Journal: Experimental & Molecular Medicine
Article Title: Targeting TGF-β–Smad2/3–JNK1-mediated SIRT1 activity overcomes the chemoresistance of KRAS mutation lung cancer
doi: 10.1038/s12276-025-01536-8
Figure Lengend Snippet: a Normal human bronchial epithelial cell (BEAS-2B) and KRAS Mut cell lines (H358, A427 and H727) were collected with lysis buffer and subjected to western blotting with anti-pERK, ERK and β-actin antibodies. b A luciferase assay was performed to assess the AP-1-mediated transcriptional regulatory activity with cell lysates in Fig. 4a. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. c TGFB1 mRNA expression was measured by RT–qPCR with same cell lines as in a . RPL32 was used as internal control and for normalization. Student’s t -test, mean ± s.d.; n = 6, * P < 0.05. d The medium of four cell lines were changed by FBS-free medium before cell collection at 24 h. Conditioned medium was collected and concentrated using an Amicon Ultra-15 tube, and total TGF-β1 levels were measured by ELISA. e KRAS Mut cell lines (H358, A427 and H727) were transfected with pcDNA , KRAS G12C , G12D and G12V plasmids (2 μg). TGF-β1 levels were measured under the same method as in d . f H358, A427 and H727 cells were transplanted with pcDNA , KRAS G12C , G12D and G12V plasmids, siCon and siSmad2/3 (80 nM) for 48 h, and then the activity of Smad2/3, JNK1 and KRAS was measured. g The cell lysates of each different KRAS Mut cell lines (H358, A427 and H727) under KWN-C with indicated dosage for 24 h were transferred by immunoblotting assay with anti-pSmad2/3, anti-Smad2/3, pJNK1, JNK1, anti-pSIRT1 Ser27 , pSIRT1 Ser47 , SIRT1, KRAS–GTP-bound and β-actin antibodies. h , H358, A427 and H460 cells were treated with DMSO or KWN-C (10 μM), and cell extracts were then immunoprecipitated using immunoglobulin G, anti-KRAS, and RAF-1 agarose bead antibodies. Immunoblotting was performed using anti-acetyl, anti-KRAS–GTP-bound, anti-SIRT1, anti-KRAS and β-actin antibodies.
Article Snippet: KRAS WT (#75282), KRAS G12C (#58901), KRAS G12D (#58902), KRAS G12V (#46746) and JNK1-GFP (#86830) plasmids were purchased from Addgene.
Techniques: Lysis, Western Blot, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation
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![A The indicated HPNE isogenic cell lines were subcutaneously implanted into the flanks of athymic mice and monitored for tumor growth over a 10-week period. Tumor size was measured weekly using a digital caliper. For mice implanted with cells expressing Dox-inducible PR55α, Dox (1 mg/ml) was administered in the drinking water and refreshed every 48 h. As a positive control, CD18/HPAF pancreatic cancer cells were implanted into athymic nude mice and monitored for tumor growth in parallel. The table summarizes the tumor formation rates for each cell line. In a separate cohort, CA3 treatment (1 mg/kg) was initiated 10 days after implantation of HPNE/PR55α + KRAS G12D cells and administered via intraperitoneal injection three times per week for three weeks, as described previously [ 41 ]. Following treatment, mice remained in remission for an additional six weeks. B Representative images of tumor-bearing mice and excised tumor samples at the end of the experiment. C Line graph depicts the number of mice with tumors over time. Statistical significance ( p -value) was assessed using Student’s t-test with SigmaPlot software. D Line graph shows average tumor volume measured weekly for 8 weeks following implantation. E At the study endpoint, tumor xenografts from mice implanted with PK and BK cells were excised and weighed. F Tumor xenograft and adjacent normal tissues were analyzed by IHC analysis for expression of Ki67 (a proliferation biomarker), PR55α, and YAP, as described in Materials and Methods . Histological analysis was also performed using H&E staining. Scale bar = 50 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4529/pmc12934529/pmc12934529__nihms-2139541-f0006.jpg)
![Oncogenic mutations in the KRAS oncogene cause KRAS activation, which is present in 90–95% of PC and serves as the driver of PC. The Raf/MEK/ERK oncogenic pathway is the major downstream effector of KRAS [ 45 ]. PR55α/PP2A was shown to facilitate the activation of the Raf/MEK/ERK signaling pathway by dephosphorylating the inhibitory sites in Raf and KSR1 [ 16 , 17 ]. Raf1 activation can block MST activity in activating LATS1/2. Conversely, the activation of LATS1/2 can suppress Raf1 [ 36 – 38 ]. YAP oncogene activation is required for KRAS-driven PC progression [ 26 – 28 ]. Our studies show that PR55α-associated PP2A promotes YAP activation by inhibiting the LATS/MOB1 auto-activation loop and dephosphorylating YAP, thus stabilizing YAP protein [ 19 ]. Our previous studies also identified a positive feedback regulation of the MST/LATS cascade by PR55α expression [ 19 ]. However, this PR55α-stimulated MST/LATS signaling was abolished by oncogenic KRAS G12D .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4529/pmc12934529/pmc12934529__nihms-2139541-f0008.jpg)