Fig 2: CTC-ITB-01 cells established from a patient with metastatic ER+/HER2- breast cancer show no response to pharmacological inhibition of ER alpha (A) (a–e) Immunohistochemical stainings of formalin-fixed CTC-ITB-01 cells reveal expression of ERa, E-cadherin and modest expression of HER2. Only weak progesterone receptor and no vimentin expression was observed. HER2 CISH shows no amplification of the HER2 gene locus. (g,h) Hematoxilin and eosin (H&E) staining reveals that cells grow in a mixed epithelial-mesenchymal morphology. (a–e,g) Bar 200 µm (f,h) Bar 400 µm. (B) CTC-ITB-01 cells were cultured in the presence of the indicated fulvestrant or AZD9496 concentrations for 32 h. Western blots of protein lysates were probed with the indicated antibodies (left panel). Equal numbers of CTC-ITB-01 cells were seeded per well of a 6-well plate and treated with the indicated fulvestrant or AZD9496 concentrations. After 9 days cells were fixed and stained (right panel). Equal amounts of CTC-ITB-01 cells were seeded on 6-well culture plates and cultured either with or without the indicated concentrations of fulvestrant or AZD9496. To determine the induction of cell death and/or cell cycle arrest cells were harvested after 36 h. The percentage of SubG1, G1, S, G2/M, D cells was evaluated using propidium iodid staining and flow cytometry. Mean values ± s.d. of four independent experiments are presented (lower panel). (C) Stable knockdown of ERa within CTC-ITB-01 cells was performed by using lentiviral transfer of non-targeted or two different targeted shRNAs against ERa. After transduction reduced expression of ERa in CTC-ITB-01 ERa k.o. cells was evidenced by western blotting. Western blots of protein lysates were probed with the indicated antibodies (left panel). Equal numbers of ERa k.o.1, ERa k.o.2 and negative control scramble CTC-ITB-01 cells were seeded on 6-well culture plates. After 9 days cells fixed and stained (right panel). (D) To compare the capacity of targeted shRNAs against ERa and fulvestrant to reduce ERa levels equal amounts of lysates from negative control, ERa k.o.1, ERa k.o.2 and fulvestrant treated CTC-ITB-01 cells were analysed by western blotting. Western blots were probed with the indicated antibodies. The uncropped Western Blot Images can be found in Figure S7.

Fig 3: FTO enhances the migration and invasion of HER2-positive breast cancer cells. A-B The transfection efficiency of FTO siRNA or overexpression plasmid in SKBR3 and MDA-MB453 cells confirmed by qRT-PCR (A) and Western blotting (B). C-D The detection of cell migration by Wound healing assay in SKBR3 cells and by Transwell migration assay in MDA-MB453 cells. The healing percent and migration cells were measured at 24 h after the transfection of FTO siRNA or overexpression plasmid. E Wound healing assay of SKBR3 and MDA-MB453 cells pretreated with gradient concentrations of MA2 for 48 h. DMSO was used as solvent control. F-G Transwell invasion assay of SKBR3 and MDA-MB453 cells treated with FTO siRNA or MA2 (80 µmol/L). H Transwell invasion assay of SKBR3 and MDA-MB453 cells transfected with FTO plasmid. Experiments were terminated after seeding for 24 h. I Relative m6A content of total RNA after FTO knockdown, overexpression, or FTO inhibitor MA2 treatment. Scale bar: 200 µm. * P < 0.05, ** P < 0.01, *** P < 0.001.Abbreviations: siCtrl, negative control of siRNA; siFTO, FTO siRNA; eVec, empty plasmid vector; oeFTO, FTO clone plasmid; qRT-PCR, quantitative reverse transcription-PCR; MA2, the ethyl ester form of meclofenamic acid other abbreviations as in Figure 1

Fig 4: Kaplan–Meier survival curves of 63 patients with endometrial cancer in relation to HER-2 expression. (A) Relapse-free survival (B) Overall survival.

Fig 5: NDUFA4L2 expression in HER2-positive breast cancer patient samples and survival analysis of patients grouped by expression level of NDUFA4L2. (a) Representative pictures of immunohistochemistry (IHC) of NDUFA4L2 in tumor tissue biopsies in trastuzumab-sensitive (top) and -resistant (bottom) patients. (b) NDUFA4L2 IHC-positive cell densities in trastuzumab-sensitive versus -resistant patient samples. Horizontal line represents median positive cell density of the group; significance was tested with the Mann–Whitney’s test. (***, p-value < 0.001) (c) Optical density quantification of NDUFA4L2 IHC-positive cells in trastuzumab-sensitive versus -resistant patient samples, expressed as mean ± SD; significance was tested with the Mann–Whitney’s test. (***, p-value < 0.001) (d) Relapse-free survival plot of current cohort of patients, subgrouped by IHC NDUFA4L2 positivity. Significance was tested by log-rank test. (e) Relapse-free survival plot of KM plotter cohort, all HER2-positive patients and (f) HER2-positive, ER/PR-negative patients, subgrouped by median expression of NDUFA4L2 (probe 218484_at). Significance was tested by log-rank test.