EGF-induced Akt Phosphorylation Research Articles

Protein kinase D1 overexpression potentiates epidermal growth factor signaling pathway in MCF-7 cells.

Protein kinase D1, PKD1, is a serine-threonine kinase implicated in cell proliferation, migration, invasion, and/or apoptosis and its activation by several growth factors sets this enzyme as a key regulator of tumorigenesis and tumor progression. Despite many studies, its role in the regulation of intracellular signaling pathways remains widely disparate and needs to be clarified. By using human breast cancer cells MCF-7, overexpressing or not PKD1, we demonstrated that PKD1 expression level modulated the tumor growth-promoting epidermal growth factor (EGF) signaling pathway. We also showed that EGF acutely stimulated PKD1 phosphorylation with similar time courses both in control and PKD1-overexpressing cells. However, PKD1 overexpression specifically and markedly increased EGF-induced phosphorylation of Akt (onto T308 and S473 residues) and extracellular-regulated protein kinase (ERK1/2). Finally, pharmacological inhibition of PKD1 activity or lowering its expression level using specific siRNAs drastically reduced EGF-stimulated Akt and ERK phosphorylation in PKD1overexpressing cells, but not in control cells. Overall, these results identified the level of PKD1 expression as a key determinant in the regulation of the EGF signaling pathway highlighting its crucial role in a tumorigenic setting.

A Novel Mechanism Underlying the Inhibitory Effects of Trastuzumab on the Growth of HER2-Positive Breast Cancer Cells.

To improve the efficacy of trastuzumab, it is essential to understand its mechanism of action. One of the significant issues that makes it difficult to determine the precise mechanism of trastuzumab action is the formation of various HER receptor dimers in HER2-positive breast cancer cells. So far, studies have focused on the role of HER2-HER3 heterodimers, and little is known regarding EGFR-HER2 heterodimers. Here, we study the role of trastuzumab on the cell signaling and cell proliferation mediated by EGFR-HER2 heterodimers in BT474 and SRBR3 cells. EGF stimulates the formation of both EGFR homodimer and EGFR-HER2 heterodimer. Trastuzumab only binds to HER2, not EGFR. Therefore, any effects of trastuzumab on EGF-induced activation of EGFR, HER2, and downstream signaling proteins, as well as cell proliferation, are through its effects on EGFR-HER2 heterodimers. We show that trastuzumab inhibits EGF-induced cell proliferation and cell cycle progression in BT474 and SKBR3 cells. Interestingly trastuzumab strongly inhibits EGF-induced Akt phosphorylation and slightly inhibits EGF-induced Erk activation, in both BT474 and SKBR3 cells. These data suggest the presence of a novel mechanism that allows trastuzumab to inhibit EGR-induced Akt activation and cell proliferation, without blocking EGF-induced EGFR-HER2 heterodimerization and activation. We show that trastuzumab inhibits EGF-induced lipid raft localization of the EGFR-HER2 heterodimer. Disruption of the lipid raft with MβCD blocks HER2-mediated AKT activation in a similar way to trastuzumab. MβCD and trastuzumab synergically inhibit AKT activation. We conclude that trastuzumab inhibits EGF-induced lipid raft localization of EGFR-HER2 heterodimer, which leads to the inhibition of Akt phosphorylation and cell proliferation, without blocking the formation and phosphorylation of the EGFR-HER2 heterodimer.

A ROS/Akt/NF-κB Signaling Cascade Mediates Epidermal Growth Factor-Induced Epithelial-Mesenchymal Transition and Invasion in Human Breast Cancer Cells.

As one of the most widely used anti-diabetic drugs for type II diabetes, metformin has been shown to exhibit anti-cancer activity in recent years. Epidermal growth factor (EGF) and its receptor, EGFR, play important roles in cancer metastasis in various tumors, including breast cancer. Epithelial-mesenchymal transition (EMT) is a critical process for cancer invasion and metastasis. In this study, we use EGF as a metastatic inducer to investigate the effect of metformin on cancer cell migration, invasion and EMT. Human breast cancer MCF-7 cells were exposed to EGF with or without metformin or N-acetyl cysteine (NAC). The effects of metformin on breast cancer cell proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The production of reactive oxygen species (ROS) was tested using 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA). The migratory and invasive abilities of tumor cells were analyzed using wound healing assay and transwell invasion assay, respectively. The expressions of E-cadherin, N-cadherin and Snail were tested using real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting at mRNA and protein levels. The activation of protein kinase B (Akt) and nuclear factor kappa B (NF-κB) were measured by western blotting. Our results showed that metformin inhibited breast cancer cell proliferation in a dose-dependent manner with or without EGF. EGF-induced alterations in cell morphology that are characteristic of EMT were reversed by metformin. Metformin also inhibited the EGF-modulated expression of E-cadherin, N-cadherin and Snail and further suppressed cell invasion and migration. In addition, metformin suppressed EGF-induced phosphorylation of Akt and NF-κB. ROS is involved in EGF-induced cancer invasion and activation of phosphatidylinositol 3-kinase (PI3K)/Akt/NF-κB pathway. Taken together, these data indicate that metformin suppresses EGF-induced breast cancer cell migration, invasion and EMT through the inhibition of the PI3K/Akt/NF-κB pathway. These results provide a novel mechanism to explain the role of metformin as a potent anti-metastatic agent in breast cancer cells.

Epimagnolin targeting on an active pocket of mammalian target of rapamycin suppressed cell transformation and colony growth of lung cancer cells.

Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3β at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3β-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.

Abstract 265: Epi-magnolin suppresses EGF-induced neoplastic cell transformation by inhibition of Akt/mTOR signaling pathway in JBC Cl41 cells

Abstract The phosphatidylinositol-3-kinase (PI3K)/Akt/the mammalian target of rapamycin (mTOR) signaling pathway is frequently activated in human cancer and plays a pivotal role in cell proliferation and transformation. In this study, we reveal epi-magnolin, a phytochemical found in Magnolia flos, as an inhibitor of Akt/mTOR signaling pathway. Epi-magnolin inhibits EGF-induced Akt phosphorylation at Thr308 and Ser473, but not extracellular signal-regulated kinases (ERKs)/ribosomal S6 kinase 2 (RSK2) signaling pathway. Moreover, we demonstrate that epi-magnolin suppresses JB6 cell proliferation by impairing EGF-induced G1/S cell cycle transition in a dose-dependent manner. Notably, epi-magnolin abrogates phosphorylation of c-Jun at Ser63 and 73 as well as total c-Jun protein level, which leads to downregulation of AP-1 transactivation activity. The inhibition effect of Akt/mTOR signaling pathway and AP-1 transactivation activity by epi-magnolin suppressed EGF-induced anchorage-independent cell transformation. Based on these results, epi-magnolin might be a chemopreventive agent by targeting Akt/mTOR signaling pathway. Key words: chemoprevention, cell transformation, Akt/mTOR signaling pathway Citation Format: Cheol-Jung Lee, Sun-Mi Yoo, Seung-Min Kim, Seon-Yeon Cho, Juhee Park, Yu-Mi Shin, Yong-Yeon Cho, Hye Suk Lee. Epi-magnolin suppresses EGF-induced neoplastic cell transformation by inhibition of Akt/mTOR signaling pathway in JBC Cl41 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 265.

Pecanex functions as a competitive endogenous RNA of S-phase kinase associated protein 2 in lung cancer

Investigating the RNA-RNA interactions involving in the initiation and progression of non-small cell lung cancer (NSCLC) may provide promising diagnostic and targeted therapeutic strategies. Here, we showed that pecanex (PCNX) positively regulates the mRNA and protein expressions of S-phase kinase associated protein 2 (Skp2) in miRNA- and 3′ UTR-dependent manners. And miR-26, miR-182, miR-340 and miR-506 were verified as the common miRNAs shared by Skp2 and PCNX. Intriguingly, we initially uncovered that PCNX-3′ UTR promotes cell growth, proliferation and cell cycle progression, and suppresses apoptosis of lung cancer cells, which is consistent with the oncogenic activity of Skp2-3′ UTR. Consequently, PCNX was identified as a competitive endogenous RNA (ceRNA) of Skp2. Moreover, knockdown of PCNX inhibits EGF-induced Akt phosphorylation, which can be reversed by the silencing of Dicer. Finally, we further discovered that Skp2 and PCNX are coordinately upregulated in lung cancer tissues compared with the adjacent non-tumor tissues. Our study establishes for the first time the oncogenic property of PCNX-3′ UTR and Skp2-3′ UTR, and the PCNX-miRNA-Skp2 regulatory pattern, which may offer a molecular basis for the diagnosis and targeted therapy in NSCLC.

Oxidative stress affects retinal pigment epithelial cell survival through epidermal growth factor receptor/AKT signaling pathway.

To investigate the cross-talk between oxidative stress and the epidermal growth factor receptor (EGFR)/AKT signaling pathway in retinal pigment epithelial (RPE) cells. Human RPE cell lines (ARPE-19 cell) were treated with different doses of epidermal growth factor (EGF) and hydrogen peroxide (H2O2). Cell viability was determined by a methyl thiazolyl tetrazolium assay. Cell proliferation was examined by a bromodeoxyuridine (BrdU) incorporation assay. EGFR/AKT signaling was detected by Western blot. EGFR localization was also detected by immunofluorescence. In addition, EGFR/AKT signaling was intervened upon by EGFR inhibitor (erlotinib), PI3K inhibitor (A66) and AKT inhibitor (MK-2206), respectively. H2O2-induced oxidative stress was blocked by antioxidant N-acetylcysteine (NAC). EGF treatment increased ARPE-19 cell viability and proliferation through inducing phosphorylation of EGFR and AKT. H2O2 inhibited ARPE-19 cell viability and proliferation and also suppressed EGF-stimulated increase of RPE cell viability and proliferation by affecting the EGFR/AKT signaling pathway. EGFR inhibitor erlotinib blocked EGF-induced phosphorylation of EGFR and AKT, while A66 and MK-2206 only blocked EGF-induced phosphorylation of AKT. EGF-induced phosphorylation and endocytosis of EGFR were also affected by H2O2 treatment. In addition, antioxidant NAC attenuated H2O2-induced inhibition of ARPE-19 cell viability through alleviating reduction of EGFR, and phosphorylated and total AKT proteins. Oxidative stress affects RPE cell viability and proliferation through interfering with the EGFR/AKT signaling pathway. The EGFR/AKT signaling pathway may be an important target in oxidative stress-induced RPE cell dysfunction.

Phosphoproteomics Reveals MAPK Inhibitors Enhance MET- and EGFR-Driven AKT Signaling in KRAS-Mutant Lung Cancer.

This study highlights the unique adaptive changes in MAPK scaffolding proteins (KSR-1, GEF-H1) and in RTK signaling, leading to enhanced PI3K/AKT signaling when the MAPK pathway is inhibited. Mol Cancer Res; 14(10); 1019-29. ©2016 AACR.

Blockade of voltage-gated sodium channels inhibits invasion of endocrine-resistant breast cancer cells.

Voltage-gated Na+ channels (VGSCs) are membrane proteins which are normally expressed in excitable cells but have also been detected in cancer cells, where they are thought to be involved in malignancy progression. In this study we examined the ion current and expression profile of VGSC (Nav1.5) in estrogen receptor (ER)-positive (MCF-7) and silenced (pII) breast cancer cells and its possible influence on their proliferation, motility and invasion. VGSC currents were analysed by whole cell patch clamp recording. Nav1.5 expression and localization, in response to EGF stimulation, was examined by western blotting and immunofluorescence respectively. Cell invasion (under-agarose and Matrigel assays), motility (wound healing assay) and proliferation (MTT assay) were assessed in pII cells in response to VGSC blockers, phenytoin (PHT) and tetrodotoxin (TTX), or by siRNA knockdown of Nav1.5. The effect of PHT and TTX on modulating EGF-induced phosphorylation of Akt and ERK1/2 was determined by western blotting. Total matrix metalloproteinase (MMP) was determined using a fluorometric-based activity assay. The level of various human proteases was detected by using proteome profiler array kit. VGSC currents were detected in pII cells, but were absent in MCF-7. Nav1.5 showed cytoplasmic and perinuclear expression in both MCF-7 and pII cells, with enhanced expression upon EGF stimulation. Treatment of pII cells with PHT, TTX or siRNA significantly reduced invasion towards serum components and EGF, in part through reduction of P-ERK1/2 and proteases such as cathepsin E, kallikrein-10 and MMP-7, as well as total MMP activity. At high concentrations, PHT inhibited motility while TTX reduced cell proliferation. Pharmacological or genetic blockade of Nav1.5 may serve as a potential anti-metastatic therapy for breast cancer.

Abstract 1915: Effects of carvedilol, a β-adrenergic receptor antagonist, on UVB-induced skin cancer development and its possible mechanism of action

Abstract Recently, we proposed that carvedilol, a β-adrenergic receptor antagonist (i.e., β-blocker), can act as a chemopreventive agent against skin cancer (Chang et al., Cancer Prev. Res, 2014). Since approximately 90 percent of diagnosed non-melanoma skin cancers are associated with exposure to ultraviolet (UV) radiation from the sun, the primary form being UVB irradition, we hypothesized that carvedilol will prevent UVB-induced skin carcinogenesis. The transformable JB6 P+ cell line, an established model for studying skin cancer promotion, was used for evaluating the impact of carvedilol on UVB-induced oxidative damage. Exposures of JB6 P+ cells to 50 - 400 mJ/cm2 UVB resulted in a dose-dependent decrease in cell viability. 25 mJ/cm2 UVB induced ∼20% of cell death, and thus was chosen to study the effect of carvedilol. 0.1 to 1.0 μM carvedilol prevented UVB-induced growth inhibition and apoptosis; however, 3 to 5 μM carvedilol, although nontoxic alone, enhanced UVB-induced cell death. Contrarily, all concentrations of carvedilol tested blocked H2O2-dependent cytotoxicity of JB6 P+ cells. Thus, although both UVB and H2O2 are classic inducers of oxidative stress resulting in DNA damage and tumor promotion, carvedilol may modulate UVB and H2O2-mediated effects via different mechanisms. Cell signaling studies revealed that carvedilol inhibited EGF-induced phosphorylation of AKT at S473 and GSK3β at S9 as well as AP-1 transactivation, which are all well-known principal mediators of skin carcinogenesis. A pitfall with experiments involving UV exposure is the potential UV absorption properties of test compounds. Examination of the structure of carvedilol indicates that it will absorb UV due to the presence of a carbazole moiety and benzene rings. If not controlled in the experiments, the observed effects will be a mixture of pharmacological properties and a UV absorption effect. To identify a solution for this problem, 4-hydroxycarbazole, which mimicks the carbazole moiety of carvedilol, was found to have the same UVB absorption properties as carvedilol but does not function as a β-adrenergic receptor antagonist nor inhibit EGF-mediated colony formation of JB6 P+ cells. 4-hydroxycarbazole is currently being used to repeat the UVB experiments mentioned above to act as a sunscreen/absorption control. Additionally, 4-hydroxycarbazole should be used as a control for carvedilol for other on-going studies for prevention of UVB-induced skin cancer. Since 4-hydroxycarbazole only has the UV absorption (i.e., sunscreen) effect, we anticipate that it will have some protective effect, but that carvedilol provides greater preventive activity against UVB-induced skin carcinogenesis. Since carvedilol and other β-blockers are already FDA-approved drugs, these results may be readily translated into clinical trials as a new approach for skin cancer chemoprevention. Citation Format: Kevin M. Huang, Andy Chang, Kristan H. Cleveland, Bradley T. Andresen, Ying Huang. Effects of carvedilol, a β-adrenergic receptor antagonist, on UVB-induced skin cancer development and its possible mechanism of action. [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 1915. doi:10.1158/1538-7445.AM2015-1915

Marine lipopeptide Iturin A inhibits Akt mediated GSK3β and FoxO3a signaling and triggers apoptosis in breast cancer.

Akt kinase is a critical component of the PI3K/Akt signaling pathway, which is frequently over expressed in human cancers including breast. Therapeutic regimens for inhibiting breast cancer with aberrant Akt activity are essential. Here, we evaluated antitumor effect of a marine bacteria derived lipopeptide ‘Iturin A’ on human breast cancer in vitro and in vivo through disrupting Akt pathway. Proliferation of MDA-MB-231 and MCF-7 breast cancer cells were significantly inhibited by Iturin A and it induced apoptosis as confirmed by increased Sub G1 populations, DNA fragmentation, morphological changes and western blot analysis. Furthermore, Iturin A inhibited EGF induced Akt phosphorylation (Ser473 and Thr308) and its downstream targets GSK3β and FoxO3a. Iturin A inactivated MAPK as well as Akt kinase leading to the translocation of FoxO3a to the nucleus. Gene silencing of Akt in MDA-MB-231 and MCF-7 cells reduced the sensitivity of cancer cells to Iturin A. Interestingly, overexpression of Akt with Akt plasmid in cancer cells caused highly susceptible to induce apoptosis by Iturin A treatment. In a xenograft model, Iturin A inhibited tumor growth with reduced expressions of Ki-67, CD-31, P-Akt, P-GSK3β, P-FoxO3a and P-MAPK. Collectively, these findings imply that Iturin A has potential anticancer effect on breast cancer.

EBP50 inhibits the migration and invasion of human breast cancer cells via LIMK/cofilin and the PI3K/Akt/mTOR/MMP signaling pathway.

The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50, also known as NHERF1 or NHERF) inhibits epidermal growth factor (EGF)-induced breast cancer cell proliferation after 3 days by blocking EGF receptor (EGFR) phosphorylation. The loss of EBP50 stimulates EGFR activity and induces the appearance of epithelial-to-mesenchymal transition phenotypic features in biliary cancer cells. However, the involvement of EBP50 in breast cancer migration and invasion remains unknown. We report that EBP50 inhibits the migration and invasion of breast cancer cells by inhibiting the phosphorylation of LIN-11, Isl1, and MEC-3 protein domain kinase, as well as cofilin. This phosphorylation is a critical step in cofilin recycling and actin polymerization mediating cytoskeletal rearrangement. Additionally, EGF-induced phosphorylation of Akt and mTOR was suppressed by upregulation of EBP50. Our results indicate that EBP50 is significantly involved in breast cancer invasion/metastasis via LIMK/cofilin and the PI3K/Akt/mTOR/MMP signaling pathway.

Abstract B229: TBKI kinase inhibition blocks RANTES secretion and exhibits minimal tumor growth inhibition in oncogenic Ras-driven tumor models.

Abstract TANK-binding kinase 1 (TBK1) is a non-canonical IKK family member and plays a critical role in innate immunity by modulating cytokine production, interferon, and NF-kB signaling. It is recently reported that TBK1 directly engages Akt survival signaling to support oncogenic Ras-driven transformation. TBK1 is also identified as a synthetic lethal partner in KRas mutant NSCLC through systematic RNA interference. In this study, we have characterized LSN3090729, a 4-aryl-2-aminopyrimidine derivative as a selective TBK1 kinase inhibitor. Biochemical and cellular analyses demonstrate that LSN3090729 is a potent TBK1 kinase inhibitor, and selectively inhibits TBK1 based on in vitro activities in biochemical assays developed with a panel of protein kinases. In Panc-1, a pancreatic tumor cell line with KRas mutation, LSN3090729 inhibits EGF-induced phosphorylation of AKT at both Thr308 and Ser473 sites. Pharmacokinetic analysis shows that LSN3090729 has an over 70% of oral bioavailability with an acceptable half life in rodents. In a mouse pharmacology model, LSN3090729 blocks LPS-induced RANTES secretion in a dose-dependent manner with 67%, 79%, and 90% inhibition at 10, 30, and 100 mg/kg, respectively. LSN3090729 is assessed for its anti-proliferation activities in vitro in a panel of tumor cells with KRas mutation or other genetic background. The sensitivity of these tumor cells to LSN3090729 in two dimensional proliferation or three dimensional soft agar growth assays appears not correlated with status of KRas mutation. In xenograft models of HCT116 and Panc-1, both with a KRas mutation, treatment of LSN3090729 exhibits minimal anti-tumor growth activities, suggesting that a combination approach might be required for TBK1 kinase inhibition to be effective in cancer settings. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B229. Citation Format: Robert Van Horn, Tinggui Yin, Xiaoyi Zhang, Chunping Yu, Youyan Zhang, Xue-Qian Gong, Sean Buchanan, Xiang S. Ye, William McMillen, David Barda, Sheng-Bin Peng. TBKI kinase inhibition blocks RANTES secretion and exhibits minimal tumor growth inhibition in oncogenic Ras-driven tumor models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B229.

Sin1 phosphorylation impairs mTORC2 complex integrity and inhibits downstream Akt signalling to suppress tumorigenesis

The mechanistic target of rapamycin (mTOR) functions as a critical regulator of cellular growth and metabolism by forming multi-component, yet functionally distinct complexes mTORC1 and mTORC2. Although mTORC2 has been implicated in mTORC1 activation, little is known about how mTORC2 is regulated. Here we report that phosphorylation of Sin1 at T86 and T398 suppresses mTORC2 kinase activity by dissociating Sin1 from mTORC2. Importantly, Sin1 phosphorylation, triggered by S6K or Akt, in a cellular context-dependent manner, inhibits not only insulin/IGF-1-mediated, but also PDGF or EGF-induced Akt phosphorylation by mTORC2, demonstrating a negative regulation of mTORC2 independent of IRS-1 and Grb10. Lastly, a cancer patient-derived Sin1-R81T mutation impairs Sin1 phosphorylation, leading to hyper-mTORC2 activation via bypassing this negative regulation. Together, our work reveals a Sin1 phosphorylation-dependent mTORC2 regulation, providing a potential molecular mechanism by which mutations in the mTORC1/S6K/Sin1 signaling axis might cause aberrant hyper-activation of mTORC2/Akt that facilitates tumorigenesis.

The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer

The identification of primary molecular targets of cancer-preventive phytochemicals is essential for a comprehensive understanding of their mechanism of action. In the present study, we investigated the chemopreventive effects and molecular targets of acacetin, a flavonoid found in Robinia p seudoacacia, also known as black locust. Acacetin treatment significantly suppressed epidermal growth factor (EGF)-induced cell transformation. Immunoblot analysis revealed that acacetin attenuated EGF-induced phosphorylation of Akt and p70(S6K), which are downstream effectors of phosphatidylinositol 3-kinase (PI3-K). An immunoprecipitation kinase assay of PI3-K and pull-down assay results demonstrated that acacetin substantially inhibits PI3-K activity by direct physical binding. Acacetin exhibited stronger inhibitory effects against anchorage-dependent and -independent cell growth in cells expressing higher PI3-K activity compared with those exhibiting relatively low PI3-K activity. Binding assay data combined with computational modeling suggest that acacetin binds in an adenosine triphosphate (ATP)-competitive manner with the p110α subunit of PI3-K and interacts with Val828, Glu826, Asp911, Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900 residues. Acacetin was also found to significantly reduce SK-MEL-28 tumor growth and Akt phosphorylation in vivo. Taken together, these results indicate that acacetin is an ATP-competitive PI3-K inhibitor and a promising agent for melanoma chemoprevention.

PTTG1 promotes migration and invasion of human non-small cell lung cancer cells and is modulated by miR-186

Deeper mechanistic understanding of non-small cell lung cancer (NSCLC), a leading cause of total cancer-related deaths, may facilitate the establishment of more effective therapeutic strategies. In this study, pituitary tumor transforming gene (PTTG1) expression was associated with lymph node and distant metastasis in patients with NSCLC and was correlated with patient survival. Reduction of PTTG1 by small interfering RNA (siRNA) inhibits the migration and invasion of NSCLC cells by mediating matrix metalloproteinases expression. To the best of our knowledge, this study is the first to report that PTTG1 promotes epidermal growth factor (EGF) induced the phosphorylation of LIN-11, Isl1 and MEC-3 protein domain kinase and cofilin, a critical step in cofilin recycling and actin polymerization. Additionally, EGF-induced Akt phosphorylation was suppressed through knockdown of PTTG1. Interestingly, miR-186 can modulate PTTG1 protein expression. As observed from the animal experiment in this study, knockdown of PTTG1 through siRNA and overexpression of miR-186 inhibited invasive activity of NSCLC cells toward the SCID mice lung. In summary, our in vitro and in vivo results indicate that PTTG1 modulated by miR-186 has an important function in NSCLC invasion/metastasis. This study identified both PTTG1 and miR-186 as potential anti-invasion targets for therapeutic intervention in NSCLC.

Migration of retinal pigment epithelial cells is EGFR PI3K AKT dependent

Abnormal migration of retinal pigment epithelium (RPE) contributes to a variety of disorders such as proliferative vitreoretinopathy. Here, the effect of epidermal growth factor (EGF), and signaling by its receptor (ERGR)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) on RPE cell migration was studied. The in vitro wound healing and migration of the human RPE cell line, ARPE19 cell was accelerated, in a dose dependent manner, in response to EGF stimulation, while pretreatment with EGFR, PI3K or AKT inhibitor, inhibited both events. Exposure of cells to EGF activated the AKT phosphorylation, whereas EGFR and PI3K inhibitors blocked EGF-induced AKT phosphorylation in a dose-dependent manner. These data suggest that EGF mediate ARPE-19 cell migration through EGFR/PI3K/AKT signaling pathway.

Abstract P5-19-03: Olaparib plus carboplatin in combination with vandetanib inhibited the growth of BRCA-wt triple negative breast tumors in mice: Outside BRCA-box

Abstract Introduction: PARP inhibition has emerged as one of the most exciting and promising ‘targeted’ therapeutic strategies in the treatment of advanced triple negative breast cancer (TNBC) – the intended ‘target,’ being DNA repair (Anders CK, 2010). Although olaparib is known to have antitumor activity in BRCA-related TNBC cells, a limited number of preclinical and clinical studies have shown antitumor efficacy of olaparib in non-BRCA-related BC (Shimo T, 2012). Understanding the biology of TNBC cells has identified molecular targets including RTK(s), such as EGFR. Purpose: Here we tested the combination of a PARP inhibitor, olaparib (O) (AstraZeneca) plus carboplatin (C) with the EGFR/VEGFR inhibitor, vandetanib (V) (AstraZeneca) in a BRCA-wt TNBC model. Methods: Athymic mice bearing TNBC (BRCA-wt VEGFR expressing MDA-MB231& MDA-MB468 [EGFR amplified/overexpressed]) xenograft tumors (200 mm3) were treated with O plus C in combination with V (Arms: vehicle control 1, vehicle control 2, C+O, V, C+O+V). In vitro effects of V in combination with O plus C (or temozolomide) on clonogenic growth (3D on-TOP assay), proliferation (MTT and CelltiterGLO), apoptosis (Apoptosis Array), cell signaling marker(s) (Western Blot), and tumor cell phenotypes (fibronectin-directed migration, matrigel-invasion, and vascular mimicry) were investigated in a panel of five BRCA-wt and BRCA-mutated TNBC cell lines. The effects of V were tested on (a) cell signaling marker(s), (b) angiogenesis marker (HIF-1alpha), and (c) angiogenesis related phenotypes (vitronectin-directed migration, and cord formation) in HUVEC. Results: (1) O plus C in combination with V caused a regression of the in vivo growth of established tumors by 50% which was evident in both the BRCA-wt TNBC models tested. Interestingly, a marked suppression of the progression of tumor-growth was observed in the O plus C arm. (2) In vitro, V alone (10 µM) inhibited baseline as well as EGF-induced phosphorylation of AKT (S473/T308), S6RP, 4EBP1 and ERK. (3) TNBC cells exhibited higher sensitivity to V in clonogenic assays when combined with a 10 µM fixed dose of O and C/temozolomide. (4) A combination of V with O plus C increased cleaved caspase-3, PARP cleavage, and pro-apoptotic signals, while inhibiting vascular mimicry, migration, and invasion in MDA-MB231, MDA-MB468, and SUM149 cells. (5) Treatment with V blocked cord formation, migration, EGF-induced HIF-1α accumulation, and phosphorylation of AKT, 4EBP1, and ERK in HUVEC. Conclusion: A profound anti-tumor efficacy of O plus C in combination with V in BRCA-wt TNBC model can be explained by a significant anti-proliferative/pro-apoptotic and anti-migratory/anti-invasive actions of the drugs (alone or in combination), as observed both in tumor cells as well as in endothelial compartments. The combination of O plus C plus V merits further investigation in TNBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-19-03.

Growth hormone modulation of EGF-induced PI3K-Akt pathway in mice liver

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling.EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.

Resveratrol blocks Akt activation in angiotensin II- or EGF-stimulated vascular smooth muscle cells in a redox-independent manner

AimsResveratrol (RV), an antioxidant, inhibits angiotensin II (Ang II)-induced hypertrophy and Ang II- or epidermal growth factor (EGF)-induced Akt phosphorylation in rat vascular smooth muscle cells (VSMCs). Both signalling pathways are reported to utilize reactive oxygen species (ROS). The aim of this study was to show whether RV reduces the ROS level in Ang II- or EGF-activated VSMCs and whether reduction of ROS causes the impeded signalling towards Akt in the presence of RV.Methods and resultsWe show here that RV reduces intracellular ROS and extracellular H2O2 release from VSMCs as measured using 2′,7′-dichlorodihydrofluorescein-diacetate and Amplex Red™. Since NADPH oxidases (Nox) 1 and 4 are major ROS sources in VSMCs, we examined their need for Akt phosphorylation in response to Ang II or EGF. Experiments using the blocking peptide gp91ds-tat verified a role for Nox1 in Ang II signalling towards Akt, but excluded a role for Nox1 in the respective EGF signalling. A small interfering RNA-mediated knock-down of Nox4 showed that Nox4 was not required for Ang II- or EGF-induced Akt phosphorylation. Use of the flavoprotein inhibitor diphenyleneiodonium, N-acetyl-cysteine, and non-antioxidant RV derivatives revealed that the antioxidant capacity of RV is not required for the inhibition of Akt phosphorylation, in both rat and human VSMCs.ConclusionThus, although RV acts as an antioxidant, the antihypertrophic response of RV in VSMCs and the signalling downstream of the EGF receptor towards Akt seem to be largely redox independent.