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. 2020 Dec 10;10:21762. doi: 10.1038/s41598-020-78646-y

Figure 3.

Figure 3

Lapatinib suppresses 4EBP1 phosphorylation insufficiently in MDA-MB-361 cells and the antiproliferative effect of lapatinib is limited in HER2+ breast cancer cell lines with PIK3CA mutations. (a) Inhibitory effects of fulvestrant (100 nM) and lapatinib (100 nM), alone and in combination treated for 24 h, on 4EBP1 phosphorylation in ER+/HER2+ breast cancer cell lines with and without PIK3CA mutation. Full-length blots are presented in Supplementary Fig. 3. (b) Newly synthesized protein in DMSO-, fulvestrant-, lapatinib-, and combination-treated cells. BT474 (upper) and MDA-MB-361 (lower) cells were treated with the indicated inhibitors for 24 h; Click-iT HPG was incorporated into the drug-treated cells for 15 h. Green indicates the newly synthesized proteins detected by the Click-iT assay (Life Technologies) and DAPI-stained nuclei, respectively. (c) PIK3CA mutation, ER expression, HER2 amplification, PTEN Loss and PIK3R1 status of the breast cancer cell lines used in this study. Information on gene modification and expression was obtained from the COSMIC database and ATCC information. (d) Antiproliferative activity of combined fulvestrant and lapatinib in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30; red bars) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361; blue bars) HER2+ breast cancer cell lines were used. The cells were treated with fulvestrant and lapatinib in combination for 8 days (mean ± SD [n = 3]). Y-axis indicates relative amounts of ATP (%), which were calculated with a chemiluminescence assay and compared with the chemiluminescence value of DMSO treatment on day 8. The data represent the mean ± SD (n = 3). (e) Inhibitory effects of fulvestrant (100 nM) and lapatinib (100 nM) in combination treatment for 24 h, on 4EBP1 phosphorylation in HER2+ breast cancer cell lines with and without PIK3CA mutation. Three PIK3CA-wild-type (BT474, SK-BR-3, and ZR-75-30) (left panel) and three PIK3CA-mutant (UACC893, HCC1954, and MDA-MB-361) (right panel) HER2+ breast cancer cell lines were used. Full-length blots are presented in Supplementary Fig. 4.