Le Xiao-Feng, Claret Francois-Xavier, Lammayot Amy, Tian Ling, Deshpande Deepa, LaPushin Ruth, Tari Ana M, Bast Robert C
Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
J Biol Chem. 2003 Jun 27;278(26):23441-50. doi: 10.1074/jbc.M300848200. Epub 2003 Apr 16.
Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.
细胞周期蛋白依赖性激酶（CDK）抑制剂p27Kip1与细胞周期蛋白E.CDK2复合物结合，在控制细胞周期和细胞生长中起主要作用。我们小组和其他研究团队已报道，抗HER2单克隆抗体通过与p27Kip1的诱导及CDK2的减少相关的G1期细胞周期停滞，对HER2过表达的乳腺癌发挥抑制作用。然而，p27Kip1在抗HER2抗体诱导的细胞周期停滞和生长抑制中的作用仍不明确。在此，我们提供了多条证据，支持p27Kip1在抗HER2抗体诱导的G1期细胞周期停滞和肿瘤生长抑制中起关键作用。抗HER2抗体、鼠源4D5或人源化曲妥珠单抗（赫赛汀）诱导p27Kip1和G1期生长停滞具有浓度依赖性、时间依赖性、不可逆性且持久。曲妥珠单抗或4D5诱导的G1期细胞周期停滞程度与诱导的p27Kip1蛋白水平密切相关。用曲妥珠单抗处理至少14小时后，p27Kip1的上调和G1期生长停滞仍无法消除。单次处理后，抗HER2抗体诱导的p27Kip1蛋白、G1期停滞和生长抑制至少持续5天。抗HER2抗体诱导的乳腺癌细胞生长抑制程度与诱导的p27Kip1水平密切平行。外源性p27Kip1的诱导表达导致与抗HER2抗体诱导的类似的p27Kip1水平依赖性G1期细胞周期停滞和生长抑制。使用p27Kip1小干扰RNA降低p27Kip1表达可阻断抗HER2抗体诱导的p27Kip1上调和G1期停滞。用抗HER2抗体处理可显著增加p27Kip1蛋白的半衰期。抑制泛素-蛋白酶体途径而非抑制钙蛋白酶和半胱天冬酶活性，可将p27Kip1蛋白上调至与抗HER2抗体诱导的程度相当。我们进一步证明抗HER2抗体显著降低p27Kip1蛋白第187位苏氨酸（Thr-187）的磷酸化，并增加p27Kip1蛋白第10位丝氨酸（Ser-10）的磷酸化。S10A和T187A突变型p27Kip1蛋白的表达增加了G1期细胞比例，并减少了抗体进一步诱导的G1期停滞。因此，p27Kip1通过翻译后调控在抗HER2抗体诱导的G1期细胞周期停滞和肿瘤生长抑制中起重要作用。p27Kip1蛋白磷酸化的调控是抗HER2抗体上调该蛋白的翻译后机制之一。
