组蛋白去乙酰化酶抑制剂LBH589对多发性骨髓瘤细胞MM1R的抑制作用

本研究探讨新一代组蛋白去乙酰化酶抑制剂LBH589单药或者联合蛋白酶体抑制剂硼替佐米,对多发性骨髓瘤(MM)细胞的抗瘤效应。采用MTT法检测LBH589(10、20、50 nmol/L)及50 nmol/L分别联合硼替佐米(10、20 nmol/L)作用于人多发性骨髓瘤MM1R细胞24,48 h后的细胞增殖抑制作用;采用流式细胞术检测LBH589对MM1R细胞周期和细胞凋亡的影响;采用Western blot分析LBH589(10、20、50 nmol/L)作用MM1R细胞24 h后组蛋白H4乙酰化的程度。结果表明,LBH589单药及与硼替佐米联合均能够抑制MM1R细胞增殖,并与药物浓度和作用时间呈正相关。MM1R细胞经药物作用48 h后,G0/G1期细胞逐渐增多,G2/M期及S期细胞逐渐减少,细胞阻滞在G0/G1期,同时可见MM1R细胞的凋亡率增加,作用呈浓度依赖性,且LBH589与硼替佐米联合作用均较单药作用更加明显(均P<0.001);Western blot分析显示,不同浓度LBH589作用MM1R细胞24 h后组蛋白H4乙酰化的程度上调,呈浓度依赖性。结论:LBH589能够抑制MM1R细胞增殖,阻滞细胞周期,诱导细胞凋亡,且与硼替佐米联合对骨髓瘤细胞有协同作用。     

LBH589或联合硼替佐米逆转髓系白血病耐药及其分子机制研究

目的 探讨新一代组蛋白去乙酰化酶抑制剂LBH589联合蛋白酶体抑制剂硼替佐米逆转急性髓系白血病(AML)细胞耐药效应及其分子机制.方法 耐药AML细胞株HL-60/ADM和难治性AML原代细胞与不同浓度LBH589、硼替佐米或两者联合进行体外培养,采用MTT法检测细胞增殖活力,Hoechst33342染色和Annexin V-FITC/PI双染流式细胞术检测细胞凋亡,用阿霉素摄取率并结合细胞增殖抑制率评估逆转耐药效应.进一步检测处理前后细胞MRP1及信号通路蛋白变化.结果 LBH589、硼替佐米均能够抑制HL-60/ADM和难治性AML原代细胞增殖,诱导凋亡,其中21 nmol/LLBH589和12 nmol/L硼替佐米联合时作用最强,经Calcusyn软件分析证明两者联合具有明显的协同作用(CI值分别为0.531和0.498).联合组MRP1阳性率为(57.78±3.34)%,显著低于LBH589、硼替佐米单药组[(76.06±5.17)%和(71.83±4.53)%,P值均＜0.05],而单药组与对照组之间差异也有统计学意义(P值均＜0.01).联合组细胞内阿霉素摄取率为(64.81±3.69)%,明显高于单药组[(28.96±2.52)%和(37.29±3.71)%](P值均＜0.05).LBH589联合硼替佐米可以明显抑制磷酸化Akt和磷酸化IKKα/β蛋白的表达,降低PI3K/Akt/NF-κB通路的活性,明显上调P53蛋白的表达,抑制NF-κB P65、Bcl-2、XIAP和MRP1蛋白活性,促进Caspase-8、Caspase-3和PARP的裂解激活.结论 LBH589、硼替佐米能够抑制耐药AML细胞增殖和促进凋亡,并逆转耐药,两者联合具有明显的协同作用.PI3K/Akt/NF-κB信号传导通路受抑或阻断可能是其主要的作用机制.

Abstract：

Objective To investigate reversal effect of histone deacetylase inhibitor LBH589 alone or in combination with proteasome inhibitor bortezomib on drug resistance in acute myeloid leukemia(AML) and its mechanism.Methods Ex vivo cultures of HL-60/ADM cells and fresh refractory AML cells were treated with LBH589, bortezomib or their combination at varying concentrations.Proliferation capacity, apoptosis rate and reversal of drug resistance were evaluated by MTT assay, dual staining of Hoechst33342 and AnnexinVFITC/PI by flow cytometry, and adriamycin uptake rate with proliferation inhibition, respectively.The change of signal pathway at protein level was analyzed by Western blot.Results Synergistic cytotoxicity was observed in the combination treatment with LBH589 and bortezomib against HL-60/ADM cells, as well as the fresh AML cells, the most powerful synergy being observed at 21 nmol/L LBH589 plus 12 nmol/L bortezomib,with CI values of 0.531 and 0.498, respectively by Calcusyn software analysis.Moreover, the accumulation of adriamycin in HL-60/ADM cells was increased more in combination treatment[(64.81 +3.69)%]than in either LBH589[( 28.96 + 2.52 ) %]or bortezomib[( 37.29 ± 3.71 ) %]alone ( P ＜ 0.05 ), and so did the uptake rate of adriamycin being (64.81 ± 3.69 ) %, ( 28.96 ± 2.52 ) % and ( 37.29 ± 3.71 ) % respectively (P ＜ 0.05 ).The combination treatment induced multiple apoptotic molecules co-action and intracellular drug accumulation contributed to the synergistic cytotoxity, including caspase activation, PARP cleavage, XIAP downregulation, p53-dependent suppression of Bcl-2 and MRP1 expression via the inhibition of phosphoinositide 3-kinase ( PI3K )/Akt/nuclear factor-κB ( NF-κB ) signaling pathway. Conclusions Combination treatment of drug resistant AML cells with LBH589 and bortezomib produces a synergistic effect of in creasing sensitivity to chemotherapy.The mechanism may be mainly resulted from inhibition of PI3K/Akt/NF-κB signaling pathway.     

Chemical ablation of androgen receptor in prostate cancer cells by the histone deacetylase inhibitor LAQ824

Androgen receptor plays a critical role in the development of primary as well as advanced hormone-refractory prostate cancer. Therefore, ablation of androgen receptor from prostate cancer cells is an interesting concept for developing a new therapy not only for androgen-dependent prostate cancer but also for metastatic hormone-refractory prostate cancer, for which there is no effective treatment available. We report here that LAQ824, a cinnamyl hydroxamatic acid histone deacetylase inhibitor currently in human clinical trials, effectively depleted androgen receptor in prostate cancer cells at nanomolar concentrations. LAQ824 seemed capable of depleting both the mutant and wild-type androgen receptors in either androgen-dependent and androgen-independent prostate cancer cells. Although LAQ824 may exert its effect through multiple mechanisms, several lines of evidence suggest that inactivation of the heat shock protein-90 (Hsp90) molecular chaperone is involved in LAQ824-induced androgen receptor depletion. Besides androgen receptor, LAQ824 reduced the level of Hsp90 client proteins HER-2 (ErbB2), Akt/PKB, and Raf-1 in LNCaP cells. Another Hsp90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), also induced androgen receptor diminution. LAQ824 induced Hsp90 acetylation in LNCaP cells, which resulted in inhibition of its ATP-binding activity, dissociation of Hsp90-androgen receptor complex, and proteasome-mediated degradation of androgen receptor. Consequently, LAQ824 blocked androgen-induced prostate-specific antigen production in LNCaP cells. LAQ824 effectively inhibited cell proliferation and induced apoptosis of these prostate cancer cells. These results reveal that LAQ824 is a potent agent for depletion of androgen receptor and a potential new drug for prostate cancer.     

Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer

EGFR is frequently mutated and amplified in lung adenocarcinomas sensitive to EGFR inhibitors gefitinib and erlotinib. A secondary mutation, T790M, has been associated with acquired resistance but has not been shown to be sufficient to render EGFR mutant/amplified lung cancers resistant to EGFR inhibitors. We created a model for studying acquired resistance to gefitinib by prolonged exposure of a gefitinib-sensitive lung carcinoma cell line (H3255; EGFR mutated and amplified) to gefitinib in vitro. The resulting resistant cell line acquired a T790M mutation in a small fraction of the amplified alleles that was undetected by direct sequencing and identified only by a highly sensitive HPLC-based technique. In gefitinib-sensitive lung cancer cells with EGFR mutations and amplifications, exogenous introduction of EGFR T790M effectively conferred resistance to gefitinib and continued ErbB-3/PI3K/Akt signaling when in cis to an activating mutation. Moreover, continued activation of PI3K signaling by the PIK3CA oncogenic mutant, p110alpha E545K, was sufficient to abrogate gefitinib-induced apoptosis. These findings suggest that allelic dilution of biologically significant resistance mutations may go undetected by direct sequencing in cancers with amplified oncogenes and that restoration of PI3K activation via either a T790M mutation or other mechanisms can provide resistance to gefitinib.     

Mitotic catastrophe cell death induced by heat shock protein 90 inhibitor in BRCA1-deficient breast cancer cell lines

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in folding, assembly, maturation, and stabilization of the client proteins that regulate survival of malignant cells. As previous reports correlate high Hsp90 expression with decreased survival in breast cancer, Hsp90 may be a favorable target for investigational therapy in breast cancer. In our study, we have examined the response of a panel of both BRCA1-null (UACC 3199, HCC 1937, and MBA-MD-436) and BRCA1-wt breast cancer cell lines (MCF-7, MBA-MD-157, and Hs578T) to determine the proteins governing response to Hsp90 inhibitor 17-allyloamino-17-demethoxy-geldanamycin. On treatment with the drug, cells arrested at G(2)-M phase and entered aberrant mitosis in a BRCA1-dependent manner. Failure to arrest the cells at or before mitosis resulted in formation of micronucleated cells, aberrant segregation of chromosomes, microtubule misalignment, and multicentrosomes, leading in eventual mitotic catastrophe cell death. Our observations show that BRCA1 mediates G(2)-M transition mainly through chek1 on 17-allyloamino-17-demethoxy-geldanamycin treatment.     

Rosemary extract reduces Akt/mTOR/p70S6K activation and inhibits proliferation and survival of A549 human lung cancer cells

Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Rosemary extract contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt, mammalian target of rapamycin (mTOR) and p70S6K, and the apoptotic protein poly ADP ribose polymerase (PARP) are key modulators of cancer cell growth and survival.In this study, we examined the effects of rosemary extract on proliferation, survival and apoptosis of human non-small cell lung cancer (NSCLC) cells and its influence on signaling events.Human NSCLC adenocarcinoma A549 cells were used. Cell proliferation and clonogenic survival were assessed using specific assays. Immunoblotting was used to examine total and phosphorylated levels of Akt, mTOR and p70S6K, and cleavage of PARP.Rosemary extract dose-dependently inhibited cell proliferation and reduced clonogenic survival of A549 cells, while PARP cleavage, an indicator of apoptosis, was enhanced. Rosemary extract significantly reduced total and phosphorylated/activated Akt, mTOR and p70S6K levels.In conclusion, rosemary extract inhibited proliferation, blocked clonogenic survival, and enhanced apoptosis of A549 lung cancer cells. These effects were associated with inhibition of Akt and downstream mTOR and p70S6K activity. Our data suggest that rosemary extract may have considerable anti-tumor and chemoprevention properties in lung cancer and deserves further systematic investigation in animal models of lung cancer.     

Targeted therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade

The outcome for patients with lung cancer has not changed significantly for more than two decades. Several studies show that the overexpression of vascular endothelial growth factor (VEGF)/vascular permeability factor and epidermal growth factor (EGF) and their receptors correlates with the clinical outcome for lung cancer patients. However, clinical trials of agents that target either of these pathways alone have been disappointing. We hypothesize that targeting both the tumor and its vasculature by simultaneously blocking the VEGFR and EGFR pathways will improve the treatment of locoregional lung cancer. Human lung cancer specimens were first examined for the activation of VEGF receptor 2 (VEGFR2) and EGF receptor (EGFR) for tumor and tumor-associated endothelial cells, and both were found to be activated. The effects of ZD6474 (ZACTIMA), a small-molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were then studied in vitro using human lung cancer and microvascular endothelial cells. In vitro, ZD6474 inhibited EGFR, VEGFR2, mitogen-activated protein kinase and Akt phosphorylation, EGF- and VEGF-induced proliferation, and endothelial cell tube formation and also induced apoptosis. ZD6474 was further studied in vivo using an orthotopic mouse model of non-small cell lung cancer using NCI-H441 human lung adenocarcinoma cells. The inhibition of both VEGFR2 and EGFR signaling pathways by ZD6474 resulted in profound antiangiogenic, antivascular, and antitumor effects. These results provide a basis for the development of clinical strategies for the combination of selective protein tyrosine kinase inhibitors that block both EGFR and VEGFR signaling as part of the management of locally advanced lung cancer.     

Features of tumor-microenvironment images predict targeted therapy survival benefit in patients with EGFR-mutant lung cancer

Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) are effective for many patients with lung cancer with EGFR mutations. However, not all patients are responsive to EGFR TKIs, including even those harboring EGFR-sensitizing mutations. In this study, we quantified the cells and cellular interaction features of the tumor microenvironment (TME) using routine H&E-stained biopsy sections. These TME features were used to develop a prediction model for survival benefit from EGFR TKI therapy in patients with lung adenocarcinoma and EGFR-sensitizing mutations in the Lung Cancer Mutation Consortium 1 (LCMC1) and validated in an independent LCMC2 cohort. In the validation data set, EGFR TKI treatment prolonged survival in the predicted-to-benefit group but not in the predicted-not-to-benefit group. Among patients treated with EGFR TKIs, the predicted-to-benefit group had prolonged survival outcomes compared with the predicted not-to-benefit group. The EGFR TKI survival benefit positively correlated with tumor-tumor interaction image features and negatively correlated with tumor-stroma interaction. Moreover, the tumor-stroma interaction was associated with higher activation of the hepatocyte growth factor/MET-mediated PI3K/AKT signaling pathway and epithelial-mesenchymal transition process, supporting the hypothesis of fibroblast-involved resistance to EGFR TKI treatment.     

Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreatic, colon, and breast tumors

The receptor for epidermal growth factor (EGFR) is overexpressed in many cancers. One important signaling pathway regulated by EGFR is the phosphatidylinositol 3'-kinase (PI3K)-phosphoinositide-dependent kinase 1-Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. Akt also regulates the mammalian target of rapamycin (mTOR)-S6K-S6 pathway to control cell growth in response to growth factors and nutrients. Recent reports have shown that the sensitivity of non-small-cell lung cancer cell lines to EGFR inhibitors such as erlotinib (Tarceva, OSI Pharmaceuticals) is dependent on inhibition of the phosphatidylinositol 3'-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway. There can be multiple inputs to this pathway as activity can be regulated by other receptors or upstream mutations. Therefore, inhibiting EGFR alone may not be sufficient for substantial inhibition of all tumor cells, highlighting the need for multipoint intervention. Herein, we sought to determine if rapamycin, an inhibitor of mTOR, could enhance erlotinib sensitivity for cell lines derived from a variety of tissue types (non-small-cell lung, pancreatic, colon, and breast). Erlotinib could inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that were the most sensitive. Rapamycin could fully inhibit S6 in all cell lines, but this was accompanied by activation of Akt phosphorylation. However, combination with erlotinib could down-modulate rapamycin-stimulated Akt activity. Therefore, in select cell lines, inhibition of both S6 and Akt was achieved only with the combination of erlotinib and rapamycin. This produced a synergistic effect on cell growth inhibition, observations that extended in vivo using xenograft models. These results suggest that combining rapamycin with erlotinib might be clinically useful to enhance response to erlotinib.     

Altered Hsp90 function in cancer: a unique therapeutic opportunity

Molecular chaperones or so-called heat shock proteins serve as central integrators of protein homeostasis within cells. In performing this function, they guide the folding, intracellular disposition, and proteolytic turnover of many key regulators of cell growth, differentiation, and survival. Recent data show essential roles for the chaperones in facilitating malignant transformation at the molecular level and support the concept that their altered utilization during oncogenesis is critical to the development of human cancers. The field is evolving rapidly, but it has become apparent that chaperones can serve as biochemical buffers at the phenotypic level for the genetic instability that is characteristic of many human cancers. Chaperone proteins thus allow tumor cells to tolerate the mutation of multiple critical signaling molecules that would otherwise be lethal. Much of the recent progress in understanding the complex role of heat shock proteins in tumorigenesis has been made possible by the discovery of several natural product antitumor antibiotics that selectively inhibit the function of the chaperone Hsp90. These agents have been used as probes to define the biological functions of Hsp90 at the molecular level and to validate it as a novel target for anticancer drug action. One of these agents, 17-allylamino,17-demethoxygeldanamycin (NSC 330507) has begun phase II clinical trials, and several second-generation compounds are now in late preclinical development. The best way to use Hsp90 inhibitors as anticancer agents remains to be defined. Trials accomplished to date, however, serve as proof of principle that Hsp90 function can be modulated pharmacologically without undue toxicity in humans. Given the redundancy and complexity of the signaling pathway abnormalities present in most cancers, the ability of Hsp90 inhibitors to alter the activity of multiple aberrant signaling molecules instead of just one or two (as most current-generation molecular therapeutics have been designed to do) may prove of unique therapeutic benefit.     

HSP抑制剂对食管鳞癌细胞株 TE-凋亡的影响及作用机制研究

目的探讨抑制热休克蛋白90（Hsp90）对食管鳞癌细胞株TE-1凋亡的影响。方法采用不同浓度（0.25、0.5、1.0、2.0 μmol/L）、不同时间（24、48、72 h）HSP90抑制剂17-烯丙胺-17-脱甲氧格尔德霉素(17-AAG)处理食管鳞癌细胞株TE-1,分别采用MTT法、流式细胞仪及western blot检测细胞增殖、细胞凋亡及Hsp90、Hsp70、Akt、Fas蛋白表达。结果17-AAG对TE-1细胞体外增殖抑制率和细胞凋亡率有明显促进作用,并呈现时间-剂量依赖性。17-AAG 处理后TE-1 细胞Hsp90、Akt 蛋白表达明显降低,Hsp70、Fas蛋白表达明显上调。结论17-AAG可显著抑制TE-1增殖,促进凋亡,其机制可能是通过抑制Hsp90活性、影响其相关信号通路所致。     

Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells

Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer.     

Dicoumarol down-regulates human PTTG1/Securin mRNA expression through inhibition of Hsp90

Securin, the natural inhibitor of sister chromatid untimely separation, is a protooncogene overexpressed in tumors. Its protein levels correlate with malignancy and metastatic proneness. Dicoumarol, a long-established oral anticoagulant, is a new Hsp90 inhibitor that represses PTTG1/Securin gene expression and provokes apoptosis through a complex trait involving both intrinsic and extrinsic pathways. Dicoumarol activity as an Hsp90 inhibitor is confirmed by smaller levels of Hsp90 clients in treated cells and inhibition of in vivo heat shock luciferase activity recovery assays. Likewise, established Hsp90 inhibitors (17-allylamino-geldanamycin and novobiocin) repress PTTG1/Securin gene expression. Also, overexpression of human Hsp90 in yeast makes them hypersensitive to dicoumarol. Both apoptosis and PTTG1/Securin gene repression exerted by dicoumarol in cancer cells are independent of three of the most important signaling pathways affected by Hsp90 inhibition: nuclear factor-kappaB, p53, or Akt/protein kinase B signaling pathways. However, effects on PTTG1/Securin could be partially ascribed to inhibition of the Ras/Raf/extracellular signal-regulated kinase pathway. Overall, we show that expression of PTTG1/Securin gene is Hsp90 dependent and that dicoumarol is a bona fide Hsp90 inhibitor. These findings are important to understand the mode of action of Hsp90 inhibitors, mechanisms of action of dicoumarol, and Securin overexpression in tumors.     

Therapeutic anti-EGFR antibody 806 generates responses in murine de novo EGFR mutant-dependent lung carcinomas

Activating EGFR mutations occur in human non-small cell lung cancer (NSCLC), with 5% of human lung squamous cell carcinomas having EGFRvIII mutations and approximately 10%-30% of lung adenocarcinomas having EGFR kinase domain mutations. An EGFR-targeting monoclonal antibody, mAb806, recognizes a conformational epitope of WT EGFR as well as the truncated EGFRvIII mutant. To explore the anticancer spectrum of this antibody for EGFR targeted cancer therapy, mAb806 was used to treat genetically engineered mice with lung tumors that were driven by either EGFRvIII or EGFR kinase domain mutations. Our results demonstrate that mAb806 is remarkably effective in blocking EGFRvIII signaling and inducing tumor cell apoptosis, resulting in dramatic tumor regression in the EGFRvIII-driven murine lung cancers. Another EGFR-targeting antibody, cetuximab, failed to show activity in these lung tumors. Furthermore, treatment of murine lung tumors driven by the EGFR kinase domain mutation with mAb806 also induced significant tumor regression, albeit to a less degree than that observed in EGFRvIII-driven tumors. Taken together, these data support the hypothesis that mAb806 may lead to significant advancements in the treatment of the population of NSCLC patients with these 2 classes of EGFR mutations.     

Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are present in lung adenocarcinomas that respond to the EGFR inhibitors gefitinib and erlotinib. Two types of mutations account for approximately 90% of mutated cases: short in-frame deletions in exon 19 and a specific point mutation in exon 21 at codon 858 (L858R). Screening for these mutations has been based mainly on direct sequencing. We report here the development and validation of polymerase chain reaction-based assays for these two predominant types of EGFR mutations. The assay for exon 19 mutations is based on length analysis of fluorescently labeled polymerase chain reaction products, and the assay for the exon 21 L858R mutation is based on a new Sau96I restriction site created by this mutation. Using serial dilutions of DNAs from lung cancer cell lines harboring either exon 19 or 21 mutations, we detected these mutations in the presence of up to approximately 90% normal DNA. In a test set of 39 lung cancer samples, direct sequencing detected mutations in 25 cases whereas our assays were positive in 29 cases, including 4 cases in which mutations were not apparent by sequencing. These assays offer higher sensitivity and ease of scoring and eliminate the need for sequencing, providing a robust and accessible approach to the rapid identification of most lung cancer patients likely to respond to EGFR inhibitors.     

Constitutive and inducible Akt activity promotes resistance to chemotherapy,trastuzumab, or tamoxifen in breast cancer cells

To evaluate the role of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in breast cancer cell survival and therapeutic resistance, we analyzed a panel of six breast cancer cell lines that varied in erbB2 and estrogen receptor status. Akt activity was constitutive in four cell lines and was associated with either PTEN mutations or erbB2 overexpression. Akt promoted breast cancer cell survival because a PI3K inhibitor, LY294002, or transient transfection of a dominant-negative Akt mutant inhibited Akt activity and increased apoptosis. When combined with therapies commonly used in breast cancer treatment, LY294002 potentiated apoptosis caused by doxorubicin, trastuzumab, paclitaxel, or etoposide. Potentiation of apoptosis by LY294002 correlated with induction of Akt by doxorubicin or trastuzumab alone that occurred before the onset of apoptosis. Similar results were observed with tamoxifen. Combining LY294002 with tamoxifen in estrogen receptor-positive cells greatly potentiated apoptosis, which was correlated with tamoxifen-induced Akt phosphorylation that preceded apoptosis. To confirm that the effects of LY294002 on chemotherapy-induced apoptosis were attributable to inhibition of Akt, we transiently transfected breast cancer cells with dominant-negative Akt and observed increased doxorubicin-induced apoptosis. Conversely, stably transfecting cells with constitutively active Akt increased Akt activity and attenuated doxorubicin-induced apoptosis. These studies show that endogenous Akt activity promotes breast cancer cell survival and therapeutic resistance, and that induction of Akt by chemotherapy, trastuzumab, or tamoxifen might be an early compensatory mechanism that could be exploited to increase the efficacy of these therapies.