Inhibition of DNA methyltransferase as a novel therapeutic strategy to overcome acquired resistance to dual PI3K/mTOR inhibitors

Dual PI3K/mTOR(phosphatidylinositol 3-kinase/mammalian target of rapamycin) inhibitors are being evaluated clinically for the treatment of tumors with a hyperactivated PI3K/mTOR pathway. However, unexpected outcomes were obtained in clinical studies of cancer patients with an aberrant PI3K pathway. In clinical trials, applicable combination regimens are not yet available. In this study, using an integrated analysis of acquired BEZ235-resistant nasopharyngeal carcinoma cells, we demonstrate that DNA methyltransferase is a key modulator and a common node upstream of the AKT/mTOR and PDK1/MYC pathways, which are activated in cancer cells with acquired BEZ235 resistance. DNA methyltransferases were upregulated and induced PTEN and PPP2R2B gene hypermethylation, which downregulated their expression in BEZ235-resistant cancer cells. Reduced PTEN and PPP2R2B expression correlated with activated AKT/mTOR and PDK1/MYC pathways and conferred considerable BEZ235 resistance in nasopharyngeal carcinoma. Targeting methyltransferases in combination with BEZ235 sensitized BEZ235-resistant cells to BEZ235 in vitro and in vivo, suggesting the potential clinical application of this strategy to overcome BEZ235 resistance.     

Compensatory activation of Akt in response to mTOR and Raf inhibitors – A rationale for dual-targeted therapy approaches in neuroendocrine tumor disease

Several studies have established a link between aberrant PI(3)K–Akt–mTOR- and Ras–Raf–MEK–Erk1/2 signaling and neuroendocrine tumor disease. In this study, we comparatively investigate the antitumor potential of novel small-molecule inhibitors targeting mTOR (RAD001), mTOR/PI(3)K (NVP-BEZ235) and Raf (Raf265) on human NET cell lines of heterogeneous origin. All inhibitors induced potent antitumor effects which involved the induction of apoptosis and G0/G1 arrest. However, the dual mTOR/PI(3)K inhibitor NVP-BEZ235 was more efficient compared to the single mTOR inhibitor RAD001. Consistently, NVP-BEZ235 prevented the negative feedback activation of Akt as observed after treatment with RAD001. Raf265 inhibited Erk1/2 phosphorylation but strongly induced Akt phosphorylation and VEGF secretion, suggesting the existence of a compensatory feedback loop on PI3K-Akt signaling. Finally, combined treatment with RAD001 or NVP-BEZ235 and Raf265 was more efficient than single treatment with either kinase inhibitor. Together, our data provide a rationale for dual targeting of PI(3)K–Akt–mTOR- and Ras–Raf–MEK–Erk1/2 signaling in NET disease.     

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.     

Superior efficacy of co-treatment with dual PI3K/mTOR inhibitor NVP-BEZ235 and pan-histone deacetylase inhibitor against human pancreatic cancer

Genetic alterations activating K-RAS and PI3K/AKT signaling are also known to induce the activity of mTOR kinase through TORC1 and TORC2 complexes in human pancreatic ductal adenocarcinoma (PDAC). Here, we determined the effects of the dual PI3K and mTOR inhibitor, NVP-BEZ235 (BEZ235), and the pan-histone deacetylase inhibitor panobinostat (PS) against human PDAC cells. Treatment with BEZ235 or PS inhibited cell cycle progression with induction of the cell cycle inhibitory proteins, p21 waf1 and p27 kip1. BEZ235 and PS also dose dependently induced loss of cell viability of the cultured PDAC cells, associated with depletion of phosphorylated (p) AKT, as well as of the TORC1 substrates 4EBP1 and p70S6 kinase. While inhibiting p-AKT, treatment with PS induced the levels of the pro-apoptotic proteins BIM and BAK. Co-treatment with BEZ235 and PS synergistically induced apoptosis of the cultured PDAC cells. This was accompanied by marked attenuation of the levels of p-AKT and Bcl-x_L but induction of BIM. Although in vivo treatment with BEZ235 or PS reduced tumor growth, co-treatment with BEZ235 and PS was significantly more effective in controlling the xenograft growth of Panc1 PDAC cells in the nude mice. Furthermore, co-treatment with BEZ235 and PS more effectively blocked tumor growth of primary PDAC heterotransplants (possessing K-RAS mutation and AKT2 amplification) subcutaneously implanted in the nude mice than each agent alone. These findings demonstrate superior activity and support further in vivo evaluation of combined treatment with BEZ235 and PS against PDAC that possess heightened activity of RAS-RAF-ERK1/2 and PI3K-AKT-mTOR pathways.     

Overcoming acquired resistance to letrozole by targeting the PI3K/AKT/mTOR pathway in breast cancer cell clones

Development of resistance to endocrine therapy is a clinical issue in estrogen receptor (ER)-positive breast cancer. Here we show that persistent activation of AKT/mTOR signaling is crucial to the acquisition of letrozole resistance in cell clones generated from MCF-7/AROM-1 aromatase-expressing breast cancer cells after prolonged letrozole exposure. ERα plays a marginal role in this context. As a proof of concept, the association between PI3K/AKT/mTOR signaling and insensitivity to endocrine therapies was confirmed in breast cancer patients who developed early letrozole resistance in neoadjuvant setting. In addition our results suggest that, regardless of the mechanism mediating the activation of AKT/mTOR pathway, either RAD001 or NVP-BEZ235 treatment may represent a promising strategy to overcome acquired resistance to letrozole in breast cancers dependent on AKT/mTOR signaling.     

The novel phosphoinositide 3‐kinase–mammalian target of rapamycin inhibitor,BEZ235, circumvents erlotinib resistance of epidermal growth factor receptor mutant lung cancer cells triggered by hepatocyte growth factor

Acquired resistance to epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR–TKIs), such as gefitinib and erlotinib, is a critical problem in the management of patients with EGFR mutant lung cancer. Several mechanisms have been reported involved in this acquired resistance, including hepatocyte growth factor (HGF) activation of an alternative pathway. PI3K and mTOR are downstream molecules of receptor tyrosine kinases, such as EGFR and Met, and are thought to be ideal targets for controlling various tumor types. We assessed whether BEZ235, a dual inhibitor of PI3K and mTOR, could overcome the EGFR–TKI resistance induced by HGF in an EGFR mutant lung cancer model. Exogenous and endogenous HGF triggered resistance to erlotinib in the PC‐9 and HCC827, EGFR mutant lung cancer cell lines. BEZ235 alone inhibited the viability of PC‐9 and HCC827 cells in vitro, irrespective of the presence or the absence of HGF. Using a xenograft model of severe combined immunodeficient mice with HGF‐gene‐transfected PC‐9 cells (PC‐9/HGF), we found that BEZ235 inhibited tumor growth, whereas erlotinib did not. BEZ235 monotherapy also inhibited the phosphorylation of Akt and p70S6K/S6RP, downstream molecules of PI3K and mTOR, respectively, as well as suppressing tumor‐cell proliferation and angiogenesis of PC‐9/HGF tumors. These results suggest that BEZ235, even as monotherapy, may be useful in managing HGF‐induced EGFR–TKI resistance in EGFR mutant lung cancer.     

The dual PI3K and mTOR inhibitor NVP-BEZ235 exhibits anti-proliferative activity and overcomes bortezomib resistance in mantle cell lymphoma cells

Mantle cell lymphoma (MCL) is one of the most difficult B-cell lymphomas to be treated. The phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway is constitutively activated in MCL and plays a critical role in tumor growth and survival. However, single targeted agent mTOR has limited efficacy in treating MCL. Here, we investigate for the first time potential efficacy of NVP-BEZ235 (BEZ235) in treating MCL by simultaneously targeting Akt and mTOR. In this study, phosphorylated Akt and mTOR level were elevated in tissue samples from MCL patients and in MCL cell lines. We also generated bortezomib-resistant MCL cell lines and found increased phosphorylation of Akt and mTOR. Individual inhibition of PI3K or mTOR had limited anti-proliferative effects, whereas dual inhibition with BEZ235 effectively inhibited cell growth. The effect of BEZ235 was synergistic and sensitized the cells to the cytotoxic effects of conventional agents. Furthermore, BEZ235 could overcome acquired resistance to bortezomib in MCL cells and suppress the activated Akt/mTOR pathway. Therefore, these data suggest that the Akt/mTOR pathway plays a key role in the growth and survival of MCL cells and that these proteins may need to be simultaneously targeted for effective treatment of the disease. Our findings suggest that BEZ235 may be an effective agent for the treatment of MCL.     

MCL-1-independent mechanisms of synergy between dual PI3K/mTOR and BCL-2 inhibition in diffuse large B cell lymphoma

The PI3K/AKT/mTOR axis promotes survival and is a frequently mutated pathway in cancer. Yet, inhibitors targeting this pathway are insufficient to induce cancer cell death as single agents in some contexts, including diffuse large B cell lymphoma (DLBCL). In these situations, combinations with inhibitors targeting BCL-2 survival proteins (ABT-199 and ABT-263) may hold potential. Indeed, studies have demonstrated marked synergy in contexts where PI3K/mTOR inhibitors suppress expression of the pro-survival protein, MCL-1. In this study, we use BH3 profiling to confirm that BCL-2 and BCL-X_L support survival following PI3K pathway inhibition, and that the dual PI3K/mTOR inhibitor BEZ235 strongly synergizes with BCL-2 antagonists in DLBCL. However, we identify an alternative mechanism of synergy between PI3K/mTOR and BCL-2 inhibitors, independent of MCL-1 down-regulation. Instead, we show that suppression of AKT activation by BEZ235 can induce the mitochondrial accumulation of pro-apoptotic BAD and BIM, and that expression of a constitutively active form of AKT prevents sensitization to BCL-2 antagonism. Thus, our work identifies an additional mechanism of synergy between PI3K pathway inhibitors and BCL-2 antagonists that strengthens the rationale for testing this combination in DLBCL.     

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     

PI3K/AKT/mTOR pathway activation in primary and corresponding metastatic breast tumors after adjuvant endocrine therapy

Both preclinical and clinical data suggest that activation of the PI3K/AKT/mTOR pathway in response to hormonal therapy results in acquired endocrine therapy resistance. We evaluated differences in activation of the PI3K/AKT/mTOR pathway in estrogen receptor α (ERα) positive primary and corresponding metastatic breast cancer tissues using immunohistochemistry for downstream activated proteins, like phosphorylated mTOR (p‐mTOR), phosphorylated 4E Binding Protein 1 (p‐4EBP1) and phosphorylated p70S6K (p‐p70S6K). For p‐mTOR and p‐4EBP1, the proportion of immunostained tumor cells (0–100%) was scored. Cytoplasmic intensity (0–3) was assessed for p‐p70S6K. The difference between expression of these activated PI3K/AKT/mTOR proteins‐ in primary and metastatic tumor was calculated and tested for an association with adjuvant endocrine therapy. In patients who had received endocrine therapy (N = 34), p‐mTOR expression increased in metastatic tumor lesions compared to the primary tumor (median difference 45%), while in patients who had not received adjuvant endocrine therapy (N = 37), no difference was found. Similar results were observed for p‐4EBP1 and p‐p70S6K expression. In multivariate analyses, adjuvant endocrine therapy was significantly associated with an increase in p‐mTOR (p = 0.01), p‐4EBP1 (p = 0.03) and p‐p70S6K (p = 0.001), indicating that compensatory activation of the PI3K/AKT/mTOR pathway might indeed be a clinically relevant resistance mechanism resulting in acquired endocrine therapy resistance.     

Dual PI3K/mTOR inhibitor NVP-BEZ235 suppresses hypoxia-inducible factor (HIF)-1α expression by blocking protein translation and increases cell death under hypoxia

The PI3K/Akt pathway is activated in many cancers; therefore, we investigated NVP-BEZ235, a dual PI3K/mTOR inhibitor. BEZ235 was more potent than either the mTOR inhibitor rapamycin or the PI3K inhibitor LY294002 in blocking HIF-1α induction. BEZ235 decreases protein translation, and 7-methyl GTP chromatography showed that the drug induced robust recruitment of 4E-BP1 to eIF4E and a near absence of binding of eIF4G. BEZ235 also decreased expression of other proteins known to be regulated by eIF4E including cyclin B1 and D1 and vascular endothelial growth factor (VEGF). BEZ235 also decreased the level of eIF4G but not eIF4E. As HIF-1α has been associated with adaptation to hypoxic stress, we examined the effect of the drug on cell survival in low pO 2. BEZ235 increased killing of cells under hypoxia, measured by short-term (MTT) and long-term (clonogenic) assays. To understand the underlying mechanism, we examined BEZ235's effect on the expression of factors associated with cell survival. Under normoxia, Akt Ser473 phosphorylation decreased within an hour of BEZ235 treatment, but then increased by 24 h. In contrast, under hypoxia, BEZ235 caused prolonged suppression of Akt Ser473 phosphorylation. Furthermore, there was greater PARP cleavage in hypoxic cells than in normoxic cells, consistent with increased apoptosis. BEZ235 increased autophagy as measured by LC3-I to LC3-II conversion under both normoxic and hypoxic conditions, but our data indicate that this is actually a pro-survival mechanism. In conclusion, we have found that BEZ235 blocks HIF-1α induction by decreasing protein translation and increases cell killing under hypoxia, likely by increasing apoptosis.     

Preclinical evaluation of the mTOR–PI3K inhibitor BEZ235 in nasopharyngeal cancer models

The dual PI3K–mTOR inhibitor BEZ235 was evaluated in preclinical models of nasopharyngeal carcinoma (NPC). The IC₅₀ value of BEZ235 for growth was in the nanomolar range in vitro, induce G1 cycle arrest and apoptosis, and inhibited AKT and mTOR signaling in most NPC cell lines. No synergistic effect was observed when BEZ235 was combined with chemotherapy. BEZ235 increased MAPK activation in vitro but not in vivo. A daily schedule was more effective than a weekly schedule on tumor growth and inhibition of downstream mTOR signaling in vivo. The activity of BEZ235 maybe independent of the PIK3CA amplification and mutation status.     

Antitumor activity of selective MEK1/2 inhibitor AZD6244 in combination with PI3K/mTOR inhibitor BEZ235 in gefitinib-resistant NSCLC xenograft models

Purpose

Although the EGF receptor tyrosine kinase inhibitors (EGFR-TKI) gefitinib have shown dramatic effects against EGFR mutant lung cancer, patients become resistant by various mechanisms, including gatekeeper EGFR-T790M mutation, MET amplification, and KRAS mutation, thereafter relapsing. AZD6244 is a potent, selective, and orally available MEK1/2 inhibitor. In this study, we evaluated the therapeutic efficacy of AZD6244 alone or with BEZ235, an orally available potent inhibitor of phosphatidylinositol 3–kinase (PI3K) and mammalian target of rapamycin (mTOR), in gefitinib-resistant non-small cell lung carcinoma (NSCLC) models.

Experimental design

NCI-H1975 with EGFR-T790M mutation, NCI-H1993 with MET amplification and NCI-H460 with KRAS/PIK3CA mutation human NSCLC cells were subcutaneous injected into the athymic nude mice respectively. Mice were randomly assigned to treatment with AZD6244, BEZ235, AZD6244 plus BEZ235, or control for 3 weeks, then all mice were sacrificed and tumor tissues were subjected to western blot analyses and immunohistochemical staining.

Results

AZD6244 could inhibit the tumor growth of NCI-H1993, but slightly inhibit the tumor growth of NCI-1975 and NCI-H460. Combining AZD6244 with BEZ235 markedly enhanced their antitumor effects and without any marked adverse events. Western blot analysis and immunohistochemical staining revealed that AZD6244 alone reduced ERK1/2 phosphorylation, angiogenesis, and tumor cell proliferation. Moreover, MEK1/2 inhibition resulted in decreased AKT phosphorylation in NCI-H1993 tumor model. BEZ235 also inhibited AKT phosphorylation as well as their downstream molecules in all three tumor models. The antiangiogenic effects were substantially enhanced when the agents were combined, which may due to the reduced expression of matrix metallopeptidase-9 in tumor tissues (MMP-9).

Conclusions

In this study, we evaluated therapy directed against MEK and PI3K/mTOR in distinct gefitinib-resistant NSCLC xenograft models. Combining AZD6244 with BEZ235 enhanced their antitumor and antiangiogenic effects. We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation, MET amplification, and KRAS/PIK3CA mutation. This new therapeutic strategy may be a practical approach in the treatment of these patients.     

Combinatorial Effect of Abiraterone Acetate and NVP-BEZ235 on Prostate Tumor Progression in Rats

Use of drug combinations that target different pathways involved in the development and progression of prostate cancer (PCa) has emerged as an alternative to overcome the resistance caused by drug monotherapies. The antiandrogen abiraterone acetate and the PI3K/Akt inhibitor NVP-BEZ235 (BEZ235) may be suitable options for the prevention of drug resistance and the inhibition of PCa progression. The aim of the present study was to evaluate whether abiraterone acetate and BEZ235 achieve superior therapeutic effects to either drug administered as monotherapy, in the early stages of PCa in an androgen-dependent system. Our study showed that each drug might impair tumor growth by reducing proliferation and increasing cell death when administered as monotherapy. However, tumor growth continued to progress with each drug monotherapy and some important side effects were related to BEZ. Conversely, when used in combination, the drugs impaired the inflammatory response, decreased hyperplastic lesions, and blocked tumor progression from premalignant to a malignant stage. Our data showed that the strategy to block the androgenic and PI3K/AKT/mTOR pathway is an effective therapeutic option and should be investigated including distinct PI3K pathway inhibitors.     

Inhibition of constitutively activated phosphoinositide 3‐kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1‐defective breast cancer cells

Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1‐defective human breast cancer cells. Levels of phospho‐AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho‐AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de‐sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI‐103, and BEZ235, showed anti‐proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti‐proliferative effects of gemcitabine by enhancing caspase‐3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1‐defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers. © 2012 Wiley Periodicals, Inc.     

BEZ235对肝癌细胞HepG2放射敏感性的影响

目的:评价双重PI3K/mTOR抑制剂BEZ235对肝癌细胞HepG2放射敏感性的影响。方法:通过MTT测定确定不同BEZ235浓度的生长抑制效果;焦点形成测量和克隆形成测定评估对放射敏感性的影响;通过Annexin-FITC/PI分析BEZ235对辐射诱导细胞凋亡的影响,以及Western blot分析BEZ235对缺氧诱导因子-1α（HIF-1α）蛋白水平的影响。结果:BEZ235以剂量依赖性方式抑制HepG2细胞增殖;10 nmol/L BEZ235增加HepG2细胞的放射敏感性;BEZ235与辐射组合增加了DNA双链断裂;BEZ235联合辐射与单独辐射相比,显著增加了HepG2细胞的凋亡率。BEZ235 降低了HIF-1α蛋白水平。结论:BEZ235增强了人肝癌HepG2细胞的放射敏感性,这一发现与抑制HIF-1α表达有关。提示BEZ235可能是潜在的放疗增敏药物。     

Multipoint targeting of the PI3K/mTOR pathway in mesothelioma

Background:

Mesothelioma is a notoriously chemotherapy-resistant neoplasm, as is evident in the dismal overall survival for patients with those of asbestos-associated disease. We previously demonstrated co-activation of multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET, and AXL in mesothelioma cell lines, suggesting that these kinases could serve as novel therapeutic targets. Although clinical trials have not shown activity for EGFR inhibitors in mesothelioma, concurrent inhibition of various activated RTKs has pro-apoptotic and anti-proliferative effects in mesothelioma cell lines. Thus, we hypothesised that a coordinated network of multi-RTK activation contributes to mesothelioma tumorigenesis.

Methods:

Activation of PI3K/AKT/mTOR, Raf/MAPK, and co-activation of RTKs were evaluated in mesotheliomas. Effects of RTK and downstream inhibitors/shRNAs were assessed by measuring mesothelioma cell viability/growth, apoptosis, activation of signalling intermediates, expression of cell-cycle checkpoints, and cell-cycle alterations.

Results:

We demonstrate activation of the PI3K/AKT/p70S6K and RAF/MEK/MAPK pathways in mesothelioma, but not in non-neoplastic mesothelial cells. The AKT activation, but not MAPK activation, was dependent on coordinated activation of RTKs EGFR, MET, and AXL. In addition, PI3K/AKT/mTOR pathway inhibition recapitulated the anti-proliferative effects of concurrent inhibition of EGFR, MET, and AXL. Dual targeting of PI3K/mTOR by BEZ235 or a combination of RAD001 and AKT knockdown had a greater effect on mesothelioma proliferation and viability than inhibition of individual activated RTKs or downstream signalling intermediates. Inhibition of PI3K/AKT was also associated with MDM2-p53 cell-cycle regulation.

Conclusions:

These findings show that PI3K/AKT/mTOR is a crucial survival pathway downstream of multiple activated RTKs in mesothelioma, underscoring that PI3K/mTOR is a compelling target for therapeutic intervention.     

Selective PI3K inhibition by BKM120 and BEZ235 alone or in combination with chemotherapy in wild-type and mutated human gastrointestinal cancer cell lines

Purpose

New targeted agents like antibodies or small molecules against tyrosine and lipid kinases clearly expand the standard therapy options in oncology. However, tumour resistance is still a challenge, often induced by mutations in growth-related signalling cascades. Twenty and ten percentage of all patients with colorectal and gastric cancers, respectively, carry phosphatidyl-3-kinase (PI3K) mutations and do not respond to receptor-blocking therapies. Recently, selective kinase inhibitors have been generated, which block the PI3K signalling pathway in tumour cells. So far, their therapeutic role for the treatment of mutated versus wild-type human gastrointestinal cancers has not been clarified in detail.

Methods

To define the inhibitory and pro-apoptotic effects of the two PI3K inhibitors BEZ235 and BKM120 in three human colon cancer (HT-29, HCT-116 and DLD-1) and three gastric cancer (NCI-n87, AGS and MKN-45), cell lines with different PIK3CA gene mutation status were used. Firstly, viability, apoptosis and caspase assays were performed during incubation with either the inhibitors alone or combined with different cytotoxic agents. Secondly, the molecular consequences for the cell cycle and signalling pathways were analysed by defining the protein levels by FACS and Western blot analysis.

Results

Both the PI3K inhibitors BEZ235 and BKM120 induced a clear concentration-dependent reduction in cell viability and an increase in apoptotic cell death, with the mutated cells being more sensitive to treatment. However, single-agent BEZ235 caused a G1 arrest in tumour cells, whilst BKM120 induced a G2 shift in a half of the gastrointestinal cancer cell lines. There was a clear downregulation in the protein levels of the PI3K–AKT pathway at the concentrations of 100nM for both agents and for BEZ235 the additional inhibition of the mTOR pathway. Furthermore, BEZ235 caused synergistic induction of apoptosis when combined with irinotecan in colon cancer cell lines. Human gastric cancer cells were less sensitive to both BEZ235 and BKM120.

Conclusions

BEZ235 and BKM120 induced pro-apoptotic effects in all cell lines and especially with an increased response in the PI3KCA mutated cells. Our data support the clinical development of these PI3K inhibitors for patients with wild-type or mutated colon cancers.     

Equivalent benefit of mTORC1 blockade and combined PI3K-mTOR blockade in a mouse model of tuberous sclerosis

Background  

Tuberous sclerosis (TSC) is a hamartoma syndrome in which renal and lung tumors cause the greatest morbidity. Loss of either TSC1 or TSC2 in TSC hamartomas leads to activation of mTORC1 and suppression of AKT. Recent studies indicate that inhibition of mTORC1 with RAD001 (everolimus) leads to rebound activation of AKT, which could protect tumors from drug-induced cell death. Here we examine the potential benefit of inhibition of both mTOR and AKT signaling in a mouse model of TSC, using a dual pan class I PI3K/mTOR catalytic small molecule inhibitor NVP-BEZ235.     

Targeting PI3K and mTORC2 in metastatic renal cell carcinoma: New strategies for overcoming resistance to VEGFR and mTORC1 inhibitors

With the advent of molecularly targeted agents, treatment of metastatic renal cell carcinoma (mRCC) has improved significantly. Agents targeting the vascular endothelial growth factor receptor (VEGFR) and the mammalian target of rapamycin complex 1 (mTORC1) are more effective and less toxic than previous standards of care involving cytotoxic and cytokine therapies. Unfortunately, many patients relapse following treatment with VEGFR and mTORC1 inhibitors as a result of acquired resistance mechanisms, which are thought to lead to the reestablishment of tumor vasculature. Specifically, the loss of negative feedback loops caused by inhibition of mTORC1 leads to upregulation of downstream effectors of the phosphoinositide 3‐kinase (PI3K)/AKT/mTOR pathway and subsequent activation of hypoxia‐inducible factor, an activator of angiogenesis. De novo resistance involving activated PI3K signaling has also been observed. These observations have led to the development of novel agents targeting PI3K, mTORC1/2 and PI3K/mTORC1/2, which have demonstrated antitumor activity in preclinical models of RCC. Several agents—BKM120, BEZ235 and GDC‐0980—are being investigated in clinical trials in patients with metastatic/advanced RCC, and similar agents are being tested in patients with solid tumors. The future success of mRCC treatment will likely involve a combination of agents targeting the multiple pathways involved in angiogenesis, including VEGFR, PI3K and mTORC1/2.