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: Activation of STING in pancreatic cancer-associated fibroblasts exerts an antitumor effect by enhancing tumor immunity

doi: 10.1038/s41598-024-68061-y

Figure Lengend Snippet: STING expression in human PDAC stroma and CAFs. ( a ) Groups categorized by positive, weakly positive, and negative STING expression in PDAC stroma. Representative IHC analysis of STING using a human PDAC tissue array (scale bar, 100 μm). ( b ) Percentages of the indicated groups in 40 PDAC samples. ( c ) Co-immunofluorescence staining for α-SMA (red) and STING (green) in STING positive or weakly positive or negative in stroma. ( d ) 5-AZA (0.1 nM) was administered to CAF1 and CAF2 derived from murine PDACs and incubated for 24 h.

Article Snippet: The human PDAC tissue array was purchased commercially from TissueArray.com (No.PA482a).

Techniques: Expressing, Immunofluorescence, Staining, Derivative Assay, Incubation

Journal: Nature cell biology

Article Title: Pancreatic cancer cells upregulate LPAR4 in response to isolation stress to promote an ECM-enriched niche and support tumor initiation

doi: 10.1038/s41556-022-01055-y

Figure Lengend Snippet: a, Immunohistochemistry staining of LPAR4 in a PDAC tissue array consisted of 15 PDAC samples and 4 samples of normal pancreas. Scale bar is 50 μM. Representative images showing normal pancreas (n=4 biological samples), low-LPAR4 PDAC (n=9 biological samples), and high-LPAR4 PDAC (n=6 biological samples). Bar graph shows the relative percentage of LPAR4-low and LPAR4-high patients in the PDAC tissue array. b, Quantitative RT-PCR confirming the manipulation of LPAR4 expression in 2 pancreatic cancer cell lines (Colo-357, MIA PaCa-2) and 2 patient-derived cancer cells (79E, 34E). Data were shown as mean ± s.d. (n=4 independent experiments for Colo-357 and 79E cells with LPAR4 stable knockdown, n=3 for Colo-357, 79E, and MiaPaCa2 cells with LPAR4 ectopic expression, n=3 for 34E cells with LPAR4 stable knockdown, and n=4 for 34E with LPAR4 ectopic expression). c, Non-invasive Bioluminescence images showing tumors formed at day 10 for Colo-357+sh-CTRL+luciferase or Colo-357+sh-R4.1+luciferase of various number implanted in the pancreas of nu/nu mice. Right panel showing the luminescence intensity in a blue-to-red spectrum. d, f, Trypan blue exclusion assay showing the relative number of viable cells with or without LPAR4 expression manipulation grown in 10% serum and 2D at day 4 and day 7. e, Tumor growth rates for 1 million Colo-357 cells with or without LPAR4 expression manipulation in a subcutaneous tumor model. Data were presented as mean ± s.d. for n=8 independent samples for each group. g, Quantitative RT-PCR confirming the knockdown of LPAR1 in Colo-357 cells. h, Effects of LPAR1 knockdown using siRNA on 2D or 3D (methylcellulose sphere forming) cell growth. Data were presented as mean ± s.d. for n=3 independent experiments (d,f,g, and h). Statistical analyses were performed using two tailed unpaired one sample t-test (b,g, and h) and one-way ANOVA (d-f). Source numerical data are available in source data.

Article Snippet: Immunohistochemistry staining of LPAR4 or FN1 in human PDAC tissue array (US Biomax PA484a) or formalin-fixed, paraffin-embedded xenograft tumors were performed using the LPAR4 antibody (Thermo Fisher, #PA5–49727) or FN1 antibody (CST, #26836, E5H6X) following the manufacturer’s protocol.

Techniques: Immunohistochemistry, Staining, Quantitative RT-PCR, Expressing, Derivative Assay, Luciferase, Trypan Blue Exclusion Assay, Two Tailed Test

Journal: Oncotarget

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

doi:

Figure Lengend Snippet: 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: The PDAC tissue array was purchased from amsbio (Cambridge, MA).

Techniques: Staining, Expressing, Western Blot, Derivative Assay, Amplification, Mutagenesis, Activation Assay, Translocation Assay, Migration