BEZ235 Research Articles

BEZ235 Increases the Sensitivity of Hepatocellular Carcinoma to Sorafenib by Inhibiting PI3K/AKT/mTOR and Inducing Autophagy.

Acquired resistance of hepatocellular carcinoma (HCC) to sorafenib (SFB) is the main reason for the failure of SFB treatment of the cancer. Abnormal activation of the PI3K/AKT/mTOR pathway is important in the acquired resistance of SFB. Therefore, we investigated whether BEZ235 (BEZ) could reverse acquired sorafenib resistance by targeting the PI3K/mTOR pathway. A sorafenib-resistant HCC cell line Huh7R was established. MTT assay, clone formation assay, flow cytometry, and immunofluorescence were used to analyze the effects of BEZ235 alone or combined with sorafenib on cell proliferation, cell cycle, apoptosis, and autophagy of Huh7 and Huh7R cells. The antitumor effect was evaluated in animal models of Huh7R xenografts in vivo. Western blot was used to detect protein levels of the PI3K/AKT/mTOR pathway and related effector molecules. In vitro results showed that the Huh7R had a stronger proliferation ability and antiapoptosis effect than did Huh7, and sorafenib had no inhibitory effect on Huh7R. SFB + BEZ inhibited the activation of the PI3K/AKT/mTOR pathway caused by sorafenib. Moreover, SFB + BEZ inhibited the proliferation and cloning ability, blocked the cell cycle in the G0/G1 phase, and promoted apoptosis in the two cell lines. The autophagy level in Huh7R cells was higher than in Huh7 cells, and BEZ or SFB + BEZ further promoted autophagy in the two cell lines. In vivo, SFB + BEZ inhibited tumor growth by inducing apoptosis and autophagy. We concluded that BEZ235 enhanced the sensitivity of sorafenib through suppressing the PI3K/AKT/mTOR pathway and inducing autophagy. These observations may provide the experimental basis for sorafenib combined with BEZ235 in trial treatment of HCC.

The PI3K/Akt/mTOR signaling pathway regulates lipid metabolism mediated by endoplasmic reticulum stress in goose primary hepatocytes

Abstract We have previously demonstrated that the PI3K/Akt/mTOR signaling pathway was closely linked to the lipid metabolism homeostasis in goose primary hepatocytes. Recently, it was demonstrated that endoplasmic reticulum stress (ERS) can not only affect lipogenesis, but also is related to the PI3K/Akt/mTOR signaling pathway, so it was hypothesised that ERS can affect the lipid metabolism through the PI3K/Akt/mTOR signaling pathway. The ERS model was established by treating goose primary hepatocytes with tunicamycin (TM), and then incubated with PI3K/Akt/mTOR signaling pathway inhibitors LY294002 (LY), Rapamycin (Ra) or NVP-BEZ235 (NVP). Hepatocytes were assigned into 5 groups, including control group, TM group, TM+LY group, TM+Ra group, and TM+NVP group. The results showed that compared with the control group, the mRNA expression and the protein content of BIP/GRP78 significantly increased ( P<Â 0.05) in the TM group, indicating that the ERS model was established successfully. The result of Oil Red O staining revealed that TM caused an increase ( P<Â 0.05) in lipid accumulation and in the mRNA expression of PI3K and S6K, implying that ERS could activate the PI3K/Akt/mTOR signaling pathway. Compared with the TM group, the mRNA expression of genes involved in the PI3K/Akt/mTOR signaling pathway ( PI3K, Akt1, mTOR and S6K), lipogenesis ( ACCα, FAS and SREBP-1), VLDL-TG assembly and secretion ( MTTP, DGAT1 and DGAT2) significantly decreased ( P<Â 0.05) in the TM+LY group, the TM+Ra group, and the TM+NVP group, whereas the protein content of CPT1 and MTTP significantly increased ( P<Â 0.05) in the TM+LY group, the TM+Ra group, and the TM+NVP group. These findings suggest that inhibition of the PI3K/Akt/mTOR signaling pathway can effectively decrease the steatosis mediated by ERS in goose hepatocytes.

Abstract 75: BCL-xL inhibition enhances therapeutic response of MTORC1/2 inhibition and induction of apoptosis in PIK3CA mutant colorectal cancer

Abstract Colorectal cancer (CRC) is a leading cause of cancer related death with PIK3CA mutations occurring in ~18% of all cases. Mutations in this gene lead to constitutive activation of the phosphoinositide-3 kinase (PI3K) oncogene. Previously we've shown that MTORC1/2 inhibition is sufficient to induce a therapeutic response both in vitro and in vivo with minimal induction of apoptosis. BCL-xL is a well-known negative regulator of apoptosis in solid tumors. We therefore investigated whether inhibition of the BCL-2 family, and more specifically BCL-xL, would enhance therapeutic response and induction of apoptosis. Murine-derived cancer organoids (MDCOs) were generated from invasive colon adenocarcinomas of Apc and Pik3ca transgenic mice (F1 (FVBxB6) Apcfl/+ Pik3caH1047R). MDCOs were allowed to mature for 24 hours, baseline brightfield imaging performed and therapeutic agents added at concentrations outlined below. Median relative change in organoid diameter after 48 hours of treatment was determined. In vivo response was measured in F1 (FVBxB6) Apcfl/+ Pik3caP110* mice as change in endoscopic tumor lumen occlusion over 14 days. Immunoblotting (IB) and immunofluorescence (IF) were utilized to evaluate for induction of apoptosis. Navitoclax (BCL-2/BCL-xL/BCL-w inhibitor, 250nM) was evaluated alone and in combination with a panel of MTORC1/2 inhibitors (BEZ-235 (BEZ), TAK-228 (TAK), copanlisib (Cop), 200nM). Navitoclax did not induce a treatment response as a single agent. Enhanced response was seen with the combination compared to the MTORC1/2 inhibitors alone (Bez 56% vs combo -100%, p&amp;lt;0.001; TAK -27% vs combo -100%, p&amp;lt;0.001; Cop -16% vs -100%; p&amp;lt;0.001). Results were confirmed in vivo with BEZ-235 (30mg/kg/day), navitoclax (80mg/kg/day), or the combination with the greatest reduction in lumen occlusion of colon tumors in the combination therapy (control +15%, navitoclax +1%, BEZ -15%, and combo -42%, p&amp;lt;0.003 BEZ vs combo). IB of cleaved PARP, a main cleavage target of cleaved caspase 3 (CC3) once apoptosis is induced, and IF of CC3 confirmed induction of apoptosis was highest in the combination therapy in both in vitro and in vivo studies. This induction was found as early as 6 hours post treatment in the MDCOs. To confirm inhibition of BCL-xL was the primary anti-apoptotic protein necessary for this induction of apoptosis, MDCOs were treated with copanlisib (200nM) alone or in combination with WEHI-539 (BCL-xL inhibitor, 250nM) or ABT-199 (BCL-2 inhibitor, 250nM). An enhanced sensitivity was observed when MTORC1/2 inhibition was combined with the inhibition of BCL-xL compared to BCL-2. These studies indicate that BCL-xL signaling reduces MTORC1/2 inhibitor response and targeting BCL-xL in combination with MTORC1/2 enhances both the treatment response and the induction of apoptosis in PIK3CA mutant CRC. Citation Format: Rebecca A. DeStefanis, Alyssa DeZeeuw, Gioia Sha, Susan N. Payne, Christopher P. Babiarz, Devon Miller, Demetra K. Korkos, Cheri A. Pasch, Linda Clipson, Kristina Matkowskyj, Dustin A. Deming. BCL-xL inhibition enhances therapeutic response of MTORC1/2 inhibition and induction of apoptosis in PIK3CA mutant colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 75.

PI3K/Akt/mTOR and CDK4 combined inhibition enhanced apoptosis of thyroid cancer cell lines

Introduction Thyroid cancer is a malignant disease with poor prognosis. The PI3K/Akt/mTOR and Cyclin-Dependent Kinase 4 (CDK4) pathways are vital regulators of tumor cell proliferation and survival. Therefore the present study was designed to use dual inhibition of such pathways to kill thyroid cancer cells. Methods and materials The effects of each inhibitors on human ATC and BCPAP cell lines were evaluated by MTT assay. The suitable concentrations of inhibitors were determined and synergistic effects of such inhibitors were evaluated by bax/bcl-2 mRNA ratio, Caspase-3, and Caspase-9 activity assay as well as Akt, mTOR, and CDK4. Results:Our finding showed that both ATC and BCPAP cell proliferation is significantly inhibited by PD-332991(PD) and NVP-BEZ235 (NVP) in a time and concentration-dependent manner (P

Enhanced Sensitivity to NVP-BEZ235 by Inhibition of p62/SQSTM1 in Human Bladder Cancer KoTCC-1 Cells Both In Vitro and In Vivo.

The prognosis of patients with invasive bladder cancer remains poor. The objective of this study was to evaluate the efficacy of NVP-BEZ235 (NVP), a dual PI3K/mTOR inhibitor, combined with the inactivation of p62/SQSTM1 (p62) in a human bladder cancer KoTCC-1 model. An expression plasmid with short hairpin RNA targeted against p62 was transfected into KoTCC-1 cells (KoTCC-1/sh-p62). The antitumor effects of NVP on KoTCC-1/sh-p62 were investigated in comparison with those on KoTCC-1 transfected with a control plasmid alone (KoTCC-1/C). KoTCC-1/sh-p62 showed significantly higher sensitivity to NVP than KoTCC-1/C. Treatment of both cell lines with NVP markedly inactivated the PI3K/Akt/mTOR signaling pathway. However, NVP treatment stimulated the autophagic pathway in KoTCC-1/C, but not in KoTCC-1/sh-p62. Furthermore, compared with KoTCC-1/C, NVP treatment induced apoptosis of KoTCC-1/sh-p62 cells, which was accompanied by significant downregulation of c-IAP-1 and XIAP as well as upregulation of Bax. Moreover, the in vivo growth of KoTCC-1/sh-p62 tumors was significantly suppressed by treatment with NVP compared to KoTCC-1/C tumors. Inhibition of p62 expression combined with NVP may represent an effective therapeutic approach for patients with invasive bladder cancer.

Synthesis of Dactolisib

Key words dactolisib - palladium scavenger - Suzuki–Miyaura cross-coupling

Downregulated ATF3 expression inhibited growth of adrenocortical carcinoma cells and investigation of its mechanisms

Objective To investigate the effect of downregulated activating transcription factor 3 (ATF3) expression on proliferation of adrenocortical carcinoma cells and its mechanisms. Methods Immunohistochemistry and Western blotting were used to detect the expression of ATF3 in human adrenocortical tumor tissues and cells. Adrenocortical carcinoma cells, Sw-13, and NCI-H259R cells, were transfected with siATF3 using lipidosome 2000, and expression of ATF3 mRNA was determined using RT-PCR; expression of ATF3, cleaved caspase 3, caspase 3, cleaved PARP, and PARP proteins were detected using Western blotting; cell growth inhibition rate and apoptosis rate were monitored using MTT and AnnexinV-FITC/PI, respectively. Sw-13 and NCI-H259R cells were treated with NVP-BEZ235, Perifosine, BKM120, IWP-2, PP2, KN93, Everolimus respectively followed by detected expression of ATF3 mRNA by realtime PCR. The effect of ATF3 on cell proliferation after inhibition of related signaling pathways were detected by MTT. Results The ATF3 in human adrenocortical gland tumor tissues and cells showed high expression. The levels of ATF3 mRNA and protein in Sw-13 and NCI-F259R cells transfected with siATF3 were significantly reduced. Compared with the negative control group (NC siRNA), siATF3 transfection significantly inhibited the proliferation of Sw-13 and NCI-F259R cells (P<0.05), and increased the apoptosis rate (P<0.05). Western blotting shown that the levels of cleaved caspase 3 and cleaved PARP protein in siATF3 transfected cells increased significantly; and realtime PCR results indicated that the expression of ATF3 mRNA was dramatically inhibited by PP2, KN93, and IWP-2 in NCI-F259R cells compared with control group (DMSO); but ATF3 significantly promoted the proliferation activity of NCI-F259R cells which treated by PP2, KN93, and IWP-2 signaling inhibitors. Conclusion High expression of ATF3 is existed in adrenocortical carcinoma cells. Downregulated ATF3 expression may inhibit cell proliferation and activate apoptosis pathway, resulting in apoptosis in Sw-13 and NCI-F259R cells, this mechanism of action is related to activating Wnt/β-catenin, CaMKI, and SRC pathway. (Chin J Endocrinol Metab, 2018, 34: 738-745) Key words: Adrenocortical carcinoma; Activating transcription factor 3; Signal pathway; Apoptosis; Mechanism

Correction: The dual PI3K/mTOR inhibitor dactolisib elicits anti-tumor activity in vitro and in vivo.

[This corrects the article DOI: 10.18632/oncotarget.23091.].

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

Dual phosphatidylinositol 3 kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 enhances apoptosis of colon cancer cells induced by oxaliplatin

Objective To investigate the effect of phosphatidylinositol 3 kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 on apoptosis induced by oxaliplatin in human colon cancer HCT116 cells. Methods Colon cancer HCT116 cells were cultured in vitro with different concentrations of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235. Methyl thiazol tetrazolium (MTT) assay was used to detect the effects of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on cell viability of HCT116 cells. Flow cytometry (FCM) was used to detect the effect of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on HCT116 cells. Immunofluorescence was used to detect the effect of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on the nuclear morphology of HCT116 cells. Western blotting was used to detect the effects of oxaliplatin and/or dual PI3K/mTOR inhibitor NVP-BEZ235 on DNA damage marker gene gamma histone variant H2A histone family member X (γ-H2AX) and apoptosis-related protein expression. Results The results of MTT showed that 2, 4, 8, 16, 32 and 64 μg/ml oxaliplatin inhibited proliferation of HCT116 cells in a dose-dependent manner, and about 16 μg/ml oxaliplatin platinum could decrease 50% HCT116 cells on the growth. The results of flow cytometry revealed that the apoptosis rate of HCT116 cells treated with 4, 16 and 64 μg/ml oxaliplatin was 11%, 36% and 80% respectively (P=0.026). The results of immunofluorescence demonstrated that the combination of oxaliplatin and NVP-BEZ235 obviously induced the apoptosis of HCT116 cells, and the nuclear shrinkage of HCT116 cells. Western blotting indicated that the expression of Cleaved cysteinyl aspartate-specific protease (Caspase)-3 was significantly increased (P=0.026), and the level of γ-H2AX was also up-regulated (P=0.017). Conclusion Dual PI3K/mTOR inhibitor NVP-BEZ235 significantly enhanced the apoptosis of colon cancer cells induced by oxaliplatin and enhanced the DNA damage of colon cancer cells induced by oxaliplatin. Key words: Colon cancer; Dual phosphatidylinositol 3 kinase/mammalian target of rapamycin; Oxaliplatin; Apoptosis; DNA damage

Antitumor efficacy of liposome-encapsulated NVP-BEZ 235 in combination with irreversible electroporation

Irreversible electroporation (IRE) is an emerging minimally invasive tumor ablation technique that delivers short pulses of strong electric fields and kills cancer cells by disrupting their cell membranes with the electric pulses. However, clinical studies report that more than 10% of local tumor recurrences occur at the original ablated site. NVP BEZ-235 (BEZ) is a dual PI3K/mTOR inhibitor that has substantial anticancer effects. However, the clinical trials of BEZ was not satisfactory because of its low bioavailability and high toxicity, which stemmed from the use of oral administration of high doses over a long period of time. In this research, we prepared a liposomal formulation of BEZ (L-BEZ) for intratumoral injection and studied its antitumor efficacy alone and in combination with IRE. We hypothesized that IRE could release BEZ from the liposomes and that the combination could decrease tumor viability. Our results show that IRE released BEZ from its liposomal encapsulation. The combination of L-BEZ and IRE killed more Hep3B tumor cells in vitro than did L-BEZ or IRE alone and also inhibited cancer cell proliferation in nude mice bearing Hep3B xenografts. Combination of chemotherapeutic agent loaded nanoparticles could enhance the antitumor efficacy of IRE.

Research and application of NVP-BEZ235 in lung cancer

NVP-BEZ235 is a newly developed dual phosphatidyl linositol kinase 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitor. Studies have revealed that NVP-BEZ235 combinate with other drugs were effective in the treatment of lung cancer. NVP-BEZ235 might have a potential application value for the treatment of lung cancer of EGFR mutation subtype and KRAS mutation subtype. Key words: Lung neoplasms; Mutation; NVP-BEZ235

Research status quo and progression in targeted therapy for advanced gastric cancer

With the deeper research of the proliferation, invasion and metastasis mechanisms of the gastric cancer, targeted therapy has become a hot spot in this field. The exploration of targeted agents for gastric cancer is mainly concentrated upon the drugs that target human epidermal growth factor receptor (HER) family, the vascular endothelial growth factor (VEGF), the phosphatidylinositol 3-kinase-protein kinase/mammalian target of rapamycin (PI3K/mTOR) and the NF-κB signaling pathways. The targeted drugs relevant to HER family include the Cetuximab, Nimotuzumab, Matuzumab, Panitumumab and Erlotinib which are aimed at HER-1, the Trastuzumab, Pertuzumab and T-DM1 (trastuzumab emtansine) which are aimed at HER-2, and the Lapatinib and Afatinib which are the multi-target agents of HER. The agents which target VEGF signaling pathway include the anti-VEGF monoclonal antibody (Bevacizumab), the anti-VEGFR drugs (Ramucirumab, Apatinib, Sorafenib, Sunitinib and cediranib), and the recombinant fusion protein (Aflibercept). The LY294002, BEZ235 and Everolimus which are aimed at PI3K/mTOR signaling pathway have shown great promise. Bortezomib has been researched more as a new agent which targets NF-κB signaling pathway. Currently, only Trastuzumab, Ramucirumab and Apatinib have been completed the stage 3 clinical trials and succeeded. In future, researches should focus on multi-target agents or applications in combination treatment. This review collects the recent researches and the clinical trials to summarize the current state and progress of research on gastric cancer targeted therapy.

Differential effects of rapalogues, dual kinase inhibitors on human ovarian carcinoma cells in vitro.

Ovarian cancer is the second most common gynaecological malignancy and was diagnosed in over 7,000 women in 2011 in the UK. There are currently no reliable biomarkers available for use in a regular screening assay for ovarian cancer and due to characteristic late presentation (78% in stages III and IV) ovarian cancer has a low survival rate (35% after 10 years). The mTOR pathway is a central regulator of growth, proliferation, apoptosis and angiogenesis; providing balance between available resources such as amino acids and growth factors, and stresses such as hypoxia, to control cellular behaviour accordingly. Emerging data links mTOR with the aetiopathogenesis of ovarian cancer. We hypothesised that mTOR inhibitors could play a therapeutic role in ovarian cancer treatment. In this study we began by validating the expression of four main mTOR pathway components, mTOR, DEPTOR, rictor and raptor, at gene and protein level in in vitro models of endometrioid (MDAH-2774) and clear cell (SKOV3) ovarian cancer using qPCR and ImageStream technology. Using a wound healing assay we show that inhibition of the mTOR pathway using rapamycin, rapalogues, resveratrol and NVP BEZ-235 induces a cytostatic and not cytotoxic response up to 18 h in these cell lines. We extended these findings up to 72 h with a proliferation assay and show that the effects of inhibition of the mTOR pathway are primarily mediated by the dephosphorylation of p70S6 kinase. We show that mTOR inhibition does not involve alteration of mTOR pathway components or induce caspase 9 cleavage. Preclinical studies including ovarian tissue of ovarian cancer patients, unaffected controls and patients with unrelated gynaecological conditions show that DEPTOR is reliably upregulated in ovarian cancer.

MP61-11 ENHANCED SENSITIVITY TO NVP-BEZ235 BY INHIBITION OF P62/SQSTM1 IN HUMAN BLADDER CANCER KOTCC-1 CELLS BOTH IN VITRO AND IN VIVO

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2016MP61-11 ENHANCED SENSITIVITY TO NVP-BEZ235 BY INHIBITION OF P62/SQSTM1 IN HUMAN BLADDER CANCER KOTCC-1 CELLS BOTH IN VITRO AND IN VIVO Bing Liu, Hideaki Miyake, and Masato Fujisawa Bing LiuBing Liu More articles by this author , Hideaki MiyakeHideaki Miyake More articles by this author , and Masato FujisawaMasato Fujisawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.885AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To date, a number of studies have shown that activation of the cascade mediating mTOR signal transduction may play an important role in the progression of a wide variety of malignant tumors, including bladder cancer. However, several studies have shown the adaptive induction of autophagy by inhibitors of the PI3K/AKT/mTOR pathway, which promotes the survival of cancer cells, resulting in the potential limitation of their own efficacies. Therefore, we herein evaluated changes in the phenotypes of human bladder cancer KoTCC-1 cells both in vitro and in vivo after the RNA interference-mediated knockdown of the p62/SQSTM1 (p62), a multifunctional protein that has been implicated in selective autophagy, with a focus on the antitumor activity of NVP-BEZ235 (NVP), a recently developed dual PI3K/mTOR inhibitor. METHODS We established KoTCC-1 cells in which an expression vector containing short hairpin RNA (shRNA) targeting p62 was introduced (KoTCC-1/sh-p62). Antitumor activity of NVP in KoTCC-1/sh-p62 was compared with those in KoTCC-1 transfected with a control vector alone (KoTCC-1/C). RESULTS When cultured in standard medium, no significant difference was observed in growth patterns between KoTCC-1/sh-p62 and KoTCC-1/C; however, the sensitivity of KoTCC-1/sh-p62 to NVP was significantly higher than that of KoTCC-1/C, reducing the IC50 of NVP in KoTCC-1/sh-p62 by approximately 90% compared with that in KoTCC-1/C. A treatment with NVP strongly inactivated the PI3K/Akt/mTOR pathway in both cell lines. However, the treatment with NVP resulted in the activation of autophagy in KoTCC-1/C, but not in KoTCC-1/sh-p62, while the treatment of KoTCC-1/sh-p62 with NVP strongly induced apoptosis, which was accompanied by significantly lower amounts of c-IAP-1 and XIAP and larger amount of Bax than those in KoTCC-1/C. Despite the lack of a significant difference in the growth patterns of KoTCC-1/sh-p62 and KoTCC-1/C tumors without any treatment, the in vivo administration of NVP significantly inhibited the growth of KoTCC-1/sh-p62 tumors compared wioth that of KoTCC-1/C tumors, and the apoptotic index in KoTCC-1/sh-p62 tumors treated with NVP was significantly greater than that in KoTCC-1/C tumors. CONCLUSIONS These findings suggest that targeting p62 using shRNA interfering technology significantly enhanced the sensitivity of KoTCC-1 cells to NVP both in vitro and in vivo. Accordingly, the suppression of p62 in combination with NVP may be a promising therapeutic strategy for patients with muscle-invasive bladder cancer. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e807 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Bing Liu More articles by this author Hideaki Miyake More articles by this author Masato Fujisawa More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Dual inhibition of DNA double strand break (DSB) repair and PI3K pathway with BEZ235 (BEZ) to sensitize refractory metastatic (met) triple negative breast cancer (TNBC) to nab paclitaxel/cisplatin (pac/cis) in a patient with an exceptional response (ExRx).

156 Background: Whether EGFR is a critical target in met TNBC is unknown. Here we report the clinical history &amp; tumor molecular alterations in a patient with refractory metTNBC who had an ExRx to pac/cis. Methods: Following IRB-approved informed consent, targeted NGS (Foundation Medicine) and WGS (TGEN) was performed on the pt’s FFPE primary TNBC and 2 recurrent lymph nodes to characterize all classes of genomic alterations in cancer-related genes. RPPA was performed at a CLIA-certified laboratory (Theranostics Health) and George Mason Univ where immunostaining was directed against HER1/2/3 pathway and other proteins. Results: At age 58 in 2006, pt had T1c 1+ node TNBC treated with FAC/T. Between 2008 and 2011 she had 4 chemotherapy-refractory recurrences in axilla, supraclavicular (SC), internal mammary (IM) LNs treated unsuccessfully with surgery, radiation and multiple cytotoxic agents including carboplatin. In 2011, following SC LN biopsy, she was treated with BEZ, a PI3K/mTOR, ATM, ATR, DNA-PKcs inhibitor, had a 3 mo response, followed by rapidly enlarging progressive disease (PD) in IM LNs pushing sternum anteriorly. She was treated with pac/cis and had an ongoing complete response (CR) of 2.5+ yrs. NGS of 2011 SC LN (pre-BEZ) &amp; 2012 IM LN (post-BEZ): TP53 &amp; BRCA2 (somatic 15% mutant allele freq) mutations, FOXM1 amplification; SMARCA4 (BRG1) deletion RPPA (GMU) 2011 SC LN (Pre-BEZ): 3+ EGFR; 2+ p-EGFR, p-AKT, p-MEK1/2, p-mTOR RPPA (Theranostics) 2012 IM LN (post-BEZ): 3+ p-MEK1/2 (EGFR &amp; p-EGFR 0) (AR-). Conclusions: Strong EGFR signaling associated with chemo-resistant metTNBC in 2011 SC LN was not present in post-BEZ rapid PD in IM LN which then had durable CR with pac/cis. BEZ inhibits DSB repair, sensitizing cancers to DNA damaging agents (Gil del Alcazar, Clin Ca Res 20:1235, 2014). Progression of p-AKT-activated TNBC following response to inhibitors of PI3K &amp; DNA repair shows DSB repair-deficiency and MAPK activation (Juvekar, Cancer Dis 2:1048, 2012). A prospective trial of BEZ followed at PD by pac/cis in metTNBC is warranted.

Abstract A56: A dual PI3K-mTOR inhibitor induced cardiac hypertrophy in mice: Role of the insulin signaling

Abstract Studies from us and others showed that mice with cardiomyocyte-specific overexpression of a dominant negative PI3Kp110α had smaller hearts but maintained normal cardiac function. Yet, these mice developed systolic heart failure in the presence of cardiac stress, such as chemotherapy drug doxorubicin and aortic stenosis. Currently, drugs targeting the PI3K pathway are being tested in clinical trials for cancer therapy. Therefore, it is important to assess the cardiac effects of pharmacological inhibition of this pathway. Here, we report that a dual PI3K-mTOR inhibitor, BEZ235 (BEZ), induced cardiac hypertrophy and subsequent heart failure in mice when it was used alone or in combination with doxorubicin. This effect of BEZ was associated with increased hepatic gluconeogenesis, hyperglycemia, hyperinsulinemia and increased activation of the IGFR/insulin receptor (IR) in the heart. In isolated cardiomyocyte culture, BEZ induced feedback activation of IGFR/IR, but this effect was negligible compared to the IGFR/IR activation in the presence of insulin. Injections of insulin in mice lowered blood glucose, improved glucose and pyruvate tolerance but further increased the activation of IGFR/IR in the heart and aggravated BEZ-induced cardiac dysfunction; whereas OSI-906, an IGFR/IR inhibitor, alleviated this cardiac dysfunction. In addition, low dose Metformin significantly prolonged survival and improved cardiac function in BEZ-treated mice. Taken together, these results identified hyperinsulinemia, a systematic effect of the drug, as one of the mechanisms of BEZ-induced cardiac hypertrophy, arguing animal studies are indispensable for evaluating drug toxicity. They also suggest that Metformin but not insulin should be considered for controlling hyperglycemia and preventing the cardiac side effects that may be caused by PI3K pathway inhibitors in clinic. Citation Format: Jillian Onufrak, Yongayo Yang, Juyong Lee, John Fuseler, Maoyun Sun, Sharath Sasi, J. Tyson McDonald, Lynn Hlatky, Robert L. Price, Joseph Carrozza, David Goukassian, Thomas K. Borg, James P. Morgan, Xinhua Yan. A dual PI3K-mTOR inhibitor induced cardiac hypertrophy in mice: Role of the insulin signaling. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A56.

Targeted Inhibition of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Sensitizes Pancreatic Cancer Cells to Doxorubicin without Exacerbating Cardiac Toxicity.

Pancreatic cancer has the lowest 5-year survival rate of all major cancers despite decades of effort to design and implement novel, more effective treatment options. In this study, we tested whether the dual phosphoinositide 3-kinase/mechanistic target of rapamycin inhibitor BEZ235 (BEZ) potentiates the antitumor effects of doxorubicin (DOX) against pancreatic cancer. Cotreatment of BEZ235 with DOX resulted in dose-dependent inhibition of the phosphoinositide 3-kinase/mechanistic target of rapamycin survival pathway, which corresponded with an increase in poly ADP ribose polymerase cleavage. Moreover, BEZ cotreatment significantly improved the effects of DOX toward both cell viability and cell death in part through reduced Bcl-2 expression and increased expression of the shorter, more cytotoxic forms of BIM. BEZ also facilitated intracellular accumulation of DOX, which led to enhanced DNA damage and reactive oxygen species generation. Furthermore, BEZ in combination with gemcitabine reduced MiaPaca2 cell proliferation but failed to increase reactive oxygen species generation or BIM expression, resulting in reduced necrosis and apoptosis. Treatment with BEZ and DOX in mice bearing tumor xenographs significantly repressed tumor growth as compared with BEZ, DOX, or gemcitabine. Additionally, in contrast to the enhanced expression seen in MiaPaca2 cells, BEZ and DOX cotreatment reduced BIM expression in H9C2 cardiomyocytes. Also, the Bcl-2/Bax ratio was increased, which was associated with a reduction in cell death. In vivo echocardiography showed decreased cardiac function with DOX treatment, which was not improved by combination treatment with BEZ. Thus, we propose that combining BEZ with DOX would be a better option for patients than current standard of care by providing a more effective tumor response without the associated increase in toxicity.

ErbB2 signaling activates the Hedgehog pathway via PI3K–Akt in human esophageal adenocarcinoma: Identification of novel targets for concerted therapy concepts

The Hedgehog pathway plays an important role in the pathogenesis of several tumor types, including esophageal cancer. In our study, we show an expression of the ligand Indian hedgehog (Ihh) and its downstream mediator Gli-1 in primary resected adenocarcinoma tissue by immunohistochemistry and quantitative PCR in fifty percent of the cases, while matching healthy esophagus mucosa was negative for both proteins. Moreover, a functionally important regulation of Gli-1 by ErbB2–PI3K–mTORC signaling as well as a Gli-1-dependent regulation of Ihh in the ErbB2 amplified esophageal adenocarcinoma cell line OE19 was observed. Treatment of OE19 cells with the Her2 antibody trastuzumab, the PI3K–mTORC1 inhibitor NVP BEZ235 (BEZ235) or the knockdown of Akt1 resulted in a downregulation of Gli-1 and Ihh as well as in a reduction of viable OE19 cells in vitro. Interestingly, the Hedgehog receptor Smo, which acts upstream of Gli-1, was not expressed in OE19 cells and in the majority of primary human esophageal adenocarcinoma, suggesting a non-canonical upregulation of Gli-1 expression by the ErbB2–PI3K axis. To translate our findings into a therapeutic concept, we targeted ErbB2–PI3K–mTORC1 by trastuzumab and BEZ235, combining both compounds with the Gli-1/2 inhibitor GANT61. The triple combination led to significantly stronger reduction of tumor cell viability than cisplatinum or each biological alone. Therefore, concomitant blockage of the ErbB2–PI3K pathway and the Hedgehog downstream mediator Gli-1 may provide a new therapeutic strategy for esophageal cancer.

Abstract 16201: Combined Treatment of the PI3k/mTOR Inhibitor BEZ235 With Doxorubicin Does Not Increase Cardiotoxicity While Improving Efficacy in Breast Cancer Cells

Background: Cardiotoxicity is a major clinical limitation with doxorubicin (DOX). Also, the PI3K/Akt survival pathway is one of the most commonly mutated pathways in cancer. Therefore, the combination of kinase inhibitors and chemotherapy, such as DOX, are necessary for achieving cancer control. The PI3K pathway is also critical for protecting the heart from stress. Therefore, we examined the combined effect of BEZ235 (BEZ), a dual PI3k/mTOR inhibitor, with DOX on cardiac function and killing of breast cancer cells. Methods and results: c57 mice were treated with BEZ (40 mg/kg daily oral gavage) and DOX (3 IV injections of 5 mg/kg) either alone or in combination. Systolic function was assessed 12 weeks after initial treatment using echocardiography. Decreased ejection fraction was observed after DOX treatment. Interestingly, the combined treatment with BEZ and DOX did not exacerbate DOX cardiotoxicity (Fig. A). Western blot analysis showed significant increase in AKT (thr 308) and ERK phosphorylation with BEZ and DOX as compared individual treatment with BEZ or DOX in the heart (Fig. B). To address the anti-cancer effects of these drugs, MDA-MB-231 breast cancer cells were treated with BEZ and DOX either alone or in combination for 48 hours. Necrosis, measured using the trypan blue assay, showed a significant increase in dead cells compared to either drug alone (Fig. C). Conclusion: Concurrent treatment with DOX and BEZ did not exacerbate cardiac toxicity as demonstrated by similar level of deficit in ejection fraction between DOX as well as DOX plus BEZ. This could be due to enhanced survival signaling in the combination group. BEZ also sensitized breast cancer cells to DOX resulting in increased cell death. We propose that this combination strategy might lead to improved long term outcomes for patients with breast cancer without additional complications related to cardiac dysfunction.