Migration Of Pancreatic Stellate Cells Research Articles

COMP promotes pancreatic fibrosis by activating pancreatic stellate cells through CD36-ERK/AKT signaling pathways

BackgroundPancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP) and pancreatic stellate cells (PSCs) are the key cells of fibrosis. As an extracellular matrix (ECM) glycoprotein, cartilage oligomeric matrix protein (COMP) is critical for collagen assembly and ECM stability and recent studies showed that COMP exert promoting fibrosis effect in the skin, lungs and liver. However, the role of COMP in activation of PSCs and pancreatic fibrosis remain unclear. We aimed to investigate the role and specific mechanisms of COMP in regulating the profibrotic phenotype of PSCs and pancreatic fibrosis. MethodsELISA method was used to determine serum COMP in patients with CP. Mice model of CP was established by repeated intraperitoneal injection of cerulein and pancreatic fibrosis was evaluated by Hematoxylin-Eosin staining (H&E) and Sirius red staining. Immunohistochemical staining was used to detect the expression changes of COMP and fibrosis marker such as α-SMA and Fibronectin in pancreatic tissue of mice. Cell Counting Kit-8, Wound Healing and Transwell assessed the proliferation and migration of human pancreatic stellate cells (HPSCs). Western blotting, qRT-PCR and immunofluorescence staining were performed to detect the expression of fibrosis marker, AKT and MAPK family proteins in HPSCs. RNA-seq omics analysis as well as small interfering RNA of COMP, recombinant human COMP (rCOMP), MEK inhibitors and PI3K inhibitors were used to study the effect and mechanism of COMP on activation of HPSCs. ResultsELISA showed that the expression of COMP significantly increased in the serum of CP patients. H&E and Sirius red staining analysis showed that there was a large amount of collagen deposition in the mice in the CP model group and high expression of COMP, α-SMA, Fibronectin and Vimentin were observed in fibrotic tissues. TGF-β1 stimulates the activation of HPSCs and increases the expression of COMP. Knockdown of COMP inhibited proliferation and migration of HPSCs. Further, RNA-seq omics analysis and validation experiments in vitro showed that rCOMP could significantly promote the proliferation and activation of HPSCs, which may be due to promoting the phosphorylation of ERK and AKT through membrane protein receptor CD36. rCOMP simultaneously increased the expression of α-SMA, Fibronectin and Collagen I in HPSCs. ConclusionIn conclusion, this study showed that COMP was up-regulated in CP fibrotic tissues and COMP induced the activation, proliferation and migration of PSCs through the CD36-ERK/AKT signaling pathway. COMP may be a potential therapeutic candidate for the treatment of CP. Interfering with the expression of COMP or the communication between COMP and CD36 on PSCs may be the next direction for therapeutic research.

ITGB6 promotes pancreatic fibrosis and aggravates the malignant process of pancreatic cancer via JAK2/STAT3 signaling pathway.

Integrin β6 (ITGB6) is upregulated in multiple tumor types and elevated ITGB6 levels have been detected in patients with chronic pancreatitis. However, the role of ITGB6 in pancreatic fibrosis and cancer remains to be elucidated. In the present study, ITGB6 expression was assessed using western blotting and qRT-PCR. Besides, cell proliferation, cycling, migration, and invasion were evaluated using CCK-8, flow cytometry, wound healing, and transwell assays, respectively. The expression of fibrosis and JAK2/STAT3 signaling markers was detected by western blotting and immunofluorescence analysis. Moreover, nude mice were subcutaneously injected with co-cultured cell suspensions to establish an in vivo model. The results showed that ITGB6 was highly expressed in pancreatic cancer tissues and TGF-β-induced pancreatic stellate cells (PSCs). Inhibition of ITGB6 expression in PSCs resulted in clear inhibition of activated PSC proliferation, migration, and fibrogenesis. Additionally, reduced ITGB6 expression inhibits the JAK2/STAT3 signaling pathway. Interestingly, activators of the JAK2/STAT3 signaling pathway reversed the effects of ITGB6 disruption on PSCs. Activated PSCs notably promoted the proliferation, invasion, and migration of pancreatic cancer cells in a co-culture assay. In contrast, activated PSCs with low ITGB6 expression failed to significantly affect the malignancy of pancreatic cancer cells. Moreover, in vivo results showed that interference with ITGB6 inhibited the activation of PSCs and promoted the development of pancreatic cancer. Silencing ITGB6 inhibited the proliferation, migration, and fibrosis-like effects of activated PSCs and indirectly inhibited the metastasis and malignant process of pancreatic cancer by inhibiting the JAK2/STAT3 signaling pathway. Therefore, ITGB6 is a potential candidate target for pancreatic cancer prevention and treatment.

The context-dependent role of the Na+/Ca2+-exchanger (NCX) in pancreatic stellate cell migration

Pancreatic stellate cells (PSCs) that can co-metastasize with cancer cells shape the tumor microenvironment (TME) in pancreatic ductal adenocarcinoma (PDAC) by producing an excessive amount of extracellular matrix. This leads to a TME characterized by increased tissue pressure, hypoxia, and acidity. Moreover, cells within the tumor secrete growth factors. The stimuli of the TME trigger Ca2+ signaling and cellular Na+ loading. The Na+/Ca2+ exchanger (NCX) connects the cellular Ca2+ and Na+ homeostasis. The NCX is an electrogenic transporter, which shuffles 1 Ca2+ against 3 Na+ ions over the plasma membrane in a forward or reverse mode. Here, we studied how the impact of NCX activity on PSC migration is modulated by cues from the TME. NCX expression was revealed with qPCR and Western blot. [Ca2+]i, [Na+]i, and the cell membrane potential were determined with the fluorescent indicators Fura-2, Asante NaTRIUM Green-2, and DiBAC4(3), respectively. PSC migration was quantified with live-cell imaging. To mimic the TME, PSCs were exposed to hypoxia, pressure, acidic pH (pH 6.6), and PDGF. NCX-dependent signaling was determined with Western blot analyses. PSCs express NCX1.3 and NCX1.9. [Ca2+]i, [Na+]i, and the cell membrane potential are 94.4 nmol/l, 7.4 mmol/l, and − 39.8 mV, respectively. Thus, NCX1 usually operates in the forward (Ca2+ export) mode. NCX1 plays a differential role in translating cues from the TME into an altered migratory behavior. When NCX1 is operating in the forward mode, its inhibition accelerates PSC migration. Thus, NCX1-mediated extrusion of Ca2+ contributes to a slow mode of migration of PSCs.

Nintedanib Alleviates Chronic Pancreatitis by Inhibiting the Activation of Pancreatic Stellate Cells via the JAK/STAT3 and ERK1/2 Pathways.

Nintedanib (Ninte) has been approved for the treatment of pulmonary fibrosis, and whether it can ameliorate chronic pancreatitis (CP) is unknown. This study was conducted to investigate the effect and molecular mechanism of Ninte on pancreatic fibrosis and inflammation in vivo and in vitro. The caerulein-induced CP model of murine was applied, and Ninte was orally administered. Pathological changes in pancreas were evaluated using hematoxylin & eosin, Sirius Red, Masson's trichrome, and anti-Ki-67 staining. For in vitro studies, the effects of Ninte on cell viability, apoptosis, and migration of pancreatic stellate cells (PSCs) were determined by CCK-8, flow cytometry, and wound healing assays, respectively. The potential molecular mechanisms of the effects of Ninte on PSCs were analyzed by RNA-Seq and verified at the gene expression and protein activity levels by qRT-PCR and Western Blot. Ninte significantly alleviated the weight loss in mice with caerulein-induced CP and simultaneously attenuated the pancreatic damage, as evidenced by reduced acinar atrophy, collagen deposition, infiltration of inflammatory cells, and inhibited cell proliferation/regeneration. Besides, Ninte markedly suppressed the transcription of fibrogenic and proinflammatory genes in pancreatic tissues. Further in vitro studies showed that Ninte significantly inhibited the transcription and protein expression of genes corresponding to fibrogenesis and proliferation in PSCs. The results of RNA-Seq analysis and subsequent verification assays indicated that Ninte inhibited the activation and proliferation of PSCs via the JAK/STAT3 and ERK1/2 pathways. These findings indicate that Ninte may be a potential anti-inflammatory and anti-fibrotic therapeutic agent for CP.

Abstract 1328: Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system

Abstract Despite the use of multi-agent conventional chemotherapy regimens, metastatic pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal solid tumors with a 5-year survival of 9%. PDAC is characterized by a complex tumor microenvironment with multilayered interplay between neoplastic and stromal cells and the natural extracellular matrix (ECM). To date, models that recapitulate the complexity of these features are only partially reproduced ex vivo and lack the capacity to recreate PDAC tumor microenvironment physiology, fluid flow and the ECM architecture. To address these issues, we developed Curiochips MPS platform for metastatic PDAC on-a-chip. To replicate an accurate PDAC complex tumor microenvironment, we investigated human pancreatic cancer cell lines with/without SMAD4 mutation as a test model. SMAD4 is among the most frequently altered genes in PDAC and its mutation accelerates pancreatic cancer cell migration. To tailor cell-ECM interactions and recreate natural biochemical environment of tumors, we examined various ECM conditions. ECM substitutes such as type I collagen and fibrin gel offer mechanical properties to closely mimic the tumor ECM. We accessed the effect of ECM condition on the pancreatic stellate cells (PSC) activation and pancreatic cancer cell migration using PANC-1 (SMAD4-WT) and BxP3 (SMAD4-null) cell lines. Notably, type I collagen induced more PSC activation than fibrin gel. Interestingly BxP3 spheroids migrated toward to the endothelial compartment compared to PANC-1. Angiogenesis of endothelial cells was more prominent in co-culture with BxP3 than PANC-1. Furthermore, we are building the metastatic model using PDAC patient derived organoids and co-culture with primary PSC in different types of ECM. Preliminary analysis showed functional responses such as 3D organoid migration and aggregation, PSC activation and migration controlled by specific properties of EMC. Our data highlight the importance of the ECM composition on PDAC migration, PSC activation and angiogenesis in the context of genetic heterogeneity and presence/absence oncogenic mutation. The 3D co-culture platform, Curiochips offers suitable and tailored solution to investigate tumor-stroma interaction and potentially can be used as a platform for drug discovery and precision oncology to target PDAC desmoplastic tumor microenvironment. Citation Format: Sumin Kim, Yeongmin Choi, Jihye Baek, Bharti Garg, Jan Pencik, Andrew M. Lowy, Herve Tiriac, Sanghee Yoo. Building a 3D co-culture model for metastatic pancreatic ductal cancer using Curiochips microphysiological system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1328.

Tcf21 Alleviates Pancreatic Fibrosis by Regulating the Epithelial-Mesenchymal Transformation of Pancreatic Stellate Cells.

The activation of pancreatic stellate cells (PSCs) plays a key role in the occurrence and development of chronic pancreatitis (CP) and pancreatic fibrosis, which is related to the process of epithelial-mesenchymal transition (EMT). This study was designed to investigate the effect and mechanism of Tcf21 (one of tumor suppressor genes) on pancreatic inflammation and fibrosis in vivo and in vitro. C57BL/6 male mice were intraperitoneally injected with caerulein for 6weeks to establish CP animal model. Fixed pancreatic tissue paraffin-embedded sections were used for immunohistochemistry staining of Tcf21, fibrosis-related markers (α-SMA), interstitial markers (Vimentin) and epithelial markers (E-cadherin). Western blotting and qRT-PCR assay were performed to analyze the change of expression of the above markers after stimulation of TGF-β1 or overexpressed Tcf21 lentivirus transfection in human pancreatic stellate cells (HPSCs). The pancreatic expression of α-SMA and Vimentin of CP mice significantly increased, while the expression of Tcf21 and E-cadherin significantly decreased. TGF-β1 could promote activation and EMT process of HPSCs, and inhibited the expression of Tcf21. Overexpression of Tcf21 could significantly down-regulate the expression of α-SMA, Fibronectin and Vimentin, and up-regulated the expression of ZO-1 of HPSCs. Cell Counting Kit-8 assay and scratch wound-healing assay results showed that overexpression of Tcf21 could significantly inhibit the cell migration and proliferation of HPSCs. Overexpression of Tcf21 could significantly alleviate the activation, proliferation, migration of PSCs by regulating the EMT process. Tcf21 had a potential prospect of a new target for CP therapy.

5-FU-miR-15a Inhibits Activation of Pancreatic Stellate Cells by Reducing YAP1 and BCL-2 Levels In Vitro.

Chronic pancreatitis is characterized by chronic inflammation and fibrosis, processes heightened by activated pancreatic stellate cells (PSCs). Recent publications have demonstrated that miR-15a, which targets YAP1 and BCL-2, is significantly downregulated in patients with chronic pancreatitis compared to healthy controls. We have utilized a miRNA modification strategy to enhance the therapeutic efficacy of miR-15a by replacing uracil with 5-fluorouracil (5-FU). We demonstrated increased levels of YAP1 and BCL-2 (both targets of miR-15a) in pancreatic tissues obtained from Ptf1aCreERTM and Ptf1aCreERTM;LSL-KrasG12D mice after chronic pancreatitis induction as compared to controls. In vitro studies showed that delivery of 5-FU-miR-15a significantly decreased viability, proliferation, and migration of PSCs over six days compared to 5-FU, TGFβ1, control miR, and miR-15a. In addition, treatment of PSCs with 5-FU-miR-15a in the context of TGFβ1 treatment exerted a more substantial effect than TGFβ1 alone or when combined with other miRs. Conditioned medium obtained from PSC cells treated with 5-FU-miR-15a significantly inhibits the invasion of pancreatic cancer cells compared to controls. Importantly, we demonstrated that treatment with 5-FU-miR-15a reduced the levels of YAP1 and BCL-2 observed in PSCs. Our results strongly suggest that ectopic delivery of miR mimetics is a promising therapeutic approach for pancreatic fibrosis and that 5-FU-miR-15a shows specific promise.

ITGA5 inhibition in pancreatic stellate cells re-educates the in vitro tumor-stromal crosstalk.

The interaction between pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs) promotes aggressive progression of pancreatic cancer, and disrupting the tumor-stromal crosstalk is a promising therapeutic strategy. Integrin α5 (ITGA5) is specifically overexpressed in pancreatic cancer stroma and activated PSCs. ITGA5 acts as a mediator in PCCs-PSCs interaction, but its role in regulating biological behaviors of PSCs and PCCs is still not quite clear. In this study, ITGA5 in PSCs was inhibited using its specific inhibitor AV3 peptide or siRNA knockdown technique. Pancreatic cancer SW1990 cells conditioned medium (SW1990-CM) and an indirect co-culture system were used to mimic the environment of the in vitro tumor-stromal crosstalk. Our results showed that ITGA5 inhibition impaired the proliferation and migration of PSCs, but enhanced autophagy. After co-culture with PSCs, SW1990 cells gained some cancer stem cells (CSCs)-like characteristics, such as increased drug resistance, migration and invasion ability, but PSCs with ITGA5 knockdown were incapable of producing these effects. The present results suggested that ITGA5 was involved in the development of the malignant biological behaviors of PSCs and PCCs, and ITGA5 inhibition in PSCs might benefit the treatment of pancreatic cancer by re-educating PCCs-PSCs interaction.

Exosomal miR-485-3p derived from pancreatic ductal epithelial cells inhibits pancreatic cancer metastasis through targeting PAK1.

Cell competition is an important feature in pancreatic cancer (PC) progression, but the underlying mechanism remains elusive. This study aims to explore the role of exosomes derived from normal pancreatic ductal epithelial cells involved in PC progression. PC cells and pancreatic stellate cells (PSCs) were treated with exosomes isolated from pancreatic ductal epithelial cells. Cell proliferation was assessed by CCK8 assays. Cell migration and invasion were assessed by Transwell assays. PC and matched adjacent non-tumor tissue specimens were obtained from 46 patients pathologically diagnosed with PC at Peking University First Hospital from 2013 to 2017. Tissue miR-485-3p and p21-activated kinase-1 (PAK1) expression was examined by real-time polymerase chain reaction (RT-PCR), and the relationship of the two was analyzed using Pearman's product-moment correlation. The clinical significance of miR-485-3p was analyzed using the Chi-square test, Wilcoxon rank-sum test, and Fisher exact probability, respectively. The binding of miR-485-3p to PAK1 5'-untranslated region (5'-UTR) was examined by luciferase assay. PC cells were xenografted into nude mice as a PC metastasis model. Exosomes from pancreatic ductal epithelial cells suppressed PC cell migration and invasion as well as the secretion and migration of PSCs. MiR-485-3p was enriched in the exosomes of pancreatic ductal epithelial cells but deficient in those of PC cells and PSCs, in accordance with the lower level in PSCs and PC cells than that in pancreatic ductal cells. And the mature miR-485-3p could be delivered into these cells by the exosomes secreted by normal pancreatic duct cells, to inhibit PC cell migration and invasion. Clinical data analysis showed that miR-485-3p was significantly decreased in PC tissues (P < 0.05) and was negatively associated with lymphovascular invasion (P = 0.044). As a direct target of miR-485-3p, PAK1 was found to exert an inhibitory effect on PC cells, and there was a significantly negative correlation between the expression levels of miR-485-3p and PAK1 (r = -0.6525, P < 0.0001) in PC tissues. Moreover, miR-485-3p could suppress PC metastasis in vivo by targeting p21-activated kinase-1. Exosomal miR-485-3p delivered by normal pancreatic ductal epithelial cells into PC cells inhibits PC metastasis by directly targeting PAK1. The restoration of miR-485-3p by exosomes or some other vehicle might be a novel approach for PC treatment.

P-element-Induced Wimpy-Testis-Like Protein 1 Regulates the Activation of Pancreatic Stellate Cells Through the PI3K/AKT/mTOR Signaling Pathway.

Pancreatic fibrosis is the main pathological characteristic of chronic pancreatitis (CP) and pancreatic cancer. Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. Any targets that may have an impact on the activation of PSCs could become potential treatment candidates for CP and pancreatic cancer. Our goal was to investigate the effect of P-element-induced wimpy-testis (PIWI) protein 1 (PIWIL1) on PSC activation. Lentivirus-based RNA interference (RNAi) and overexpression vector construction were used to knock down and over-express the PIWIL1 protein. Immunocytofluorescent staining, western blotting, wound healing assay, transwell assay, and phalloidin staining were used to investigate the effects of PIWIL1 on the secretion of extracellular matrix components (EMC), actin cytoskeleton, and on the invasion and migration abilities of primary PSCs isolated from C57BL/6 mice. Moreover, pancreatic fibrosis was induced by L-arginine in C57BL/6 mice. The expression of PIWIL1 and collagen deposition in vivo were tested by western blotting and Sirius red staining. Expression levels of collagen I, collagen III, and α-smooth muscle actin were significantly decreased in the LV-PIWIL1 group. Compared with the si-PIWIL1 group, significant differences were observed in the expression of desmin, p-PI3K, p-AKT, and p-mTOR in the LV-PIWIL1 group. Furthermore, PIWIL1 suppressed the PSCs' invasion and migration abilities. In a rescue experiment, the PI3K/AKT/mTOR signaling pathway was found to be the underlying mechanism in PSCs activation mediated by PIWIL1. Our findings suggest that PIWIL1 inhibits the activation of PSCs via the PI3K/AKT/mTOR signaling pathway. PIWIL1 is a potential therapeutic target for pancreatic fibrosis.

Puerarin Ameliorates Caerulein-Induced Chronic Pancreatitis via Inhibition of MAPK Signaling Pathway.

Pancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP), and pancreatic stellate cells (PSCs) are considered to be the key cells. Puerarin is the most important flavonoid active component in Chinese herb Radix Puerariae, and it exhibited anti-fibrotic effect in various fibrous diseases recently. However, the impact and molecular mechanism of puerarin on CP and pancreatic fibrosis remain unknown. This study systematically investigated the effect of puerarin on CP and pancreatic fibrosis in vivo and in vitro. H&E staining, Sirius Red staining, qRT-PCR and Western blotting analysis of fibrosis and inflammation related genes of pancreatic tissues showed that puerarin notably ameliorated pancreatic atrophy, inflammation and fibrosis in a model of caerulein-induced murine CP. Western blotting analysis of pancreatic tissues showed the phosphorylation level of MAPK family proteins (JNK1/2, ERK1/2 and p38 MAPK) significantly increased after modeling of cerulein, while puerarin could inhibit their phosphorylation levels to a certain extent. We found that puerarin exerted a marked inhibition on the proliferation, migration and activation of PSCs, determined by CCK-8 assay, transwell migration assay, scratch wound-healing assay and expression levels of α-SMA, Fibronectin, Col1α1 and GFAP. Western blotting result demonstrated that puerarin markedly inhibited the phosphorylation of MAPK family proteins (JNK1/2, ERK1/2 and p38 MAPK) of PSCs in a dose-dependent manner whether or not stimulated by platelet-activating factor. In conclusion, the present study showed that puerarin could be a potential therapeutic candidate in the treatment of CP, and the MAPK pathway might be its important target.

TGF-β1 induced proliferation, migration, and ECM accumulation through the SNHG11/miR-34b/LIF pathway in human pancreatic stellate cells

Chronic pancreatitis (CP) is a chronic inflammatory and fibrotic disease of the pancreas, and activated pancreatic stellate cells (PSCs) play a vital role in the progression of pancreatic fibrosis in CP. It has been reported that long non-coding RNA small nucleolar RNA host gene 11 (SNHG11) is highly expressed in chronic pancreatitis (CP) patients. However, the role of SNHG11 in CP progression is unclear. The purport of the study was to survey the role of SNHG11 in CP. We employed transforming growth factor (TGF)-beta1 (TGF-β1) to activate human pancreatic stellate cells (PSCs). Expression of SNHG11 was assessed with qRT-PCR. Loss-of-function experiments were executed to evaluate the effects of SNHG11 on the proliferation and migration of TGF-β1-treated PSCs. Some protein levels were detected by western blotting. The regulatory mechanism of SNHG11 was verified by the dual-luciferase reporter and RIP assays. As a result, SNHG11 was upregulated in plasma of CP patients and TGF-β1-treated PSCs. Also, SNHG11 inhibition reduced TGF-β1-induced proliferation, migration, and ECM accumulation in PSCs. Mechanistically, SNHG11 regulated leukemia inhibitory factor (LIF) expression by sponging miR-34b. Furthermore, miR-34b inhibitor abolished SNHG11 silencing-mediated effects on TGF-β1-treated PSC proliferation, migration, and ECM accumulation. LIF overexpression counteracted the repressive influence of miR-34b mimic on proliferation, migration, and ECM accumulation of TGF-β1-treated PSCs. In conclusion, SNHG11 knockdown reduced TGF-β1-induced PSC proliferation, migration, and ECM accumulation by the miR-34b/LIF axis.

ASIC1 inhibition impairs the proliferation and migration of pancreatic stellate cells induced by pancreatic cancer cells.

Activated pancreatic stellate cells (PSCs) with an increased proliferation and migration ability are the partners in crime with pancreatic cancer cells. Acid-sensing ion channel 1 (ASIC1) is expressed in pancreatic cancer and PSCs, and especially, it mediates the activation of PSCs. However, whether ASIC1 is involved in pancreatic cancer cells-induced biological behavior re-educating of PSCs is unclear. In this study, the change of ASIC1 expression in PSCs and pancreatic cancer Panc-1 cells after in-direct co-culture was detected by western blotting, and the proliferation and migration of PSCs with ASIC1 knockdown under Panc-1 cells-conditioned medium (Panc-1-CM) was assessed. The results showed that pancreatic cancer cells induced ASIC1 overexpression, and the enhanced proliferation and migration of PSCs was weakened by ASIC1 inhibition. In addition, the extracellular signal-regulated kinase (ERK) expression in PSCs remained stable, but the phosphorylated ERK (p-ERK) expression in PSCs treated with Panc-1-CM increased, which was suppressed by ASIC1 knockdown. These results indicate that ASIC1 participates in the regulation of PSCs proliferation and migration induced by cancer cells via the ERK pathway, and ASIC1 inhibition may be beneficial to pancreatic cancer treatment.

Curcumin inhibits pancreatic cancer cell invasion and EMT by interfering with tumor‑stromal crosstalk under hypoxic conditions via the IL‑6/ERK/NF‑κB axis

Hypoxic microenvironment and pancreatic stellate cells (PSCs) play important roles in pancreatic cancer progression. PSCs secrete a number of soluble factors, such as interleukin (IL)‑6, to facilitate cancer metastasis. Our previous study revealed that curcumin inhibited the invasive ability of pancreatic cancer cells by modulating epithelial‑to‑mesenchymal transition (EMT)‑related factors. However, whether curcumin could suppress tumor‑stromal crosstalk in pancreatic cancer and the underlying mechanisms have yet to be fully elucidated. The aim of the present study was to evaluate whether curcumin could affect pancreatic cancer cell invasion and EMT by interfering with tumor‑stromal interaction under hypoxic conditions. The PSCs were treated with curcumin under hypoxic conditions. The activation of PSCs was detected by testing the expression of α‑smooth muscle actin by western blotting and immunofluorescence analysis. The wound healing assay was used to evaluate the migratory potential of PSCs. The secretion and expression of IL‑6 by PSCs was detected by ELISA and reverse transcription‑quantitative PCR (RT‑qPCR) analysis. BxPC‑3 and Panc‑1 cells were treated with PSC‑conditioned media (PSC‑CM), IL‑6, IL‑6‑neutralizing antibody or curcumin under conditions of normoxia or hypoxia. Transwell invasion assay was used to examine the invasive potential of pancreatic cancer cells. The activation of phosphorylated (p‑) extracellular signal‑regulated kinase (ERK) and p‑nuclear factor (NF)‑κB were measured by western blot analysis. The expression of EMT‑related genes at the mRNA and protein levels was detected by RT‑qPCR and western blot analysis, respectively. The results of the present study demonstrated that curcumin inhibited the activation and migration of PSCs under hypoxic conditions. Curcumin also suppressed the secretion and expression of IL‑6 in PSCs. In addition, curcumin and IL‑6‑neutralizing antibody treatment suppressed PSC‑CM‑modulated pancreatic cancer invasion, EMT and the changes in the expression of E‑cadherin, vimentin and matrix metallopeptidase‑9. Furthermore, the increase in the levels of p‑ERK and p‑NF‑κB induced by PSC‑CM could be counterbalanced by both curcumin and IL‑6‑neutralizing antibody treatment under hypoxic conditions. Taken together, these data indicate that curcumin plays an important role in suppressing tumor‑stromal crosstalk and pancreatic cancer metastasis by inhibiting the IL‑6/ERK/NF‑κB axis. Blocking the IL‑6/ERK/NF‑κB axis by curcumin may be a promising therapeutic strategy for the treatment of pancreatic cancer.

Protonation of Piezo1 Impairs Cell-Matrix Interactions of Pancreatic Stellate Cells.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an acidic and fibrotic stroma. The extracellular matrix (ECM) causing the fibrosis is primarily formed by pancreatic stellate cells (PSCs). The effects of the altered biomechanics and pH landscape in the pathogenesis of PDAC, however, are poorly understood. Mechanotransduction in cells has been linked to the function of mechanosensitive ion channels such as Piezo1. Here, we tested whether this channel plays crucial roles in transducing mechanical signals in the acidic PDAC microenvironment. We performed immunofluorescence, Ca2+ influx and intracellular pH measurements in PSCs and complemented them by live-cell imaging migration experiments in order to assess the function of Piezo1 channels in PSCs. We evaluated whether Piezo1 responds to changes of extracellular and/or intracellular pH in the pathophysiological range (pH 6.6 and pH 6.9, respectively). We validated our results using Piezo1-transfected HEK293 cells as a model system. Indeed, acidification of the intracellular space severely inhibits Piezo1-mediated Ca2+ influx into PSCs. In addition, stimulation of Piezo1 channels with its activator Yoda1 accelerates migration of PSCs on a two-dimensional ECM as well as in a 3D setting. Furthermore, Yoda1-activated PSCs transmit more force to the surrounding ECM under physiological pH, as revealed by measuring the dislocation of microbeads embedded in the surrounding matrix. This is paralleled by an enhanced phosphorylation of myosin light chain isoform 9 after Piezo1 stimulation. Intriguingly, upon acidification, Piezo1 activation leads to the initiation of cell death and disruption of PSC spheroids. In summary, stimulating Piezo1 activates PSCs by inducing Ca2+ influx which in turn alters the cytoskeletal architecture. This results in increased cellular motility and ECM traction, which can be useful for the cells to invade the surroundings and to detach from the tissue. However, in the presence of an acidic extracellular pH, although net Ca2+ influx is reduced, Piezo1 activation leads to severe cell stress also limiting cellular viability. In conclusion, our results indicate a strong interdependence between environmental pH, the mechanical output of PSCs and stromal mechanics, which promotes early local invasion of PDAC cells.

Overexpression of Gremlin�1 by sonic hedgehog signaling promotes pancreatic cancer progression

Sonic hedgehog (SHH) signaling is an important promotor of desmoplasia, a critical feature in pancreatic cancer stromal reactions involving the activation of pancreatic stellate cells (PSCs). Gremlin 1 is widely overexpressed in cancer-associated stromal cells, including activated PSCs. In embryonic development, SHH is a potent regulator of Gremlin 1 through an interaction network. This subtle mechanism in the cancer microenvironment remains to be fully elucidated. The present study investigated the association between Gremlin 1 and SHH, and the effect of Gremlin 1 in pancreatic cancer. The expression of Gremlin 1 in different specimens was measured using immunohistochemistry. The correlations among clinico-pathological features and levels of Gremlin 1 were evaluated. Primary human PSCs and pancreatic cancer cell lines were exposed to SHH, cyclopamine, GLI family zinc finger-1 (Gli-1) small interfering RNA (siRNA), and Gremlin 1 siRNA to examine their associations and effects using an MTT assay, reverse transcription-quantitative polymerase chain reaction analysis, western blot analysis, and migration or invasion assays. The results revealed the overexpression of Gremlin 1 in pancreatic cancer tissues, mainly in the stroma. The levels of Gremlin 1 were significantly correlated with survival rate and pT status. In addition, following activation of the PSCs, the expression levels of Gremlin 1 increased substantially. SHH acts as a potent promoter of the expression of Gremlin 1, and cyclopamine and Gli-1 siRNA modulated this effect. In a screen of pancreatic cancer cell lines, AsPC-1 and BxPC-3 cells expressed high levels of Gremlin 1, but only AsPC-1 cells exhibited a high expression level of SHH. The results of the indirect co-culture experiment suggested that paracrine SHH from the AsPC-1 cells induced the expression of Gremlin 1 in the PSCs. Furthermore, Gremlin 1 siRNA negatively regulated the proliferation and migration of PSCs, and the proliferation, invasion and epithelial-mesenchymal transition of AsPC-1 and BxPC-3 cells. Based on the data from the present study, it was concluded that an abnormal expression level of Gremlin 1 in pancreatic cancer was induced by SHH signaling, and that the overexpression of Gremlin 1 enabled pancreatic cancer progression.

Regulation of pancreatic stellate cell activation by Notch3

BackgroundActivated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC.MethodsThe expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student’s t-test.ResultsNotch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells.ConclusionsNotch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the PaSC-induced pro-tumorigenic effect. Therefore, Notch3 silencing in PaSCs is a potential novel therapeutic option for patients with PDAC.

Resveratrol Inhibits ROS-Promoted Activation and Glycolysis of Pancreatic Stellate Cells via Suppression of miR-21.

Activation of pancreatic stellate cells (PSCs) initiates pancreatic fibrosis in chronic pancreatitis and furnishes a niche that enhances the malignancy of pancreatic cancer cells (PCCs) in pancreatic ductal adenocarcinoma (PDAC). Resveratrol (RSV), a natural polyphenol, exhibits potent antioxidant and anticancer effects. However, whether and how RSV influences the biological properties of activated PSCs and the effects of these changes on tumor remain unknown. In the present study, we found that RSV impeded hydrogen peroxide-driven reactive oxygen species- (ROS-) induced activation, invasion, migration, and glycolysis of PSCs. In addition, miR-21 expression in activated PSCs was downregulated after RSV treatment, whereas the PTEN protein level increased. miR-21 silencing attenuated ROS-induced activation, invasion, migration, and glycolysis of PSCs, whereas the overexpression of miR-21 rescued the responses of PSCs treated with RSV. Moreover, RSV or N-acetyl-L-cysteine (NAC) administration or miR-21 knockdown in PSCs reduced the invasion and migration of PCCs in coculture, and the effects of RSV were partly reversed by miR-21 upregulation. Collectively, RSV inhibits PCC invasion and migration through suppression of ROS/miR-21-mediated activation and glycolysis in PSCs. Therefore, targeting miR-21-mediated glycolysis by RSV in tumor stroma may serve as a new strategy for clinical PDAC prevention or treatment.

Abstract 2961: Targeting stromal-derived Dickkopf-3 (DKK3) for the treatment of pancreatic ductal adenocarcinoma (PDAC)

Abstract Introduction: We and others have shown that pancreatic stellate cells (PSCs) in the tumor-associated stroma of PDAC promote tumor progression and resistance to therapy but the precise mechanisms are unclear. We investigated the role of PSC-derived DKK3, a member of the Dickkopf family of glycoproteins, in PDAC progression, metastasis and response to chemotherapy. Methods: We evaluated expression of DKK3 in human PDAC tissue and cell lines, human PSCs and in a genetically engineered mouse model (GEMM) of PDAC. The paracrine and autocrine effects of DKK3 on PDAC and PSCs were examined by treatment with exogenous DKK3 and gain- and loss of function assays for proliferation, migration, invasion, and gemcitabine-induced apoptosis. The effects of DKK3 on PDAC progression and metastasis were determined by shRNA neutralization and genetic ablation in orthotopic xenograft models and the KPC autochthonous model of PDAC. We developed novel monoclonal antibodies (mAbs) against DKK3 and tested their ability to neutralize DKK3 and prolong survival in mouse models of PDAC. Results: DKK3 was expressed at 4.5 times higher levels in human PDAC by Affymetrix profiling compared to normal pancreas and was present in 99% (118/119) of samples on a tissue microarray with moderate to high expression in 59%. In a GEMM of PDAC, DKK3 appeared early with preneoplastic PanIN lesions with increased expression in invasive carcinoma. DKK3 was strongly expressed by PSCs with minimal to no expression in PDAC cells and knockdown by shRNA reduced PSC proliferation and migration by 60% and 84% compared to controls (p&amp;lt;0.001). Treatment of Panc1 and BxPC3 cells with DKK3 stimulated migration and invasion by 100-300% (p&amp;lt;0.001) and proliferation of DKK3-silenced Panc1 cells was decreased by 80% (p&amp;lt;0.001). Overexpression of DKK3 in L3.6pl cells increased colony formation in gemcitabine by &amp;gt;90% (p&amp;lt;0.001) with 65% reduction in apoptosis (p&amp;lt;0.01), indicating that DKK3 contributes to PDAC resistance to chemotherapy. When we ablated DKK3 in KPC mice by breeding with DKK3-knockout mice, tumor growth was inhibited and survival increased by 45% (p=0.0002). In addition, fewer PanIN lesions developed in DKK3-null mice suggesting that DKK3 may contribute to the early developmental stages of PDAC. DKK3 neutralizing mAbs abrogated DKK3-mediated induction of PDAC cell migration, invasion and resistance to gemcitabine in vitro. Furthermore, treatment with DKK3 mAb significantly inhibited primary tumor growth, reduced peritoneal metastases and prolonged survival in an orthotopic model of PDAC by 43% compared to control mAb (p=0.005; HR 0.24, 95% CI 0.01-0.30). Conclusions: These data are the first report, to our knowledge, of a tumor-promoting function of DKK3 and our results suggest that neutralization of DKK3 may be an effective approach as a primary treatment for PDAC and to enhance responsiveness to chemotherapy. Citation Format: Liran Zhou, Hongmei Husted, Todd Moore, Mason Lu, Defeng Deng, Yan Liu, Vijaya Ramachandran, Thiruvengadam Arumugam, Baoan Ji, Huamin Wang, Jeffrey E. Lee, Craig D. Logsdon, Rosa F. Hwang. Targeting stromal-derived Dickkopf-3 (DKK3) for the treatment of pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2961. doi:10.1158/1538-7445.AM2017-2961

Ion channels in control of pancreatic stellate cell migration.

Pancreatic stellate cells (PSCs) play a critical role in the progression of pancreatic ductal adenocarcinoma (PDAC). Once activated, PSCs support proliferation and metastasis of carcinoma cells. PSCs even co-metastasise with carcinoma cells. This requires the ability of PSCs to migrate. In recent years, it has been established that almost all “hallmarks of cancer” such as proliferation or migration/invasion also rely on the expression and function of ion channels. So far, there is only very limited information about the function of ion channels in PSCs. Yet, there is growing evidence that ion channels in stromal cells also contribute to tumor progression. Here we investigated the function of KCa3.1 channels in PSCs. KCa3.1 channels are also found in many tumor cells of different origin. We revealed the functional expression of KCa3.1 channels by means of Western blot, immunofluorescence and patch clamp analysis. The impact of KCa3.1 channel activity on PSC function was determined with live-cell imaging and by measuring the intracellular Ca2+ concentration ([Ca2+]i). KCa3.1 channel blockade or knockout prevents the stimulation of PSC migration and chemotaxis by reducing the [Ca2+]i and calpain activity. KCa3.1 channels functionally cooperate with TRPC3 channels that are upregulated in PDAC stroma. Knockdown of TRPC3 channels largely abolishes the impact of KCa3.1 channels on PSC migration. In summary, our results clearly show that ion channels are crucial players in PSC physiology and pathophysiology.