Everolimus

Three SMIs, including everolimus (mTOR inhibitor), MK-2206 (AKT inhibitor), and ibrutinib (BTK inhibitor), were applied in this study (Cayman, USA). These chemical reagents were dissolved in dimethyl sulfoxide (DMSO) (Merck, Germany), and aliquots were frozen. The MCF-7 cells were seeded in 96-well culture plates (SPL, South Korea) in a 200 μL fully supplemented medium in the presence of various drugs at concentrations ranging from 16 to 8192 nM and incubated for 48 hours to determine the half-maximal inhibitory concentration (IC₅₀) values for all SMIs. Subsequently, each well was filled with 20 μL of freshly prepared 5 mg/mL of MTT solution (Sigma-Aldrich, USA) and incubated at 37 °C for 4 hours. Following incubation, the medium was carefully removed, and the reduced formazan crystals were dissolved in 150 μL DMSO by incubating at 37°C for 30 minutes in the dark. Optical density (OD) values were measured at 570 and 630 nm using an enzyme-linked immunosorbent assay plate reader (Synergy H1 BioTek, USA). All the experiments were performed in triplicates. The relative cell proliferation was calculated by dividing the mean OD values of each group by the OD values obtained from the control group.

The human PC cell lines Capan1 (HBT-79), MiaPaca2 (CRL-1420), Panc1 (CRL-1469) and Cos7 (CRL-1651) cell lines were purchased from American Tissue Cell Culture (Manassas, Virginia, USA) and maintained under culture with DMEM (Gibco) High Glucose containing 2 mM glutamine (Invitrogen), 5000 U/mL Penicillin-Streptomycin (Gibco) and 10% heat-inactivated dialyzed fetal bovine serum (Invitrogen). For drug treatment, Everolimus (11597, Cayman Chemical), CB-839 (22038, Cayman Chemical) or BPTES (S7753, Aurogene) were added the day after seeding in culture media. Cells were counted at the indicated time point with CellTiter-Fluor Cell Viability Assay (Promega). Human cell lines authentication was performed by BMR Genomics. All cell lines were routinely tested for Mycoplasma contamination using PCR.

MG132 (S2619), BI-D1870 (S2843), and cycloheximide (S6611) were purchased from Selleck. Puromycin (P8833), Blasticidin (15205), iodonitrotetrazolium chloride (I10406), MTT (475989), anti-Flag agarose beads (A-2220), anti-HA agarose beads (A-2095), and glutathione agarose beads (G4510) were purchased from Sigma-Aldrich. Rapamycin (13346), Torin2 (14185), MK2206 (11593), GSK2110 (17988), S6K1-I (15018), Pazopanib (12097), Crizotinib (12087), Everolimus (11597), and Temsirolimus (11590) were purchased from Cayman Chemical. Active RSK3 protein (50-199-8827) was purchased from Thermo Fisher Scientific. JR-AB2-011 (HY-122022) was purchased from MedChemExpress.

Sources of pharmacological agents were as follows: WST‐1 (Dojindo, Kumamoto, Japan), T16inh‐A01 (Tocris Bioscience, Bristol, UK), everolimus (Cayman Chemical, Ann Arbor, MI, USA), AZD 5363 (Cayman Chemical), MHY 1485 (Cayman Chemical), and DPP (Cayman Chemical). Other agents were obtained from Sigma‐Aldrich (St Louis, MO, USA) or Wako Pure Chemical Industries (Osaka, Japan).