Migration Of Pancreatic Cells Research Articles

Cover Image, Volume 123, Number 1, January 2022

Front Cover: The cover image is based on the Research Article BAG3 induces fibroblasts to release key cytokines involved in pancreatic cell migration by Beatrice Dufrusine et al., https://doi.org/10.1002/jcb.30172.

Abstract PO-082: Delineating the molecular basis of early dissemination of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced stages rendering the already limited therapeutic options ineffective for many patients. Earlier diagnosis permits for “curative” surgery; however, 95% of these patients experience recurrence locally or distally and die within ten years of treatment. One hypothesis that can explain high recurrence is the early dissemination of PDAC progression. At the time of surgery, systemic subclinical dissemination of predominantly dormant cells found ubiquitously within patient livers has already occurred prior to the development of overt metastatic disease or bona fide recurrent PDAC. However, remarkably little is known about early PDAC cell dissemination despite these dismal outcomes, mainly due to the lack of human models to study disease progression. We previously demonstrated the cellular reprogramming “proof-of-principle” to model human PDAC progression (designated “10-22 cells”) and its use as a discovery tool for early biomarkers by unveiling a marker for stage 1 PDAC in human patients. We further analyzed transcriptomes of genetically tagged 10-22 cells progressing from pancreatic intraepithelial neoplasia (PanIN) to PDAC in mice and validated the results using the TCGA PDAC dataset, human clinical PanIN, PDAC tissues, and a well-established murine PDAC model. We found that extracellular vesicle transport and neuronal cell differentiation pathways were derepressed in the progression of PanINs to PDAC. HMG-Box Transcription Factor 1 (HBP1) emerged as a potential master factor regulating dynamically expressed genes in neuronal cell differentiation and dissemination during PDAC progression. HBP1 expression are aberrantly regulated in human and murine PanINs and PDAC tissue samples, and their mRNA expressions are inversely correlated with PDAC patients' prognosis. Ectopic overexpression of HBP1 increased proliferation and migration of normal and cancerous pancreatic cells. Conversely, knocking out HBP1 in PDAC cells decreased proliferation and migration. Altogether, our data indicate that HBP1 may confer the cell dissemination capacity in early PDAC progression. We are on the way to further dissect the mechanisms by which HBP1 regulates pancreatic cell migration and confers neuronal migratory phenotypes and how these signaling influence PDAC progression and prognosis. Citation Format: Taelor J. Ekstrom, Dmytro Grygoryev, Terry Morgan, Kenneth S. Zaret, Jungsun Kim. Delineating the molecular basis of early dissemination of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-082.

BAG3 induces fibroblasts to release key cytokines involved in pancreatic cell migration.

Pancreatic ductal adenoma carcinoma (PDAC) is considered one of the deadliest solid cancers as it is usually diagnosed in advanced stages and has a poor response to treatment. The enormous effort made in the last 2 decades in the oncology field has not led to significant progress in improving early diagnosis or therapy for PDAC. The stroma of PDAC plays an active role in tumour initiation and progression and includes immune cells and stromal cells. We previously reported that Bcl2‐associated athanogene (BAG3) secreted by PDAC cells activates tumour‐associated macrophages to promote tumour growth. The disruption of this tumour–stroma axis by the anti‐BAG3 H2L4 therapeutic antibody is sufficient to delay tumour growth and limit metastatic spreading in different PDAC preclinical models. In the present study, we examined the role of BAG3 to activate human fibroblasts (HF) in releasing cytokines capable of supporting tumour progression. Treatment of fibroblasts with recombinant BAG3 induced important changes in the organisation of the cytoskeleton of these cells and stimulated the production of interleukin‐6, monocyte chemoattractant protein‐1/C–C motif chemokine ligand 2, and hepatocyte growth factor. Specifically, we observed that BAG3 triggered a depolymerisation of microtubules at the periphery of the cell while they were conserved in the perinuclear area. Conversely, the vimentin‐based intermediate filaments increased and spread to the edges of the cells. Finally, the conditioned medium (CM) collected from BAG3‐treated HF promoted the survival, proliferation, and migration of the PDAC cells. Blocking of the PDAC‐fibroblast axis by the H2L4 therapeutic anti‐BAG3 antibody, resulted in inhibition of cytokine release and, consequently, the inhibition of the migratory phenotype conferred by the CM to PDAC cells.

Study on the molecular mechanism of miR-200b-3p regulates the proliferation, invasion, migration and apoptosis of pancreatic cancer cells by down-regulating VEGFA

Objective To explore the molecular mechanism of miR-200b-3p regulates the proliferation, invasion, migration and apoptosis of pancreatic cancer cells by targeting vascular endothelial growth factor A (VEGFA). Methods The expression of miR-200b-3p in pancreatic cancer tissues and cell lines was detected by quantitative real-time fluorescence PCR (qRT-PCR). Pancreatic cancer PANC-1 cells were divided into NC group, miR-200b-3p mimic group, si-VEGFA group and si-VEGFA+ miR-200b-3p inhibitor group. The proliferation, migration and invasion of PANC-1 cells were measured by CCK-8 and Transwell assay. The apoptosis of PANC-1 cells was detected by Annexin V-FITC/PI double staining flow cytometry assay. The targeted relationship of miR-200b-3p and VEGFA was estimated by dual luciferase reporter gene assay and Western blotting. Results The expression of miR-200b-3p in pancreatic cancer tissues and cell lines was decreased. After miR-200b-3p was overexpressed in PANC-1 cells for 48 h, the cell viabilities of PANC-1 cells in NC group and miR-200b-3p mimic group were 1.250±0.028 and 0.983±0.044, the numbers of migrated cells were 402.700±21.530 and 158.000±17.620, the numbers of invaded cells were 478.300±31.050 and 170.000±32.470, and the cell apoptosis rates were (5.280±0.352)% and (7.430±0.393)%. The cell viability, migration and invasion of PANC-1 cells in miR-200b-3p mimic group were significantly decreased than those in NC group (t=5.060, P=0.007; t=8.796, P=0.001; t=6.863, P=0.002). The cell apoptosis rate in miR-200b-3p mimic group was significantly higher than that in NC group (t=4.076, P=0.015). The fluorescence intensity in VEGFA-WT group was 1.000±0.027, which was significantly higher than that in VEGFA-WT+ miR-200b-3p mimic group (0.632±0.048; t=6.637, P=0.003). The fluorescence intensities in VEGFA-MUT group and VEGFA-MUT + miR-200b-3p mimic group were 1.000±0.049 and 0.868±0.047, with no statistically significant difference (t=1.944, P=0.124). After miR-200b-3p was overexpressed in PANC-1 cells for 48 h, the expressions of VEGFA in NC group and miR-200b-3p mimic group were 1.000±0.058 and 0.762±0.020, respectively. The expression level in miR-200b-3p mimic group was lower than that in NC group (t=3.908, P=0.017). After transfection of PANC-1 cells with si-VEGFA or si-VEGFA + miR-200b-3p inhibitor for 48 h, the cell viabilities of PANC-1 cells in NC group, si-VEGFA group and si-VEGFA + miR-200b-3p inhibitor group were 1.300±0.058, 0.943±0.047 and 1.143±0.023, the numbers of migrated cells were 446.000±17.350, 206.300±19.360 and 428.300±30.330, and the numbers of invaded cells were 510.300±24.550, 175.700±24.290 and 473.700±35.530, with statistically significant differences (F=15.830, P=0.004, F=33.530, P=0.001, F=38.860, P<0.001). The cell viability, migration and invasion of PANC-1 cells in si-VEGFA group were significantly decreased than those in NC group (P=0.003, P<0.001, P<0.001). There was no significant difference between si-VEGFA + miR-200b-3p inhibitor group and NC group (P=0.107, P=0.854, P=0.671). The cell apoptosis rates in NC group, si-VEGFA group and si-VEGFA+ miR-200b-3p inhibitor group were (3.810±0.577)%, (7.373±0.482)% and (3.650±0.514)%, with a statistically significant difference (F=16.020, P=0.004). The cell apoptosis rate in si-VEGFA group was significantly higher than that in NC group (P=0.007), but there was no significantly difference between si-VEGFA + miR-200b-3p inhibitor group and NC group (P=0.975). Conclusion miR-200b-3p suppresses the proliferation, invasion and migration and promotes the apoptosis of pancreatic cells by down-regulating VEGFA. Key words: Pancreatic neoplasms; MicroRNA-200b-3p; Vascular endothelial growth factor A

Abstract 3855: Refolded albumin inhibits IL-17B-driven pancreatic cancer migration

Abstract Folding and refolding a protein may change its physical, chemical and biological properties. Here, we used a novel protein refolding protocol to convert globular albumin into another form, namely f-albumin, with melting point 10oC higher than albumin (83oC vs 72oC). f-albumin, unlike albumin, inhibited the migration/invasion of a variety of cancer cells. Recently, anti-IL-17RB antibodies have been shown to block the IL-17B-IL17RB signaling-mediated migration/metastasis of pancreatic cancer cells via silencing multiple chemokines, notably CCL20, CXCL1, IL-8 and TFF1. We thus examined whether f-albumin is effective in blocking the stimulatory effects of IL-17B. We found that f-albumin inhibited the migration of pancreatic cancer cells (BxPC3, MIA-paca2 and Panc1) with IC50 around 0.2 μM. Although IL-17B (50 ng/ml) increased the migration of pancreatic cells, this effect of IL-17B was abolished by f-albumin (0.2 μM). The inhibitory effect of f-albumin, however, was not necessarily correlated with its inhibition of CCL20, CXCL1, IL-8 or TFF1. Additional study revealed that IL-17B increased the phosphorylation of PI3K, Akt and MAPK in pancreatic cancer cells. f-albumin modulated not only the IL-17B-mediated phosphorylation of Akt but also the IL-17B-mediated phosphorylation of MAPK in an integrin-dependent manner. These findings demonstrated that f-albumin downregulates Akt/MAPK signaling via integrin to play a braking role in the IL-17B-driven pancreatic cancer migration. Citation Format: Chi-Ming Liang, Ching-Chun Liao, Shu-Mei Liang. Refolded albumin inhibits IL-17B-driven pancreatic cancer migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3855.

Functional study of miRNA-301a-3p in pancreatic cancer

Objective To investigate the expression of miRNA-301a-3p in pancreatic cancer and to correlate the expression on invasion, migration and colony formation of pancreatic cancer cells. Methods The expression of miRNA-301a-3p in 20 paired pancreatic cancer tissues and matched adjacent tissues, and pancreatic cancer cell lines and normal pancreatic ductal cells were detected by real-time PCR. miRNA-301a-3p mimics or inhibitors were used to up-regulate or down-regulate the miRNA-301a-3p level in pancreatic cancer cell lines in order to figure out the effects of miRNA-301a-3p on cell invasion, migration and colony formation of pancreatic cancer cells, respectively. Results In pancreatic cancer tissues and cell lines, miRNA-301a-3p was significantly up-regulated when compared with the matched adjacent tissues (P<0.05) and normal pancreatic ductal cells (P<0.05), respectively. Overexpression or downexpression of miRNA-301a-3p enhanced or suppressed colony formation, invasion and migration abilities of pancreatic cancer cells in vitro. Upregulation of miRNA-301a-3p promoted tumorigenesis in vivo. Conclusion miRNA-301a-3p might function as an oncogene to promote tumorigenesis in pancreatic cancer. Key words: miRNA; Pancreatic cancer; Oncogene

Effects of secreted protein acidic and rich in cysteine on proliferation, migration and invasion ability of pancreatic cancer cells

Objective To investigate the effects of exogenous secreted protein acidic and rich in cysteine (SPARC) on proliferation,migration and invasion ability of human pancreatic cancer cell lines.Methods The cuhured Mia PaCa-2 and PANC-1 pancreatic cancer cells were established in the presence of different concentrations (0,0.5,1.0,3.0,5.0,10.0 mg/L) or absence of exogenous SPARC as experimental and control groups.The change of proliferation was measured by methyl thiazol tetrazolium (MTT) assay.The 24 h cultured Mia PaCa-2 and PANC-1 pancreatic cancer cells were established in the presence (5 mg/L) or absence of exogenous SPARC as experimental and control groups,and the change of migration ability was detected by single cell migration assay and the change of invasion ability by Matrigel invasion assay respectively.Results Exogenous SPARC could inhibit the proliferation of cells in a concentration-dependent manner.Cell proliferation was inhibited in the presence of 10.0 mg/L exogenous SPARC by (32.8 ± 7.4) ％ and (32.5 ± 1.8) ％ in Mia PaCa-2 and PANC-1,respectively.5.0 mg/L SPARC reduced the distance covered within 24 h by 26％ and 39％,and in the presence of 5.0 mg/L SPARC the number of invasive cells was inhibited by 30％ and 56％ in 24 h,respectively.Conclusion Exogenous SPARC could inhibit the proliferation,migration and invasion ability in pancreatic cancer cells,suggesting extracelluar matrix may affect biological functions of malignant tumors. Key words: Secreted protein acidic and rich in cysteine; Extrcelluar matrix; Pancreatic cancer

The role of Gli1 in the invasion and migration of pancreatic cancer cells

To study the role and possible mechanism of glioma-associated oncogene-1 (Gli1) in regulating the cell invasion and migration of pancreatic cancer cells. Quantitative real-time (qRT) -PCR was used to detect the effect of siRNA interference on Gli1, murine double minute 2 (MDM2) and p53 genes. Cell invasion and migration assays were used to observe the effect of Gli1, MDM2 and p53 silence on cell invasion and migration in p53 wild-type Capan-2 pancreatic cancer cells, respectively. Meanwhile, immunoblotting (IB) was used to detect the protein level of matrix metalloproteinase (MMP) -9, phospho-excelluar signal-regulated kinase (pERK) and phosphorylation protein kinase B (pAKT) in Gli1-silencing Capan-2 cells. The data were analyzed by paired t-test. qRT-PCR showed that the expression of Gli1, MDM2 and p53 is down-regulated 70.5% and 74.5%, 61.8% and 65.3%, and 73.8% and 78.2% after siRNA interference, compared with the mock and siRNA control groups, respectively. Cell invasion (94 ± 8) and migration (143 ± 8) in p53 wild-type Capan-2 cells transfected with Gli1siRNA were significantly decreased, compared with the siRNA control group (150 ± 7, 190 ± 10) (t = 6.584, P = 0.022; t = 8.266, P = 0.014) , while MDM2 silence inhibited cell invasion (experiment group:85 ± 12, control group: 138 ± 6) and migration (experiment group: 127 ± 9, control group:180 ± 10) in the same cells, respectively (t = 5.097, P = 0.036;t = 4.860, P = 0.040). However, cell invasion (experiment group: 153 ± 11, control group: 106 ± 7) and migration (experiment group: 209 ± 13, control group: 164 ± 8) in p53-silencing Capan-2 cells were significantly enhanced (t = 4.669, P = 0.043; t = 4.990, P = 0.038). IB showed that Gli1 silence down-regulated MMP-9 but not pERK and pAKT protein expression. Gli1 might contribute to the cell invasion and migration in pancreatic cancer via the regulation of MDM2, p53 and MMP-9 expression.

Trefoil factor 3 (TFF3) expression is regulated by insulin and glucose

Introduction: Trefoil factors are effector molecules in gastrointestinal tract physiology. They are classified into three groups: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Previous studies have shown that trefoil factors are located and expressed in human endocrine pancreas suggesting that TFF3 play a role in: a) pancreatic cells migration, b) β-cell mitosis, and c) pancreatic cells regeneration. We speculated that the presence of TFF3 in pancreas, could be associated to a possible regulation mechanism by insulin and glucose. To date, there are not reports whether the unbalance in carbohydrate metabolism observed in diabetes could affect the production or expression of TFF3.Methods: We determined the TFF3 levels and expression by immunoassay (ELISA) and semi-quantitative RT-PCR technique respectively, of intestinal epithelial cells (HT-29) treated with glucose and insulin. Also,Real Time-PCR (RTq-PCR) was done.Results: Increasing concentrations of glucose improved TFF3 expression and these levels were further elevated after insulin treatment. Insulin treatment also led to the up-regulation of human sodium/glucose transporter 1 (hSGLT1), which further increases intracellular glucose levels. Finally, we investigated theTFF3 levels in serum of diabetes mellitus type 1 (T1DM) and healthy patients. Here we shown that serum TFF3 levels were down-regulated in T1DM and this levels were up-regulated after insulin treatment. Also, the TFF3 levels of healthy donors were up-regulated 2 h after breakfast.Conclusion: Our fi ndings suggest for the fi rst time that insulin signaling is important for TFF3 optimal expression in serum and intestinal epithelial cells.

Role of connective tissue growth factor (CTGF) in proliferation and migration of pancreatic cancer cells

To explore the expression of connective tissue growth factor (CTGF) in pancreatic cancer and its influence on the proliferation and migration of cancer cells. The expression of CTGF in pancreatic cell line PANC-1 cells was analyzed by real-time PCR and in pancreatic carcinoma (50 cases) tissues by immunohistochemistry. The ability of proliferation and migration in vitro of PANC-1 cells was tested by MTT assay, scratch test and Boyden chamber test after the CTGF gene was overexpressed by Ad5-CTGF or silenced with Ad5-siCTGF transfection. CTGF was overexpressed in both pancreatic cancer cells and tissues. Overxpression of CTGF leads to increased proliferation and migration of PANC-1 cells. The CTGF-transfected PANC-1 cells showed apparent stronger proliferation ability and scratch-repair ability than that of empty vector controls. The results of Boyden chamber test showed that there were 34 cells/field (200× magnificantion) of the CTGF-transfected overexpressing cells, much more than the 11 cells/field of the empty vector control cells; and 6 cells/microscopic field of the Ad5-siCTGF-transfected silenced cells, much less than the 15 cells/field of the control cells. CTGF is overexpressed in both pancreatic cancer cells in vitro and in vivo, indicating that it may play an important role in the cell proliferation and migration in pancreatic cancer.

Expression of Slug in pancreatic cancer and inhibitory effects of anti-Slug on invasion and migration of pancreatic carcinoma cells

Objective To investigate expression of slug and E-cadherin in pancreatic cancer tissues and determine the inhibitory effects of anti-Slug, an anti-sense plasmid, on the invasion of pancreatic cancer cell lines in vitro. Methods Slug and E-cadherin protein and mRNA was analyzed by IHP and RT-PCR in 36 cases of pancreatic cancer. Then anti-Slug plasmid was transfected into herin and Slug expression. The inhibitory effects of anti-sense Slug were also detected by Transwell motility assay and Matrigel invasion assay. Results The expression of Slug and mRNA in metastatic pancreatic cancer tissue was higher than that in non-metastatic tissue. E-cadherin and mRNA was lower in metastasis tissues(P＜0.05). The inverse relationships were further observed by transient transfection of anti-Slug into SW1990H4 cells. The downregulated expression of Slug and re-expression of E-cadherin were found. The Slug mRNA levels were 0.985±0.016,0.973±0.014, 0. 554±0. 011 after 0, 48 h of transfection of anti-sense Slug, and that of E-cadherin were 0.120±0.001, 0.360±0.002, 0. 727±0. 006, respectively. The diference was significant between different time points (P＜0.05). The Slug mRNA levels were 0. 206±0.017, 0.968±0.015, and that of E-cadherin were 0. 18±0.002,0.727±0.006 after stable transfection of anti-sense Slug, and control plasmid, respectively. The diference was significant (P＜0.05). The motility activity(393±28, 352±24, 96 ±13 )and the invasion activity (223 ± 69, 202 ± 64, 65 ±19) of1 antisense Slug transfectant cells were significantly decreased as compared with those of control cells (P＜0.05). Conclusions Higher expression of slug and lower expression of E-cadherin is related to the invasion and metastasis in pancreatic cancer. A reverse corelation of E-cadherin and Slug expression exists in pancreatic cancer. Slug is possibly a potential target for cancer gene therapy blocking invasion and metastasis in human pancreatic cancer. Key words: Pancreatic carcinoma; Slug; Cadherins; Transfection; Metastasis