Feedback Activation Of AKT Research Articles

Targeting feedback activation of signaling transduction pathways to overcome drug resistance in cancer.

Dysregulation or aberrant signaling transduction contributes to tumorigenesis. Targeting these abnormal signaling pathways becomes an effective anticancer strategy. However, feedback activation or crosstalk between signaling pathways drives adaptive drug resistance which causes failure of cancer therapy. In this review article, we summarized treatments that cause feedback activation of AKT, ERK, STAT3, EGFR, FGFR, and HER2/3 signaling pathways and the combination therapy to enhance anti-tumor effect or to overcome drug resistance, to explore the underlying mechanisms that define the protein molecules participated or regulated the feedback activation. In addition, we reviewed clinical trials that employ combination treatments to suppress feedback activation and improve therapeutic efficacy of cancer treatments.

Jolkinolide B sensitizes bladder cancer to mTOR inhibitors via dual inhibition of Akt signaling and autophagy.

The monotherapy of mTOR inhibitors (mTORi) in cancer clinical practice has achieved limited success due to the concomitant activation of compensatory pathways, such as Akt signaling and cytoprotective autophagy. Thus, the combination of mTORi and the inhibitors of these pro-survival pathways has been considered a promising therapeutic strategy. Herein, we report the synergistic effects of a natural anti-cancer agent Jolkinolide B (JB) and mTORi (temsirolimus, rapamycin, and everolimus) for the effective treatment of bladder cancer. A mechanistic study revealed that JB induced a dual inhibition of Akt feedback activation and cytoprotective autophagy, potentiating the anti-proliferative efficacy of mTORi in both PTEN-deficient and cisplatin-resistant bladder cancer cells. Meanwhile, mTORi augmented the pro-apoptotic and pro-paraptotic effects of JB by reinforcing JB-activated endoplasmic reticulum stress and MAPK pathways. These synergistic mechanisms were related to cellular reactive oxygen species accumulation. Our study suggests that dual inhibition of Akt feedback activation and cytoprotective autophagy is an effective strategy in mTORi-based therapy, and JB+mTORi combination associated with multiple anti-cancer mechanisms and good tolerance in mouse models may serve as a promising treatment for bladder cancer.

KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer.

Endometrial cancer (EC) often exhibit aberrant activation of PI3K/Akt/mTOR signaling and targeted therapies using mTOR inhibitors showed limited success. The epigenetic modifier, lysine-specific histone demethylase-1A (KDM1A/LSD1) is overexpressed in EC, however, the mechanistic and therapeutic implications of KDM1A in EC are poorly understood. Here, using 119 FDA-approved drugs screen, we identified that KDM1A inhibition is highly synergistic with mTOR inhibitors. Combination therapy of KDM1A and mTOR inhibitors potently reduced the cell viability, survival, and migration of EC cells. Mechanistic studies demonstrated that KDM1A inhibition attenuated the activation of mTOR signaling cascade and abolished rapamycin induced feedback activation of Akt. RNA-seq analysis identified that KDM1A inhibition downregulated the expression of genes involved in rapamycin induced activation of Akt, including the mTORC2 complex. Chromatin immunoprecipitation experiments confirmed KDM1A recruitment to the promoter regions of mTORC2 complex genes and that KDM1A inhibition promoted enrichment of repressive H3K9me2 marks at their promoters. Combination therapy of KDM1A inhibitor and rapamycin reduced the tumor growth in EC xenograft and patient derived xenograft models in vivo and patient derived tumor explants ex vivo. Importantly, in silico analysis of TCGA EC patients data sets revealed that KDM1A expression positively correlated with the levels of PI3K/Akt/mTOR genes. Collectively, our results provide compelling evidence that KDM1A inhibition potentiates the activity of mTOR inhibitors by attenuating the feedback activation of Akt survival signaling. Furthermore, the use of concurrent KDM1A and mTOR inhibitors may be an attractive targeted therapy for EC patients.

Potentiation of the Anticancer Effects by Combining Docetaxel with Ku-0063794 against Triple-Negative Breast Cancer Cells.

PurposemTORC1 and mTORC2 inhibition by Ku-0063794 could confer profound anticancer effects against cancer cells because it eliminates feedback activation of Akt. Herein, we aimed to determine anticancer effects of docetaxel and Ku-0063794, individually or in combination, against breast cancer cells, especially triple-negative breast cancer (TNBC) cells.Materials and MethodsMCF-7 breast cancer and MDA-MB-231 TNBC cell lines for in vitro studies and mouse xenograft model for in vivo studies were used to investigate the effect of docetaxel, Ku-0063794, or their combination.ResultsIn the in vitro experiments, combination therapy synergistically reduced cell viability and induced higher apoptotic cell death in breast cancer cells than the individual monotherapies (p < 0.05). Western blot analysis and flow cytometric analysis showed that the combination therapy induced higher apoptotic cell death than the individual monotherapies (p < 0.05). In the in vivo experiment, docetaxel and Ku-0063794 combination therapy reduced the growth of MDA-MB-231 cells xenografted in the nude mice better than in the individual monotherapies (p < 0.05). Immunohistochemistry showed that the combination therapy induced the highest expression of cleaved caspase-3 and the lowest expression of Bcl-xL in the MDA-MB-231 cells xenografted in the nude mice (p < 0.05). Western blot analysis and immunofluorescence, incorporating both in vitro and in vivo experiments, consistently validated that unlike individual monotherapies, docetaxel and Ku-0063794 combination therapy significantly inhibited epithelial-mesenchymal transition (EMT) and autophagy (p < 0.05).ConclusionThese data suggest that docetaxel and Ku-0063794 combination therapy has higher anticancer activities over individual monotherapies against MDA-MB-231 TNBC cells through a greater inhibition of autophagy and EMT.

Abstract 3452: TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model

Abstract Background: mTOR is an atypical Ser/Thr kinase involved in regulating major cellular functions, such as nutrients sensing, growth, proliferation and angiogenesis. Current FDA approved clinical mTOR inhibitors only inhibit mTORC1 and have several limitations. First, inhibiting mTORC1 releases the negative feedback of PI3K-AKT signaling and therefore activates upstream AKT signaling. Secondly, allosteric inhibitors of rapalogs block the phosphorylation of some but not all mTORC1 downstream substrates. In particular phosphorylation of 4EBP1 is largely insensitive to rapalogs. In order to address these issues, “second generation” ATP competitive catalytic inhibitors including TAK228 against mTORC1/C2 have also been developed and are now in active in various stages of clinical trials. Purpose: Resistance of breast cancers (BC) to targeted hormone receptor inhibitors often occurs through deregulation of the PI3K-AKT-mTOR pathway. Mutations or loss of function of upstream regulators such as TSC1/2, LKB1, or components of the PI3K pathway such as PIK3CA, AKT, or PTEN have been reported in most types of human tumors including BC. Here, we investigated the effects of TAK228 alone or in conjunction with letrozole in ER+ BC cell lines. Methodology: Anti-proliferative, apoptotic, cell cycle, and intracellular signaling effects of TAK228 alone and in combination with letrozole were evaluated in ER +/PIK3CA mutated and ER+/PTEN mutated BC cell lines. Results: 1) TAK228 caused a strong differential growth inhibition in ER+/PIK3CA or ER+/PTEN mutated or ER+ (WT of PI3K pathway molecules) BC cell lines by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom. Inhibition is greater when TAK228 was combined with letrozole. 2) Administration of TAK228 induced cell cycle G0/G1 arrest and resulted in increased apoptosis in a dose-dependent manner. 3) qRT-PCR data showed that pro-apoptotic transcript, PUMA and cell survival transcripts SURVIVIN &amp; STATHMIN mRNA expressions were significantly increased and decreased respectively following the treatment of TAK228. 4) CYCLIN D1 mRNA expression was significantly decreased in both 24 &amp; 48 hrs following the treatment of TAK228. 5) TAK228 is mechanistically distinct from everolimus by a more potent and comprehensive inhibition of the PI3K-AKT-mTOR signaling network: a) avoided S6K inhibition-mediated feedback activation of PI3K-AKT and b) abrogated 4EBP1 phosphorylation. Conclusion: Data showed that TAK228 effectively inhibited proliferation and enhanced apoptosis via inhibition of the PI3K-AKT-mTORC1/C2 signaling pathway and dual TORC1/2 inhibition also mitigated the feedback activation of AKT caused by selective inhibition of mTORC1, and because of this, TAK228 may yield greater therapeutic benefit in BC patients. Citation Format: Pradip De, Jennifer H. Carlson, Tyler Jepperson, Casey Williams, Nandini Dey, Brian Leyland Jones. TAK228, a kinase inhibitor of mTORC1 and mTORC2, is highly effective in ER+ breast cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3452.

The dual PI3K/mTOR inhibitor GSK2126458 is effective for treating solid renal tumours in Tsc2+/- mice through suppression of cell proliferation and induction of apoptosis.

Tuberous sclerosis (TSC) is an inherited tumour syndrome caused by mutations in TSC1 or TSC2 that lead to aberrant activation of mTOR. Tumour responses in TSC patients to rapamycin, an allosteric inhibitor of mTOR, or its analogs are partial and reversible probably due to feedback activation of Akt. In this study, we examined the efficacy of GSK2126458, an ATP-competitive dual inhibitor of PI3K/mTOR, in comparison to rapamycin for treatment of renal tumours in genetically engineered Tsc2+/- mice. We found that both GSK2126458 and rapamycin caused significant reduction in number and size of solid renal tumours. GSK2126458 also significantly reduced the number and size of all lesions (cystic, papillary and solid) although to a lesser extent compared to rapamycin. GSK2126458 inhibited both PI3K and mTOR while rapamycin exerted stronger inhibitory effect on mTORC1 in renal tumours. Furthermore, GSK2126458 and rapamycin suppressed proliferation of tumour cells. Importantly, GSK2126458 increased apoptosis of solid tumours but rapamycin did not. Further investigations are therefore needed to test whether rapamycin in combination with GSK2126458 could promote apoptosis and thus improve therapy of TSC-associated renal tumours.

Synergistic antitumor activity of the combination of salubrinal and rapamycin against human cholangiocarcinoma cells.

Less is known about the roles of eukaryotic initiation factor alpha (eIF2α) in cholangiocarcinoma (CCA). Here, we report that eIF2α inhibitor salubrinal inhibits the proliferation of human CCA cells. Clinical application of mammalian target of rapamycin (mTOR) inhibitors only has moderate antitumor efficacy. Therefore, combination approaches may be required for effective clinical use of mTOR inhibitors. Here, we investigated the efficacy of the combination of salubrinal and rapamycin in the treatment of CCA. Our data demonstrate a synergistic antitumor effect of the combination of salubrinal and rapamycin against CCA cells. Rapamycin significantly inhibits the proliferation of CCA cells. However, rapamycin initiates a negative feedback activation of Akt. Inhibition of Akt by salubrinal potentiates the efficacy of rapamycin both in vitro and in vivo. Additionally, rapamycin treatment results in the up-regulation of Bcl-xL in a xenograft mouse model. It is notable that salubrinal inhibits rapamycin-induced Bcl-xL up-regulation in vivo. Taken together, our data suggest that salubrinal and rapamycin combination might be a new and effective strategy for the treatment of CCA.

Rapamycin Decreases the Osteogenic Response in Aortic Valve Interstitial Cells Through the Stat3 Pathway

Calcific aortic valve disease (CAVD) is an age-related and slowly progressive valvular disorder. We have previously found that the increased inflammatory and osteogenic responses to Toll-like receptor 4 (TLR4) stimulation is correlated with lower signal transducer and activator of transcription 3 (Stat3) activity in aortic valve interstitial cells (AVICs). Rapamycin, a drug used clinically, induces feedback activation of Akt. Akt in turn may upregulate Stat3. Therefore we hypothesized that rapamycin will decrease TLR4-induced osteogenic response in human AVICs through modulation of Stat3 activity. AVICs were isolated from normal valves taken from the explanted hearts of patients undergoing transplantation. Cells were treated with TLR4 ligand lipopolysaccharide (LPS) or rapamycin, or both. The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and bone morphogenetic protein 2 (BMP-2), as well as activation ofStat3 and its associated signaling molecules, were analyzed. LPS induces the expression of RUNX2, ALP, and BMP-2. Rapamycin decreased both the baseline and LPS-induced expression of RUNX2, ALP, and BMP-2. Rapamycin also decreased calcium deposit formation. Rapamycin activated both Stat3 and Akt in AVICs. Suppression of Akt resulted in abolishment of Stat3 activation. Inhibition of Stat3 enhanced expression of RUNX2, ALP, and BMP-2 at baseline and in response to LPS. Rapamycin inhibits TLR4-induced osteogenic responses in AVICs by activation of Stat3 through Akt. Rapamycin may alleviate inflammation-induced initiation and progression of CAVD.

N-Hydroxyphthalimide exhibits antitumor activity by suppressing mTOR signaling pathway in BT-20 and LoVo cells.

BackgroundN-Hydroxyphthalimide (NHPI), an important chemical raw material, was found to have potent and selective anti-proliferative effect on human breast carcinoma BT-20 cells, human colon adenocarcinoma LoVo and HT-29 cells during our screening for anticancer compounds. The purpose of this study is to assess the antitumor efficacy of NHPI in vitro and in vivo and to explore the underlying antitumor mechanism.MethodsCell cytotoxicity of NHPI was evaluated using MTS assay and cell morphological analysis. After NHPI treatment, cell cycle, apoptosis and mitochondrial membrane potential were analyzed using flow cytometer. The subcellular localization of eukaryotic initiation factor 4E (eIF4E) was analyzed by immunofluorescence assay. The antitumor efficacy of NHPI in vivo was tested in BT-20 xenografts. The underlying antitumor mechanisms of NHPI in vitro and in vivo were investigated with western blot analysis in NHPI-treated cancer cells and tumor tissues. Statistical significance was determined using Student’s t-test.ResultsIn vitro, NHPI selectively inhibited the proliferation and induced G2/M phase arrest in BT-20 and LoVo cells, which was attributed to the inhibition of cyclin B1 and cdc2 expressions. Furthermore, NHPI induced apoptosis via mitochondrial pathway. Of note, NHPI effectively inhibited mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2) signaling, and overcame the feedback activation of Akt and extracellular signal-regulated kinase (ERK) caused by mTORC1 inhibition in BT-20 and LoVo cells. In vivo, NHPI inhibited tumor growth and suppressed mTORC1 and mTORC2 signaling in BT-20 xenografts with no obvious toxicity.ConclusionsWe found for the first time that NHPI displayed antitumor activity which is associated with the inhibition of mTOR signaling pathway. Our findings suggest that NHPI may be developed as a promising candidate for cancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.

Combinatorial Antitumor Effect of Rapamycin and β-Elemene in Follicular Thyroid Cancer Cells.

Background. mTOR signaling would be a promising target for thyroid cancer therapy. However, in clinical trials, objective response rate with mTOR inhibitor monotherapy in most cancer types was modest. A new focus on development of combinatorial strategies with rapalogs is increasing. Objective. Investigating the combinatorial antitumor effect of rapamycin and β-elemene in follicular thyroid cancer cells. Methods. MTT assay was used to determine the FTC-133 cell proliferation after culturing with rapamycin and/or β-elemene. To analyze their combinatorial effect, immunoblotting was performed to analyze the activation status of AKT. Moreover, β-elemene attenuated rapamycin-induced immunosuppression was tested in mice. Results. Combination of rapamycin and β-elemene exerted significant synergistic antiproliferative effects in FTC-133 cell lines in vitro, based on inhibiting the AKT feedback activation induced by rapamycin. In vivo, the β-elemene could attenuate rapamycin-induced immunosuppression via reversing imbalance of Treg/Th17, with the underlying mechanism needed to be declared. Conclusions. We demonstrate that the novel combination of mTOR inhibitor with β-elemene synergistically attenuates tumor cell growth in follicular thyroid cancer, which requires additional preclinical validation.

Abstract LB-027: Inhibition of mammalian target of rapamycin by Torin2, an ATP-competitive inhibitor, induces growth inhibition in adult T cell leukemia

Abstract Adult T cell leukemia (ATL) is one of the aggressive malignant lymphomas induced by the infection of human T-cell Lymphotropic virus-1. Advanced stages of ATL patients still have a poor prognosis and novel therapeutic approaches are needed. Since mammalian target of rapamycin (mTOR) is a key molecule in cell growth and survival in a number of hematological malignancies, we here focused on mTOR signaling pathway consisting from mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). It has been already reported that inhibition of mTOR with rapamycin, a classical mTOR inhibitor, or its derivatives induces growth inhibition in ATL cells, and we found that Torin2, a second generation ATP-competitive mTOR inhibitor, has more beneficial effects than rapamycin does. Although Torin2 showed weaker growth inhibitory effect against ATL cell lines than rapamycin did at low concentrations (&amp;lt; 10 nM), its effect exceeded at higher concentrations (&amp;gt; 10nM) in CCK-8 assay and Trypan blue staining assay. To understand differences of mechanism of action between Torin2 and rapamycin, we first studied apoptosis and cell cycle arrest of the ATL cell line stained with annexin-FITC; Although both Torin2 and rapamycin caused G1 cell cycle arrest, a small number of ATL cells died in apoptotic manner at higher concentration of Torin2 but not of rapamycin. It is important to note that inhibition of mTOR pathway with rapamycin induced feedback activation of Akt (phosphorylation of Akt at Ser471), down stream of mTORC2, at high concentrations (&amp;gt; 50 nM) but Torin2 inhibited phosphorylation of Akt dose-dependently. Based on these results, we think Torin2 inhibits mTORC1 and mTORC2 followed by G1 cell cycle arrest, however rapamycin inhibits only mTORC1 and it induces feedback activation of Akt at high concentration resulting in resistance to apoptotic cell death. Thus, inhibition of mTOR pathway with Torin2 is new potent therapeutic approach in the treatment of ATL. Citation Format: Tatsuro Watanabe, Naoko Aragane, Eisaburo Sueoka. Inhibition of mammalian target of rapamycin by Torin2, an ATP-competitive inhibitor, induces growth inhibition in adult T cell leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-027. doi:10.1158/1538-7445.AM2015-LB-027

Inhibition of mTORC2 Induces Cell-Cycle Arrest and Enhances the Cytotoxicity of Doxorubicin by Suppressing MDR1 Expression in HCC Cells.

mTOR is aberrantly activated in hepatocellular carcinoma (HCC) and plays pivotal roles in tumorigenesis and chemoresistance. Rapamycin has been reported to exert antitumor activity in HCC and sensitizes HCC cells to cytotoxic agents. However, due to feedback activation of AKT after mTOR complex 1 (mTORC1) inhibition, simultaneous targeting of mTORC1/2 may be more effective. In this study, we examined the interaction between the dual mTORC1/2 inhibitor OSI-027 and doxorubicin in vitro and in vivo. OSI-027 was found to reduce phosphorylation of both mTORC1 and mTORC2 substrates, including 4E-BP1, p70S6K, and AKT (Ser473), and inhibit HCC cell proliferation. Similar to OSI-027 treatment, knockdown of mTORC2 induced G0-G1 phase cell-cycle arrest. In contrast, rapamycin or knockdown of mTORC1 increased phosphorylation of AKT (Ser473), yet had little antiproliferative effect. Notably, OSI-027 synergized with doxorubicin for the antiproliferative efficacy in a manner dependent of MDR1 expression in HCC cells. The synergistic antitumor effect of OSI-027 and doxorubicin was also observed in a HCC xenograft mouse model. Moreover, AKT was required for OSI-027-induced cell-cycle arrest and downregulation of MDR1. Our findings provide a rationale for dual mTORC1/mTORC2 inhibitors, such as OSI-027, as monotherapy or in combination with cytotoxic agents to treat HCC. Mol Cancer Ther; 14(8); 1805-15. ©2015 AACR.

Basal Autophagy and Feedback Activation of Akt Are Associated with Resistance to Metformin-Induced Inhibition of Hepatic Tumor Cell Growth.

While accumulating evidence has shown that the use of the diabetic drug metformin may be beneficial against various tumors in some epidemiological studies, a few studies failed to show the same beneficial effects. The molecular and cellular mechanisms for these conflicting observations are not clear. In this study, we compared the inhibitory effects of cell growth by metformin on several hepatic tumor cell lines: SMMC-7721, HCC-97L, HCC-LM3 and HepG2. While metformin inhibited cell growth in all these cells, we found that SMMC-7721, HCC-97L and HCC-LM3 cells were more resistant than HepG2 cells. Mechanistically, we found that metformin inhibited mTOR in all these hepatic tumor cells. However, SMMC-7721 cells had higher levels of basal autophagy and mTORC2-mediated feedback activation of Akt than HepG2 cells, which may render SMMC-7721 cells to be more resistant to metformin-induced inhibition of cell growth. Similarly, HCC-97L and HCC-LM3 cells also had higher feedback activation of AKT than HepG2 cells, which may also account for their resistance to metformin-induced inhibition of cell growth. Therefore, the various basal autophagy and mTOR activity in different cancer cells may contribute to the controversial findings on the use of metformin in inhibition of cancers in humans.

LKB1 gene inactivation does not sensitize non-small cell lung cancer cells to mTOR inhibitors in vitro.

Previous study has shown that endometrial cancers with LKB1 inactivation are highly responsive to mTOR inhibitors. In this study we examined the effect of LKB1 gene status on mTOR inhibitor responses in non-small cell lung cancer (NSCLC) cells. Lung cancer cell lines Calu-1, H460, H1299, H1792, and A549 were treated with the mTOR inhibitors rapamycin or everolimus (RAD001). The mTOR activity was evaluated by measuring the phosphorylation of 4EBP1 and S6K, the two primary mTOR substrates. Cells proliferation was measured by MTS or sulforhodamine B assays. The basal level of mTOR activity in LKB1 mutant A549 and H460 cells was significantly higher than that in LKB1 wild-type Calu-1 and H1792 cells. However, the LKB1 mutant A549 and H460 cells were not more sensitive to the mTOR inhibitors than the LKB1 wild-type Calu-1 and H1792 cells. Moreover, knockdown of LKB1 gene in H1299 cells did not increase the sensitivity to the mTOR inhibitors. Treatment with rapamycin or RAD001 significantly increased the phosphorylation of AKT in both LKB1 wild-type and LKB1 mutant NSCLC cells, which was attenuated by the PI3K inhibitor LY294002. Furthermore, RAD001 combined with LY294002 markedly enhanced the growth inhibition on LKB1 wild-type H1792 cells and LKB1 mutant A549 cells. LKB1 gene inactivation in NSCLC cells does not increase the sensitivity to the mTOR inhibitors. The negative feedback activation of AKT by mTOR inhibition may contribute to the resistance of NSCLC cells to mTOR inhibitors.

Targeting Focal Adhesion Kinase and Resistance to mTOR Inhibition in Pancreatic Neuroendocrine Tumors.

Focal adhesion kinase (FAK) mediates survival of normal pancreatic islets through activation of AKT. Upon malignant transformation of islet cells into pancreatic neuroendocrine tumors (PanNETs), AKT is frequently overexpressed and mutations in the AKT/mTOR pathway are detected. Because mTOR inhibitors rarely induce PanNET tumor regression, partly because of feedback activation of AKT, novel combination strategies are needed to target FAK/AKT/mTOR signaling. We characterized the activation of FAK in PanNETs using immunohistochemistry and Western blot analysis and tested the FAK inhibitor PF-04554878 in human PanNET cells in vitro and in vivo (at least three mice per group). In addition, we evaluated the effect of combined FAK and mTOR inhibition on PanNET viability and apoptosis. All statistical tests were two-sided. We found that FAK is overexpressed and hyperphosphorylated in human PanNETs and that PF-04554878 strongly inhibited FAK (Tyr397) autophosphorylation in a dose-dependent manner. We found that PF-04554878 inhibited cell proliferation and clonogenicity and induced apoptosis in PanNET cells. Moreover, oral administration of PF-04554878 statistically significantly reduced tumor growth in a patient-derived xenograft model of PanNET (P = .02) and in a human PanNET xenograft model of peritoneal carcinomatosis (P = .03). Importantly, PF-04554878 synergized with the mTOR inhibitor everolimus by preventing feedback AKT activation. We demonstrate for the first time that FAK is overexpressed in PanNETs and that inhibition of FAK activity induces apoptosis and inhibits PanNET proliferation. We found that the novel FAK inhibitor PF-04554878 synergizes with everolimus, a US Food and Drug Administration-approved agent for PanNETs. Our findings warrant the clinical investigation of combined FAK and mTOR inhibition in PanNETs.

Abstract P5-07-07: Combination of IGF1R/FAK inhibition and PI3K/mTOR inhibition in triple-negative breast cancers

Abstract Introduction: Triple-negative breast cancers (TNBCs) account for 15%-20% of all breast cancers with limited treatment options and poor prognosis. The poor outcomes seen with TNBCs are in part due to a lack of viable therapeutic targets. Overexpression of insulin-like growth factor 1 receptors (IGF1R) and focal adhesion kinase (FAK) are closely associated with invasive breast carcinomas. However, in our previous study, we found that the combined use of IGF1R inhibitor and FAK inhibitor had produced limited effects on TNBC cells inhibition. NVP-BEZ235 is a potent PI3K/mTOR dual inhibitor and has been shown to be effective in TNBC cell lines, especially for the mesenchymal-like and luminal-androgen receptor subtypes. Unlike rapamycin, which produces a feedback activation of Akt, NVP-BEZ235 alone successfully blocks the Akt activation and effectively inhibits the cells proliferation. Our hypothesis is that the combined inhibition of IGF1R/FAK and PI3K/mTOR produces greater suppression on TNBC cell growth. Methods: We examined the effects of NVP-TAE226, the dual inhibitor of IGF1R and FAK, in combination with NVP-BEZ235 on human MDA-MB-231 and BT549 TNBC cell lines. SRB cell survival assays were performed following NVP-TAE226 or NVP-BEZ235 treatments alone and NVP-TAE226 in combination with NVP-BEZ235. Western blotting was used to detect expression and phosphorylation of down-stream signaling proteins and epithelial to mesenchymal transition (EMT)-related markers. Matrigel invasion chamber assay was performed to evaluate the TNBC cells invasion patterns under treatments of either NVP-TAE226 or NVP-BEZ235 alone or the two drugs in combination. Spheroid migration assay will also be used to assess combination effect on the metastatic nature of TNBC cells. Results: The combined IGF1R/FAK inhibition with NVP-TAE226 and PI3K/mTOR inhibition with NVP-BEZ235 resulted in significantly greater cytotoxicity than either single agent alone in MDA-MB231 and BT549 cell lines (P&amp;lt;0.05). The combination of IGF1R/FAK and PI3K/mTOR inhibition suppressed the PI3K/Akt and MEK/ERK signaling cascades, reduced FAK and ZEB1 activity and significantly decreased the cell invasion for TNBC cell lines (p&amp;lt;0.05). Our data indicated that the combination treatment targeting both PI3K/Akt pathway and EMT related protein molecules (IGF1R/FAK) lead to greater cytotoxic effect and suppression of EMT and invasion. Conclusion: These results suggest that the combined inhibition of IGF1R/FAK and PI3K/mTOR may be an effective strategy for TNBC and warrant further investigation in in vivo animal studies. Citation Format: LaTonia D Taliaferro-Smith, Tongrui Liu, Tiffanie Y Alcaide, Tanisha Z McGlothen, Ruth M O'Regan. Combination of IGF1R/FAK inhibition and PI3K/mTOR inhibition in triple-negative breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-07.

Abstract P5-05-04: Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer

Abstract Background: The addition of the selective mTORC1 inhibitor everolimus (RAD001/Afinitor®), to exemestane significantly prolongs progression free survival in patients with advanced hormone receptor (ER+) positive breast cancer. However, despite these improved outcomes, the majority of patients that achieve an initial clinical benefit will eventually go on to develop progressive disease. In order to prevent or delay the onset of this acquired resistance, it is essential to identify the mechanisms by which continued proliferation is being driven in these resistant tumors. In this study, we used reverse phase protein array (RPPA) technology to compare cell signaling in ER+ breast cancer cell line xenografts that were responsive to everolimus versus those that had acquired resistance to everolimus. Materials and Methods: Xenograft models were established from three ER+ breast cancer cell lines containing different molecular alterations that confer activated PI3K/mTOR signaling; MCF7 and KPL-1 (both PIK3CA mutant) and ZR75-1 (PTEN-null). Mice were treated daily with either vehicle or 10 mg/kg everolimus until progression. Snap frozen tissue samples (n = 3) were collected from vehicle control tumors, from everolimus resistant tumors and from everolimus responsive tumors. Whole cell lysates were prepared from tumor tissues and processed for RPPA analysis. A customized panel of 27 known cancer/PI3K pathway associated signaling proteins were selected for the RPPA analysis. Results: In the vehicle controls, significantly higher levels of pAKT, pPRAS40 and p4EBP1 were detected in the PTEN-null ZR75-1 tumors compared to the PIK3CA mutant tumors, indicating potential variations in intensity of PI3K/AKT pathway signaling depending on the initiating molecular alteration. Complete inhibition of tumor proliferation was observed in response to everolimus in all 3 xenograft models for up to 7-weeks of treatment, until tumors showed signs of progression. Significantly lower S6 phosphorylation was observed in tumors responding to everolimus, which was accompanied by feedback activation of pAKT. Significant inhibition of S6 phosphorylation was also observed in each of the ZR75-1 and MCF7 tumors that were progressing on everolimus, indicating that although the tumor cells have acquired resistance to the anti-proliferative activity of everolimus, mTORC1 signaling is still being successfully blocked. Feedback activation of pAKT was observed in the ZR75-1 resistant model, however no feedback activation of AKT occurred in the MCF7 resistant model. The KPL-1 model showed the expected loss of S6 pathway inhibition upon resistance. No significant signaling changes were observed in any of the other signaling proteins measured, including ER, HER2, EGFR, MEK and ERK. Ongoing studies to measure the mRNA expression changes in these tumors using mRNA microarrays may identify signaling pathways that are driving the proliferation of these resistant tumors. Discussion: These preclinical data show that multiple pathways of resistance can develop in response to long-term everolimus treatment and resistance can occur despite continued inhibition of PI3K/mTOR signaling. Data from mRNA microarray experiments will be presented at the time of the meeting. Citation Format: Neil A O'Brien, Dylan Conklin, Michaela Ching, Luo Tong, Raul Ayala, Shawnt Issakhanian, Sara A Hurvitz, Emmanuelle di Tomaso, Ronald Linnartz, Richard S Finn, Samit Hirawat, Dennis J Slamon. Acquired resistance to everolimus occurs independently of mTORC1 inhibition in preclinical in vivo models of ER+ breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-05-04.

Abstract LB-200: Potent antitumor activity of MLN0128, an investigational selective mTORC1/2 kinase inhibitor, in preclinical models of rhabdomyosarcoma

Abstract Phosphatidylinositol 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis regulates growth and homeostasis, and is often dysregulated in rhabdomyosarcoma. Agents that target mammalian target of rapamycin complex 1 (mTORC1), one of the two multi-protein complexes comprising the serine-threonine kinase mTOR, have been disappointing in clinical trials, and novel therapeutics are urgently needed to treat patients with relapsed, refractory, or metastatic alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. It is hypothesized that modest clinical results with mTORC1 only inhibitors may be attributed to the lack of inhibition of the second mTOR complex, mTORC2, and its downstream effectors, as well as release of mTOR-mediated negative feedback of the pathway that leads to activation of receptor tyrosine kinases and feedback activation of AKT. In the present preclinical study, MLN0128, a selective and highly potent inhibitor of both mTORC1 and mTORC2, was tested for its antitumor activity in vitro and in a mouse xenograft model. Data suggest that the anti-proliferative effect of MLN-0128 was superior to rapamycin, a selective mTORC1 inhibitor, in a broad panel of ERMS and ARMS cell lines where it was found to be highly effective at low concentrations from 5 to 175 nanomolar. Western immunoblot analysis of treated cells showed inhibition of phosphorylation of mTORC1 and mTORC2 targets including phosphorylated S6 ribosomal protein (pS6), phospho-p70-S6K, phospho-AKT-S473, phospho-AKT-T308, and phospho-4E-BP1. Apoptosis in both ERMS and ARMS cell lines was confirmed by the presence of cleaved poly ADP ribose. Additionally, MLN0128 was found to have superior anti-tumor effect in a xenograft model of rhabdomyosarcoma without significant toxicity when compared with rapamycin. These pre-clinical studies support further research of MLN0128 as a potential therapeutic approach for patients with ARMS and ERMS. Citation Format: Emily Slotkin, Parag Patwardhan, Shyamprasad Vasudeva Deraje, Gary Schwartz, Tap William. Potent antitumor activity of MLN0128, an investigational selective mTORC1/2 kinase inhibitor, in preclinical models of rhabdomyosarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2014-LB-200

Combined SFK/mTOR inhibition prevents rapamycin-induced feedback activation of AKT and elicits efficient tumor regression.

Resistance to receptor tyrosine kinase (RTK) blockade in breast cancer is often mediated by activation of bypass pathways that sustain growth. Src and mammalian target of rapamycin (mTOR) are two intrinsic targets that are downstream of most RTKs. To date, limited clinical efficacy has been observed with either Src or mTOR inhibitors when used as single agents. Resistance to mTOR inhibitors is associated with loss of negative feedback regulation, resulting in phosphorylation and activation of AKT. Herein, we describe a novel role for Src in contributing to rapalog-induced AKT activation. We found that dual activation of Src and the mTOR pathway occurs in nearly half of all breast cancers, suggesting potential cross-talk. As expected, rapamycin inhibition of mTOR results in feedback activation of AKT in breast cancer cell lines. Addition of the Src/c-Abl inhibitor, dasatinib, completely blocks this feedback activation, confirming convergence between Src and the mTOR pathway. Analysis in vivo revealed that dual Src and mTOR inhibition is highly effective in two mouse models of breast cancer. In a luminal disease model, combined dasatinib and rapamycin is more effective at inducing regression than either single agent. Furthermore, the combination of dasatinib and rapamycin delays tumor recurrence following the cessation of treatment. In a model of human EGFR-2-positive (HER2(+)) disease, dasatinib alone is ineffective, but potentiates the efficacy of rapamycin. These data suggest that combining mTOR and Src inhibitors may provide a new approach for treating multiple breast cancer subtypes that may circumvent resistance to targeted RTK therapies.

Targeting PI3K/mTOR overcomes resistance to HER2-targeted therapy independent of feedback activation of AKT.

Altered PI3K/mTOR signaling is implicated in the pathogenesis of a number of breast cancers, including those resistant to hormonal and HER2-targeted therapies. The activity of four classes of PI3K/mTOR inhibitory molecules, including a pan-PI3K inhibitor (NVP-BKM120), a p110α isoform-specific PI3K inhibitor (NVP-BYL719), an mTORC1-specific inhibitor (NVP-RAD001), and a dual PI3K/mTORC1/2 inhibitor (NVP-BEZ235), was evaluated both in vitro and in vivo against a panel of 48 human breast cell lines. Each agent showed significant antiproliferative activity in vitro, particularly in luminal estrogen receptor-positive and/or HER2(+) cell lines harboring PI3K mutations. In addition, monotherapy with each of the four inhibitors led to significant inhibition of in vivo growth in HER2(+) breast cancer models. The PI3K/mTOR pathway inhibitors were also effective in overcoming both de novo and acquired trastuzumab resistance in vitro and in vivo. Furthermore, combined targeting of HER2 and PI3K/mTOR leads to increased apoptosis in vitro and induction of tumor regression in trastuzumab-resistant xenograft models. Finally, as previously shown, targeting mTORC1 alone with RAD001 leads to consistent feedback activation of AKT both in vitro and in vivo, whereas the dual mTOR1-2/PI3K inhibitor BEZ235 eliminates this feedback loop. However, despite these important signaling differences, both molecules are equally effective in inhibiting tumor cell proliferation both in vitro and in vivo. These preclinical data support the findings of the BOLERO 3 trial that shows that targeting of the PI3K/mTOR pathway in combination with trastuzumab is beneficial in trastuzumab-resistant breast cancer.