Activity of the novel dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 against osteosarcoma

Recent studies have identified that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling is an important feature of osteosarcoma, where it promotes cell proliferation, survival, and chemo-resistance. Here, we studied the therapeutic potential of NVP-BEZ235, a novel dual PI3K/mTOR dual inhibitor, on osteosarcoma cells in vivo and in vitro. NVP-BEZ235 was cytotoxic and cytostatic to a panel of osteosarcoma lines (MG-63, U2OS and SaOs-2), where it induce apoptosis and cell-cycle arrest. At the molecular level, NVP-BEZ235 inhibited PI3K-AKT-mTORC1 activation and downregulated cyclin D1/cyclin B1 expressions, while increasing MEK/Erk phosphorylation in osteosarcoma cells. MEK/Erk inhibitors PD98059 and MEK-162 increased NVP-BEZ235 activity on osteosarcoma cells. In vivo, oral NVP-BEZ235 inhibited U2OS xenograft in SCID mice, and its anti-tumor efficiency was further enhanced by MEK-162 co-administration. Taken together, our findings indicate that dual inhibition of PI3K and mTOR with NVP-BEZ235, either alone or in combination with MEK/Erk inhibitors, may be an efficient treatment for osteosarcoma.     

The phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 is effective in inhibiting regrowth of tumour cells after cytotoxic therapy

PurposeRegrowth of tumour cells between cycles of chemotherapy is a significant clinical problem. Treatment strategies where antiproliferative agents are used to inhibit tumour regrowth between chemotherapy cycles are attractive, but such strategies are difficult to test using conventional monolayer culture systems.MethodsWe used the in vitro tumour spheroid model to study regrowth of 3-D colon carcinoma tissue after cytotoxic therapy. Colon carcinoma cells with wild-type or mutant phosphatidyl inositol 3-kinase catalytic subunit (PI3KCA) or KRAS alleles were allowed to form multicellular spheroids and the effects of different pharmacological compounds were studied after sectioning and staining for relevant markers of cell proliferation and apoptosis.ResultsStudies using colon cancer cells with gene disruptions suggested that the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was essential for proliferation in 3-D culture. The dual PI3K-mTOR inhibitor NVP-BEZ235, currently in clinical trials, was found to inhibit phosphorylation of the mTOR target 4EBP1 in 3-D cultured cells. The ability of NVP-BEZ235 to inhibit tumour cell proliferation and to induce apoptosis was markedly more pronounced in 3-D cultures compared to monolayer cultures. It was subsequently found that NVP-BEZ235 was effective in inhibiting regrowth of 3-D cultured cells after treatment with two cytotoxic inhibitors of the ubiquitin–proteasome system (UPS), methyl-13-hydroxy-15-oxokaurenoate (MHOK) and bortezomib (Velcade®).ConclusionsThe dual PI3K-mTOR inhibitor NVP-BEZ235 was found to reduce cell proliferation and to induce apoptosis in 3-D cultured colon carcinoma cells, NVP-BEZ235 is a promising candidate for use in sequential treatment modalities together with cytotoxic drugs to reduce the cell mass of solid tumours.     

Growth inhibition by NVP-BEZ235, a dual PI3K/mTOR inhibitor, in hepatocellular carcinoma cell lines

Dysregulation of the phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway frequently occurs in human tumors, and is therefore considered to be a good molecular target for treatment. In hepatocellular carcinoma (HCC), overexpression of p-Akt and decrease of PTEN expression have been reported. NVP-BEZ235 is a novel dual inhibitor of PI3K and mTOR; however, its effect on HCC has not been documented. Consequently, we investigated the effects of NVP-BEZ235 on the PLC/PRF/5, HLE, JHH7 and HepG2 HCC cell lines in vitro and in vivo. NVP-BEZ235 decreased the levels of p-Akt and p-p70S6K and inhibited cell proliferation in all HCC cell lines in a dose-dependent manner. Flow cytometric analysis revealed that inhibition of cell proliferation by NVP-BEZ235 was accompanied by G1 arrest in all cell lines, and that NVP-BEZ235 induced apoptosis in PLC/PRF/5 and HLE cells. Tumor growth was suppressed without body weight loss when NVP-BEZ235 was orally administered to JHH-7 tumor-bearing mice for 11 days. These results suggest that NVP-BEZ235 is a potential new candidate for targeted HCC therapy.     

NVP-BEZ235, a dual PI3K/mTOR inhibitor, prevents PI3K signaling and inhibits the growth of cancer cells with activating PI3K mutations

Phosphatidylinositol-3-kinase (PI3K) pathway deregulation is a common event in human cancer, either through inactivation of the tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 or activating mutations of p110-alpha. These hotspot mutations result in oncogenic activity of the enzyme and contribute to therapeutic resistance to the anti-HER2 antibody trastuzumab. The PI3K pathway is, therefore, an attractive target for cancer therapy. We have studied NVP-BEZ235, a dual inhibitor of the PI3K and the downstream mammalian target of rapamycin (mTOR). NVP-BEZ235 inhibited the activation of the downstream effectors Akt, S6 ribosomal protein, and 4EBP1 in breast cancer cells. The antiproliferative activity of NVP-BEZ235 was superior to the allosteric selective mTOR complex inhibitor everolimus in a panel of 21 cancer cell lines of different origin and mutation status. The described Akt activation due to mTOR inhibition was prevented by higher doses of NVP-BEZ235. NVP-BEZ235 reversed the hyperactivation of the PI3K/mTOR pathway caused by the oncogenic mutations of p110-alpha, E545K, and H1047R, and inhibited the proliferation of HER2-amplified BT474 cells exogenously expressing these mutations that render them resistant to trastuzumab. In trastuzumab-resistant BT474 H1047R breast cancer xenografts, NVP-BEZ235 inhibited PI3K signaling and had potent antitumor activity. In treated animals, there was complete inhibition of PI3K signaling in the skin at pharmacologically active doses, suggesting that skin may serve as surrogate tissue for pharmacodynamic studies. In summary, NVP-BEZ235 inhibits the PI3K/mTOR axis and results in antiproliferative and antitumoral activity in cancer cells with both wild-type and mutated p110-alpha.     

Effect of BRAF-mediated PI3K/Akt/mTOR pathway on biological characteristics and chemosensitivity of NSCLC A549/DDP cells

The present study aimed to explore the biological characteristics of non-small cell lung cancer (NSCLC) cells and the mechanism of chemosensitivity through the role of the PI3K/Akt/mTOR signaling pathway mediated by BRAF gene silencing. Following cell transfection and grouping, an MTT assay detected the activity of NSCLC cells, a scratch wound test assessed the migration ability, flow cytometry using PI staining detected the cell cycle phase, TUNEL and flow cytometry through Annexin V-PI staining assessed the apoptosis, and colony formation was used to detect the sensitivity of lung cancer cells to cisplatin chemotherapy. Furthermore, the relative expression levels of BRAF, PTEN, PI3K, mTOR mRNA were assessed by RT-qPCR, and the protein expression levels of BRAF, PTEN, PI3K, phosphorylated (p)-PI3K, Akt, p-Akt, mTOR, p-mTOR, cisplatin resistance-related enzymes ERCC1 and BRCA1, apoptotic proteins Bax and Bcl-2 were assessed by western blotting. Compared with the control group and NC group, there were differences in decreased BRAF mRNA expression levels in the small interfering (si)BRAF group and siBRAF + IGF-1 group (both P<0.05). In addition, compared with the control group, the siBRAF, NVP-BEZ235 and siBRAF + NVP-BEZ235 groups had significant decreased cell viability at 2–6 days, decreased migration ability, shortened proportion of S-phase cells, increased proportion of G₁/G₀-phase cells, increased apoptosis rate, decreased number of colony-forming cells, decreased mRNA expression of PI3K, Akt and mTOR, increased PTEN mRNA expression, decreased protein expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, ERCC1, BRCA1 and Bcl-2, and increased protein expression levels of PTEN and Bax (all P<0.05); and more obvious trends were revealed in the siBRAF + NVP-BEZ235 group (all P<0.05); whereas opposite results were detected in the siBRAF + IGF-1 group when compared with the siBRAF group and NVP-BEZ235 group (all P<0.05). Silencing of BRAF gene expression to inhibit the activation of the PI3K/Akt/mTOR signaling pathway exerted a synergistic effect decreasing cell viability, inhibiting the cell cycle and migration, increasing the apoptosis rate, decreasing the number of colony-forming cells and increasing chemosensitivity of NSCLC. Activation of the PI3K/Akt/mTOR signaling pathway may reverse the role of silencing of BRAF gene expression, providing a potential approach for improving the chemosensitivity of NSCLC. The present study for the first time, to the best of our knowledge, clarified the possible mechanism of NSCLC cell biological characteristic changes and chemosensitivity from the perspective of BRAF gene silencing and PI3K/Akt/mTOR signaling pathway activation, providing a potential reference for suppressing tumor aggravation and improving the therapeutic outcomes of NSCLC at the genetic level.     

Dual PI3K/mTOR inhibition is required to effectively impair microenvironment survival signals in mantle cell lymphoma

Phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway activation contributes to mantle cell lymphoma (MCL) pathogenesis and drug resistance. Antitumor activity has been observed with mTOR inhibitors. However, they have shown limited clinical efficacy in relation to drug activation of feedback loops. Selective PI3K inhibition or dual PI3K/mTOR catalytic inhibition are different therapeutic approaches developed to achieve effective pathway blockage. Here, we have performed a comparative analysis of the mTOR inhibitor everolimus, the pan-PI3K inhibitor NVP-BKM120 and the dual PI3K/mTOR inhibitor NVP-BEZ235 in primary MCL cells. We found NVP-BEZ235 to be more powerful than everolimus or NVP-BKM120 in PI3K/Akt/mTOR signaling inhibition, indicating that targeting the PI3K/Akt/mTOR pathway at multiple levels is likely to be a more effective strategy for the treatment of MCL than single inhibition of these kinases. Among the three drugs, NVP-BEZ235 induced the highest change in gene expression profile. Functional validation demonstrated that NVP-BEZ235 inhibited angiogenesis, migration and tumor invasiveness in MCL cells. NVP-BEZ235 was the only drug able to block IL4 and IL6/STAT3 signaling which compromise the therapeutic effect of chemotherapy in MCL. Our findings support the use of the dual PI3K/mTOR inhibitor NVP-BEZ235 as a promising approach to interfere with the microenvironment-related processes in MCL.     

Comparison of the effects of the PI3K/mTOR inhibitors NVP-BEZ235 and GSK2126458 on tamoxifen-resistant breast cancer cells

Background

Treatment with anti-estrogens or aromatase inhibitors is commonly used for patients with estrogen receptor-positive (ER⁺) breast cancers; however resistant disease develops almost inevitably, requiring a choice of secondary therapy. One possibility is to use inhibitors of the PI3K/mTOR pathway and several candidate drugs are in development. We examined the in vitro effects of two inhibitors of the PI3K/mTOR pathway on resistant MCF-7 cells.

Results

The derived sub-lines showed increased resistance to tamoxifen but none exhibited concomitantly increased sensitivity to the PI3K inhibitors. NVP-BEZ235 and GSK2126458 acted mainly by induction of cell cycle arrest, particularly in G₁-phase, rather than by induction of apoptosis. The lines varied considerably in their utilization of the AKT, p70S6K and ERK pathways. NVP-BEZ235 and GSK2126458 inhibited AKT signaling but NVP-BEZ235 showed greater effects than GSK2126458 on p70S6K and rpS6 signaling with effects resembling those of rapamycin.

Methods

We cultured MCF-7 cells for prolonged periods either in the presence of the anti-estrogen tamoxifen (three sub-lines) or in estrogen free medium (two sub-lines) to mimic the effects of clinical treatment. We then analyzed the effects of two dual PI3K/mTOR phosphoinositide-3-kinase inhibitors, NVP-BEZ235 and GSK2126458, on the growth and signaling pathways of these MCF-7 sub-lines. The functional status of the PI3K, mTOR and ERK pathways was analyzed by measuring phosphorylation of AKT, p70S6K, rpS6 and ERK.

Conclusion

Increased resistance to tamoxifen in these MCF-7 sub-lines is not associated with hypersensitivity to PI3K inhibitors. While both drugs inhibited AKT signaling, NVP-BEZ235 resembled rapamycin in inhibiting the mTOR pathway.Key words: breast cancer, PI3K, mTOR, BEZ235, GSK2126458, estrogen receptor, MCF-7     

NVP-BEZ235, a dual PI3K/mTOR inhibitor,inhibits osteosarcoma cell proliferation and tumor development in vivo with an improved survival rate

Despite recent improvements in chemotherapy and surgery, the problem of non-response osteosarcoma to chemotherapy remains, and is a parameter that is critical for prognosis. The present work investigated the therapeutic value of NVP-BEZ235, a dual class I PI3K/mTOR inhibitor. NVP-BEZ235 inhibited osteosarcoma cell proliferation by inducing G0/G1 cell cycle arrest with no caspase activation. In murine pre-clinical models, NVP-BEZ235 significantly slowed down tumor progression and ectopic tumor bone formation with decreased numbers of Ki67⁺ cells and reduced tumor vasculature. Finally, NVP-BEZ235 considerably improved the survival rate of mice with osteosarcoma. Taken together, the results of the present work show that NVP-BEZ235 exhibits therapeutic interest in osteosarcoma and may be a promising adjuvant drug for bone sarcomas.     

High frequency of loss of PTEN expression in human solid salivary adenoid cystic carcinoma and its implication for targeted therapy

Salivary gland tumor (SGT) is one of the least studied cancers due to its rarity and heterogeneous histological types. Here, we reported that loss of PTEN expression was most frequently found in the poorly differentiated, high grade solid adenoid cystic carcinomas. Loss of PTEN expression correlated with activation of mTOR by increased phosphorylated S6 ribosome protein. We further functionally studied the role of PTEN in a pair of human SACC cell lines, SACC-83 and SACC-LM. Reduced PTEN level was correlated with the metastasis potential. When we knocked down PTEN in the SACC-83 cell line, we observed increased proliferation and enhanced migration/invasion in vitro, and increased tumor size in vivo. We further tested the therapeutical effect by applying a PI3K/mTOR inhibitor NVP-BEZ235 to both SACC cell lines. Decreased cell proliferation, increased apoptosis, as well as reduced cell migration/invasion were observed in both cell lines upon the NVP-BEZ235 treatment. Moreover, the NVP-BEZ235 treatment in a SGT xenograft mouse model significantly reduced primary tumor size and lung metastasis. Taken together, our results demonstrated that PTEN is a potent tumor suppressor in human SGTs, and targeting PI3K/mTOR pathway may be effective in the targeted therapy for human SGT patients with loss of PTEN expression.     

Concurrent inhibition of PI3-Kinase and mTOR induces cell death in diffuse large B cell lymphomas,a mechanism involving down regulation of Mcl-1

Phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling is frequently dysregulated in diffuse large B cell lymphoma (DLBCL) including the favorable germinal centre B-cell (GCB) and the unfavorable activated B-cell (ABC) subtypes. mTOR promotes cap-dependent translation of proteins, like Mcl-1, through inhibitory phosphorylation of the eukaryotic translation initiation factor 4E binding protein 1 (4EBP1). Inhibition of mTOR by RAD001 reduces proliferation but fails to dephosphorylate 4EBP1 and to induce cell death in either DLBCL subtype. In contrast, concurrent inhibition of PI3K and mTOR with NVP-BEZ235 inhibits proliferation, dephosphorylates 4EBP1, and induces cells death, notably more pronounced in CGB cells. Small RNA interference identifies Mcl-1 as a crucial cell death mediator of both DLBCL subtypes. Inhibition of the PI3K/mTOR/4EBP1 by NVP-BEZ235 results in suppression of the cap-dependent translation initiation complex and concomitant downregulation of Mcl-1 in GCB cell lines. In ABC cell lines, this suppression is possibly compensated by NF-κB- or Pim kinase-mediated signaling.     

Vascular responses to radiotherapy and androgen-deprivation therapy in experimental prostate cancer

Background

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated in tumor cells and promotes tumor cell survival after radiation-induced DNA damage. Because the pathway may not be completely inhibited after blockade of PI3K itself, due to feedback through mammalian target of rapamycin (mTOR), more effective inhibition might be expected by targeting both PI3K and mTOR inhibition.

Materials and methods

We investigated the effect of two dual PI3K/mTOR (both mTORC1 and mTORC2) inhibitors, NVP-BEZ235 and NVP-BGT226, on SQ20B laryngeal and FaDu hypopharyngeal cancer cells characterised by EGFR overexpression, on T24 bladder tumor cell lines with H-Ras mutation and on endothelial cells. Analysis of target protein phosphorylation, clonogenic survival, number of residual ??H2AX foci, cell cycle and apoptosis after radiation was performed in both tumor and endothelial cells. In vitro angiogenesis assays were conducted as well.

Results

Both compounds effectively inhibited phosphorylation of Akt, mTOR and S6 target proteins and reduced clonogenic survival in irradiated tumor cells. Persistence of DNA damage, as evidenced by increased number of ??H2AX foci, was detected after irradiation in the presence of PI3K/mTOR inhibition, together with enhanced G2 cell cycle delay. Treatment with one of the inhibitors, NVP-BEZ235, also resulted in decreased clonogenicity after irradiation of tumor cells under hypoxic conditions. In addition, NVP-BEZ235 blocked VEGF- and IR-induced Akt phosphorylation and increased radiation killing in human umbilical venous endothelial cells (HUVEC) and human dermal microvascular dermal cells (HDMVC). NVP-BEZ235 inhibited VEGF-induced cell migration and capillary tube formation in vitro and enhanced the antivascular effect of irradiation. Treatment with NVP-BEZ235 moderately increased apoptosis in SQ20B and HUVEC cells but not in FaDu cells, and increased necrosis in both tumor and endothelial all cells tumor.

Conclusions

The results of this study demonstrate that PI3K/mTOR inhibitors can enhance radiation-induced killing in tumor and endothelial cells and may be of benefit when combined with radiotherapy.     

Effects of the dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 on the tumor vasculature: implications for clinical imaging

Dysregulated angiogenesis and high tumor vasculature permeability, two vascular endothelial growth factor (VEGF)-mediated processes and hallmarks of human tumors, are in part phosphatidylinositol 3-kinase (PI3K) dependent. NVP-BEZ235, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, was found to potently inhibit VEGF-induced cell proliferation and survival in vitro and VEGF-induced angiogenesis in vivo as shown with s.c. VEGF-impregnated agar chambers. Moreover, the compound strongly inhibited microvessel permeability both in normal tissue and in BN472 mammary carcinoma grown orthotopically in syngeneic rats. Similarly, tumor interstitial fluid pressure, a phenomenon that is also dependent of tumor permeability, was significantly reduced by NVP-BEZ235 in a dose-dependent manner on p.o. administration. Because RAD001, a specific mTOR allosteric inhibitor, was ineffective in the preceding experiments, we concluded that the effects observed for NVP-BEZ235 are in part driven by PI3K target modulation. Hence, tumor vasculature reduction was correlated with full blockade of endothelial nitric oxide (NO) synthase, a PI3K/Akt-dependent but mTORC1-independent effector involved in tumor permeability through NO production. In the BN472 tumor model, early reduction of permeability, as detected by K(trans) quantification using the dynamic contrast-enhanced magnetic resonance imaging contrasting agent P792 (Vistarem), was found to be a predictive marker for late-stage antitumor activity by NVP-BEZ235.     

PI3K／Akt／mTOR在表阿霉素抑制Jurkat细胞增殖和诱导凋亡中的作用

目的 探讨PI3K／Akt／mTOR信号通路在表阿霉素抑制人T细胞淋巴瘤细胞株Jurkat细胞增殖和诱导凋亡中的作用。方法 用0、1.25、2.5、5、10μmol／L表阿霉素和0、0.25、0.5、1、2μmol／L PI3K／mTOR双重抑制剂（NVP-BEZ235）对Jurkat细胞作用48h后,CCK-8试剂盒检测Jurkat细胞株增殖抑制情况;采用AnnexinⅤ／PE双染法流式细胞术检测上述药物作用Jurkat细胞48h的凋亡率以及5μmol／L表阿霉素和2μmol／L NVP BEZ235单独及联合作用Jurkat细胞0、12、24、36、48h的凋亡率;Western blotting法检测5、10μmol/L的表阿霉素作用Jurkat细胞0、6、12、24、48h,以及5μmol/L表阿霉素与2μmol/L NVP BEZ235单独及联合作用Jurkat细胞24、48h的PI3K／Akt／mTOR信号通路中Akt、mTOR、p70s6k等表达变化。结果 表阿霉素能够抑制Jurkat细胞增殖并诱导其凋亡,且凋亡作用呈浓度依赖性,5μmol／L表阿霉素作用Jurkat细胞48h的凋亡率为57.72％。在表阿霉素诱导Jurkat细胞凋亡过程中伴随Akt、mTOR、p70s6k的表达变化, NVP-BEZ235能够降低Jurkat细胞Akt、p70s6k的磷酸化水平,显著提高表阿霉素诱导Jurkat细胞凋亡的作用,5μmol／L表阿霉素和2μmol／L NVP-BEZ235联合作用Jurkat细胞48h的凋亡率达78.31％,明显高于5μmol／L表阿霉素的57.72％。结论 表阿霉素抑制Jurkat细胞增殖和诱导凋亡与PI3K／Akt／mTOR信号通路有关,当该通路抑制剂与表阿霉素联用时,Jurkat细胞对于表阿霉素敏感性有一定程度的提高。     

Antitumor activity of the combination of an HSP90 inhibitor and a PI3K/mTOR dual inhibitor against cholangiocarcinoma

The PI3K/Akt/mTOR pathway is overactivated and heat shock protein (HSP) 90 is overexpressed in common cancers. We hypothesized that targeting both pathways can kill intrahepatic cholangiocarcinoma (CCA) cells. HSP90 and PTEN protein expression was evaluated by immunohistochemical staining of samples from 78 patients with intrahepatic CCA. CCA cell lines and a thioacetamide (TAA)-induced CCA animal model were treated with NVP-AUY922 (an HSP90 inhibitor) and NVP-BEZ235 (a PI3K/mTOR inhibitor) alone or in combination.Both HSP90 overexpression and loss of PTEN were poor prognostic factors in patients with intrahepatic CCA. The combination of the HSP90 inhibitor NVP-AUY922 and the PI3K/mTOR inhibitor NVP-BEZ235 was synergistic in inducing cell death in CCA cells. A combination of NVP-AUY922 and NVP-BEZ235 caused tumor regression in CCA rat animal model. This combination not only inhibited the PI3K/Akt/mTOR pathway but also induced ROS, which may exacerbate the vicious cycle of ER stress. Our data suggest simultaneous targeting of the PI3K/mTOR and HSP pathways for CCA treatment.     

Exogenous IGFBP-2 promotes proliferation,invasion, and chemoresistance to temozolomide in glioma cells via the integrin β1-ERK pathway

Background:

Insulin-like growth factor binding protein-2 (IGFBP-2) is significantly increased in the serum of patients with malignant gliomas. High plasma IGFBP-2 levels are correlated with poor prognosis in glioma patients. However, the exact role of exogenous IGFBP-2 in gliomas is unclear.

Methods and results:

Using the MTT cell viability assay, cell cycle analysis, and the transwell migration assay, it was demonstrated that IGFBP-2 treatment stimulated proliferation and invasion in U87 and U251 cell lines and primary SU3 glioma cells. Western blot analysis and immunofluorescence staining revealed that IGFBP-2 promoted ERK phosphorylation and nuclear translocation. Moreover, blocking ERK activation using the inhibitor PD98059 markedly reduced the effects of IGFBP-2 in glioma cells. As IGFBP-2 has an integrin-binding domain, the contribution of integrin β1 to these IGFBP-2-mediated processes was examined. Neutralisation or knockdown of the expression of integrin β1 inhibited IGFBP-2-induced ERK activation, cell proliferation, and cell invasion. Significantly, IGFBP-2 induced temozolomide resistance in glioma cells in an integrin β1/ERK-dependent manner.

Conclusions:

Exogenous IGFBP-2 induces proliferation, invasion, and chemoresistance in glioma cells via integrin β1/ERK signaling, suggesting that targeting this pathway could represent a potential therapeutic strategy for the treatment of gliomas. The identification of this pathway in glioma progression provides insight into the mechanism by which serum IGFBP-2 levels can predict the prognosis of glioma patients.     

The Dual PI3K/mTOR Inhibitor NVP-BEZ235 Is a Potent Inhibitor of ATM- and DNA-PKCs-Mediated DNA Damage Responses

Inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy owing to the frequent mutational activation of the PI3K-Akt-mTORC1 pathway in many cancers, including glioblastomas (GBMs). NVP-BEZ235 is a novel and potent dual PI3K/mTOR inhibitor that is currently in phase 1/2 clinical trials for advanced solid tumors. Here, we show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the two major kinases responding to ionizing radiation (IR)-induced DNA double-strand breaks (DSBs). Consequently, NVP-BEZ235 blocks both nonhomologous end joining and homologous recombination DNA repair pathways resulting in significant attenuation of DSB repair. In addition, phosphorylation of ATMtargets and implementation of the G₂/M cell cycle checkpoint are also attenuated by this drug. As a result, NVP-BEZ235 confers an extreme degree of radiosensitization and impairs DSB repair in a panel of GBM cell lines irrespective of their Akt activation status. NVP-BEZ235 also significantly impairs DSB repair in a mouse tumor model thereby validating the efficacy of this drug as a DNA repair inhibitor in vivo. Our results, showing that NVP-BEZ235 is a potent and novel inhibitor of ATM and DNA-PKcs, have important implications for the informed and rational design of clinical trials involving this drug and also reveal the potential utility of NVP-BEZ235 as an effective radiosensitizer for GBMs in the clinic.     

Recombinant viral capsid protein VP1 suppresses lung cancer metastasis by inhibiting COX-2/PGE2 and MIG-7

Recombinant capsid protein VP1 (rVP1) of foot-and-mouth disease virus binds to integrins to modulate Akt/GSK3-β signaling and suppress migration/invasion and metastasis of cancer cells, but the underlying molecular mechanism is unclear. Here, we showed that the rVP1-mediated inhibition of Akt/GSK3-β signaling and cell migration/invasion was accompanied by downregulation in phosphatidylinositol (3,4,5)-triphosphate (PIP3), integrin-linked kinase (ILK) and IKK/NF-κB signaling as well as suppression of COX-2/PGE2 and MIG-7. Addition of PIP3 or overexpression of ILK reversed the rVP1-induced inhibition of IKK/NF-κB signaling, COX-2 and MIG-7. The rVP1-mediated downregulation of COX-2/PGE2 and MIG-7 led to not only attenuation of epithelial-mesenchymal transition, MMP2 activity and invasion of lung cancer cells in vitro but also decreased tumor growth and metastasis of lung cancer in xenograft mice. Moreover, downregulation of COX-2/PGE2 and MIG-7 significantly prolonged the overall and disease-free survival of lung cancer-bearing mice. These results suggest that rVP1 inhibits cancer invasion/metastasis, partly if not mainly, via downregulating integrin/PI3K/Akt, ILK and IKK/NF-κB signaling to suppress expression of COX-2/PGE2 and MIG-7.     

Inhibition of phosphoinositide-3 kinase pathway down regulates ABCG2 function and sensitizes malignant pleural mesothelioma to chemotherapy

Malignant pleural mesothelioma (MPM) is a relatively chemoresistant malignancy. Diverse biological targets are under investigation to develop new therapeutic options. One of these targets, namely the phosphoinositide-3-kinase (PI3K) pathway, has been shown to be a regulator of the side population (SP) phenotype in different cancers. The SP phenotype is due to drug efflux abilities providing drug-resistant properties. The presence of a SP fraction in MPM was recently observed in our laboratory. The aim of this study was to investigate the role of the PI3K pathway in the regulation of the SP phenotype in MPM. Treatment of overnight serum-starved cells with IGF increased phosphorylation of downstream target AKT, S6 and 4EBP1 and SP fraction in ZL55, ZL34 and SDM103T2 MPM cell lines. The PI3K/mTOR inhibitor NVP-BEZ235 and PI3K inhibitor wortmannin reduced the phosphorylation of downstream target AKT, S6 and 4EBP1 and decreased the SP fraction. Chemotherapy resistance mediated by drug efflux was tested by treating the cells with mitoxantrone. NVP-BEZ235 increased mitoxantrone cytotoxicity and this effect was mimicked by fumitremorgin C, a specific ABCG2 inhibitor, although not to the same extent, indicating that ABCG2-mediated drug efflux participates to chemoresistance. The involvement of ABCG2 in drug efflux was confirmed by determination of ABCG2-mediated decrease of intracellular mitoxantrone accumulation and silencing experiments. NVP-BEZ235-mediated decrease in drug efflux was associated with a significant decrease of ABCG2 present at the cell surface in ZL55 and SDM103T2 cells. In conclusion, the PI3K pathway is playing an important role in regulating the SP phenotype in MPM cells and inhibition of this activity may contribute to a more efficient cancer treatment.     

Inhibition of mTOR reduce Stat3 and PAI related angiogenesis in salivary gland adenoid cystic carcinoma

Angiogenesis is a complex biological process, which is involved in tumorigenesis and progression. However, the molecular mechanism of underlying angiogenesis remains largely unknown. In this study, we accessed the expression of proteins related angiogenesis by immunohistochemical staining of human tissue microarray which contains 72 adenoid cystic carcinoma (AdCC), 12 pleomorphic adenoma (PMA) and 18 normal salivary gland (NSG) using digital pathological scanner and scoring system. We found that the expression of p-S6^S235/236 (a downstream molecule of mTOR), p-Stat3^T705, PAI, EGFR, and HIF-1α was significantly increased in AdCC as compared with PMA and (or) NSG (p < 0.05). While, the expression of these proteins was not associated with pathological type of human AdCC (p > 0.05). Correlation analysis of these proteins revealed that p-S6^S235/236 up-regulates the expression of EGFR/p-Stat3^T705 (p < 0.05) and HIF-1α/PAI (p < 0.05). Moreover, the activation of p-S6^S235/236, EGFR/p-Stat3^T705 and HIF-1α/PAI associated with angiogenesis (CD34) and proliferation (Ki-67). In vitro, Rapamycin suppressed the expression of p-S6^S235/236, EGFR, p-Stat3^T705, HIF-1α and PAI. Further more, target inhibition of mTOR by rapamycin effectively reduced tumor growth of SACC-83 cells line nude mice xenograft and decreased the expression of p-S6^S235/236, EGFR/p-Stat3^T705 and HIF-1α/PAI. Taken together, these data revealed that mTOR signaling pathway regulates tumor angiogenesis by EGFR/p-Stat3^T705 and HIF-1α/PAI. Inhibition of mTOR by rapamycin could effectively reduced tumor growth. It is likely that mTOR inhibitors may be a potential candidate for treatment of AdCC.