Fig 2: Proteasome mediates METTL3 cleavage in an mTOR-dependent manner.(a) Immunoblot of T47D cells treated with indicated inhibitors. Immunoblot of T47D (b) and MDA-MB-231 (c) cells treated with MG132 for 8 hr. (d) Immunoblot of T47D cells infected with METTL3-Flag followed by treatment with MG132 (10 µM) for 8 hr. (e) Immunoblot of purified recombinant METTL3-His protein incubated with or without T47D cell lysates wherein with or without MG132 treatment (20 µM) at 37°C for 1 hr. (f) List of the proteasome components identified by liquid chromatography–tandem mass spectrometry (LC–MS)/MS analysis of the tandem affinity purification with METTL3 antibody in 293T cells. (g) Immunoblot of whole cell extracts (WCE) and immunoprecipitations (IP) of T47D cells infected with METTL3-Flag or control vector. Immunoblot of MDA-MB-231 cells infected with shControl, shPSMC3 (h), shPSMC5 (i), and shPSMD10 (j). (k) Immunoblot of the purified HA-METTL3-Flag WT or indicated mutant from 293T cells using HA-magnetic beads followed by incubation with 26S at 37°C for 1 hr. (l, m) Immunoblot of MDA-MB-231 cells infected with shmTOR or shControl. (N) Immunoblot of T47D cells or MDA-MB-231 cells with time course treatment of rapamycin (100 nM) as indicated. (o) Immunoblot of Tsc2 WT (Tsc2+/+) or Tsc2 KO (Tsc2-/-) MEF cells treated with or without MG132 (10 µM) for 8 hr. (p) A model for METTL3a-mediated methyltransferase complex (MTC) assembly. FL indicates the full-length of METTL3. The short forms are labeled as a and b. se and le indicated short exposure and long exposure, respectively. Figure 7—source data 1.Unedited western blot images for Figure 7.

Fig 3: Expression of proteins involved in the HER2/AKT/mTOR and cyclinD1/CDK4 signaling pathways in BT474 cell lines treated with lapatinib and/or palbociclib.a Western blot analysis of proteins involved in the HER2/AKT/mTOR signaling pathway after treatment with lapatinib and/or palbociclib for 48 h. b Western blot analysis of proteins involved in the cyclinD1/CDK4 signaling pathway after treatment with lapatinib and/or palbociclib for 48 h. c p-HER2 expression levels under treatment with different drugs in the same cell. d p-HER2 expression levels in TSC2-NC, WT, and MT groups treated with the same drugs. e p-TSC2 expression levels under treatment with different drugs in the same cell. f p-TSC2 expression levels in TSC2-NC, WT, and MT groups treated with the same drugs. g p-mTOR expression levels under treatment with different drugs in the same cell. h p-mTOR expression levels in TSC2-NC, WT, and MT groups treated with the same drugs. i p-P70S6K expression levels under treatment with different drugs in the same cell. j p-P70S6K expression levels in TSC2-NC, WT, and MT groups treated with the same drugs. Data are shown as mean ± SD; One-way ANOVA was used to analyze the data in (d, f, h, j). Error bars are SEM. *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001.

Fig 4: Palbociclib reverses anti-HER2 resistance induced by the TSC2 c.4349 C > G (p.Pro1450Arg) variant.a Phosphorylation level of TSC2 p.Pro1450Arg mutant on Thr1462 increased, which led to activation of the mTOR pathway. In addition, phosphorylation levels of mTOR and P70S6K increased. P70S6K partially suppresses the RTK signal of EGFR family members via negative feedback. The potential mechanism of increased phosphorylation in the TSC2 p.Pro1450Arg variant on Thr1462 might be caused by cyclin D1/CDK4 binding to and regulating the phosphorylation level of TSC2. b After palbociclib treatment, the activation of CDK4 was inhibited, leading to decreased phosphorylation of TSC2, mTOR, and P70S6K. As a result, the feedback inhibition of the EGFR family by P70S6K was attenuated. Moreover, palbociclib effectively inhibited Rb phosphorylation, resulting in some cells stagnating in the G1 phase. (p: phosphorylation).

Fig 5: Magnetic resonance imaging (MRI) reveals a clinical response to neoadjuvant therapy containing trastuzumab in a breast cancer patient with the TSC2 c.4349 C > G (p.Pro1450Arg) variant and mutant TSC2 conferring resistance to lapatinib.a Left: Breast MRI scan captured before trastuzumab treatment initiation. Right: Follow-up image captured after trastuzumab treatment initiation, showing considerable tumor enlargement after four treatment cycles. b Three groups of lentiviruses were added to the BT474 culture to validate the transfection efficacy using fluorescence microscopy. Images in b are at ×20 magnification. Scale bar for each image is 1 cm. c Expression of the FLAG tag in the TSC2-NC, TSC2-WT, and TSC2-MT groups was determined via western blotting. d Expression of EGFR and HER2 in BT474, TSC2-NC, TSC2-WT, and TSC2-MT cells was determined via western blotting. e Relative expression of EGFR and HER2 in BT474, TSC2-NC, TSC2-WT, and TSC2-MT. (f) Expression of EGFR and HER2 in TSC2-NC, TSC2-WT, and TSC2-MT cells was determined by flow cytometry. g Mean fluorescence intensity (MFI) values of EGFR and HER2 in TSC2-NC, TSC2-WT, and TSC2-MT cells. h IC50 values of lapatinib for the TSC2-NC, TSC2-WT, and TSC2-MT groups are 0.2503 µM, 0.2919 µM, and 1.416 µM, respectively. Overexpression of TSC2-MT enhanced lapatinib resistance compared with that of TSC2-WT (P < 0.001). i Inhibition curve of the CDK4/6 inhibitor palbociclib. j Under combined treatment with palbociclib and lapatinib, palbociclib concentration was fixed (IC20 = 5 µM). IC50 values of lapatinib for the TSC2-NC, TSC2-WT, and TSC2-MT groups are 0.1838 µM, 0.2506 µM, and 0.2764 µM, respectively. Data are shown as mean± (Standard Deviation) SD; Data in (E, G, H, I, J) were analyzed with one-way ANOVA. Error bars are standard error of mean (SEM). **P < 0.05, ****P < 0.0001.