Journal: Oncotarget

Article Title: Fendiline inhibits proliferation and invasion of pancreatic cancer cells by interfering with ADAM10 activation and β-catenin signaling.

doi: 10.18632/oncotarget.5933

Figure Lengend Snippet: Figure 8: PDAC tumor tissue array show enhanced expression of ADAM10: A.-C. Tumor tissue array containing normal, islet cell tumor and Grade I, II and III PDAC tissue samples were immunostained using an ADAM10 antibody A. and intensity of the stained sections were measured by Dr. Coppola, Senior Pathologist at Moffitt Cancer Center. The stain was semiquantitatively scored based on the intensity of the stain as negative (0), weak (1), moderate (2) and strong (3). In all cases at least 34% of the tumor was positive, which is shown in B. The bargraph in C. shows that ADAM10 levels are increased in tumor tissues, with Grade 1 tumors showing a significant increase. D.-J. Expression of vimentin, c-Myc and ADAM10 are significantly increased in PDAC: PDAC tissue samples and samples from normal pancreas were analyzed by western blot using vimentin (D. and H.), c-Myc (E. and I.) and ADAM10 (F. and J.) antibodies and blots were reprobed with GAPDH antibody for normalization of proteins. K. Graph plotted using the data derived from TCGA portal show that PDAC human samples show increased alterations, especially amplification and/or mutation, in ADAM10, β-catenin (CTNNB1), cyclin D1 (CCND1), CD44, Myc (MYC) and vimentin (VIM). L. Proposed signaling mechanisms by which calcium dysregulation enhances ADAM10-mediated tumor progression: Based on our data with fendiline we hypothesize that calcium influx induces ADAM10 activation, leading to enhanced cadherin cleavage, release of β-catenin, its nuclear translocation and activation of TCF/LEF containing promoters. This enhances expression of genes associated with proliferation, epithelial mesenchymal transition and metastasis of cancers such as c-Myc, cyclin D1 and CD44. Additionally, β-catenin/TCF signaling has been shown to enhance ADAM10 expression thereby playing a feed-forward role in ADAM10-mediated downstream signaling and promotion of oncogenic cycle. In addition to this indirect activation of β-catenin-TCF signaling, ADAM10-mediated cleavage of substrates such as cadherins and CD44 allow detachment of cell-cell and cell-substratum adhesions, migration and invasion of cancer cells. Our data indicate that inhibitors of calcium channels prevent ADAM10- dependent signaling and expression of c-Myc, cyclin D1 and CD44, by stabilizing cadherin-catenin interaction at the cell membrane, enhancing adherens junction formation, subsequently reducing p-catenin-TCF/LEF signaling and target gene expression.

Article Snippet: Immunohistochemical analysis of human PDAC tissue tumor microarray The PDAC tissue array was purchased from amsbio (Cambridge, MA).

Techniques: Expressing, Staining, Western Blot, Derivative Assay, Amplification, Mutagenesis, Activation Assay, Translocation Assay, Migration, Membrane, Targeted Gene Expression

Journal: Scientific Reports

Article Title: Role of stromal activin A in human pancreatic cancer and metastasis in mice

doi: 10.1038/s41598-021-87213-y

Figure Lengend Snippet: Activin A levels are significantly higher in tumor tissue and correlate to worse prognosis in PDAC. ( A ) Representative cores of pancreatic tumors from a human pancreatic tissue microarray stained with H&E to assess the percentage of epithelial versus stromal cells in each core. Adjacent sections were immunostained for activin A expression. ( B ) Quantification of the percentage of epithelial cells (left panel) or stromal cells (right panel) in normal tissue (white bar) versus tumor tissue (black bar). Statistical analysis is unpaired t-test. ( C ) Quantification of activin A in TMA (n = 63). An average of the percentage of epithelial and stromal fraction was calculated and scored in each TMA. Comparing epithelial (left panel) and stromal fraction (right panel) in normal tissue (white bar) versus tumor tissue (black bar). Statistical analysis 2-way ANOVA; mean ± SEM, ***(p < 0.001). ( D ) Kaplan–Meier plots of patient overall survival versus expression of activin A in either epithelial cells (left panel) or stromal cells (right panel). Statistical analysis Log-rank (Mantel-Cox) test, ** (p < 0.01), ***(p < 0.001).

Article Snippet: PDAC tissue microarray was commercially purchased from US Biomax Inc.

Techniques: Microarray, Staining, Expressing

Journal: Archives of Medical Science : AMS

Article Title: Comprehensive analysis of differentially expressed circRNAs revealed a ceRNA network in pancreatic ductaladenocarcinoma

doi: 10.5114/aoms.2019.85204

Figure Lengend Snippet: GO Analysis of differentially expressed circRNA-mediated ceRNAs in PDAC. We conducted GO analysis to obtain meaningful annotation of genes by using the MAS system provided by CapitalBio Corporation (Molecule Annotation System, http://bioinfo.capitalbio.com/mas3/ ). P < 0.05 was used as the criterion for statistical significance. A – The potential biological processes that were regulated by up-regulated circRNA-mediated ceRNA network. B – The potential biological processes that were regulated by down-regulated circRNA-mediated ceRNA network

Article Snippet: DHX9 protein expression in PDAC tissues was reviewed in Human Protein Atlas ( http://www.proteinatlas.org/ ) [ ].

Techniques:

Journal: Archives of Medical Science : AMS

Article Title: Comprehensive analysis of differentially expressed circRNAs revealed a ceRNA network in pancreatic ductaladenocarcinoma

doi: 10.5114/aoms.2019.85204

Figure Lengend Snippet: Higher expression level of DHX9 was significantly associated with shorter overall survival time. A – Relative DHX9 mRNA levels in GSE28735. DHX9 mRNA is up-regulated in PDAC with p < 0.01. B – The Kaplan-Meier curve analysis showed that higher expression level of DHX9 was significantly associated with shorter overall survival time. C – Higher protein expression of PDAC utilizing antibody staining

Article Snippet: DHX9 protein expression in PDAC tissues was reviewed in Human Protein Atlas ( http://www.proteinatlas.org/ ) [ ].

Techniques: Expressing, Staining

Journal: Cell Death Discovery

Article Title: ALYREF-JunD-SLC7A5 axis promotes pancreatic ductal adenocarcinoma progression through epitranscriptome-metabolism reprogramming and immune evasion

doi: 10.1038/s41420-024-01862-2

Figure Lengend Snippet: A The mRNA expression profiles of m 5 C regulators in PDAC and normal tissues were based on TCGA+GTEx databases. B The mRNA expression of ALYREF in PDAC and adjacent tissues were obtained from GSE15471 and GSE16515 datasets. C ALYREF protein levels were analyzed in PDAC and paired normal tissues by western blotting (n = 9). D Representative IHC images and IHC scores of ALYREF staining in 20 pairs PDAC and normal tissues (scale bar, 250 μm (100×), 50 μm (400 ×)). E , F mIHC was performed on a tissue microarray to detect the relationship between ALYREF and CD8 + T cells ( n = 135, scale bar, 50 μm). G Kaplan–Meier curve of overall survival was performed based on the follow-up data of TMA assay. H Multivariate analysis of several factors was performed in TMA assay. Data are presented the mean ± SD. **** p < 0.0001.

Article Snippet: The human PDAC tissue microarray was created by Wuhan Servicebio technology (Wuhan, China) using 156 PDAC tissue specimens from the First Affiliated Hospital, School of Medicine, Zhejiang University, China.

Techniques: Expressing, Western Blot, Staining, Microarray

Journal: Cell Death Discovery

Article Title: ALYREF-JunD-SLC7A5 axis promotes pancreatic ductal adenocarcinoma progression through epitranscriptome-metabolism reprogramming and immune evasion

doi: 10.1038/s41420-024-01862-2

Figure Lengend Snippet: A Venn Diagram showing the intersection of RIP-BisSeq, RNA-seq and JASPAR database. JunD is identified as a potential target. B , C Expression of JunD following ALYREF knockdown was detected by RT-qPCR and western blotting. D RIP assay was used to validate the direct binding between ALYREF and JunD. E The mRNA decay rate of JunD in shNC and shALYREF PDAC cells treated with Actinomycin D. F The dot blot assay was used to assess the change of mRNA m 5 C level after NSUN2 knockdown in PDAC cells. G MeRIP-qPCR assay was conducted using m 5 C-specific antibody to measure the m 5 C levels. H RIP-qPCR assay was performed using anti-ALYREF antibody to determine the binging efficiency between ALYREF and JunD mRNA. I , J Expression of SLC7A5 following JunD knockdown was detected by RT-qPCR and western blotting. K Schematic illustration showing the position of ChIP- qPCR primers. L , M ChIP assay was performed to test whether JunD could bind to the promoter of SLC7A5. Dual-luciferase assays verified that whether JunD could promote the transcription of SLC7A5 mRNA. Data are presented the mean ± SD of 3 independent experiments, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The human PDAC tissue microarray was created by Wuhan Servicebio technology (Wuhan, China) using 156 PDAC tissue specimens from the First Affiliated Hospital, School of Medicine, Zhejiang University, China.

Techniques: RNA Sequencing, Expressing, Knockdown, Quantitative RT-PCR, Western Blot, Binding Assay, Dot Blot, ChIP-qPCR, Luciferase

Journal: Cell Death Discovery

Article Title: ALYREF-JunD-SLC7A5 axis promotes pancreatic ductal adenocarcinoma progression through epitranscriptome-metabolism reprogramming and immune evasion

doi: 10.1038/s41420-024-01862-2

Figure Lengend Snippet: A The expression levels of ALYREF, JunD and SLC7A5 in TMA were measured by multiplex immunohistochemical and two groups of representative multiplexes immunohistochemical images are shown (scale bar, 50 μm). B The correlation of among the expression levels of ALYREF, JunD and SLC7A5 in TMA samples was analyzed by Pearson correlation coefficient ( n = 139). C Overall survival analysis based on TMA cohort showed that patients with high co-expression of ALYREF, JunD and SLC7A5 have poor prognosis. D The graphic illustration of ALYREF modulating tumor progression and immune escape in PDAC.

Article Snippet: The human PDAC tissue microarray was created by Wuhan Servicebio technology (Wuhan, China) using 156 PDAC tissue specimens from the First Affiliated Hospital, School of Medicine, Zhejiang University, China.

Techniques: Expressing, Multiplex Assay, Immunohistochemical staining

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: (A) PDAC cell lines were infected by lentivirus vectors encoding nonspecific (NS) control or distinct shRNAs targeting ARAF, BRAF, CRAF, or KRAS sequences. Colonies were stained by crystal violet ~10 days after plating. Data are presented as median. All p values shown are in comparison to the vehicle control for the individual graph. Adjusted p values are from Dunnett’s multiple comparison test. Adjusted p values: Pa01C ( ARAF-sh1 = 0.6792, ARAF-sh2 = 0.6726, BRAF-sh1 = 0.8883, BRAF-sh2 = 0.0592, CRAF-sh1 = *, 0.0024, CRAF-sh2 = 0.6350), ( KRAS-sh = ***, 0.0010). Pa02C ( ARAF-sh1 = 0.7153, ARAF-sh2 = 0.0692, BRAF-sh1 = 0.8788, BRAF-sh2 = 0.1990, CRAF-sh1 = 0.0539, CRAF-sh2 = 0.2738), ( KRAS-sh = **, 0.0068). Pa14C ( ARAF-sh1 = ****, < 0.0001, ARAF-sh2 = ****, < 0.0001, BRAF-sh1 = *, 0.0103, BRAF-sh2 = ****, < 0.0001, CRAF-sh1 = ****, < 0.0001, CRAF-sh2 = ****, < 0.0001, KRAS-sh = ****, < 0.0001). Pa16C ( ARAF-sh1 = 0.9995, ARAF-sh2 = 0.7500, BRAF-sh1 = 0.9977, BRAF-sh2 = ****, < 0.0001, CRAF-sh1 = ****, < 0.0001, CRAF-sh2 = ****, < 0.0001, KRAS-sh = ****, < 0.0001). MIA PaCa-2 ( ARAF-sh1 = 0.5520, ARAF-sh2 = **, 0.0051, BRAF-sh1 = 0.2316, BRAF-sh2 = **, 0.0076, CRAF-sh1 = **, 0.0086, CRAF-sh2 = **, 0.0098). PANC-1 ( ARAF-sh1 = 0.3213, ARAF-sh2 = ****, < 0.0001, BRAF-sh1 = 0.0586, BRAF-sh2 = **, 0.0025, CRAF-sh1 = ****, < 0.0001, CRAF-sh2 = ****, < 0.0001). (B) PDAC cell lines were treated with RAFi (0.04–10 μM, 72 h). Cell lysates were immunoblotted to determine levels of the indicated proteins. Data are representative of three independent experiments. (C) PDAC cell lines were treated with RAFi (0.01–2.5 μM, 72 h). Proliferation was measured by Calcein AM cell viability assay. Data are the mean average of three independent experiments. Error bars are shown as ± SEM. (D) CRISPR screen. PDAC cell lines were infected with the CRISPR library and treated with vehicle control or RAFi (w2, 2 weeks; w4, 4 weeks; a and b indicate replicate samples). The enrichment score indicates either enrichment (red) or depletion (blue) of the indicated genes in cells treated with RAFi relative to vehicle control. (E) Cell lines were infected by lentivirus vectors encoding NS or two distinct ARAF shRNAs (72 h) and treated with RAFi (0.01–2.5 μM, 120 h). Proliferation was measured by Calcein AM cell viability assay. Data are the mean average of three technical replicates. Error bars are shown as ± SEM. Summary of GI 50 values.

Article Snippet: CFPAC-1 KRAS mutant PDAC rat xenograft tumor tissue , Provided by Eli Lilly , section of this paper .

Techniques: Infection, Control, Staining, Comparison, Viability Assay, CRISPR

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: (A) RPPA analyses of PDAC cell lines treated with vehicle control, RAFi (0.3 μM), ERKi (0.04 μM), or the combination for multiple time points (0.25, 1, 8, 24 and 72 h). RAFi/ERKi-treated PDAC cells were normalized to their respective vehicle control. Proteins with significant phosphorylation or expression changes at 72-h time point are plotted as fold changes. Red, increased fold change; blue, decreased fold change; white, no change. (B) GSEA of the cell lines shown in (A). Enriched or depleted gene sets treated with RAFi/ERKi compared to RAFi (upper graph) or ERKi (below graph) are shown (24 h). (C) Pa16C cells were treated with vehicle control, RAFi (0.3 μM) and ERKi (0.04 μM) alone, or the combination (120 h). Cell lysates were immunoblotted to determine levels of pERK, total ERK, total MYC, and vinculin. Data are representative of three independent experiments.

Article Snippet: CFPAC-1 KRAS mutant PDAC rat xenograft tumor tissue , Provided by Eli Lilly , section of this paper .

Techniques: Control, Phospho-proteomics, Expressing

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: (A) KRAS mutant cancer cell lines were treated with RAFi (0.01–2.5 μM) and ERKi (0.08–1.25 μM) alone or in combination for 120 h. Proliferation was measured by Calcein AM cell viability assay. Representative bliss synergy score heatmaps for three independent experiments is shown (left). Red, synergy; green, antagonism; white, no effect. The averaged dose response curves of three independent experiments are shown (right). Error bars are ± SEM. Synergy avg = average bliss synergy score. (B) KRAS mutant PDAC (10 days) and CRC organoids (5 days) were treated with RAFi (0.01–2.5 μM) and ERKi (0.04–0.63 μM) alone or in combination. Proliferation was measured by CellTiter-Glo 3D cell viability assay. Dose response curves and bliss synergy scores were calculated and represented as in (A). (C) Representative images of PDAC organoid hM1A treated with the vehicle control DMSO or RAFi (0.16 μM) or ERKi (0.04 μM) alone or in combination (left). Scale bar, 200 μm. Representative images of CRC organoid OT238 treated with DMSO or RAFi (0.31 μM) or ERKi (0.04 μM) alone or in combination (right). Scale bar, 100 μm. (D) Relative tumor volume of the NIH nude rats with implanted HPAF-II cells were treated with RAFi (20 mpk, BID) alone or in combination with the ERKi LY3214996 (LY ERKi, 10 mpk, QD) for 36 days (left). Body weight changes are shown (right). Error bars are shown as ± SEM. (E) Quantitation of blot analysis to determine levels of pRSK of tumor lysates (n = 5 animals per group). Error bars are shown as ± SEM.

Article Snippet: CFPAC-1 KRAS mutant PDAC rat xenograft tumor tissue , Provided by Eli Lilly , section of this paper .

Techniques: Mutagenesis, Viability Assay, Control, Quantitation Assay

Journal: Cell reports

Article Title: Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers

doi: 10.1016/j.celrep.2020.107764

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CFPAC-1 KRAS mutant PDAC rat xenograft tumor tissue , Provided by Eli Lilly , section of this paper .

Techniques: Virus, Mutagenesis, Recombinant, Cell Viability Assay, Viability Assay, CellTox Assay, Cytotoxicity Assay, In Situ, CRISPR, Protein Array, RNA Sequencing, Mass Spectrometry, shRNA, Sequencing, Software