair in a panel of human GBM lines resulting in significant radiosensitization. This radiosensitizing effect is more potent than that seen with much higher concentrations (10 μm) of current inhibitors of DNA-PKcs or ATM that are being optimized for clinical testing. In order to examine the effects of NVP-BEZ235 in vivo, we sub-cutaneously injected mice with radioresistant U87 glioma cells ectopically expressing EGFRvIII and treated with clinically-relevant doses of the drug. We find that NVP-BEZ235 blocks the activation of both DNA-PKcs and ATM in irradiated tumors resulting in significant attenuation of DSB repair. Tumors treated with a combination of IR and NVP-BEZ235 exhibit elevated DSBs and reduced Ki67 positivity compared to tumors treated with only IR or NVP-BEZ235. Consequently, a combination of IR and NVP-BEZ235 results in dramatic inhibition of tumor growth, in contrast to almost no inhibition with either treatment modality alone. Finally, we established that NVP-BEZ235 can cross the blood-brain barrier and therefore examined its effectiveness in an orthotopic brain tumor model. We find that NVP-BEZ235 given with IR can attenuate the growth of intracranial tumors generated from U87-EGFRvIII cells, thereby extending survival of tumor-bearing mice. Importantly, NVP-BEZ235 also sensitizes subcutaneous and intracranial tumors to temozolomide by attenuating the repair of secondary (replication-associated) DSBs that are generated upon temozolomide treatment. In sum, this study provides support for the potential use of NVP-BEZ235 as a radio- and chemo-sensitizer for improving the outcome of GBM therapy. Citation Format: Carlos Gil Del Alcazar, Molly Gillam, Nozomi Tomimatsu, Xiaohuan Gao, Bipasha Mukherjee, Sandeep Burma. Inhibition of DNA repair by the dual PI3K/mTOR inhibitor NVP-BEZ235 as a novel radiosensitization strategy for glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-359. doi:10.1158/1538-7445.AM2013-LB-359

1318 HYPOXIC MICROENVIRONMENT PROMOTES PI3K/AKT/MTOR SIGNALING PATHWAYS IN HUMAN CASTRATION RESISTANT PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: Recently we demonstrated that the activated PI3K-Akt-mTOR signaling pathway induced by androgen deprivation therapy or docetaxel explains at least in part the aggressiveness in CRPC. However microenviromental regulation of PI3K/Akt signaling pathways or the efficacy of PI3K/Akt inhibitors in hypoxic condition has not been fully characterized yet. METHODS: Human CRPC cell lines: C4-2 and C4-2AT6 were used in this study. The C4-2AT6 cell line was established from C4-2 cells grown in androgen ablated serum for six months. We examined the change of phosphorylated Akt (pAkt) and phosphorylated S6 expression under hypoxic condition (1-5%O2) using hypoxic chamber. We explore the efficacy of PI3K/Akt/mTOR inhibitors: NVP-BEZ235 and everolimus in hypoxic condition. Epithelial mesenchymal transition (EMT) related markers or invasive activity under normoxic and hypoxic conditions was quantified. RESULTS: We investigated the expression of phosphorylated pAkt and pS6 in C4-2AT6 cultured under normoxia or hypoxia. Although under normoxia C4-2AT6 expressed elevated pAkt and pS6 accompanied by elevated HIF-1a expression, 1-5% hypoxic condition further induced pAkt and pS6 expression in C4-2AT6. These results indicates HIF-1 or Akt, which is induced in aggressive PCa, are induced moreover under hypoxic condition. Next we examined the inhibitory effect of PI3K/Akt/mTOR inhibitor. The expression of pAkt and pS6 under hypoxia of 1% O2 was significantly inhibited by at the dose of 500nM NVP-BEZ235 for 24 hr culture period. The expression of pAkt and pS6 were inhibited by NVP-BEZ235 at the dose dependent manner. These results suggested that NVP-BEZ235 inhibited PI3K/Akt/ mTOR signaling pathway under hypoxia as well as normoxia despite the further up-regulated pAkt under hypoxia. The cytotoxic effect of NVP-BEZ235 under hypoxia was examined by direct cell count. The number of cells under hypoxia of 1% O2 was 4.08 0.75 10 / ml and the number of cells treated with 500nM NVP-BEZ235 under hypoxia of 1% O2 was 2.72 0.82 10 / ml. The difference of the cell number was significant (p 0.05). The morphological change of cancer cells from mesenchymal to epithelial was induced under hypoxia at 24 hr, suggesting the induction of EMT, although E-cadherin expression was not significantly changed for 24 h culture period. CONCLUSIONS: Hypoxic microenvironment promotes PI3K/ Akt/mTOR signaling pathways and morphological change in human CRPC.

NVP-BEZ235 alone and in combination in mantle cell lymphoma: an effective therapeutic strategy

Objectives: Mantle cell lymphoma (MCL) is a distinct subtype of B-cell lymphoma; the complete response rate for standard therapies in use today is 85 – 90%. NVP-BEZ235 inhibits the PI3K/Akt/mTOR signaling axis at the level of both PI3K and mTOR. In this study, we analyzed the inhibitory effects of NVP-BEZ235 on mantle cell lines and its effects in combination with enzastaurin, everolimus and perifosine.Methods: The effects of NVP-BEZ235 on cell proliferation and apoptosis were evaluated using MTT assay and flow cytometry analysis. The cell cycle analysis was performed applying BrdU incorporation. Western blot analysis was utilized for phosphorylation status evaluation of protein kinases. The interaction between NVP-BEZ235 and enzastaurin, everolimus and perifosine was examined by Chou-Talalay method.Results: NVP-BEZ235 induced significant increase of apoptosis, both via intrinsic and extrinsic pathways. We found that NVP-BEZ235 inhibited mantle cells growth by induction of G1 arrest. NVP-BEZ235 exerts its antitumor activity even when mantle cells were in contact with bone marrow microenvironment. Enzastaurin, everolimus and perifosine enhanced the cytotoxicity triggered by NVP-BEZ235.Conclusions: The above results encourage clinical development of NVP-BEZ235 in combination and the possible inclusion of patients with mantle lymphoma in Phase I/II clinical trials.

Abstract 2800: Dual PI3K/mTOR inhibitor NVP BEZ-235 suppresses translation of hypoxia-inducible factor (HIF)-1α and increases cell death under hypoxia

Abstract Our goal was to investigate the effects of the Novartis compound NVP-BEZ235, a dual PI3K/mTOR inhibitor, on hypoxia-inducible factor-1 alpha (HIF-1α) expression. NVP-BEZ235 was much more potent than either the mTOR inhibitor rapamycin or the PI3K inhibitor LY294002 in blocking the hypoxic induction of HIF-1α in vitro, due to decreased protein translation. 7-methyl GTP chromatography showed that NVP-BEZ235 led to a robust recruitment of 4E-BP1 to eIF4E and a near absence of binding of eIF4G, consistent with inhibition of protein translation. NVP-BEZ235 also decreased expression of several proteins regulated by eIF4E including cyclin B1 and D1, survivin, and vascular endothelial growth factor. NVP-BEZ235 specifically decreased eIF4G but not eIF4E expression. As HIF-1α has been associated with adaptation under hypoxia, we examined the effect of NVP-BEZ235 on cell survival in low pO2 conditions. The drug increased killing of cells under hypoxia as measured by both short-term (MTT) and long-term (clonogenic) assays. To understand the mechanism behind this observation, we performed immunoblotting for factors associated with cell survival. In normoxia, Akt S473 phosphorylation decreased within an hour of NVP-BEZ235 treatment, but then increased by 24 hours. In contrast, under hypoxia, NVP-BEZ235 caused a prolonged suppression of Akt phosphorylation. Furthermore, we found a greater increase in PARP cleavage in hypoxic cells treated 1-16 hours after drug treatment than in normoxic cells, consistent with increased apoptosis. This was confirmed using an ELISA assay that measures cytoplasmic oligonucleosomes. We also found that NVP-BEZ235 increases autophagy as measured by (i) LC3-I to LC3-II conversion on immunoblotting, (ii) degradation of p62 and (iii) GFP-LC3 subcellular localization detected by immunofluorescence. Treatment of cells with the drug under hypoxia further increased autophagy, although it is unclear at this point whether this actually contributes to cell death or is a pro-survival mechanism. In conclusion, we have found that NVP-BEZ235 blocks HIF-1α induction and increases cell killing under hypoxia, which may have important implications for the use of this drug in cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2800. doi:1538-7445.AM2012-2800

Effect of NVP-BEZ235, a dual PI3K/mTOR inhibitor, on chemotherapy and antiangiogenic response in pancreatic cancer.

243 Background: The phosphatidylinositol-3-kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathway dysregulation is a prominent feature of pancreatic ductal adenocarcinoma (PDAC). Gemcitabine (GEM), a standard systemic treatment for PDAC, has limited clinical benefits. The present study investigated the effects of NVP-BEZ235 (BEZ235), a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Methods: Protein expression and cell proliferation were analyzed by Western blotting and WST-1 assay. Animal experiments were performed in murine xenografts. Results: BEZ235 inhibited phospho-AKT (Ser473) and phospho-mTOR (Ser2448) expression in PDAC (AsPC-1), endothelial (HUVECs) and fibroblast (WI-38) cells. NVP-BEZ235 also caused a significant dephosphorylation of downstream mTORC1 target proteins phospho-p70 S6K (Thr389) and phospho-4E-BP1 (Thr37/46). In vitro 72-hour proliferation of four PDAC cell lines was significantly inhibited by BEZ235. Additive effects on proliferation inhibition were observed in the BEZ235 and GEM combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI-38 cells. BEZ235, alone or in combination with GEM and EMAP, induced apoptosis in AsPC-1, HUVECs and WI-38 cells as observed by increased expression of cleaved poly (ADP-ribose) polymerase-1 (PARP-1) and caspase-3 proteins. PDAC in vivo therapy demonstrated that compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and GEM monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235+GEM (30 days, p=0.007), BEZ235+EMAP (27 days, p=0.02), GEM+EMAP (31 days, p=0.001) and BEZ235+GEM +EMAP (33 days, p=0.004). Conclusions: BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that can be enhanced in combination with cytotoxic (GEM) and antiendothelial (EMAP) agents. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment.