Ho J C S, Nadeem A, Rydström A, Puthia M, Svanborg C
Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Lund, Sweden.
Oncogene. 2016 Feb 18;35(7):897-907. doi: 10.1038/onc.2015.144. Epub 2015 Jun 1.
HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.
哈姆雷特（人α-乳白蛋白对肿瘤细胞具有致死性）可广泛杀伤肿瘤细胞，这表明保守的生存途径受到了干扰。我们现在确定核苷酸结合蛋白为哈姆雷特的结合伴侣，在包含8000种人类蛋白质的蛋白质微阵列中，它们约占所有哈姆雷特靶点的35%。靶点激酶存在于激酶组树的所有分支中，包括26种酪氨酸激酶、10种类酪氨酸激酶、13种酵母不育激酶的同源物、4种酪蛋白激酶1激酶、15种含蛋白激酶A、蛋白激酶G、蛋白激酶C家族的激酶、15种钙/钙调蛋白依赖性蛋白激酶激酶以及13种来自细胞周期蛋白依赖性激酶、丝裂原活化蛋白激酶、糖原合成酶激酶3、酪蛋白激酶家族的激酶。在对347种野生型、93种突变型、19种非典型和17种脂质激酶的筛选中，哈姆雷特在体外表现为一种广泛的激酶抑制剂。在经哈姆雷特处理的肺癌细胞提取物中也检测到了磷酸化的抑制。此外，哈姆雷特识别24种Ras家族蛋白，并在质膜的胞质面与Ras、RasL11B和Rap1B结合。通过免疫共沉淀证实了哈姆雷特与包括Braf在内的活化Ras家族成员之间的直接细胞相互作用。结果，致癌性Ras和Braf活性受到抑制，哈姆雷特和Braf抑制剂协同增加了对哈姆雷特产生反应的肿瘤细胞死亡。与大多数小分子激酶抑制剂不同，哈姆雷特在体外和体内对肿瘤细胞表现出选择性。这些结果确定核苷酸结合蛋白为哈姆雷特的靶点，并表明在肿瘤细胞最初选择性识别哈姆雷特之后，ATP酶/激酶/鸟苷三磷酸酶机制的失调导致细胞死亡。因此，这些发现为哈姆雷特通过激酶和致癌性鸟苷三磷酸酶的失调产生的保守杀肿瘤作用提供了分子基础，而肿瘤细胞对这些酶存在依赖性。
