Fig 1: SIRT1 inhibitor treatment is required for the synergistic therapeutic efficacy of EGFR TKIs in the KRASG12C lung orthotopic tumor model.A H358 cells were injected intratracheally into nude mice (1 × 106 cells/mouse). Tumors were allowed to establish for 10 days before the mice were randomized into treatment groups. Survival curves of lung orthotopic tumor-bearing mice. n = 10; *p < 0.05. Two-way ANOVA. The mouse survival curves were generated and visualized using the Kaplan‒Meier method. Therapeutic candidate drugs, including EX527 (10 mg/kg/3 times/week, i.p.) and erlotinib (15 mg/kg/2 times/week, i.p.) were administered as specified. Mice were euthanized at the first indication of morbidity, and the lungs were excised and stained with Bouin’s fixative. B Median survival times (days) and P values were calculated by the log-rank test and Gehan-Breslow-Wilcoxon test, respectively. *p < 0.001. On the basis of Student’s t test. C Tumor weights were measured after tumor excision from the mice. Student’s t test was performed for statistical analysis, and the values are shown as the means ± SEMs; n = 8; *p < 0.05. D The colonies formed in the lungs were counted based on colony size: less than 1 mm, between 1 mm and 2 mm, and more than 2 mm. Student’s t test was performed for statistical analysis, and the values are shown as the means ± SEMs; n = 8; *p < 0.05. E Schematic overview of the chemoresistance mechanism of KRASMut-induced SIRT1 and definition of a rational combination strategy to overcome chemoresistance in KRASMut cancers.
Fig 2: SIRT1 upregulation is mediated by c-Myc downstream of KRAS.A HEK293T cells were transfected with pcDNA and KRASG12C plasmids (2 μg). H460 cells were transfected with siCon and siKRAS (80 nM). The cells were harvested with lysis buffer and subjected to western blotting. B, C KRASMut cells (H358, NCIH23, SKLU-1, SW900, A427, H727), KRASWT cells, and KRASG12C cells (H1299G12C) were transfected with siCon, c-Myc specific siRNA (80 nM), pcDNA, or c-Myc plasmid (2 μg) for 48 h, and the levels of the KRAS downstream effectors c-Myc and SIRT1 were measured. D H358 cells were transfected with KRASG12C and siCon or sic-Myc, and cell extracts were immunoprecipitated with an anti-KRAS antibody and immunoblotted with anti-SIRT1, anti-c-Myc, and anti-KRAS antibodies. E Chromatin immunoprecipitation-qPCR analysis of KRAS, SIRT1, and SIRT2 was performed in H358 cells transfected with siCon or siKRAS (80 nM) for 48 h and then immunoprecipitated using an anti-c-Myc antibody or mouse IgG as a negative control. The relative enrichment was calculated by normalizing the qPCR signals. The data are plotted as the mean values determined from at least two independent chromatin immunoprecipitation assays and three independent amplification reactions. Student’s t test, mean ± SD; n = 6; *p < 0.05. F H358 cells were transfected with siCon and siKRAS (80 nM) for 48 h and then fixed after 4 h. c-Myc expression was detected with an RFP emission filter, and SIRT1 expression was detected with a GFP emission filter.
Fig 3: SIRT1 deacetylates lysine 104 of KRASG12C.A H358 cell extracts were immunoprecipitated with anti-KRAS and anti-SIRT1 antibodies and immunoblotted with anti-KRAS and anti-SIRT1 antibodies. B H358 cells were transfected with pcDNA and SIRT1 (4 μg), siCon, and siSIRT1 (80 nM) and then immunoblotted with anti-acetylated lysine, anti-KRAS-GTP, and anti-KRAS antibodies. C The amino acid sequence of KRASG12C is shown. Lysine acetylation residues are marked as K in bold font. D HEK293T cells were transfected with GFP-E.V., GFP-KRASG12C (with three of the four lysine acetylation residues [K101, K104, K128, and K147] sequentially mutated to arginine), Flag-SBP-E.V., Flag-SBP-SIRT1, siCon, and/or siSIRT1 and incubated for 48 h. Protein lysates were subjected to immunoprecipitation with an anti-GFP antibody and then immunoblotted using anti-acetylated lysine, anti-KRAS, and anti-GFP antibodies.
Fig 4: SIRT1 is aberrantly upregulated in KRASMut NSCLC cell lines and tumors.A SIRT1 protein expression was measured in one noncancerous lung epithelial cell line (BEAS-2B), five KRASMut cell lines (H358, H460, NCIH23, SKLU-1, and SW960), a KRASMut/EGFRMut cell line (H1650), five KRASWT/EGFRWT cell lines (HCC1666, H322M, H522, Calu-3, and H1650), and three EGFRMut cell lines (H1975, HCC827, and HCC2279). The area density of each band was measured with ImageJ software. The data were normalized to actin and are presented as the ratio with respect to the area density of actin. The data are plotted relative to the values obtained in the BEAS-2B cell line with SIRT1 expression. Student’s t test, mean ± SD; n = 3; *p < 0.05. B Representative immunohistochemical staining for SIRT1 and H&E staining in KrasLA2_WT and KrasLA2_G12D Tg mouse lungs at 16 weeks of age. Representative images are shown. Scale bar, 100 μm. The high-magnification images correspond to the areas marked by the black box. C SIRT1 mRNA expression was measured by quantitative real-time PCR in the same cell lines shown in Fig. 1A. RPL32 was used as an internal control and for normalization. Student’s t test, mean ± SD; n = 3; *p < 0.05. D The mRNA expression of Sirt1 was measured by RT‒qPCR in cancerous and adjacent noncancerous lung tissues of KrasLA2_G12D Tg mice. Rpl32 was used as an internal control and for normalization. Student’s t test, mean ± SEM; n = 3; *p < 0.05.
Fig 5: Sirt1 potentiates the anticancer effect of chemotherapy and EGFR TKI treatment against KrasG12D-induced lung tumorigenesis.A Schematic of genetic manipulation of LSL-KrasG12D/+ and/or Sirt1co/co mice before and after adenoviral Cre administration. B PET images of mice between 22 and 23 weeks of age. All images were normalized to the same maximal standard uptake value (SUVmax) to facilitate the comparison of PET lesions. The yellow arrow indicates the tumor region. Cisplatin (7 mg/kg/1 time/week, i.p.) and erlotinib (15 mg/kg/2 times/week, i.p.) were administered beginning 13 weeks after Ad-cre virus injection. C Quantification of the tumor uptake value (SUVmax) and mean standardized uptake value (SUVmax) in the lung. Student’s t test, mean ± SEM; n = 6; *p < 0.05. D Representative H&E-stained lung sections from LSL-KrasG12D/+;Sirt1+/+ and LSL-KrasG12D/+;Sirt1co/co mice. The bars represent 800 μm. E Average tumor number per lung area in specimens collected from LSL-KrasG12D/+;Sirt1+/+ (n = 6) and LSL-KrasG12D/+;Sirt1co/co (n = 6) mice between 24 and 27 weeks. Student’s t test, mean ± SEM; n = 6; *p < 0.05. F Survival rates of LSL-KrasG12D/+;Sirt1+/+ (n = 6) and LSL-KrasG12D/+;Sirt1co/co (n = 6) mice following Cre induction (log-rank test). G Median survival times (days) and P values were calculated using the log-rank test and Gehan-Breslow-Wilcoxon test on the basis of Student’s t test, respectively.
Supplier Page from Abcam for Recombinant human SIRT1 protein (Active) (GST tag N-Terminus)