Fig 2: PSCs harboring KRASG12D protein activate KRAS-related signaling pathways while promoting proliferation and motility during activation(A) Western blot analysis of KRASG12D and KRAS proteins in PSC-transfected KRASWT and KRASG12D plasmids. (B) Western blot analysis of identified markers and KRASG12D and KRAS proteins in EVs derived from BxPC3-KRASWT and BxPC3-KRASG12D cells. (C) Western blot analysis of KRAS downstream proteins, including c-RAF, p-c-RAF, AKT, p-AKT, ERK, p-ERK, and PI3K110α in PSC-KRASG12D/PANC1 EV-treated PSCs and control cells. (D) Viability of PSCs overexpressing KRASWT and KRASG12D (n = 5, ∗∗∗p < 0.001, two-way analysis of variance [ANOVA], multiple comparisons). (E) Viability of PSCs treated with KRASWT and KRASG12D EVs derived from BxPC3 cells overexpressing KRASWT and KRASG12D (n = 5, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, two-way ANOVA, multiple comparisons). (F–H) Representative images and statistical results of cell migration assay of PSC-KRASWT, PSC-KRASG12D, and control cells at 24 and 48 h, respectively (n = 4, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, two-tailed t test). (I–K) Representative images and statistical results of the cell migration assay of PSCs treated with EVs derived from PANC1 cells for 24 and 48 h, respectively (n = 5, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, two-tailed t test).

Fig 3: The effects of KRAS, HRAS, and NRAS in HCC. (A) A Kaplan–Meier survival curve for overall survival in liver cancer patients according to the tumor expression of KRAS, HRAS, and NRAS. (B) Immunohistochemical analyses of KRAS were examined in paracancerous tissues and cancer tissues. Quantitative analyses were examined. Scale bar: 50 μm (up) or 100 μm (down). n = 3. (C) The SMMC‐7721 and HepG2 cells were analyzed under KRAS knockdown for 7 days. Clone formation and quantitative analyses were examined. n = 3. (D) Dynamic proliferation profiling was detected in SMMC‐7721 and HepG2 cells treated with KRAS knockdown over 80 h using the xCELLigence system. (E) The qualitative and quantitative expression of KRAS, HRAS, and NRAS were examined in SMMC‐7721, BEL‐7402, and HepG2 cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: NSC48160 induces cell death by targeting KRAS. (A) Structure models of HRASG60A (PDB ID: 1XCM) and superposition of the two structures as ribbon (top) and surface (bottom) representations. (B) The interaction between NSC48160 and HRASG60A was analyzed using a surface plasmon resonance (SPR) assay. A graph of equilibrium RIU responses versus compound concentrations was plotted (right). (C) CETSA was used to evaluate the binding between NSC48160 and KRAS at the thermodynamic level. The expression of KRAS was detected by western blotting. (D–G) Cell growth curves of BEL‐7402, SMMC7721, HepG2 cells, and HL7702 cells for 48 h were treated with different doses of NSC48160; n = 3, performed in triplicate.

Fig 5: NSC48160‐SLC7A11 knockdown combination remodels metabolic phenotype. (A) The top 20 pathways in rich factors of gene pathway enrichment. The rich factor indicates the ratio of the number of differentially enriched genes to the number of annotated genes in this pathway, and the number indicates the number of genes enriched in this pathway. (B and C) Effect of knockdown KRAS on the real‐time mitochondrial respiration and the real‐time glycolysis with or without NSC48160. HepG2 cells treated with NSC48160 (1.25 μM) for 24 h were evaluated for mitochondrial OCR and the extracellular acidification rate in Seahorse testing. (D and E) The effect of NSC48160 synergy with knockdown SLC7A11 on the Seahorse metabolic analysis was performed to examine the OCR and ECAR in HepG2 cells. Quantitative data are expressed as mean ± SD. *p < 0.05, as compared with the control. (F) Flow cytometric analysis of intracellular ROS in HepG2 and SMMC‐7721 cells treated with DMSO, NSC48160 with or without knockdown SLC7A11. (G) The lipid peroxidation detection in the above group. (n = 3, *p < 0.05).