Functional characterization of PI3K C2 domain mutations detected in breast cancer circulating tumor cells and metastatic cells

• Published in: Cellular signalling Vol. 121; p. 111270
• Main Authors: Smit, Daniel J., Brauer, Helena, Horn, Stefan, Yigit, Gökhan, Haider, Marie-Therese, Pogenberg, Vivian, Schumacher, Udo, Pantel, Klaus, Jücker, Manfred
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01.09.2024
• Subjects: Breast cancer; C2 domain; Circulating tumor cells; PI3K; C2 domain; Breast cancer; Circulating tumor cells; PI3K
• ISSN: 0898-6568; 1873-3913; 1873-3913
• DOI: 10.1016/j.cellsig.2024.111270

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. 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. 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. 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. •PIK3CA C2 domain mutations are rare mutations with unknown impact in breast cancer.•The PIK3CA E418K and E453K mutation have been detected in CTCs and metastatic cells.•Both C2 domain mutations activate PI3K/AKT/mTOR pathway signaling through AKT.•For the E453K mutation, a disruption of p85α/p110α interaction can be proposed.•CTC-ITB-01 cells show a high susceptibility for dual targeting of AKT and mTOR.