Journal: Science Advances

Article Title: CASTOR1 functional inactivation drives KRAS -mutant NSCLC and therapy resistance by a noncanonical mechanism of tumor suppression

doi: 10.1126/sciadv.aeb9836

Figure Lengend Snippet: ( A ) Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice 5 months after intranasal inoculation with Adeno-Cre (left). Tumor incidence, size, and burden (tumor area/total lung area × 100) were quantified (second to fourth panels, respectively). Data presented as means and SD. Statistical analysis was performed by one-way ANOVA and Student’s t test. ( B ) Representative IHC staining of CASTOR1 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice. ( C ) Representative IHC staining images of Ki67 in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left) and quantification of Ki67-positive cells (middle and right panels). Statistical analysis was performed by one-way ANOVA and Student’s t test. ( D and E ) Representative IHC staining images of p-4EBP1 (D) and p-S6 (E) of lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (left panels) and quantification of positive cells (middle and right panels). Statistical analysis was performed by one-way ANOVA and Student’s t test. ( F ) Immunoblotting analysis of mTORC1 downstream targets p-S6K and p-4EBP1, KRAS downstream target ERK and AKT, and their phosphorylated forms in normal lung tissues from KRAS WT ;C1 WT and KRAS WT ;C1 KO mice and tumors from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO mice (left panels). Three samples per group were analyzed and quantified (right panels). Statistical analyses were performed using nested-ANOVA followed by Tukey’s post hoc test for multiple comparisons. ( G ) Representative IHC staining images of p-ERK in lung tumors from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice (top panels) and quantification of positive cells (bottom). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test or Student’s t test, as appropriate. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: Primary antibodies included CASTOR1 (1:50 dilution; Abclonal, catalog no. A20710), p-ERK (1:100 dilution; Cell Signaling Technology, catalog no. 4370, RRID: AB_2315112), p-4EBP1 (1:100 dilution; Cell Signaling Technology, catalog no. 2855, RRID: AB_560835), p-S6K (1:100 dilution; Abclonal, catalog no. AP0502, RRID: AB_2863907), p-S6 (1:100 dilution; Cell Signaling Technology, catalog no. 2211, RRID: AB_331679), p-AKT–S473 (1:100 dilution; Cell Signaling Technology, catalog no. 4060, RRID: AB_2315049), and pCASTOR1 (1:100).

Techniques: Analysis, Immunohistochemistry, Western Blot

Journal: Science Advances

Article Title: CASTOR1 functional inactivation drives KRAS -mutant NSCLC and therapy resistance by a noncanonical mechanism of tumor suppression

doi: 10.1126/sciadv.aeb9836

Figure Lengend Snippet: ( A to C ) CASTOR1 loss enhances resistance to KRAS G12D -specific inhibitor MRTX1133 in tumors. Mice were intranasally inoculated with Adeno-Cre for 4 weeks and then treated with MRTX1133 or vehicle for 4 weeks. Representative H&E images of lungs collected from KRAS G12D ;C1 WT or KRAS G12D ;C1 KO mice were shown (A). Tumor size (B) and burden (tumor area/total lung area × 100) (C) were quantified. Results presented as mean and SD. Statistical analysis was performed by nested ANOVA, followed by Tukey’s post hoc test for multiple comparisons. ( D ) KRAS G12D ;C1 WT tumor organoids had heterogeneous response to MRTX1133. Representative pictures of nine KRAS G12D ;C1 WT organoids after 1 week of treatment with MRTX1133 or vehicle. Statistical analysis was performed using Student’s t test. ( E ) Immunoblotting analysis of KRAS G12D protein, mTORC1 downstream targets S6K and 4EBP1, KRAS downstream target ERK, and their phosphorylated forms in KRAS G12D ;C1 WT tumor organoids. ( F ) Regression analysis of KRAS G12D , S6K, 4EBP1, and ERK and their phosphorylated forms versus organoid viability, showing the strongest correlation with p-S6K, followed by p-4EBP1. ( G ) KRAS G12D ;C1 KO tumor organoids were resistant to MRTX1133. Representative pictures of four KRAS G12D ;C1 KO organoids after 1 week of treatment with MRTX1133 or vehicle. Statistical analysis was performed using Student’s t test. ( H ) Comparison of sensitivity to MRTX1133 between organoids derived from KRAS G12D ;C1 WT and KRAS G12D ;C1 KO tumors. * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: Primary antibodies included CASTOR1 (1:50 dilution; Abclonal, catalog no. A20710), p-ERK (1:100 dilution; Cell Signaling Technology, catalog no. 4370, RRID: AB_2315112), p-4EBP1 (1:100 dilution; Cell Signaling Technology, catalog no. 2855, RRID: AB_560835), p-S6K (1:100 dilution; Abclonal, catalog no. AP0502, RRID: AB_2863907), p-S6 (1:100 dilution; Cell Signaling Technology, catalog no. 2211, RRID: AB_331679), p-AKT–S473 (1:100 dilution; Cell Signaling Technology, catalog no. 4060, RRID: AB_2315049), and pCASTOR1 (1:100).

Techniques: Analysis, Organoids, Western Blot, Comparison, Sensitivity, Derivative Assay

Journal: Science Advances

Article Title: CASTOR1 functional inactivation drives KRAS -mutant NSCLC and therapy resistance by a noncanonical mechanism of tumor suppression

doi: 10.1126/sciadv.aeb9836

Figure Lengend Snippet: ( A ) Knockdown of CASTOR1 enhanced cell proliferation of human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines following siRNA knockdown of CASTOR1 . ( B ) Overexpression of CASTOR1 inhibited cell proliferation of human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines following the overexpression of CASTOR1 . ( C ) Knockdown of CASTOR1 enhanced resistance to mTORC1 inhibitor Torin1 in human lung cancer cell lines. Clonogenic assay and immunoblotting of human lung cancer cell lines treated with Torin1 following siRNA knockdown of CASTOR1 . ( D ) Overexpression of CASTOR1 sensitized human lung cancer cell lines to mTORC1 inhibitors Torin1 and rapamycin. Clonogenic assay and immunoblotting of human lung cancer cell lines with mTOR inhibitors Torin1 and rapamycin following the overexpression of CASTOR1 . Rapamycin was used at 20 nM, while Torin1 was used at 0.2 μM.

Article Snippet: Primary antibodies included CASTOR1 (1:50 dilution; Abclonal, catalog no. A20710), p-ERK (1:100 dilution; Cell Signaling Technology, catalog no. 4370, RRID: AB_2315112), p-4EBP1 (1:100 dilution; Cell Signaling Technology, catalog no. 2855, RRID: AB_560835), p-S6K (1:100 dilution; Abclonal, catalog no. AP0502, RRID: AB_2863907), p-S6 (1:100 dilution; Cell Signaling Technology, catalog no. 2211, RRID: AB_331679), p-AKT–S473 (1:100 dilution; Cell Signaling Technology, catalog no. 4060, RRID: AB_2315049), and pCASTOR1 (1:100).

Techniques: Knockdown, Clonogenic Assay, Western Blot, Over Expression