ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines

Therapies that target the EGF receptor (EGFR), such as gefitinib (IRESSA), are effective in a subset of patients with advanced non-small cell lung cancer (NSCLC). The differences in intracellular signaling networks between gefitinib-sensitive and -resistant NSCLCs remain poorly understood. In this study, we observe that gefitinib reduces phospho-Akt levels only in NSCLC cell lines in which it inhibits growth. To elucidate the mechanism underlying this observation, we compared immunoprecipitates of phosphoinositide 3-kinase (PI3K) between gefitinib-sensitive and -resistant NSCLC cell lines. We observe that PI3K associates with ErbB-3 exclusively in gefitinib-sensitive NSCLC cell lines. Gefitinib dissociates this complex, thereby linking EGFR inhibition to decreased Akt activity. In contrast, gefitinib-resistant cells do not use ErbB-3 to activate the PI3K/Akt pathway. In fact, abundant ErbB-3 expression is detected only in gefitinib-sensitive NSCLC cell lines. Two gefitinib-sensitive NSCLC cell lines with endogenous distinct activating EGFR mutations (L858R and Del747-749), frequently observed in NSCLC patients who respond to gefitinib, also use ErbB-3 to couple to PI3K. Down-regulation of ErbB-3 by means of short hairpin RNA leads to decreased phospho-Akt levels in the gefitinib-sensitive NSCLC cell lines, Calu-3 (WT EGFR) and H3255 (L858R EGFR), but has no effect on Akt activation in the gefitinib-resistant cell lines, A549 and H522. We conclude that ErbB-3 is used to couple EGFR to the PI3K/Akt pathway in gefitinib-sensitive NSCLC cell lines harboring WT and mutant EGFRs.     

非小细胞肺癌中PI3K/Akt信号通路通过Cdh1调控Skp2表达的机制研究

目的探讨非小细胞肺癌（NSCLC）中Cdc20同源蛋白1（Cdh1）参与磷脂酰肌醇三羟基激酶（PI3K）/Akt信号通路对S期激酶相关蛋白2（Skp2）表达调控的机制。方法体外培养NSCLC细胞系A549、LK2和H460,LY294002特异性阻断PI3K/Akt信号通路后,Western blot检测Skp2、Cdh1及p-Akt蛋白表达的变化,免疫荧光（IF）检测Cdh1在NSCLC中的定位变化。结果 LY294002处理后,与对照组相比3种细胞中Skp2蛋白表达和Akt磷酸化水平均降低（P〈0.01）,Cdh1在3种细胞的核内表达均增多。结论 NSCLC中PI3K/Akt信号通路抑制剂LY294002使Skp2蛋白表达下调与Cdh1由细胞质向细胞核转位有关。     

Epidermal growth factor receptor mutations in non-small cell lung cancer influence downstream Akt, MAPK and Stat3 signaling

Purpose  The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC) has been linked to activating mutations in the EGFR gene. So far these mutations have been extensively characterized in established cell lines. The aim of this study was to determine the effects of EGFR mutations on downstream signaling in human tumor specimens. Methods  We have looked for mutations of the EGFR gene in specimens of 67 patients with NSCLC and correlated these with EGFR phosphorylation and the activity of its three main downstream signaling cascades Akt, MAPK and Stat3 by immunohistochemistry. Results  We show that the phosphorylation of tyrosine residues 922 and 1173, but not 1068, are primarily affected by the activating EGFR mutations. Akt activity was significantly higher in patients with EGFR mutations but we found no difference in Stat3 or MAPK phosphorylation. Our results suggest that EGFR mutations not only increase receptor activity, but also alter responses of downstream signaling cascades in human NSCLCs and that these finding differ from results obtained in cell lines. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Akt expression may predict favorable prognosis in cholangiocarcinoma

BACKGROUND: Overexpression of signaling proteins including epidermal growth factor receptor (EGFR), Akt, mitogen activated protein kinase (MAPK) and cyclooxygenase-2 (COX-2) occurs in cholangiocarcinoma cell lines. However, the prognostic value of these markers is unknown. No prior study correlated the expression of these signaling proteins with clinical outcome. Further, co-expression of these proteins has not been reported. Co-expression may reflect cross-talk between signaling pathways. The aim of this clinicopathological study was to investigate the overexpression and co-expression of EGFR and related signaling proteins in cholangiocarcinoma and explore their relationship to clinical outcome. METHODS: Twenty-four consecutive cases of cholangiocarcinoma treated from 1996 to 2002 at Roswell Park Cancer Institute were included. Immunohistochemical staining of paraffin-embedded tissue sections was performed using antibodies against Akt, p-Akt, MAPK, p-MAPK, COX-2, EGFR and p-EGFR. Two pathologists independently scored the protein expression. RESULTS: Cyclooxygenase-2, Akt, and p-MAPK were commonly expressed in biliary cancers (100%, 96% and 87% of malignant cells, respectively). EGFR (60%) and p-EGFR (22%) overexpression was also detected. There was a significant association between EGFR and p-EGFR (P = 0.027) and between Akt and p-Akt (P = 0.017) expression in tumor tissue. A noteworthy association was shown between MAPK and p-Akt (P = 0.054). Multivariate analysis using the Cox proportional hazard model identified the use of chemotherapy (hazard ratio [HR] = 0.039, P = 0.0002), radiation (HR = 0.176, P = 0.0441) and Akt expression (HR = 0.139, P = 0.006) as the best predictors of overall prognosis. CONCLUSION: Epidermal growth factor receptor signaling intermediates are commonly expressed in cholangiocarcinoma. Expression of Akt and use of systemic chemotherapy or radiation may correlate with improved survival.     

Alisol B acetate induces apoptosis of SGC7901 cells via mitochondrial and phosphatidylinositol 3-kinases/Akt signaling pathways

AIM： To examine the effect of alisol B acetate on the growth of human gastric cancer cell line SGC7901 and its possible mechanism of action.
METHODS： The cytotoxic effect of alisol B acetate on SGC7901 cells was measured by 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MI-I-） assay. Phase-contrast and electron microscopy were used to observe the morphological changes. Cell cycle and mitochondrial transmembrane potential （A~Pm） were determined by flow cytometry. Western blotting was used to detect the expression of apoptosis-regulated gene Bcl-2, Bax, Apaf-1, caspase-3, caspase-9, Akt, P-Akt and phosphatidylinositol 3-kinases （PI3K）.
RESULTS： Alisol B acetate inhibited the proliferation of SGC7901 cell line in a time- and dose-dependent manner. PI staining showed that alisol B acetate can change the cell cycle distribution of SGC7901, increase the proportion of cells in G0-G1 phase and decrease the proportion of S phase cells and G2-M phase cells. Alisol B acetate at a concentration of 30 pmol/L induced apoptosis after 24, 48 and 72 h incubation, with occurrence rates of apoptotic cells of 4.36%, 14.42% and 21.16%, respectively. Phase-contrast and electron microscopy revealed that the nuclear fragmentation and chromosomal condensed, cells shrank and attachment loss appeared in the SGC7901 treated with alisol B acetate. Apoptosis of SGC7901 cells was associated with cell cycle arrest, caspase-3 and caspase-9 activation, loss of mitochondrial membrane potential and up-regulation of the ratio of Bax/Bcl-2 and inhibition of the PI3K/Akt.
CONCLUSION： Alisol B acetate exhibits an antiproliferative effect in SGC7901 cells by inducing apoptosis. Apoptosis of SGC7901 cells involves mitochondria-caspase and PI3K/Akt dependent pathways.     

Nicotine activates cell-signaling pathways through muscle-type and neuronal nicotinic acetylcholine receptors in non-small cell lung cancer cells

Nicotinic acetylcholine receptors (nAChR) are expressed on non-neuronal cell types, including normal bronchial epithelial cells, and nicotine has been reported to cause Akt activation in cultured normal airway cells. This study documents mRNA and protein expression of subunits known to form a muscle-type nAChR in non-small cell lung cancer (NSCLC) cell lines. In one NSCLC examined, mRNA and protein for a heteropentamer neuronal-type alpha3beta2 nAChR was detected in addition to a muscle-type receptor. Protein for the alpha5 nAChR was also detected in NSCLC cells. Although, mRNA for the alpha7 nAChR subunit was observed in all cell lines, alpha7 protein was not detectable by immunoblot in NSCLC cell extracts. Immunohistochemistry (IHC) of NSCLC primary tissues from 18 patients demonstrated protein expression of nAChR alpha1 and beta1 subunits, but not alpha7 subunit, in lung tumors, indicating preferential expression of the muscle-type receptor. In addition, the beta1 subunit showed significantly increased expression in lung tumors as compared to non-tumor bronchial tissue. The alpha1 subunit also showed evidence of high expression in lung tumors. Nicotine at a concentration of 10 microM caused phosphorylation of mitogen-activated protein kinase (MAPK) (p44/42) that could be inhibited using nAChR antagonists. Inhibition was observed at 100 nM alpha-bungarotoxin (alpha-BTX) or 10 microM hexamethonium (HEX); maximal inhibition was achieved using a combination of alpha-BTX and HEX. Akt was also phosphorylated in NSCLC cells after exposure to nicotine; this effect was inhibited by the PI3K inhibitor LY294002 and antagonists to the neuronal-type nAChR, but not to the muscle-type receptor. Nicotine triggered influx of calcium in the 273T NSCLC cell line, suggesting that L-type calcium channels were activated. 273T cells also showed greater activation of p44/42 MAPK than of Akt in response to nicotine. Cultures treated with nicotine and the EGFR tyrosine kinase inhibitor gefitinib showed a significant increase in the number of surviving cells compared to gefitinib alone. These data indicate that the muscle-type nAChR, rather than the alpha7 type, is highly expressed in NSCLC and leads to downstream activation of the p44/42 MAPK pathway. Neuronal-type receptors are also present and functional, as evidenced by antagonist studies, although, the expression levels are lower than muscle-type nAChR. They also lead to downstream activation of MAPK and Akt. Nicotine may play a role in regulating survival of NSCLC cells and endogenous acetylcholine released locally in the lung and/or chronic nicotine exposure might play a role in NSCLC development. In addition, exposure of NSCLC patients to nicotine through use of nicotine replacement products or use of tobacco products may alter the efficacy of therapy with EGFR inhibitors.     

IL-2- and IL-15-induced activation of the rapamycin-sensitive mTORC1 pathway in malignant CD4+ T lymphocytes

We examined functional status, activation mechanisms, and biologic role of the mTORC1 signaling pathway in malignant CD4(+) T cells derived from the cutaneous T-cell lymphoma (CTCL). Whereas the spontaneously growing CTCL-derived cell lines displayed persistent activation of the TORC1 as well as the PI3K/Akt and MEK/ERK pathways, the IL-2-dependent cell lines activated the pathways in response to IL-2 and IL-15 but not IL-21. Activation of mTORC1 and MEK/ERK was nutrient dependent. The mTORC1, PI3K/Akt, and MEK/ERK pathways could also be activated by IL-2 in the primary leukemic, mitogen-preactivated CTCL cells. mTORC1 activation was also detected in the CTCL tissues in the lymphoma stage-dependent manner with the highest percentage of positive cells present in the cases with a large cell transformation. Rapamycin inhibited mTORC1 signaling and suppressed CTCL cell proliferation but showed little effect on their apoptotic rate when used as a single agent. Activation of the mTORC1, PI3K/Akt, and MEK/ERK pathways was strictly dependent on the Jak3 and Jak1 kinases. Finally, mTORC1 activation was transduced preferentially through the PI3K/Akt pathway. These findings document the selective gammac-signaling cytokine-mediated activation of the mTORC1 pathway in the CTCL cells and suggest that the pathway represents a therapeutic target in CTCL and, possibly, other T-cell lymphomas.     

Galphaq expression activates EGFR and induces Akt mediated cardiomyocyte survival: dissociation from Galphaq mediated hypertrophy

Our laboratory has previously shown that adenoviral-mediated overexpression of Galphaq in neonatal rat ventricular cardiomyocytes increases the phosphorylation of Akt, a well-established anti-apoptotic effector. As demonstrated here, Galphaq expression protects cardiomyocytes against apoptosis induced by treatment with 2-deoxyglucose (2DOG) and this protection is lost when Akt activation is prevented by treatment with LY294002 (an inhibitor of PI3K). Galphaq-induced Akt phosphorylation is not caused by increased Gbetagamma signaling and does not appear to involve PKC activation. Rather studies using the EGF receptor inhibitor AG1478 and the Src inhibitor PP2 implicate these tyrosine kinases in the pathway inducing Akt phosphorylation. EGFR phosphorylation is increased in cells expressing Galphaq and this effect is inhibited by PP2, placing Src upstream of EGFR phosphorylation. EGFR activation appears to be required for Galphaq-mediated protection since inhibition of Src or EGFR rendered cells susceptible to 2DOG-induced apoptosis. In contrast to the requirement for EGFR mediated Akt activation in cardioprotection, neither EGFR nor Akt activation are necessary for the hypertrophic increases in cell size or ANF content elicited by Galphaq overexpression. These data demonstrate that increased Galphaq activity can provide anti-apoptotic signals by eliciting EGFR phosphorylation and subsequent Akt activation, independent of the well-known ability of Galphaq signaling to elicit hypertrophy.     

Highly prevalent genetic alterations in receptor tyrosine kinases and phosphatidylinositol 3-kinase/akt and mitogen-activated protein kinase pathways in anaplastic and follicular thyroid cancers

CONTEXT: Genetic alterations in receptor tyrosine kinases (RTKs) and phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways have not been fully defined in anaplastic and follicular thyroid cancers [anaplastic thyroid cancer (ATC), follicular thyroid cancer (FTC)]. OBJECTIVE: The objective of the study was to explore a wide-range genetic basis for the involvement of these pathways in ATC. DESIGN: We examined mutations and copy number gains of a large panel of genes in these pathways and corresponding phosphorylation of ERK (p-ERK) and Akt. RESULTS: We found frequent copy gains of RTK genes, including EGFR, PDGFRalpha and -beta, VEGFR1 and 2, KIT, and MET and in PIK3Ca, PIK3Cb, and PDK1 genes in the PI3K/Akt pathway. Mutations of Ras, PIK3Ca, PTEN, and BRAF genes and RET/PTC rearrangements were common, whereas mutations in PDK1, Akt1, Akt2, and RTK genes were uncommon in ATC. Overall, 46 of 48 ATC (95.8%) harbored at least one genetic alteration, and coexistence of two or more was seen in 37 of 48 ATC (77.1%). These genetic alterations were somewhat less common in FTC. Genetic alterations that could activate both the PI3K/Akt and MAPK pathways were found in 39 of 48 ATC (81.3%). RTK gene copy gains were preferentially associated with p-Akt, suggesting their dominant role in activating the PI3K/Akt pathway. The phosphorylation of Akt was far more common than p-ERK in FTC, and both were relatively common and often coexisted in ATC. CONCLUSIONS: Genetic alterations in the RTKs and PI3K/Akt and MAPK pathways are extremely prevalent in ATC and FTC, providing a strong genetic basis for an extensive role of these signaling pathways and the development of therapies targeting these pathways for ATC and FTC, particularly the former.     

Transcriptional and posttranslational up-regulation of HER3 (ErbB3) compensates for inhibition of the HER2 tyrosine kinase

Sustained and complete inhibition of HER3 and its output to PI3K/Akt are required for the optimal antitumor effect of therapeutic inhibitors of the HER2 oncogene. Here, we show that, after inhibition of the HER2 tyrosine kinase with lapatinib, there is PI3K/Akt and FoxO3a-dependent up-regulation of HER3 mRNA and protein. Up-regulated HER3 was then phosphorylated by residual HER2 activity, thus partially maintaining P-Akt and limiting the antitumor action of lapatinib. Inhibition of HER3 with siRNA or a neutralizing HER3 antibody sensitized HER2+ breast cancer cells and xenografts to lapatinib both in vitro and in vivo. Combined blockade of HER2 and HER3 inhibited pharmacodynamic biomarkers of PI3K/Akt activity more effectively than each inhibitor alone. These results suggest that because of HER3-mediated compensation, current clinical inhibitors of HER2 and PI3K/Akt will not block the PI3K pathway completely. They also suggest that therapeutic inhibitors of HER3 should be used in combination with HER2 inhibitors and PI3K pathway inhibitors in patients with HER2- and PI3K-dependent cancers.     

The AMPK agonist AICAR inhibits the growth of EGFRvIII-expressing glioblastomas by inhibiting lipogenesis

The EGFR/PI3K/Akt/mTOR signaling pathway is activated in many cancers including glioblastoma, yet mTOR inhibitors have largely failed to show efficacy in the clinic. Rapamycin promotes feedback activation of Akt in some patients, potentially underlying clinical resistance and raising the need for alternative approaches to block mTOR signaling. AMPK is a metabolic checkpoint that integrates growth factor signaling with cellular metabolism, in part by negatively regulating mTOR. We used pharmacological and genetic approaches to determine whether AMPK activation could block glioblastoma growth and cellular metabolism, and we examined the contribution of EGFR signaling in determining response in vitro and in vivo. The AMPK-agonist AICAR, and activated AMPK adenovirus, inhibited mTOR signaling and blocked the growth of glioblastoma cells expressing the activated EGFR mutant, EGFRvIII. Across a spectrum of EGFR-activated cancer cell lines, AICAR was more effective than rapamycin at blocking tumor cell proliferation, despite less efficient inhibition of mTORC1 signaling. Unexpectedly, addition of the metabolic products of cholesterol and fatty acid synthesis rescued the growth inhibitory effect of AICAR, whereas inhibition of these lipogenic enzymes mimicked AMPK activation, thus demonstrating that AMPK blocked tumor cell proliferation primarily through inhibition of cholesterol and fatty acid synthesis. Most importantly, AICAR treatment in mice significantly inhibited the growth and glycolysis (as measured by ¹⁸fluoro-2-deoxyglucose microPET) of glioblastoma xenografts engineered to express EGFRvIII, but not their parental counterparts. These results suggest a mechanism by which AICAR inhibits the proliferation of EGFRvIII expressing glioblastomas and point toward a potential therapeutic strategy for targeting EGFR-activated cancers.     

Roles of the MEK1/2 and AKT pathways in CXCL12/CXCR4 induced cholangiocarcinoma cell invasion

AIM:To evaluate the expression of C-X-C motif chemokine receptor 4(CXCR4)and its signaling cascades,which were previously identified as a key factor for cancer cell progression and metastasis,in cholangiocarcinoma cell lines.METHODS:The expression of CXCR4 and its signaling cascades were determined in the cholangiocarcinoma cell lines(RMCCA1 and KKU100)by Western blotting.The invasion assays and the detection of actin polymerization were tested in these cholangiocarcinoma cells treated with CXC chemokine ligand-12(CXCL12).RESULTS:Expression of CXCR4 was detected in both cholangiocarcinoma cell lines and activation of CXCR4 with CXCL12 triggered the signaling via the extracellular signal-regulated kinase-1/2(ERK1/2)and phosphoinositide 3-kinase(PI3K)and induction of cholangiocarcinoma cell invasion,and displayed high levels of actin polymerization.Addition of CXCR4 inhibitor(AMD3100)abrogated CXCL12-induced phosphorylation of MEK1/2 and Akt in these cells.Moreover,treatment with MEK1/2 inhibitor(U0126)or PI3K inhibitor(LY294 002)also attenuated the effect of CXCL12-induced cholangiocarcinoma cell invasion.CONCLUSION:These results indicated that the activation of CXCR4 and its signaling pathways(MEK1/2 and Akt)are essential for CXCL12-induced cholangiocarcinoma cell invasion.This rises Implications on a potential role for the inhibition of CXCR4 or its signal cascades in the treatment of cholangiocarcinoma.     

Y-box binding protein 1 augments sorafenib resistance via the PI3K/Akt signaling pathway in hepatocellular carcinoma

BACKGROUNDSorafenib is the first-line treatment for patients with advanced hepatocellular carcinoma (HCC). Y-box binding protein 1 (YB-1) is closely correlated with tumors and drug resistance. However, the relationship between YB-1 and sorafenib resistance and the underlying mechanism in HCC remain unknown.AIMTo explore the role and related mechanisms of YB-1 in mediating sorafenib resistance in HCC.METHODSThe protein expression levels of YB-1 were assessed in human HCC tissues and adjacent nontumor tissues. Next, we constructed YB-1 overexpression and knockdown hepatocarcinoma cell lines with lentiviruses and stimulated these cell lines with different concentrations of sorafenib. Then, we detected the proliferation and apoptosis in these cells by terminal deoxynucleotidyl transferase dUTP nick end labeling, flow cytometry and Western blotting assays. We also constructed a xenograft tumor model to explore the effect of YB-1 on the efficacy of sorafenib in vivo. Moreover, we studied and verified the specific molecular mechanism of YB-1 mediating sorafenib resistance in hepatoma cells by digital gene expression sequencing (DGE-seq).RESULTSYB-1 protein levels were found to be higher in HCC tissues than in corresponding nontumor tissues. YB-1 suppressed the effect of sorafenib on cell proliferation and apoptosis. Consistently, the efficacy of sorafenib in vivo was enhanced after YB-1 was knocked down. Furthermore, KEGG pathway enrichment analysis of DGE-seq demonstrated that the phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was essential for the sorafenib resistance induced by YB-1. Subsequently, YB-1 interacted with two key proteins of the PI3K/Akt signaling pathway (Akt1 and PIK3R1) as shown by searching the BioGRID and HitPredict websites. Finally, YB-1 suppressed the inactivation of the PI3K/Akt signaling pathway induced by sorafenib, and the blockade of the PI3K/Akt signaling pathway by LY294002 mitigated YB-1-induced sorafenib resistance.CONCLUSIONOverall, we concluded that YB-1 augments sorafenib resistance through the PI3K/Akt signaling pathway in HCC and suggest that YB-1 is a key drug resistance-related gene, which is of great significance for the application of sorafenib in advanced-stage HCC.     

The role of Akt in the signaling pathway of the glycoprotein Ib-IX induced platelet activation

The platelet von Willebrand factor (vWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. We show that GPIb-IX-induced platelet aggregation and stable adhesion under flow were impaired in mouse platelets deficient in PI3K effectors, Akt1 and Akt2, and in human platelets treated with an Akt inhibitor, SH-6. Akt1 and Akt2 play important roles in early GPIb-IX signaling independent of Syk, adenosine diphosphate (ADP), or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of Akt1 or Akt2 diminished platelet spreading on vWF but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both vWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1- or Akt2-deficiency diminished vWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by exogenous cGMP. Thus, Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway.     

PI-103在体外抗急性髓细胞白血病效应的实验研究

目的研究急性髓细胞白血病（AML）细胞P13K-Akt—mTOR信号转导通路各基因的表达及PI-103在体外对AML细胞增殖、凋亡及细胞周期的影响。方法RT-PCR法检测AML细胞P13K、Akt、mTORmRNA的表达;四甲基偶氮唑蓝法检测PI-103对AML细胞的增殖作用;流式细胞仪检测PI-103对AML细胞的凋亡率和细胞周期的影响。结果①81．25％的AML患者表达P13K基因,87．5％的患者表达mTOR基因,50％的患者同时表达此两种基因。②PI-103能明显提高PI3K—Akt-mTOR信号通路持续活化的AML细胞的增殖抑制率和凋亡率、阻滞细胞于G0／G1期（P均〈0．05）,且与剂量显著相关（P均〈0．05）。结论大部分AML患者存在PI3K-Akt-mTOR信号转导通路的异常活化;PI-103可通过PI3K、mTOR信号通路抑制AML细胞增殖、促进其凋亡。     

Therapeutic strategy for advanced EGFR mutant non-small-cell lung carcinoma

Activating mutation in exons 19 or 21 of epidermal growth factor receptor (EGFR) in non-small-cell lung cancers (NSCLC) are associated with increased sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib. Cancer patients harboring activating EGFR mutations benefit from first-line TKI therapy. Yet 10% of patients present a primary TKI resistance, while 50% of the others develop a secondary resistance within 9–12 months after starting TKI. The RECIST's definition of progression appears flawed when applied to EGFR-mutated NSCLC patients. Most often, tumor volume shrinking widely exceeds 30% during TKI response and kinetics of growth is low during relapse. At present, secondary resistance mechanisms associated with progression are better known: clonal selection of EGFR resistance mutation (T790M mutation in exon 20), amplification of transmembrane receptors for other growth factors (c-met, HER family, IGF1R, or AXL), downstream molecular alterations in EGFR signaling pathway (PI3K or PTEN), and epithelial–mesenchymal transition or transdifferentiation to small-cell cancer. The best strategy for secondary resistance is not well-defined: maintaining TKI therapy, switching to chemotherapy, combining both treatments, or using new therapies targeting other signaling pathways.     

Metadherin Regulation of Vascular Endothelial Growth Factor Expression Is Dependent Upon the PI3K/Akt Pathway in Squamous Cell Carcinoma of the Head and Neck

Our previous study indicated overexpression of metadherin (MTDH) is an adverse prognostic factor in squamous cell carcinoma of the head and neck (SCCHN) and promotes SCCHN cell proliferation and invasion. However, its mechanism remains unclear. Recent studies have indicated that MTDH is a cancer-metastasis-associated molecule that participates in the process of angiogenesis. Therefore, the study is aimed to investigate that whether vascular endothelial growth factor (VEGF), as one of the most potent proangiogenic cytokines, is regulated by MTDH and the role of the phosphatidylinositide 3-kinases/Protein Kinase B (PI3K/Akt) pathway in this process of regulation and the clinical significance of both MTDH and VEGF in SCCHN.Immunohistochemistry was used to assay the expression of MTDH and VEGF in a cohort of 189 SCCHN patients with intact follow-up information. The expression of MTDH was then upregulated or inhibited by lentivirus-mediated MTDH Complementary deoxyribonucleic acid or MTDH short hairpin ribonucleic acid (shRNA) to observe the resulting alterations in VEGF expression and the PI3K/Akt signaling pathway in SCCHN cell lines. In addition, the PI3K/Akt pathway was modulated to observe the resulting changes in the MTDH-mediated expression of VEGF.The immunohistochemistry data showed that MTDH expression is positively correlated with VEGF expression in SCCHN tissues. Moreover, the overexpression of MTDH in SCCHN Tu686 and 5-8F cells led to increases in the expression of VEGF, and this effect was accompanied by activation of the PI3K/Akt pathway. Conversely, shRNA-mediated knockdown of MTDH led to decreased VEGF expression. In addition, inhibition of the Akt signaling pathway reversed the upregulation of VEGF resulting from MTDH overexpression. Moreover, the survival analysis revealed that VEGF is an independent prognostic factor, and a combined survival analysis based on both MTDH and VEGF showed synergistic effects in the prognosis evaluation of SCCHN patients.The findings of the present study demonstrate that MTDH regulates the expression of VEGF via the PI3K/Akt signaling pathway, indicating the potential role of the MTDH-mediated activation of VEGF signaling pathway in SCCHN angiogenesis and metastasis.     

Akt is a major downstream target of PI3-kinase involved in angiotensin II-induced proliferation

Different signal transduction cascades have been implicated in angiotensin II (Ang II)-mediated cell growth, such as the extracellular signal-regulated kinase 1/2 (ERK1/2) and the phosphatidylinositol 3-kinase (PI3K) pathways. To identify the downstream targets of PI3K involved in Ang II-induced proliferation, we used both rat aortic smooth muscle (RASM) cells and a CHO cell line stably expressing the rat AT1A receptor. The ERK1/2 and PI3K pathways are independently activated and implicated in Ang II-mediated DNA synthesis and cell number increase in these 2 cell lines. In addition, a specific inhibitor of Akt inhibited Ang II-induced Akt phosphorylation, DNA synthesis and proliferation in CHO-AT1A or RASM cells. A dominant-negative mutant of Akt was also found to selectively block Ang II-induced proliferation of CHO-AT1A cells. To further elucidate the signaling events leading to Akt activation, we used an AT1 receptor mutant (AT1AD74E), deficient for Gq protein coupling, and the intracellular calcium chelator BAPTA-AM. Although altered Akt and ERK1/2 activation was observed in the CHO-AT1AD74E cell line, blockade of intracellular calcium elevation did not affect phosphorylation of these kinases. These results provide the first evidence of a specific and necessary role of Akt in Ang II-induced proliferation through a Gq protein-dependent calcium-independent pathway.