Journal: Nature Communications

Article Title: Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

doi: 10.1038/s41467-024-54476-8

Figure Lengend Snippet: a Cell growth inhibition by shRNA-mediated depletion of NDRG3 expression in pancreatic and lung cancer cell lines expressing wild-type (KRAS-WT) or mutant (KRAS-Mutant) KRAS. b Quantification of the inhibition of anchorage-independent cell growth by shRNA-mediated depletion of NDRG3 expression in KRAS-WT or KRAS-Mutant pancreatic and lung cancer cell lines. (n = 4 biological replicates/cell line). c Differential inhibition of AKT and ERK1/2 activities by NDRG3 depletion in KRAS-WT and KRAS-Mutant cells. (n = 3 biological replicates/cell line). d Effects of NDRG3 depletion on the tumorous growth of KRAS-WT or KRAS-Mutant cells in nude mice (n = 5 mice/cell line). e A heatmap showing the genomic activity (Z-score) of upstream regulators for NDRG3-dependent DEGs in different pancreatic cancer cell lines. Ras-related signaling pathways are denoted in red, while the pathways not significantly enriched in IPA are shown in dark gray (left). A differential genomic activity profile of RAS-related signaling pathways between KRAS-WT and KRAS-Mutant cells is shown (right, n = 7 NDRG3-dependent RAS-related pathways). The genomic activity of an upstream pathway was estimated by IPA analysis of NDRG3-dependent DEGs (≥|1.5| fold change) for each pathway as identified from RNA-Seq. f Upstream regulatory pathways whose NDRG3-dependent genomic activity is highly correlated with cell growth inhibition caused by NDRG3 depletion. g Analysis of NDRG3-dependent changes in AKT and ERK1/2 activities induced by ectopic expression of KRAS G12D in BxPC-3 cells. h NDRG3 dependence of the tumorous growth of KRAS G12D -expressing BxPC-3 cells in nude mice (n = 8 mice/group). i Histogram of Ki-67-positive cells and p-ERK1/2-positive areas in KRAS G12D -expressing BxPC-3 tumor tissues (n = 12 areas/group). Data are presented as median (center), with the box plots extending from the 25th to 75th percentiles and the whiskers (the minimum and maximum values) ( a ), median and quartile ( b ), or mean ± SEM ( c , d , h ) or ±SD ( e , i ). Statistical analysis was done with two-sided Student’s t-Test ( a – d ), one-way ANOVA ( e , I ), or two-way ANOVA ( h ), followed by Dunnett’s multiple comparison test. Source data are provided as a Source Data file.

Article Snippet: Human lung cancer cell lines (NCI-H520, NCI-H1975, PANC-1, HCC827, A549, NCI-H2009), human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, AsPC-1) and HEK293T cell line were purchased from American Type Culture Collection (ATCC).

Techniques: Inhibition, shRNA, Expressing, Mutagenesis, Activity Assay, Protein-Protein interactions, RNA Sequencing, Comparison

Journal: Nature Communications

Article Title: Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

doi: 10.1038/s41467-024-54476-8

Figure Lengend Snippet: a Correlation analysis between the expression levels of KRAS and NDRG3 proteins in human pancreatic cancer patient tissues. Representative immunohistochemical images are shown (left). Scale bars, 200 μm and 100 μm in 40X and 200X images, respectively. H-scores of each immunohistochemical image were quantitatively measured using IHC profiler of ImageJ program, and average values of two different tissue sections of one human sample were subject to the correlation analysis (right; n = 39). Linear regression line of best fit with 95% confidence intervals is shown. b Correlation between the basal expression levels of KRAS and NDRG3 proteins in different pancreatic cancer cell lines. (n = 3 biological replicates/cell line). c KRAS expression changes in Capan-1 cells after depletion of NDRG3 expression with shRNAs targeting the CDS or 3’-UTR of the NDRG3 gene. d KRAS expression changes after CRISPR/Cas9-mediated disruption of NDRG3 gene in different cancer cell lines. e KRAS expression and downstream signaling activities in NDRG3-depleted and -reconstituted cells. f Expression of ectopically provided KRAS G12D in HEK293T cells where a fixed amount of FLAG-KRAS G12D was co-expressed with an increasing amount of NDRG3. g Kinetics of endogenous KRAS protein degradation dependent on NDRG3 expression status. KRAS protein levels were time-dependently analyzed by western blotting in MIA PaCa-2 cells after cycloheximide (CHX) treatment. (n = 3 biological replicates). h Recovery of KRAS protein level by MG-132 treatment in NDRG3-depleted cells. i Ubiquitin binding to ectopic Flag-KRAS G12D protein dependent on NDRG3 expression status. j Kras protein expression in pancreatic tissues of mice with β-cell-specific knock-out ( Ndrg3 f/f ) of Ndrg3 gene. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ) Data are presented as mean ± SEM ( b , g ). Statistical analysis was done with a two-sided Pearson correlation coefficient test ( a , b ) or two-sided Student’s t-Test ( g ). Source data are provided as a Source Data file.

Article Snippet: Human lung cancer cell lines (NCI-H520, NCI-H1975, PANC-1, HCC827, A549, NCI-H2009), human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, AsPC-1) and HEK293T cell line were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Immunohistochemical staining, CRISPR, Disruption, Western Blot, Ubiquitin Proteomics, Binding Assay, Knock-Out

Journal: Nature Communications

Article Title: Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

doi: 10.1038/s41467-024-54476-8

Figure Lengend Snippet: a Schematic representation of gene targeting constructs used to generate pancreas-specific conditional Kras G12D knock-in or/and Ndrg3 knock-out mouse models of PDAC development. b Scheme of experimental schedule for the tamoxifen-dependent activation of Cre-recombinase system followed by caerulein-mediated acute pancreatitis induction. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ). c Expression of Kras, Ndrg3 and PanIN markers in pancreatic tissues of mice with pancreas-specific Kras G12D knock-in (KC mice) or/and Ndrg3 knock-out (KNC mice). d Immunofluorescence staining to assess acinar-ductal metaplasia (ADM) lesions in pancreatic tissues of KC and KNC mice. Scale bars, 100 μm. e Quantitative assessment of ADM lesions in pancreatic tissues of KC and KNC mice based on measurement of CK19-positive and amylase-low area (n = 6 mice/group). f H&E and Alcian blue staining of pancreatic tissues from KC and KNC mice. Representative optical microscopic images are shown. Scale bars, 200 μm and 100 μm in 40X and 200X images, respectively. g Quantitative assessment of PanIN-like ductal lesions in pancreatic tissues of KC and KNC mice (n = 5, 8, and 6 for control, KC, and KNC mice, respectively). Data are presented as mean ± SD ( e, g ). Statistical analysis was done with one-way ANOVA followed by Dunnett’s multiple comparison test ( e , g ). Source data are provided as a Source Data file.

Article Snippet: Human lung cancer cell lines (NCI-H520, NCI-H1975, PANC-1, HCC827, A549, NCI-H2009), human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, AsPC-1) and HEK293T cell line were purchased from American Type Culture Collection (ATCC).

Techniques: Construct, Knock-In, Knock-Out, Activation Assay, Expressing, Immunofluorescence, Staining, Control, Comparison

Journal: Nature Communications

Article Title: Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

doi: 10.1038/s41467-024-54476-8

Figure Lengend Snippet: a Study scheme for searching NDRG3-binding proteins related to protein stability regulation. Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license ( https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en ). b Physical interaction between NDRG3 and endogenous USP9X in HEK293T cells overexpressing Flag-NDRG3. c Protein and mRNA expression of KRAS after siRNA-mediated depletion of USP9X expression in KRAS-Mutant cells. d Effects of a deubiquitinase inhibitor, WP1130 on KRAS protein expression (top) in and cell viability (bottom) of pancreatic and lung cancer cell lines (n = 3 biological replicates). e Ubiquitination of KRAS G12D protein after siRNA-mediated depletion or pharmacologic inhibition of USP9X. f Physical interaction between KRAS and endogenous USP9X in HEK293T cells overexpressing Flag- KRAS G12D . g NDRG3 dependence of the KRAS-USP9X interaction in HEK293T cells. h PLA analysis to confirm the dependence of the interaction between USP9X and KRAS on NDRG3 (left). Quantitated PLA signal (right, n = 15 nuclei/group). Scale bar = 20 μm. i Requirement for NDRG3 in USP9X-mediated up-regulation of KRAS G12D protein expression. Representative western blot result showing the NDRG3 dependence of KRAS G12D protein up-regulation by USP9X (top). Values in western blots were quantitatively measured and shown as a histogram (bottom) (n = 3 biological replicates). j Requirement for NDRG3 in USP9X-mediated KRAS deubiquitination. Data are presented as mean ± SD ( d ), ±SEM ( I ), or median (center), with the box plots extending from the 25th to 75th percentiles and the whiskers (the minimum and maximum values) ( h ). Statistical analysis was done with a one-way ANOVA test followed by Dunnett’s multiple comparison test ( d , h ) or parametric unpaired two-sided Student’s t-Test ( i ). n.s . = not significant. Source data are provided as a Source Data file.

Article Snippet: Human lung cancer cell lines (NCI-H520, NCI-H1975, PANC-1, HCC827, A549, NCI-H2009), human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, AsPC-1) and HEK293T cell line were purchased from American Type Culture Collection (ATCC).

Techniques: Binding Assay, Expressing, Mutagenesis, Ubiquitin Proteomics, Inhibition, Western Blot, Comparison

Journal: Nature Communications

Article Title: Anti-proteolytic regulation of KRAS by USP9X/NDRG3 in KRAS-driven cancer development

doi: 10.1038/s41467-024-54476-8

Figure Lengend Snippet: a Evaluation of C-terminal subdomains of NDRG3 protein for their effects on KRAS G12D protein expression. Positions of Myc-tagged NDRG3 fragments are schematically represented (top). KRAS expression levels in western blots (middle) were quantitatively measured (bottom) (n = 3 biological replicates). b Dominant-negative effect of the 173-345 aa form of NDRG3 on endogenous KRAS protein expression in KRAS-Mutant cells. c Dose-dependent suppression of KRAS G12D protein expression by NDRG3 173-345 aa form. d Dominant-negative effects of NDRG3 173-345 aa form on KRAS-NDRG3 and KRAS-USP9X interactions. Immunoprecipitated NDRG3 and USP9X proteins were quantitatively assessed (n = 4 biological replicates). e Enhancement of KRAS G12D ubiquitination by NDRG3 173-345 aa form. f Interaction between KRAS G12D and C-terminal fragments of NDRG3. g Interaction between C-terminal subdomains of NDRG3 and USP9X. h Verification of the specific domain of NDRG3 for interaction with KRAS or USP9X. i Effects of lentivirally expressed full-length or C-terminal fragments of NDRG3 on endogenous KRAS protein expression and downstream signaling in KRAS-Mutant and KRAS-WT pancreatic cancer cells. j Effect of lentivirally expressed full-length or C-terminal fragments of NDRG3 on the growth of KRAS-Mutant and KRAS-WT pancreatic cancer cells (n = 3 biological replicates). Data are represented as mean ± SEM ( a , d , j ). Statistical analysis was done with a one-way ANOVA test ( a ) or two-way ANOVA ( d , j ) followed by Dunnett’s multiple comparison test. n.s . = not significant. Source data are provided as a Source Data file.

Article Snippet: Human lung cancer cell lines (NCI-H520, NCI-H1975, PANC-1, HCC827, A549, NCI-H2009), human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, AsPC-1) and HEK293T cell line were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Western Blot, Dominant Negative Mutation, Mutagenesis, Immunoprecipitation, Ubiquitin Proteomics, Comparison

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: Antitumor activity of BiTP combinatorial chemotherapy in orthotopic PDAC models. (A) Treatment plan and schedules. (B) Schematic diagram of the KPC model. The approximate time frame of mouse tumor progression to borderline resectable (BR), locally advanced (LA), and terminal (TERM) are days 7, 14, and 21, respectively. (C) Representative tumor images of orthotopic PDAC mice receiving different therapies. (D) Tumor weights and volumes on day 30. (E) The table included the survival rate (%), tumor proliferation (T/C, %), tumor growth inhibition (TGI, %), and resectability status of orthotopic PDAC mice receiving different therapies on day 30, and the median survival time (MST) in survival assays. (F) Kaplan–Meier plot survival curve and pairwise comparison results of orthotopic PDAC mice receiving different therapies in survival assays. Data presented in the graphs represent mean±standard deviation (SD) (**** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns P >0.05). BiTP, anti-PD-L1/TGF-β antibody; CTH, chemotherapy; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death 1 ligand 1.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: Activity Assay, Inhibition, Comparison, Standard Deviation

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: Decisions about resectability status in PDAC murine models.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: Membrane

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: Preclinical assessment of neoadjuvant BiTP combinatorial chemotherapy for the treatment of PDAC. (A) Treatment plan and schematic diagram of treatment schedules. (B) Schematic diagram of distal pancreatectomy in orthotopic PDAC mice model. (C) Representative images of tumors. (D) Tumor weights and volumes of direct surgery (day 7) and post-neoadjuvant therapy resections (day 24). (E) Table of resectability status (day 24), operative mortality (OM), and median survival time (MST) of orthotopic PDAC mice receiving different therapies. (F–H) Kaplan–Meier plot survival curve and pairwise comparison results of orthotopic PDAC mice receiving different therapies in survival assays. Data presented in the graphs represent mean±standard deviation (SD). (**** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns P >0.05). AC, adjuvant chemotherapy; ACI, adjuvant chemo-immunotherapy; BiTP, anti-PD-L1/TGF-β antibody; CTH, chemotherapy; NAC, neo-adjuvant chemotherapy; NACI, neo-adjuvant chemo-immunotherapy; NAT, neo-adjuvant therapy; SRG, surgery.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: Comparison, Standard Deviation, Adjuvant

Journal: International Journal of Surgery (London, England)

Article Title: Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study

doi: 10.1097/JS9.0000000000001226

Figure Lengend Snippet: BiTP reverses TGF-β-induced EMT (epithelial–mesenchymal transition) of PDAC (pancreatic ductal adenocarcinoma) cells. (A) CCK-8 (Cell Counting Kit-8) assay to measure the effect of BiTP on TGF-β-mediated chemoresistance in PDAC cells. After 10 ng/ml TGF-β1, 10 5 pM BiTP or hIgG treatment for 24 h. HPDE6-C7, CFPAC-1, ASPC-1, and KPC cells growing in 96-well plates were exposed to serial dilutions of gemcitabine and nab-paclitaxel for 72 h and CCK-8 assays was performed. (B) The representative images of IHC (immunohistochemistry) staining of Ki67 in orthotopic PDAC mice model and statistical graph of the percentage of Ki67-positive cells. (C) The representative images of TUNEL staining for apoptosis (green) and nuclei (DAPI, blue) in orthotopic PDAC mice model and statistical graph of the percentage of apoptosis cells. (D) Transwell assays to measure the effect of BiTP on TGF-β-mediated migration and in PDAC cells. After 10 ng/ml TGF-β1, 10 5 pM BiTP or hIgG treatment for 96 h. 1×10 4 CFPAC-1 and KPC cells were seeded in the upper chambers and Transwell assays were performed. (E) Western blotting assays exploring the chemotherapy-induced EMT and the blocking effect of BiTP. (F) The representative images of IHC staining of E-cadherin in orthotopic PDAC mice model. Statistical graph of the AOD (average optical density) of E-cadherin. (G) The representative images of IF staining of Vimentin (green) and α-SMA (rose red) in orthotopic PDAC mice model. Statistical graph of the arbitrary units of Vimentin and α-SMA. (H) The representative images of Masson staining in orthotopic PDAC mice model. Statistical graph of the percentage of collagen volume fraction (%). Data presented in the graphs represent mean±standard deviation (SD). (**** P <0.0001; *** P <0.001; ** P <0.01; * P <0.05; ns P >0.05). BiTP, anti-PD-L1/TGF-β antibody; CTH, chemotherapy; KPC, LSL-Kras(+/G12D);LSL-Trp53(+/R172H);Pdx1-Cre; PDAC, pancreatic ductal adenocarcinoma; PD-L1, programmed cell death 1 ligand 1; TGF-β, transforming growth factor-β.

Article Snippet: Human PDAC cell lines ASPC-1 (with Kras G12D mutation) and CFPAC-1 (with Kras G12V mutation) and human pancreatic ductal epithelial cell line HPDE6-C7 were obtained from China Center for the Conservation of Typical Cultures (CCTCC).

Techniques: CCK-8 Assay, Cell Counting, Immunohistochemistry, Staining, TUNEL Assay, Migration, Western Blot, Blocking Assay, Standard Deviation