Reduced PTEN expression in breast cancer cells confers susceptibility to inhibitors of the PI3 kinase/Akt pathway.

The PTEN protein is a lipid phosphatase with putative tumor suppressing abilities, including inhibition of the PI3K/Akt signaling pathway. Inactivating mutations or deletions of the PTEN gene, which result in hyper-activation of the PI3K/Akt signaling pathway, are increasingly being reported in human malignancies, including breast cancer, and have been related to features of poor prognosis and resistance to chemotherapy and hormone therapy. Prior studies in different tumor models have shown that, under conditions of PTEN deficiency, the PI3K/Akt signaling pathway becomes a fundamental proliferative and survival pathway, and that pharmacological inhibition of this pathway results in tumor growth inhibition. This study aimed to explore further this hypothesis in breast cancer cells. To this end, we have determined the growth response to inhibition of the PI3K/Akt signaling pathway in a series of breast cancer cell lines with different PTEN levels. The PTEN-negative cell line displayed greater sensitivity to the growth inhibitory effects of the PI3K inhibitor, LY294002 and rapamycin, an inhibitor of the PI3K/Akt downstream mediator mTOR, compared with the PTEN-positive cell lines. To determine whether or not these differences in response are specifically due to effects of PTEN, we developed a series of cell lines with reduced PTEN protein expression compared with the parental cell line. These reduced PTEN cells demonstrated an increased sensitivity to the anti-proliferative effects induced by LY294002 and rapamycin compared with the parental cells, which corresponded to alterations in cell cycle response. These findings indicate that inhibitors of mTOR, some of which are already in clinical development (CCI-779, an ester of rapamycin), have the potential to be effective in the treatment of breast cancer patients with PTEN-negative tumors and should be evaluated in this setting.     

Deregulation of PKB influences antiapoptotic signaling by PKC in breast cancer cells

We have previously shown that novel protein kinase C (nPKC) isozymes, such as nPKCepsilon, negatively regulate TNF-induced apoptosis in breast cancer cells although the level on nPKCs did not correlate with cellular sensitivity to TNF. In the present study, we examined if the level/activation status of Akt/PKB influences antiapoptotic signaling by nPKCepsilon. While MCF-7 cells overexpressed PKB, BT-20 and SKBR-3 cells expressed constitutively phosphorylated PKB, and MDA-MB-231 cells expressed unphosphorylated PKB. Ly294002, an inhibitor of PI-3 kinase, induced cell death in SKBR-3 cells, which contained little nPKCs. Although Ly294002 by itself had only a modest effect on cell death in BT-20 and MCF-7 cells, it potentiated sensitivity of these cells to TNF. In contrast, Ly294002 either alone or in combination with TNF had little effect on cell death in MDA-MB-231 cells. These results suggest that the status of PKB in breast cancer cells influences antiapoptotic signaling by PKC.     

PI3K/Akt信号通路在RANKL诱导的MCF-7乳腺癌细胞迁移中的作用

目的：核因子-κB受体活化因子配体（RANKL）能促进表达RANK的上皮癌细胞迁移至骨,与乳腺癌骨转移密切相关。本文探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶（phosphoinositide3-kinase/serine/threonine protein kinase PI3K/Akt）信号通路在RANKL诱导的乳腺癌MCF-7细胞迁移中的作用。方法：流式细胞仪检测MCF-7细胞表面RANK蛋白的表达;Transwell法测定RANKL刺激后MCF-7细胞迁移能力的改变;Western-blot检测MCF-7细胞RANKL刺激后p-Akt及Akt的表达。SPSS 16.0软件分析实验数据。结果：MCF-7细胞表达RANK蛋白,RANKL诱导MCF-7细胞迁移能力显著增强,RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移。RANKL刺激后MCF-7细胞p-Akt表达在1、5分钟时一过性升高,PI3K抑制剂LY294002显著抑制RANKL诱导的细胞迁移。结论：PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞系MCF-7迁移。     

PI3K/Akt信号通路在RANKL诱导的MCF-7乳腺癌细胞迁移中的作用

目的 核因子-κB受体活化因子配体(RANKL)能促进表达RANK的上皮癌细胞迁移至骨,与乳腺癌骨转移密切相关.本文探讨磷脂酰肌醇-3-激酶/丝苏氨酸蛋白激酶(phosphoinositide3-kinase/serine/threonine protein kinase PI3K/Akt)信号通路在RANKL诱导的乳腺癌MCF-7细胞迁移中的作用.方法 流式细胞仪检测MCF-7细胞表面RANK蛋白的表达;Transwell法测定RANKL刺激后MCF-7细胞迁移能力的改变;Western-blot检测MCF-7细胞RANKL刺激后p-Akt及Akt的表达.SPSS 16.0软件分析实验数据.结果 MCF-7细胞表达RANK 蛋白,RANKL诱导MCF-7细胞迁移能力显著增强,RANKL的圈套受体OPG可阻断RANKL诱导的细胞迁移.RANKL刺激后MCF-7细胞p-Akt表达在1、5分钟时一过性升高,PI3K抑制剂LY294002显著抑制RANKL诱导的细胞迁移.结论 PI3K/Akt信号通路参与RANKL诱导的乳腺癌细胞系MCF-7迁移.     

Resistance to gefitinib in PTEN-null HER-overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3'-kinase/Akt pathway signaling.

PURPOSE: Tyrosine kinase (TK) inhibitors are emerging as a promising new approach to the treatment of HER overexpressing tumors, however optimal use of these agents awaits further definition of the downstream signaling pathways that mediate their effects. We reported previously that both EGFR- and Her2-overexpressing tumors are sensitive to the new EGFR-selective TK inhibitor gefitinib (ZD1839, "Iressa"), and sensitivity to this agent correlated with its ability to down-regulate Akt. However, EGFR-overexpressing MDA-468 cells, which lack PTEN function, are resistant to ZD1839, and ZD1839 is unable to down-regulate Akt activity in these cells. EXPERIMENTAL DESIGN: To study the role of PTEN function, we generated MDA468 cells with tet-inducible PTEN expression. RESULTS: We show here that the resistance of MDA-468 cells to ZD1839 is attributable to EGFR-independent constitutive Akt activation caused by loss of PTEN function in these cells. Reconstitution of PTEN function through tet-inducible expression restores ZD1839 sensitivity to these cells and reestablishes EGFR-stimulated Akt signaling. Although restoration of PTEN function to tumors is difficult to implement clinically, much of the effects of PTEN loss are attributable to overactive PI3K/Akt pathway signaling, and this overactivity can be modulated by pharmacologic approaches. We show here that pharmacologic down-regulation of constitutive PI3K/Akt pathway signaling using the PI3K inhibitor LY294002 similarly restores EGFR-stimulated Akt signaling and sensitizes MDA-468 cells to ZD1839. CONCLUSIONS: Sensitivity to ZD1839 requires intact growth factor receptor-stimulated Akt signaling activity. PTEN loss leads to uncoupling of this signaling pathway and results in ZD1839 resistance, which can be reversed with reintroduction of PTEN or pharmacologic down-regulation of constitutive PI3K/Akt pathway activity. These data have important predictive and therapeutic clinical implications.     

Metformin and erlotinib synergize to inhibit basal breast cancer

Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.     

Indole-3-carbinol-induced modulation of NF-κB signalling is breast cancer cell-specific and does not correlate with cell death

Indole-3-carbinol (I3C), a dietary chemopreventive compound, induces cell death in human breast cancer cells by modulating activities of Src and epidermal growth factor receptor (EGFR). The effect of I3C on NF-kappaB, constitutively activated in breast cancer cells, was investigated. Nuclear extracts of MDA-MB-468, MDA-MB-231 and HBL100 cells contained all of the Rel proteins with similar expression patterns in the latter two. The level of NF-kappaB-regulated reporter gene expression was in the order HBL100 < MDA-MB-468 < MDA-MB-231. Upstream inhibition, using PI3K, EGFR or IKKbeta inhibitors, resulted in cell-specific effects on expression of the NF-kappaB-regulated reporter gene and endogenous genes Bcl-xL, IkappaBalpha and IL-6, as well as on cell viability. The expression patterns of Rel and several NF-kappaB-regulated genes and the response to LY249002 in MDA-MB-468 cells contrasted with those in other cells. I3C induced NF-kappaB-regulated reporter gene expression at 12 h in MDA-MB-468 cells. Conversely, it was reduced at 24 h in HBL100 cells. I3C treatment for 6 h alone or in combination with TNFalpha induced NF-kappaB-regulated reporter gene expression, detected 5 h later, in MDA-MB-468, but not HBL100 cells. I3C induced NF-kappaB p65/p50 DNA binding at 6.5 h, preceded by association of IKKbeta with the Src/EGFR complex and increased phospho-IkappaBalpha in MDA-MB468 cells. TNFalpha increased I3C-induced apoptosis in MDA-MB-468 and MDA-MB-231 cells. It also induced apoptosis, enhanced by I3C, in HBL100 cells. Hence, regulation of constitutive NF-kappaB was cell-specific. I3C influenced the NF-kappaB pathway in a cell-specific manner, which was not related to apoptosis. However, the combination of I3C and TNFalpha increased apoptosis in all cell lines.     

阻断PI3K/AKT通路对乳腺癌细胞放射敏感性影响的研究

目的 研究抑制磷脂酰肌醇3激酶和（或）蛋白激酶B（P13K／AKT）生存传导路径是否改变乳腺癌细胞的放射敏感性。方法 用乳腺癌细胞细胞株MCF7为实验对象，分别接受单纯放射、Ly294002（P13K抑制剂）和二者结合处理。通过Western印记法证实Ly294002可下调AKT活性。采用成克隆法定量分析细胞增殖性死亡。通过半胱天冬酶-3（easpase-3）活性评估细胞凋亡。结果 单纯Ly294002（5μmol／L）可抑制AKT的磷酸化，而单纯放射对AKT的活性无明显影响，二者结合可提高对AKT活性的抑制作用。Ly294002（5μmol／L）在放射前与细胞作用1h及放射后作用10d均可提高MCF7细胞对放射的敏感性。Ly294002结合放射可增加MCF7细胞增殖性死亡，SF4值的放射增敏比为1．25，D0值的放射增敏比为1．42。Ly294002可增加MCF7细胞放射后诱导的细胞凋亡。结论 抑制P13K通过降低AKT活性，增加MCF7细胞对放射的敏感性，为筛选放射敏感剂进行临床实验提供了依据。     

Deregulation of the EGFR/PI3K/PTEN/Akt/mTORC1 pathway in breast cancer: possibilities for therapeutic intervention

The EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway plays prominent roles in malignant transformation, prevention of apoptosis, drug resistance and metastasis. The expression of this pathway is frequently altered in breast cancer due to mutations at or aberrant expression of: HER2, ERalpha, BRCA1, BRCA2, EGFR1, PIK3CA, PTEN, TP53, RB as well as other oncogenes and tumor suppressor genes. In some breast cancer cases, mutations at certain components of this pathway (e.g., PIK3CA) are associated with a better prognosis than breast cancers lacking these mutations. The expression of this pathway and upstream HER2 has been associated with breast cancer initiating cells (CICs) and in some cases resistance to treatment. The anti-diabetes drug metformin can suppress the growth of breast CICs and herceptin-resistant HER2+ cells. This review will discuss the importance of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway primarily in breast cancer but will also include relevant examples from other cancer types. The targeting of this pathway will be discussed as well as clinical trials with novel small molecule inhibitors. The targeting of the hormone receptor, HER2 and EGFR1 in breast cancer will be reviewed in association with suppression of the EGFR/PI3K/PTEN/Akt/mTORC1/GSK-3 pathway.     

A new selective AKT pharmacological inhibitor reduces resistance to chemotherapeutic drugs, TRAIL, all-trans-retinoic acid, and ionizing radiation of human leukemia cells.

It is now well established that the reduced capacity of tumor cells of undergoing cell death through apoptosis plays a key role both in the pathogenesis of cancer and in therapeutic treatment failure. Indeed, tumor cells frequently display multiple alterations in signal transduction pathways leading to either cell survival or apoptosis. In mammals, the pathway based on phosphoinositide 3-kinase (PI3K)/Akt conveys survival signals of extreme importance and its downregulation, by means of pharmacological inhibitors of PI3K, considerably lowers resistance to various types of therapy in solid tumors. We recently described an HL60 leukemia cell clone (HL60AR cells) with a constitutively active PI3K/Akt pathway. These cells were resistant to multiple chemotherapeutic drugs, all-trans-retinoic acid (ATRA), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Treatment with two pharmacological inhibitors of PI3K, wortmannin and Ly294002, restored sensitivity of HL60AR cells to the aforementioned treatments. However, these inhibitors have some drawbacks that may severely limit or impede their clinical use. Here, we have tested whether or not a new selective Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (Akt inhibitor), was as effective as Ly294002 in lowering the sensitivity threshold of HL60 cells to chemotherapeutic drugs, TRAIL, ATRA, and ionizing radiation. Our findings demonstrate that, at a concentration which does not affect PI3K activity, the Akt inhibitor markedly reduced resistance of HL60AR cells to etoposide, cytarabine, TRAIL, ATRA, and ionizing radiation. This effect was likely achieved through downregulation of expression of antiapoptotic proteins such as c-IAP1, c-IAP2, cFLIP(L), and of Bad phosphorylation on Ser 136. The Akt inhibitor did not influence PTEN activity. At variance with Ly294002, the Akt inhibitor did not negatively affect phosphorylation of protein kinase C-zeta and it was less effective in downregulating p70S6 kinase (p70S6K) activity. The Akt inhibitor increased sensitivity to apoptotic inducers of K562 and U937, but not of MOLT-4, leukemia cells. Overall, our results indicate that selective Akt pharmacological inhibitors might be used in the future for enhancing the sensitivity of leukemia cells to therapeutic treatments that induce apoptosis or for overcoming resistance to these treatments.     

PTEN suppresses breast cancer cell growth by phosphatase activity-dependent G1 arrest followed by cell death

PTEN/MMAC1/TEP1, a tumor suppressor gene, is frequently mutated in a variety of human cancers. Germ-line mutations of phosphatase and tensin homolog, deleted on chromosome ten (PTEN) are found in two inherited hamartoma tumor syndromes: Cowden syndrome, which has a high risk of breast, thyroid, and other cancers; and Bannayan-Zonana syndrome, a related disorder. PTEN encodes a phosphatase that recognizes both protein substrates and phosphatidylinositol-3,4,5-triphosphate. The lipid phosphatase activity of PTEN seems to be important for growth suppression through inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. We established clones with stable PTEN expression controlled by a tetracycline-inducible system to examine the consequences of increased levels of wild-type and mutant PTEN expression in a well-characterized breast cancer line, MCF-7. When we overexpressed PTEN in MCF-7, growth suppression was observed, but only if PTEN phosphatase activity is preserved. The initial growth suppression was attributable to G1 cell cycle arrest, whereas subsequent growth suppression was attributable to a combination of G1 arrest and cell death. Of note, the decrease in Akt phosphorylation preceded the onset-of suppression of cell growth. Treatment of MCF-7 cells with wortmannin, a PI3K inhibitor, caused cell growth inhibition in a way similar to the effects of overexpression of PTEN in this cell. In general, the inverse correlation between PTEN protein level and Akt phosphorylation was found in a panel of breast cancer cell lines. Therefore, PTEN appears to suppress breast cancer growth through down-regulating PI3K signaling, which leads to the blockage of cell cycle progression and the induction of cell death, in a sequential manner.     

水通道蛋白3在表皮生长因子诱导的乳腺癌细胞迁移中的作用机制研究

目的研究水通道蛋白3（aquapofin3,AQm）与表皮生长因子（EGF）诱导的乳腺癌细胞迁移之间的关系,并对其可能的机制进行初步探索。方法采用Westernblot法检测4种乳腺癌细胞MDA—MB-231、T47D、MCF-7、ER—ZR-70中的AQP3与EGFR的表达情况,选取高表达的细胞株进行EGF诱导,观察细胞迁移（划痕愈合实验）及AQP3表达的变化。施加CuSO。（AQm抑制剂）后观察细胞迁移及AQP3表达的变化。为进一步证明EGF通过哪条通路来调节AQF3的表达,采用LY294002（P13K／AKT通路抑制剂）及U1026（MAPK／ERK1／2通路抑制剂）分别进行阻断,观察AQP3的表达变化。结果Western blot检测发现迁移性强的MDA—MB-231细胞具有EGFR和AQP3的高表达。针对此细胞用不同浓度的EGF进行诱导,发现AQ1Y3的反应性表达与伤口愈合程度呈现一致的趋势（r=0．885,P〈0．01）,采用不同浓度的CuSO。对AQP3的表达进行抑制,MDA—MB-231细胞的伤口愈合程度明显下降（r=0．959,P〈0．01）。采用LY294002阻断P13K／AKT通路后,AQm表达下降,而U1026作用后AQP3表达无明显变化。结论在乳腺癌细胞中存在AQP3表达;在EGF诱导的乳腺癌细胞迁移中,AQP3的反应性表达起到重要的作用,而EGF可能通过P13K／AKT通路来调节AQP3的表达。     

Different signalling pathways regulate VEGF and IL-8 expression in breast cancer: implications for therapy

Elevated expression of pro-angiogenic cytokines is associated with aggressive tumour growth and decreased survival of patients with breast cancer. In general, the breast cancer cell lines with high vascular endothelial growth factor (VEGF) expression also express high levels of interleukin-8 (IL-8). The consequence of inhibiting mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K), both implicated in regulation of these cytokines, was examined in four cell lines. Treatment with the mitogen-activated protein kinase/extracellular signal-related kinase (MEK) inhibitor U0126 reduced expression of VEGF and IL-8 in MDA-MB-231 cells, partially inhibited expression in MDA-MB-468 and Hs578T cells, with minimal effects in GI101A cells. Treatment with LY294002 reduced cytokine expression in GI101A and MDA-MB-468 cells, with partial reduction in Hs578T and less effect in MDA-MB-231 cells. Thus, IL-8 and VEGF were regulated by different signalling pathways in different cell lines; this suggests that inhibition of the dominantly active pathway can downregulate both angiogenic cytokines. Recognising which signalling pathway is active may identify targets for anti-angiogenic therapy of breast cancer.     

The in vitro and in vivo anti-tumor effect of KO-202125, a sauristolactam derivative, as a novel epidermal growth factor receptor inhibitor in human breast cancer

Epidermal growth factor receptor (EGFR) is one of the most promising targets for cancer therapy. Here, we show the in vitro and in vivo anticancer effects and associated mechanisms of KO-202125, one of the synthesized aristolactam analogs, as a novel EGFR inhibitor, in EGFR-overexpressing cancer cell lines. KO-202125 showed more effective growth inhibition and apoptosis induction than gefitinib, a representative EGFR inhibitor, in various EGFR-overexpressing human cancers including estrogen receptor (ER)-negative MDA-MB-231 human breast cancer cells. Epidermal growth factor receptor phosphorylation at Tyr1068 was reduced and, consequently, the association of EGFR with p85 was decreased by KO-202125 treatment in MDA-MB-231 cell lines. This led to inactivation of the PI3K/Akt pathway, and consequently suppression of activation of the Wnt pathway and enhancement of the nuclear import of p27Kip1. KO-202125 treatment in nude mice injected with MDA-MB-231 cells showed inhibition of tumor growth without toxicity. Collectively, our results showed the possibility of KO-202125 as an effective therapy agent of EGFR-overexpressing cancer cells through reduced EGFR activity and downregulation of the Akt pathway.     

VES对MDA-MB-453细胞PI3K/Akt信号通路的影响

目的：检测维生素E琥珀酸酯（vitamin E succinate,VES）对MDA-MB-453乳腺癌细胞PI3K/Akt信号通路中PI3K及Akt表达的影响。方法：不同浓度VES作用于人乳腺癌细胞MDA-MB-453（Her-2过表达株）24h和48h,VES的浓度分别为5、10和20μg/ml。RT-PCR法检测VES作用前后PI3K及Akt的mR-NA表达变化。结果：VES对MDA-MB-453乳腺癌细胞PI3 K/Akt信号通路中PI3 K有抑制作用（P〈0.05）,并呈时间-剂量依赖关系;而对Akt无明显的抑制作用（P〉0.05）。结论：VES可能是通过作用于PI3K/Akt信号通路而发挥其对MDA-MB-453乳腺癌细胞的抑制及凋亡作用。