PMC

SHCBP1 expression is associated with β-catenin signaling activity and CSCs properties in NSCLC. a WB analyses exhibit the nuclear SHCBP1 protein levels in eight NSCLC tumor specimens (T) and their paired adjacent non-cancerous lung tissue (N). b Kaplan–Meier analysis of overall survival of 207 NSCLC patients which were grouped according to nuclear SHCBP1 expression assessed by immunohistochemistry analysis. c IHC was carried out in paraffin-embedded sections derived from 50 NSCLC patients. Correlations between nuclear SHCBP1 and Axin2, CCDN1 and survivin were analyzed by chi-squared test. d GSEA analysis to determine the correlation between SHCBP1 expression and β-catenin signaling activation. e GSEA analysis in TCGA NSCLC patients. Correlation between SHCBP1 and stemness-associated signature was identified and listed

SHCBP1-mediated cellular stemness promotes malignant phenotype of NSCLC in vivo. a A549-luc-Vector, A549-luc-EGF, and A549-luc-EGF-sh-SHCBP1 cells at indicated numbers were implanted into the inguinal folds of nude mice. Representative bioluminescent images of subcutaneous tumors grown by indicated cells after 6 weeks are shown. b Tumor-free survival time was analyzed in mice with 5 × 10⁴ tumor cells implanted using in vivo bioluminescent imaging systems. c A549-luc-Vector and A549-luc-SHCBP1 cells at indicated numbers were implanted into the inguinal folds of nude mice. Representative bioluminescent images of subcutaneous tumors grown by indicated cells at indicated time points are shown. d Subcutaneous tumors at indicated time points were dissected and photographed. e Effects of ICG-001 on NSCLC cells with EGF or SHCBP1-ectopic expression was analyzed by subcutaneous tumorigenesis assay. ICG-001 (50 mg/kg) or vehicle (PBS) was intraperitoneally injected every 3 days for 3 weeks. f Tumor-free survival time was analyzed in mice with 5 × 10⁴ tumor cells implanted according to in vivo bioluminescent imaging systems

SHCBP1 mediates EGF-induced stem cell-like properties of NSCLC cells in vitro. a Representative images of tumor cell spheres formed by cultured cells with or without SHCBP1 silenced in the presence or absence of EGF. b qRT-PCR performed to assess the effects of SHCBP1 RNA interference on EGF-enhanced expression of stemness-related genes. *P < 0.05. c Flow cytometry determines the fraction of CD44/EpCAM double-positive cells upon EGF stimulation with or without SHCBP1 deletion. d Flow cytometry determines the effects of SHCBP1 depletion on SP fractions in cultured NSCLC cells treated with EGF for 48 h. e IC₅₀ assay was carried out to assess the drug cytotoxic of cisplatin on NSCLC cell lines when SHCBP1 was silenced in the presence of EGF. *P < 0.05 vs. EGF + Vector. f Dual-luciferase reporter assay to assess the effects of SHCBP1 deletion on the TOP/FOP activity in HCC827 and HCC4006 cells. g Effects of ICG-001 on EGF-mediated increase of tumor sphere formation were analyzed. h IC₅₀ assay was carried out to assess the cytotoxicity of cisplatin when ICG-001 was used in the presence of EGF. *P < 0.05 vs. EGF + Vehicle

Upregulation of SHCBP1 expression in human cancers. a qRT-PCR and WB analyses determining the SHCBP1 mRNA (left panel) and protein (right panel) levels in eight NSCLC tumor (T) and their paired adjacent non-cancerous lung tissue specimens (N). b Next-generation sequencing data from The Cancer Genome Atlas (TCGA) dataset show SHCBP1 mRNA expression in 90 NSCLC tumor specimens compared to their paired adjacent non-cancerous lung tissue. c qRT-PCR and WB analyses to assess SHCBP1 mRNA (left panel) and protein (right panel) in indicated NSCLC cell lines as compared with benign primary lung epithelial cells (NLE). d Representative images of immunohistochemistry (IHC) from NSCLC patients show the protein expression level of SHCBP1 in all clinical stages of NSCLC in comparison with normal lung tissue. Scale bar: 50μm. e Kaplan–Meier analysis of overall survival of a cohort of 207 NSCLC patients, divided into high (>median) and low (≤median) SHCBP1 expression groups by immunohistochemistry analysis. f Kaplan–Meier analysis of overall survival of a cohort of 1926 NSCLC patients (data from online KMPLOT). g Kaplan–Meier analysis of recurrence-free survival in patients with lung cancer (GSE8894). h Kaplan–Meier analysis of overall survival of a cohort of 360 NSCLC patients (data from TCGA), divided into three groups according to EGFR mutation status and SHCBP1 expression

SHCBP1 translocates to the nucleus in response to EGF stimulation. a Following immunoprecipitation against SHC1, western blotting analysis reveals interaction between SHCBP1 and SHC1 in response to EGF treatment in the absence or presence of EGFR depletion. 293T and A549 cells were incubated with EGF (100 ng/ml) for 30 min. b WB analysis showing SHCBP1 redistribution in the cytoplasm and nucleus in response to EGF treatment. c, d WB examining nuclear SHCBP1 after EGF treatment at indicated concentrations (c) and time points (d) in 293T and A549 nuclear extracts. e Total SHCBP1 in whole-cell lysates were detected at indicated time points upon EGF treatment. f SHCBP1 expression in NSCLC patients with or without EGFR mutation was analyzed (data from TCGA). g WB results showing nuclear translocation of SHCBP1 stimulated by EGF in HepG2 liver cancer, MCF-7 breast cancer, and KYSE410 esophageal carcinoma cell lines. h Effects of SHC1 depletion on relocation of SHCBP1 to the nucleus. i WB analysis determining the impact of Ras/Erk inhibitor (U0126, 10 µM) or PI3K/Akt inhibitor (LY294002, 20 µM) on nuclear translocation of SHCBP1 in 293T cells. j SHCBP1 nuclear translocation was analyzed by WB assay in the absence or presence of EGFR tyrosine kinase inhibitor gefitinib in two NSCLC cell lines with EGFR activating mutations, HCC827 and HCC4006

Nuclear SHCBP1 facilitates the binding between β-catenin and CBP and is required for EGF-induced β-catenin transactivation. a Immunoprecipitation assays showing interaction between β-catenin and SHCBP1 in the nucleus upon EGF stimulation. b Immunoprecipitation assays detect the effect of gefitinib on interaction between nuclear SHCBP1 and β-catenin in HCC827 and HCC4006 cells. c 293T cells were transfected with SHCBP1 siRNA, followed by EGF (100 ng/ml) treatment for 30 min. Nuclear extracts were analyzed by WB. d Immunoprecipitation assay detect the tyrosine phosphorylation and lysine acetylation of nuclear β-catenin in 293T and A549 cells treated with EGF (left panel). The strip gray level was analyzed subsequently (right panel). *P < 0.05. e Interaction between CBP and β-catenin upon EGF treatment was determined by immunoprecipitation assays. f Immunoprecipitation assay performed to examine the effects of SHCBP1 depletion on binding between CBP and β-catenin upon EGF treatment. g Immunoprecipitation assay performed to examine the effects of SHCBP1 depletion on lysine acetylation of β-catenin upon EGF treatment. h Modified peptide pull-down assays demonstrate interaction between SHCBP1 and CBP in 293T extraction using Flag-SHCBP1-peptide, visualized by Coomassie staining (left panel), incubated with a 293T extraction and followed by immunoblotting analysis with an anti-Flag antibody (right panel). i 293T cell lysates with CBP-Flag ectopic expression were immunoprecipitated by anti-Flag affinity gel. The products were incubated with purified β-catenin-GST protein (100 ng) and SHCBP1-HA protein at indicated concentrations for 2 h, washed for six times, and immunoblotted for Flag, GST, and HA. j Effects of ICG-001 on EGF-induced CBP/β-catenin interaction and lysine acetylation of β-catenin were analyzed using immunoprecipitation. k TOP/FOP dual-luciferase reporter assay was performed to analyze the effect of ICG-001 on β-catenin signaling activation driven by EGF. *P < 0.05 vs. EGF + Vehicle

SHCBP1 mediates EGF activation of β-catenin signaling in NSCLC. a Western blotting assay of the nuclear and cytoplasmic extraction of 293T and A549 cells following EGF stimulation. b Relative luciferase activities of TOP/FOP dual-luciferase reporter determined to assess the effects of EGF (100 ng/ml) in both cell lines. c qRT-PCR performed to test the effects of EGF stimulation on β-catenin downstream genes in 293T and A549 cells. d Immunoprecipitation of FLAG- β-catenin in 293T cell lysates following EGF stimulation, and MS peptide sequencing identified SHCBP1 in the precipitate. e Effects of silencing SHCBP1 on cells treated with EGF, measured using TOP/FOP dual-luciferase reporter assay. f qRT-PCR performed to analyze the alteration of β-catenin downstream genes expression upon EGF stimulation without or with SHCBP1 depleted in A549 cells. g Immunoprecipitation assays to assess the interaction between β-catenin and SHCBP1 following EGF stimulation in 293T cells and A549 lung cancer cells. h Modified peptide pull-down assays were performed in 293T extraction using FLAG-β-catenin peptide, visualized by Coomassie staining (left panel), and incubated with SHCBP1-HA affinity agarose beads for 4 h at 4 °C. Beads containing affinity-bound proteins were washed followed by immunoblotting analysis with an anti-FLAG antibody (right panel). i Immunoprecipitation assays to assess the interaction between SHCBP1 and β-catenin in response to EGF stimulation. j Immunoprecipitation assay show that the N terminal amino-acid sequence (1–428) of SHCBP1 specifically interacts with β-catenin. For b, c, *P < 0.05 vs. Vehicle; for e, f, *P < 0.05 vs. EGF + NC

SHCBP1 upregulation per se facilitates nuclear translocation, β-catenin transactivation, and development of stem cell-like phenotype of cancer cells in vitro. a WB analysis showing SHCBP1 and β-catenin redistribution in the cytoplasm and the nucleus in response to ectopic SHCBP1 expression. b Relative luciferase activities of TOP/FOP dual-luciferase reporter assays to determine to the effects of SHCBP1 ectopic expression. *P < 0.05 vs. Vector. c Representative images of cultured tumor spheres in cells with SHCBP1 depletion or overexpression. d IC₅₀ assays determine the effects of SHCBP1 overexpression on cisplatin cytotoxicity in NSCLC cells. *P < 0.05 vs. Vector. e qRT-PCR quantifying Survivin expression in cells with SHCBP1 ectopically expressed. *P < 0.05. f TOP/FOP dual-luciferase reporter assay was performed to analyze the effect of ectopic SHCBP1 expression on β-catenin signaling activity in cells treated with gefitinib. *P < 0.05. g Level of nuclear SHCBP1 was determined in NSCLC spheres and non-sphere cells by WB. h CD44⁺/EpCAM⁺ cells sorted from both cultured NSCLC cell lines by flow cytometry and the expression of nuclear SHCBP1 was determined by WB compared to the corresponding parental cell lines. i Immunoprecipitation assays to determine the effects of ICG-001 on SHCBP1-mediated CBP/β-catenin interaction. j TOP/FOP dual-luciferase reporter assay was performed to analyze the effect of ICG-001 on activation of β-catenin signaling driven by ectopic SHCBP1 expression. *P < 0.05 vs. SHCBP1 + Vehicle. k Representative images of tumor cell spheres cultured with or without ICG-001 treatment when the SHCBP1 was over-expressed. l IC₅₀ assay was carried out to assess cytotoxicity of cisplatin on SHCBP1 over-expressed NSCLC cell lines in the presence of ICG-001. P < 0.05 vs. SHCBP1 + Vehicle