Fig. 1.

NCT-547 targets HER2 signaling and cancer stem-like properties in HER2-positive breast cancer cells. a Chemical structures of NCT-547. NCT-547 was synthesized as a lead-optimized product of L80, which is a C-ring truncated deguelin derivative. b HER2-positive cell lines (BT474, SKBR3, JIMT-1, and MDA-MB-453) were treated with the indicated concentrations of NCT-547 (0–20 μM) for 72 h. c-e NCT-547 inhibits HER2 signaling. c Reduced expression of full-length p185HER2, p95HER2, phospho-HER2 (Tyr1221/1222), HER3, phospho-HER3 (Tyr1289), EGFR, phospho-EGFR, and Akt observed following exposure to NCT-547 (0–10 μM, 72 h) by immunoblot analysis. d Immunocytochemical analysis and intensity profiling for HER2. BT474 cells were treated with NCT-547 (10 μM, 24 h) and immunostained for HER2 (1:100, green) with DAPI nuclear staining (blue). The intensity (y-axis, green) of HER2 signal in the plasma membrane is represented in arbitrary units as defined by the software. e BT474 and JIMT-1 cells were treated with NCT-547 (0–10 μM) for 24 h. Cells were immunoprecipitated with HER2 antibody and analyzed by immunoblotting of HER2, HER3, EGFR and HSP90 antibodies. IP, immunoprecipitation; IB, immunoblot; IgG, normal rabbit immunoglobulin G. f-k NCT-547 suppresses cancer stem-like properties. f Aldefluor-positivity in BT474 and JIMT-1 cells as measured by flow cytometry after exposure to NCT-547 (0–10 μM, 72 h) and Aldefluor-positive cells were quantified (*p < 0.05). g CD44+/CD24- population in the JIMT-1 cells was analyzed by flow cytometry after NCT-547 treatment (0–10 μM) for 72 h (**p < 0.01). h Effect of NCT-547 on mammosphere formation by BT474 and JIMT-1 cells was demonstrated by 3D-culturing cells in ultralow attachment plates in the presence or absence of NCT-547 (0–10 μM, 72 h). The numbers and sizes of BT474- and JIMT-1-mammospheres were significantly reduced after exposure to NCT-547 (**p < 0.01). i Effect of NCT-547 (10 μM, 72 h) on expression of stemness-related factors in BT474 mammospheres. Changes in HSP90, Nanog, Oct4 and Sox2, PARP and cleaved caspase-3 levels as determined by immunoblotting. Quantitative graphs of Nanog, Oct4 and Sox2 expression [**p < 0.01, adherent cells (Ad.) vs mammospheres (Mammo.); ##p < 0.01, DMSO control vs NCT-547 treatment in mammospheres]. j-k Effect of NCT-547 on mammosphere formation in a JIMT-1 xenograft model. Dissociated single cells (1 × 10⁶/ml) from primary tumors (200 ~ 250 mm³) were plated in ultralow attachment dishes and cultured in the presence or absence of NCT-547 (0–10 μM) for 8 days. The numbers and volumes of mammospheres were quantified (**p < 0.01)