Yakes F Michael, Chinratanalab Wichai, Ritter Christoph A, King Walter, Seelig Steven, Arteaga Carlos L
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Cancer Res. 2002 Jul 15;62(14):4132-41.
We have examined whether inhibition of phosphatidylinositol-3 kinase (PI3K) and its target, the serine/threonine kinase Akt, play a role in the antitumor effect of the HER2 antibody Herceptin. Herceptin inhibited colony formation, down-regulated cyclin D1, and increased p27 protein levels in the HER2 gene-amplified BT-474 and SKBR-3 human breast cancer cells. These effects were temporally associated with the inhibition of PI3K activity in vitro as well as Akt function as measured by steady-state levels of phospho-Ser473 Akt and kinase activity against glycogen synthase kinase (GSK)-3beta. These responses were not observed in MDA-361 and MDA-453 cells, which do not exhibit HER2 gene amplification and are relatively resistant to Herceptin. Treatment of BT-474 cells with Herceptin inhibited the constitutive tyrosine phosphorylation of HER3 and disrupted the basal association of HER3 with HER2 and of HER3 with p85alpha potentially explaining the inhibition of PI3K. Treatment with either Herceptin or the PI3K inhibitor LY294002 increased the levels of p27 in the nucleus>cytosol, thus increasing the ratio of p27:Cdk2 in the nucleus and inhibiting Cdk2 activity and cell proliferation. Antisense p27 oligonucleotides abrogated the increase in p27 induced by Herceptin and prevented the antibody-mediated reduction in S phase. Transduction of BT-474 cells with an adenovirus-encoding active (myristoylated) Akt (Myr-Akt), but not with a beta-galactosidase control adenovirus, prevented the Herceptin- or LY294002-induced down-regulation of cyclin D1 and of phosphorylated GSK-3beta and prevented the accumulation of p27 in the nucleus and cytosol. In addition, Myr-Akt prevented Herceptin-induced inhibition of the cell proliferation of BT-474 cells and Herceptin-induced apoptosis of SKBR-3 cells. These data suggest that (a) changes in cell cycle- and apoptosis-regulatory molecules after HER2 blockade with Herceptin result, at least in part, from the inhibition of Akt; and (b) disabling PI3K and Akt is required for the antitumor effect of HER2 inhibitors.
我们研究了磷脂酰肌醇-3激酶（PI3K）及其靶点丝氨酸/苏氨酸激酶Akt的抑制作用是否在HER2抗体赫赛汀的抗肿瘤效应中发挥作用。赫赛汀抑制了HER2基因扩增的BT-474和SKBR-3人乳腺癌细胞的集落形成，下调了细胞周期蛋白D1，并增加了p27蛋白水平。这些效应在体外与PI3K活性的抑制以及Akt功能的抑制在时间上相关，Akt功能通过磷酸化丝氨酸473的Akt的稳态水平以及针对糖原合酶激酶（GSK）-3β的激酶活性来衡量。在不表现出HER2基因扩增且对赫赛汀相对耐药的MDA-361和MDA-453细胞中未观察到这些反应。用赫赛汀处理BT-474细胞可抑制HER3的组成型酪氨酸磷酸化，并破坏HER3与HER2以及HER3与p85α的基础结合，这可能解释了PI3K的抑制。用赫赛汀或PI3K抑制剂LY294002处理可增加细胞核>细胞质中p27的水平，从而增加细胞核中p27:Cdk2的比例并抑制Cdk2活性和细胞增殖。反义p27寡核苷酸消除了赫赛汀诱导的p27增加，并阻止了抗体介导的S期减少。用编码活性（肉豆蔻酰化）Akt（Myr-Akt）的腺病毒转导BT-474细胞，但不用β-半乳糖苷酶对照腺病毒转导，可防止赫赛汀或LY294002诱导细胞周期蛋白D1和磷酸化GSK-3β的下调，并防止p27在细胞核和细胞质中的积累。此外，Myr-Akt可防止赫赛汀诱导的BT-474细胞增殖抑制和赫赛汀诱导的SKBR-3细胞凋亡。这些数据表明：（a）用赫赛汀阻断HER2后细胞周期和凋亡调节分子的变化至少部分是由于Akt的抑制；（b）HER2抑制剂的抗肿瘤效应需要使PI3K和Akt失活。
