背景:在乳腺癌中,超过三分之一的患者在PIK3CA基因中存在体细胞突变,在其肿瘤细胞中编码磷脂酰肌醇3-激酶(PI3K)的p110α催化亚基。循环肿瘤细胞(CTC)是从原发性肿瘤脱落到血流中的细胞。最近,从液体活检来源的细胞建立了具有致瘤和转移能力的长期稳定的乳腺癌CTC-ITB-01细胞系.在原发肿瘤中检测到致癌热点PIK3CA突变H1047R(激酶结构域),同一患者的CTC和转移。在CTC和阴道转移中检测到位于C2结构域内的其他PIK3CA突变(E418K和E453K),但在原发性肿瘤中未检测到。我们研究的目的是在功能上表征在原发肿瘤中未检测到的C2域内的罕见E418K和E453K突变的影响。
方法:PIK3CA突变E418K,E453K,H1047R通过定点诱变产生,并通过慢病毒转导在乳腺癌细胞中稳定过表达。通过蛋白质印迹分析检查随后的信号通路激活。使用IncucyteZoom系统通过活细胞成像研究了PIK3CA突变对生物过程的影响。在Pymol中进行结构建模。通过使用超速离心分离胞质和膜部分来评估突变体的膜定位。通过活细胞成像分析CTC-ITB-01细胞的药物敏感性。
结果:稳定过表达PIK3CA野生型(WT)或E418K之一的人MDA-MB-231,MCF-7和T47D乳腺癌细胞的蛋白质印迹分析,E453K或H1047R突变体揭示了在C2突变体(E418K和E453K)和激酶结构域突变体H1047R中AKT磷酸化的显著增加。功能分析显示过表达E453K和H1047R突变体的MDA-MB-231细胞的增殖显著增加。在所有过表达WT和每种突变体的细胞中迁移增加。有趣的是,侵袭和趋化性仅在过表达C2结构域突变体的MDA-MB-231细胞中增强,即E418K和E453K。此外,两个C2结构域突变体的膜定位增加。E453K突变的结构模型表明,p85α亚基的负调节域与p110α催化亚基之间的相互作用被破坏,这是导致观察到的PI3K/AKT/mTOR信号激活的潜在机制。通过MK2206和RAD001双重靶向AKT/mTOR通路导致非常强的协同作用(IC50MK2206:148nM,IC50RAD001:15nM)通过凋亡诱导在CTC-ITB-01细胞系中增殖。
结论:我们的结果表明,PIK3CAC2结构域突变激活PI3K下游AKT信号,并可以增加增殖,稳定的慢病毒转导后的迁移和侵袭。尽管两个研究的突变-E418K和E453K-都位于C2域内,可以提出不同的分子机制。PIK3CA突变的CTC-ITB-01显示出对AKT/mTOR双重抑制的高易感性。需要进一步的研究来充分阐明罕见PIK3CA突变的致癌潜力。
BACKGROUND: In breast cancer, over one third of all patients harbor a somatic mutation in the PIK3CA gene, encoding the p110α catalytic subunit of the phosphatidylinositol 3-kinase (PI3K) in their tumor cells. Circulating tumor cells (CTCs) are cells shed from the primary tumor into the blood stream. Recently, the long-term stable breast cancer CTC-ITB-01 cell line with tumorigenic and metastatic capacity was established from liquid biopsy derived cells. The oncogenic hotspot PIK3CA mutation H1047R (kinase domain) was detected in the primary tumor, CTCs and metastasis of the same patient. Other PIK3CA mutations located within the C2 domain (E418K and E453K) were detected in the CTCs and the vaginal metastasis but not in the primary tumor. The goal of our study was to functionally characterize the impact of the rare E418K and E453K mutations within the C2 domain that were not detected in the primary tumor.
METHODS: PIK3CA mutations E418K, E453K, H1047R were generated by site-directed mutagenesis and stably overexpressed in breast cancer cells by lentiviral transduction. Subsequent signaling pathway activation was examined by western blot analysis. The impact of PIK3CA mutations on biological processes was studied by live cell imaging using the Incucyte Zoom system. Structural modeling was conducted in Pymol. The membrane localization of the mutants was evaluated by separating the cytosolic and membrane fraction using ultracentrifugation. Drug susceptibility of CTC-ITB-01 cells was analyzed by live cell imaging.
RESULTS: Western blot analysis of human MDA-MB-231, MCF-7 and T47D breast cancer cells stably overexpressing either the PIK3CA wildtype (WT) or one of the E418K, E453K or H1047R mutants revealed a significant increase in AKT phosphorylation in both C2 mutants (E418K and E453K) and the kinase domain mutant H1047R. Functional analysis showed a significantly increased proliferation of MDA-MB-231 cells overexpressing the E453K and H1047R mutants. Migration was increased in all cells overexpressing WT and each of the mutants. Interestingly, invasion and chemotaxis were only enhanced in the MDA-MB-231 cells overexpressing the C2 domain mutants, i.e. E418K and E453K. In addition, membrane localization of the two C2 domain mutants was increased. Structural modeling of the E453K mutation suggests a disruption of the interaction between the negative regulatory domain of the p85α subunit and the p110α catalytic subunit as a potential mechanism leading to the observed activation of PI3K/AKT/mTOR signaling. Dual targeting of AKT/mTOR pathway by MK2206 and RAD001 leads to very strong synergistic effects (IC50 MK2206: 148 nM, IC50 RAD001: 15 nM) with respect to proliferation in the CTC-ITB-01 line through apoptosis induction.
CONCLUSIONS: Our results demonstrate that PIK3CA C2 domain mutations activate PI3K downstream AKT signaling and can increase proliferation, migration and invasion after stable lentiviral transduction. Although both investigated mutations - E418K and E453K - are located within the C2 domain, a different molecular mechanism can be proposed. The PIK3CA mutated CTC-ITB-01 shows a high susceptibility against dual inhibition of AKT/mTOR. Further studies are required to fully elucidate the oncogenic potential of rare PIK3CA mutations.