Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Overexpression of laminin-5 gamma-2 promotes tumorigenesis of pancreatic ductal adenocarcinoma through EGFR/ERK1/2/AKT/mTOR cascade

doi: 10.1007/s00018-022-04392-1

Figure Lengend Snippet: LAMC2 is highly upregulated and associated with worse survival in patients with PDAC. A LAMC2 transcript is overexpressed in PDAC tumor specimens compared to normal or adjacent non-cancerous pancreatic samples in the microarray data (GSE15471, GSE28735, and GSE62125). Statistical significance was calculated using GraphPad Prism (***P-value < 0.0001; two-tailed t-test). B RNA sequencing data analysis displayed higher LAMC2 transcript in PDAC and other human malignancies in TCGA Pan-Cancer datasets. C, D Kaplan–Meier plots analysis displayed elevated levels of LAMC2 transcripts were linked with overall (P-value < 0.0001) and disease-free survival (P-value < 0.012) in the patients with PDAC (TCGA data). Statistical significance was calculated using the Log-rank test

Article Snippet: Pancreatic cancer cell lines (HPAC, AsPC-1, BxPC-3, Capan-2, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, PL45, SW1990, CFPAC-1, Panc10.05 and hTERT-HPNE, human pancreatic normal epithelial cells were obtained from American Type Culture Collection, USA.

Techniques: Microarray, Two Tailed Test, RNA Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Overexpression of laminin-5 gamma-2 promotes tumorigenesis of pancreatic ductal adenocarcinoma through EGFR/ERK1/2/AKT/mTOR cascade

doi: 10.1007/s00018-022-04392-1

Figure Lengend Snippet: Overexpression of LAMC2 transcript and protein in PDAC cell lines and clinical samples. A Quantitative real-time PCR (qPCR) displayed upregulation of LAMC2 transcript in PDAC cells compared to normal human pancreatic tissue and cell line (hTERT-HPNE). Results represent mean ± SD; n = 3. **P-value < 0.001; ***P-value < 0.0001; two-tailed t-test. B Western blot analysis displayed higher LAMC2 protein expression in PDAC cell lines compared to hTERT-HPNE (normal human pancreatic epithelial) cells. C Immunofluorescence experiments demonstrated LAMC2 protein expression in the cytoplasm of PDAC cells. DAPI (4′,6′-diamino-2-phenylindole) was used for nuclear staining. D IHC (immunohistochemistry) data showed strong, moderate, and low staining for LAMC2 in the representative PDAC clinical tissue sections. No/negative staining was noticed in normal pancreatic tissue sections (original magnification, X200). Histoscore analysis displayed significant difference in the staining intensity among low and moderate/high groups. Results represent mean ± SD. ***P-value < 0.0001

Article Snippet: Pancreatic cancer cell lines (HPAC, AsPC-1, BxPC-3, Capan-2, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, PL45, SW1990, CFPAC-1, Panc10.05 and hTERT-HPNE, human pancreatic normal epithelial cells were obtained from American Type Culture Collection, USA.

Techniques: Over Expression, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Expressing, Immunofluorescence, Staining, Immunohistochemistry, Negative Staining

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Overexpression of laminin-5 gamma-2 promotes tumorigenesis of pancreatic ductal adenocarcinoma through EGFR/ERK1/2/AKT/mTOR cascade

doi: 10.1007/s00018-022-04392-1

Figure Lengend Snippet: Inhibition of LAMC2 suppressed EGFR/ERK1/2/AKT/mTOR/P70S6K cascade in PDAC. A Upregulation of EGFR transcripts in 179 PDAC samples compared to 171 non-cancerous pancreatic samples in TCGA data. B Higher expression of EGFR displayed inferior overall survival. C Pearson analysis revealed a positive correlation between LAMC2 and EFGR expression. D Epidermal growth factor (100 ng/mL) treatment induced the expression of LAMC2 in HPAC and BxPC-3 cells. E Western blotting experiments displayed robust expression of LAMC2 and EGFR in protein lysates (input) of HPAC and BxPC-3. F LAMC2 antibodies were used for immunoprecipitation in the cell lysates of HPAC and BxPC-3 and immunoprecipitated complexes were immunoblotted with EGFR antibodies. EGFR antibodies were used for immunoprecipitation and immunoprecipitated complexes were subjected to western blotting with LAMC2 antibodies. G HPAC and BxPC-3 cells (either with LAMC2 shRNA or scramble shRNA) were starved for at least 24 h followed by stimulation with EGF (100 ng/mL) for 20 min. Phosphorylation of EGFR, ERK1/2, AKT, mTOR, P70S6K, and P85S6K was demonstrated using western blotting experiments in HPAC and BxPC-3 cells. H Densitometric analysis revealed a significant decrease in phosphorylation of EGFR, ERK1/2, AKT, mTOR, P70S6K, and P85S6K

Article Snippet: Pancreatic cancer cell lines (HPAC, AsPC-1, BxPC-3, Capan-2, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, PL45, SW1990, CFPAC-1, Panc10.05 and hTERT-HPNE, human pancreatic normal epithelial cells were obtained from American Type Culture Collection, USA.

Techniques: Inhibition, Expressing, Western Blot, Immunoprecipitation, shRNA, Phospho-proteomics

Journal: British Journal of Cancer

Article Title: PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells

doi: 10.1038/bjc.2013.531

Figure Lengend Snippet: Expression of PIAS4 in human pancreatic cancer cell lines, pancreatic tumours, and normal pancreas. ( A ) Relative RNA expression levels of PIAS4 as a mean of 12 pancreatic cancer cell lines, 11 tumor samples, and 6 normal pancreas. The relative PIAS4 RNA expression level in each sample was normalised to that of GAPDH. *compared with the normal pancreas, P =0.0154. ( B ) Immunohistochemistry analysis of PIAS4 in normal pancreas and pancreatic tumor. ( C ) Western blot analysis of PIAS4 protein expression in 14 pancreatic cancer cell lines. The arrow indicates the band corresponding to the PIAS4 protein. GAPDH was used as a loading control.

Article Snippet: Fourteen pancreatic cancer cell lines (AsPc1 BxPc3, Panc1, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, Panc1005, CFPAC1, CaPan2, MiaPaCa2, PL45, and SU8686) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, RNA Expression, Immunohistochemistry, Western Blot, Control

Journal: British Journal of Cancer

Article Title: PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells

doi: 10.1038/bjc.2013.531

Figure Lengend Snippet: Effect of silencing and overexpression of PIAS4. ( A ) Four pancreatic cancer cell lines (AsPc1, BxPc3, Panc0327, and Panc1005) were transfected with either mock transfection (wild-type, wt), control siRNA (ctrl siRNA) or pooled PIAS4 siRNA. MTT assays show the effect of PIAS4 gene silencing on cell growth. Results represent the mean of three independent experiments with triplicate wells per experimental point. ( B ) Colony assays were photographed 72 h after seeding and stained with crystal violet. Bar graph shows the quantification of colonies by densitometry after colonies were solubilised and measured at OD 540 nm. ( C ) MTT assays show the effect of PIAS4 gene silencing (targeting either exon 2 (siEXON2) or exon 6 (siEXON6), or a negative control (non-target siRNA NC)) on cell growth. Real-time PCR was used to validate knock down of PIAS4 mRNA in these cells (lower panel). ( D ) Panc1 cells were transfected with either the PIAS4 overexpression construct pCMV-PIAS4 or empty vector pCMV. Alpha-Tubulin was used as a loading control in western blot analysis. ( E ) Panc1 cells were co-transfected with a Myc reporter (Myc-LUC) and pCMV-PIAS4 or pCMV. Myc reporter activity was normalised to Renilla activity and calculated as relative light units. Results represent the mean±s.d. of two experiments in triplicate wells.

Article Snippet: Fourteen pancreatic cancer cell lines (AsPc1 BxPc3, Panc1, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, Panc1005, CFPAC1, CaPan2, MiaPaCa2, PL45, and SU8686) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Over Expression, Transfection, Control, Staining, Negative Control, Real-time Polymerase Chain Reaction, Knockdown, Construct, Plasmid Preparation, Western Blot, Activity Assay

Journal: British Journal of Cancer

Article Title: PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells

doi: 10.1038/bjc.2013.531

Figure Lengend Snippet: Induction of expression of PIAS4 and HIF1 α under hypoxic conditions. Pancreatic cancer cell lines were incubated in 1% O 2 for the durations as indicated and examined for induction of gene expression. ( A ) Real-time quantitative RT–PCR measurement of PIAS4 expression (top panel) and HIF α (bottom panel) in four cell lines (BxPc3, Panc0327, AsPc1, and Panc1005). ( B ) Western blot analysis of protein expression in BxPc3 cells under hypoxic conditions (1% O 2 ) for 2 and 48 h. GAPDH was used as loading control.

Article Snippet: Fourteen pancreatic cancer cell lines (AsPc1 BxPc3, Panc1, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, Panc1005, CFPAC1, CaPan2, MiaPaCa2, PL45, and SU8686) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Incubation, Gene Expression, Quantitative RT-PCR, Western Blot, Control

Journal: British Journal of Cancer

Article Title: PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells

doi: 10.1038/bjc.2013.531

Figure Lengend Snippet: Effect of PIAS4 siRNA on expression of HIF α target genes and on cell invasion. MiaPaCa2 pancreatic cancer cells were transfected with either control siRNA or PIAS4 siRNA. ( A ) Relative RNA expression levels of PIAS4, HIF α , VEGF, and VHL in transfected cells (real-time quantitative RT–PCR). Expression of each gene in each sample was normalised to GAPDH. ( B ) Cellular invasion assays. After transfection, equal numbers of cells were applied and allowed to migrate overnight through Boyden chambers. Unmigrated cells were removed and migrated cells were fixed and stained with crystal violet for photography (dark staining represents migrating cells). Results are representative of two independent experiments, each with duplicates. ( C ) Relative RNA expression of VEGF and JMJD1A under hypoxic condition (1% O 2 ) for 2 and 48 h (real-time quantitative RT–PCR). After transfection, cells were incubated in a hypoxic chamber (1% O 2 ) and assayed for gene expression normalised to GAPDH.

Article Snippet: Fourteen pancreatic cancer cell lines (AsPc1 BxPc3, Panc1, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, Panc1005, CFPAC1, CaPan2, MiaPaCa2, PL45, and SU8686) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, Transfection, Control, RNA Expression, Quantitative RT-PCR, Staining, Incubation, Gene Expression

Journal: British Journal of Cancer

Article Title: PIAS4 is an activator of hypoxia signalling via VHL suppression during growth of pancreatic cancer cells

doi: 10.1038/bjc.2013.531

Figure Lengend Snippet: Mechanism of PIAS4 signalling in pancreatic cancer cells. In response to hypoxia, PIAS4 sumoylates and inactivates VHL which leads to HIF1 α activation. In normoxic condition, PIAS4 activates signalling pathways leading to cell proliferation, angiogenesis, and metastasis.

Article Snippet: Fourteen pancreatic cancer cell lines (AsPc1 BxPc3, Panc1, Panc0203, Panc0327, Panc0403, Panc0504, Panc0813, Panc1005, CFPAC1, CaPan2, MiaPaCa2, PL45, and SU8686) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activation Assay