Additional file 1 of Metabolic synthetic lethality by targeting NOP56 and mTOR in KRAS-mutant lung cancer
Creators: name: Yang, Zhang
name: Liang, Shun-Qing
name: Zhao, Liang
name: Yang, Haitang
name: Marti, Thomas M.
name: Hegedüs, Balazs
name: Gao, Yanyun
name: Zheng, Bin
name: Chen, Chun
name: Wang, Wenxiang
name: Dorn, Patrick
name: Kocher, Gregor J.
name: Schmid, Ralph A.
name: Peng, Ren-Wang
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Cancer
subject: Hematology

description: Additional file 1: Figure S1. NOP56 knockdown inhibits proliferation of KRAS-mutant cancer cells. A, Immunoblots of KRAS-mutant and KRAS-wild type cancer cells that were transfected with NOP56-specific siRNAs (si-NOP56) or scramble control siRNAs (si-Control). B, KRAS mutant and KRAS wild type cancer cells were transfected with control siRNAs or NOP56-specific siRNAs. Cell viability was determined 72 h post transfection. Data are presented as mean ± SD (n = 3). *p &lt; 0.05, **p &lt; 0.01, ***P &lt; 0.001, ****P &lt; 0.0001 and ns P&gt;0.05 by two-way ANOVA with Tukey’s multiple comparisons test. C, NOP56 is not a biomarker of survival in patients with KRAS-wild-type lung adenocarcinoma (LC), pancreatic cancer (PC) and colon cancer (CC). Kaplan–Meier survival analyses of patient cohorts in TCGA were stratified by the optimal cut-off value of the mRNA level of NOP56. D, Gene set enrichment analysis (GSEA) of a TCGA cohort of patients with KRAS-mutant lung (n = 141), pancreatic (n = 133) and colon cancer (n = 170). Figure S2. Stable expression of NOP56-specific shRNAs activates IRE1α-mediated UPR. A, Immunoblots of H358 and H460 cells expressing scrambled control or NOP56 shRNAs. B, Immunofluorescence of H358 and H460 cells that express scrambled control or NOP56 shRNAs. The NOP56 signal is indicated by arrowheads. C, The cell viability curve of H358 and H460 cells expressing scramble control shRNA or the NOP56-targeted shRNAs was measured at the indicated time points. D, Clongenic assay of H358 and H460 cells expressing scramble control or NOP56-targeted shRNAs. Quantification of clongenic assay were shown underneath. Data are presented as mean ± SD (n = 3). E, Growth inhibition of H358 and H460 cells expressing control shRNA or NOP56-targeted shRNA (3000 cells/well) treated for 72 h with the indicated doses of an IRE1α inhibitor (4μ8C). Data are presented as mean ± SD (n = 3). F, Apoptosis assay of H460 cells expressing scrambled control or NOP56-targeted shRNAs after transfection with IRE1α-specific or control siRNAs for 72 h. Data are presented as mean ± SD (n = 3). ***P &lt; 0.001 and ns P&gt;0.05 by two-way ANOVA with Tukey’s multiple comparisons test. Figure S3. NOP56 KD renders KRAS-mutant lung cancer cells susceptible to mTOR inhibition. A, Bar graphs illustrating the change of sensitivity to different inhibitors in H460 cells after NOP56 knockdown. Data are presented as IC50 values of the indicated inhibitors in H460 cells expressing scramble control shRNAs compared to IC50 in H460 cells expressing NOP56-targeted shRNAs. Data are shown as mean (n = 2). B, Viability assay of H460 and H358 cells expressing control shRNA or NOP56-targeted shRNA (3000 cells/well) after treated for 72 h with the indicated doses of PI3K inhibitor (LY294002) and AKT inhibitor (AZD5363). Data are presented as mean ± SD (n = 3). C, Viability assay of H460 and H358 cells expressing control shRNA or NOP56-targeted shRNA (3000 cells/well) after treated for 72 h with the indicated doses of BiP inhibitor (HA15) and ER stress inducer (bortezomib). Data are presented as mean ± SD (n = 3). D, Immunoblots of KRAS-mutant (H358, H460) and wild-type (H1703, H520) cells expressing NOP56-specific sgRNAs. E, Viability assay of the cells expressing control or NOP56-specific sgRNAs after treated with rapamycin for 72 h. Data are shown as mean ± SD (n = 3). F, NOP56 is negatively correlated with PI3K/AKT/mTOR pathway genes (PI3KCA, PDPK1, PIK3R1) in KRAS-mutant lung cancer patients. Pearson and Spearman coefficient and significance (p-value) are analyzed using R software (Cor.test function). G, Immunoblots of H460 and H1703 cells expressing control or NOP56-targeted shRNAs. H, I, Viability assay of KRAS-mutant (H) and wildtype (I) cancer cells expressing control or NOP56-specific siRNAs after treated with rapamycin. The assay was performed 72 h after drug treatment (96 h after siRNA transfection). Figure S4. NOP56 KD activates and induces dependence on the mTOR pathway in KRAS-mutant cancer cells. A, Immunoblots of H358 cells expressing control or NOP56-targeted shRNAs after treated with the AKT inhibitor (AZD5363) for 24 h. B, Clongenic assay of H358 cells expressing control or NOP56-specific shRNAs after treated with indicated doses of AZD5363. Representative images are shown. C, D, Immunoblots (C) and viability assay (D) of H358 cells expressing control or NOP56-specific shRNAs after transfected with raptor- or rictor-specific or control siRNAs for 72 h. Data are presented as mean ± SD (n = 3). E, Clongenic assay of H358 and H460 cells expressing control shRNA or NOP56-specific shRNAs after treatment with indicated doses of eIF4E inhibitor (Briciclib). Representative images are shown. F, Immunoblots of H460 cells expressing control or NOP56-target shRNAs after treated with rapamycin (1 μM) for 24 h. Figure S5. In vivo activity and selectivity of co-targeting NOP56 and mTOR in KRAS-mutant lung cancer. A, H&amp;E and IHC analysis of p-AKT(T308), p-mTOR(S2448),p-S6(S235/236), Ki67 and Caspase-3) in residual H460 xenograft tumors after the indicated treatment. Scale bars 100 μm. B, Tumor volume of H1703 xenografts in immunocompromised (NSG) mice. H1703 cells were transduced with either a control or an shRNA against NOP56 (shNOP56a). Tumors were measured every 5 days with a caliper. C, Kaplan-Meier survival curve of mice harboring H1703 xenografts from the experiment shown in B.