Migration Of Pancreatic Ductal Adenocarcinoma Cells Research Articles

TNFR2 promotes pancreatic cancer proliferation, migration, and invasion via the NF-κB signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant disease with low overall survival; chemotherapy and immunotherapy have limited efficacy. Tumor necrosis factor receptor 2 (TNFR2), a type II transmembrane protein, contributes to the development and progression of several tumors. In this study, we elucidated the effect and molecular mechanisms of TNFR2. We used The Cancer Genome Atlas and the Genotype-Tissue Expression database to compare the expression of the TNFR2 gene between normal and malignant pancreatic tissue. Using immunohistochemical staining, we divided the patients into high and low-expression groups, then investigated clinicopathologic data and survival curves of pancreatic cancer patients. We measured TNFR2 protein expression in PANC-1 and ASPC-1 pancreatic cancer cells subjected to TNFR2 small interfering RNA or negative control treatment. We performed proliferation, invasion, and migration assays to study the biological effects of TNFR2 in PDAC. The molecular mechanisms were validated using western blotting. TNFR2 was more highly expressed in PDAC cells and tissues than controls. Abundant expression of TNFR2 was associated with aggressive clinicopathologic characteristics and poor outcomes. Overexpression of TNFR2 promoted PDAC cell proliferation, migration, and invasion in vitro. Mechanistically, TNFR2 binds to TNF-α and activates the NF-κB signaling pathway. TNFR2 is a prognostic marker that facilitates the proliferation, migration, and invasion of PDAC via the NF-κB signaling pathway. TNFR2 may become a therapeutic target.

Nuclear receptor coactivator 6 (NCoA6) promotes cell proliferation, migration, and invasion in pancreatic cancer.

Nuclear receptor coactivator 6 (NCoA6) is overexpressed in various cancers and considered a multifunctional coactivator of various transcription factors and nuclear receptors. However, the role of NCoA6 in pancreatic ductal adenocarcinoma (PDAC) remains unclear. NCoA6 expression data in PDAC were extracted from TCGA and GTEx databases, and their correlation with survival outcomes were analyzed using the Kaplan-Meier plotter database. NCoA6 protein expression in PDAC tissues was evaluated using immunohistochemistry. RNA-sequencing technology was used to sequence the transcriptome of NCoA6-silenced PANC-1 cells, followed by differential expression, GO/KEGG and GSEA analyses. The effects of NCoA6 on cell proliferation, migration, invasion, cell cycle, and apoptosis were determined in two representative cell lines (PANC-1 and SW1990). Western blotting, qPCR, and co-immunoprecipitation were performed to explore the mechanism of action of NCoA6 in PDAC cells. NCoA6 expression was markedly increased in PDAC tissues, and high NCoA6 expression was associated with poor survival prognosis. However, there was no significant relationship between NCoA6 expression and metastasis in PDAC patients. Our RNA-sequencing data analysis found 1194 significant differentially expressed genes between the control and NCoA6-silenced PANC-1 cells. GO/KEGG analysis results mainly focused on cytokine production, cytokine activity, and cytokine-cytokine receptor interactions. GSEA results showed that the knockdown of NCoA6 affected the expression of histone deacetylase 1 (HDAC1) targeted genes. NCoA6 knockdown suppressed proliferation, migration, and invasion of PDAC cells. Finally, western blotting, qPCR, and co-immunoprecipitation results showed that NCoA6 interacted with HDAC1 and that NCoA6 expression was negatively correlated with F-box and WD repeat domain-containing 7 (FBW7) and caudal-related homeobox transcription factor 2 (CDX2) expression in pancreatic cancer. NCoA6 has a profound effect on cell proliferation, migration, invasion, and prognosis of PDAC and is potentially related to the expression of HDAC1, FBW7, and CDX2. Our results may provide novel therapeutic strategies for PDAC patients.

Schwann cells regulate tumor cells and cancer-associated fibroblasts in the pancreatic ductal adenocarcinoma microenvironment

Neuropathy is a feature more frequently observed in pancreatic ductal adenocarcinoma (PDAC) than other tumors. Schwann cells, the most prevalent cell type in peripheral nerves, migrate toward tumor cells and associate with poor prognosis in PDAC. To unveil the effects of Schwann cells on the neuro-stroma niche, here we perform single-cell RNA-sequencing and microarray-based spatial transcriptome analysis of PDAC tissues. Results suggest that Schwann cells may drive tumor cells and cancer-associated fibroblasts (CAFs) to more malignant subtypes: basal-like and inflammatory CAFs (iCAFs), respectively. Moreover, in vitro and in vivo assays demonstrate that Schwann cells enhance the proliferation and migration of PDAC cells via Midkine signaling and promote the switch of CAFs to iCAFs via interleukin-1α. Culture of tumor cells and CAFs with Schwann cells conditioned medium accelerates PDAC progression. Thus, we reveal that Schwann cells induce malignant subtypes of tumor cells and CAFs in the PDAC milieu.

Protein tyrosine phosphatase nonreceptor type 2 exerts antimetastatic functions in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive tumor with a dismal prognosis. Recent studies have demonstrated PTPN2 (protein tyrosine phosphatase nonreceptor type 2) as a potential target for cancer therapy. However, the functions of PTPN2 in PDAC progression remain poorly understood. In this study, we found PTPN2 expression was downregulated in PDAC tissues, and decreased PTPN2 expression was associated with unfavorable prognosis. Functional studies indicated that PTPN2 knockdown promoted the migration and invasion abilities of PDAC cells in vitro, and the liver metastasis in vivo through epithelial-mesenchymal transition process. Mechanistically, MMP-1 was identified as a downstream target of PTPN2 via RNA-seq data and was responsible for the enhanced metastasis of PDAC cells upon PTPN2 knockdown. Moreover, according to chromatin immunoprecipitation and electrophoretic mobility shift assay, PTPN2 depletion transcriptionally activated MMP-1 via regulating the interaction of p-STAT3 with its distal promoter. This study, for the first time, demonstrated that PTPN2 inhibited PDAC metastasis, and presented a novel PTPN2/p-STAT3/MMP-1 axis in PDAC progression.

SPOCK2 gene expression is downregulated in pancreatic ductal adenocarcinoma cells and correlates with prognosis of patients with pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents a widespread form of malignant pancreatic neoplasms and a leading oncologic cause of death in Europe and the USA. Despite advances in understanding its molecular biology, the 5-year survival rate remains low at 10%. The extracellular matrix in PDAC contains proteins, including SPOCK2, which are essential for tumorigenicity and drug resistance. The present study aims to explore the possible role of SPOCK2 in the pathogenesis of PDAC. Expression of SPOCK2 was evaluated in 7 PDAC cell lines and 1 normal pancreatic cell line using quantitative RT-PCR. Demethylation of the gene was carried out using 5-aza-2'-deoxycytidine (5-aza-dC) treatment with subsequent validation Western Blot analysis. In vitro downregulation of SPOCK2 gene was performed using siRNA transfection. MTT and transwell assays were employed to evaluate the impact of the SPOK2 demethylation on the proliferation and migration of PDAC cells. KM Plotter was applied to analyze a correlation between SPOCK2 mRNA expression and the survival of PDAC patients. In contrast to the normal pancreatic cell line, SPOCK2 expression was significantly downregulated in PDAC cell lines. Treatment with 5-aza-dC, led to increase in SPOCK2 expression in the cell lines tested. Importantly, compared with control cells, transfected with SPOCK2 siRNA cells exhibited increased growth rates and more migration ability. Finally, we demonstrated that a high SPOCK2 expression level correlated with longer overall survival of patients with PDAC. The expression of SPOCK2 is downregulated in PDAC as a result of hypermethylation of its corresponding gene. SPOCK2 expression as well as the demethylation of its gene could be a potential marker for PDAC.

Qingyihuaji Formula promotes apoptosis and autophagy through inhibition of MAPK/ERK and PI3K/Akt/mTOR signaling pathway on pancreatic cancer in vivo and in vitro.

Qingyihuaji Formula (QYHJ), a widely used traditional Chinese medicine (TCM), has been used to treat patients with cancer in China. However, the effect and mechanism of QYHJ on pancreatic ductal adenocarcinoma (PDAC) remains unclear. This study aimed to explore the roles and evaluate the possible underlying molecular mechanisms of QYHJ and its core component in PDAC using label-free quantitative proteomics in conjunction with network pharmacology-based analysis. By screening differentially expressed proteins (DEPs) in proteomics and QYHJ-predicted gene sets, we identified QYHJ-related PDAC targets annotated with bioinformatic analysis. A subcutaneous tumor model was established to assess the role of QYHJ in vivo. The effects of quercetin (Que), a core component of QYHJ, on cell proliferation, migration, invasion, apoptosis, and autophagy in SW1990 and PANC-1cells were investigated in vitro. Immunohistochemistry, western blotting, mRFP-GFP-LC3 adenovirus, and kinase analysis were used to determine the underlying mechanisms. Bioinformatics analysis revealed that 41 QYHJ-related PDAC targets were closely related to the cellular response to nitrogen compounds, positive regulation of cell death, regulation of epithelial cell apoptotic processes, and chemokine signaling pathways. CASP3, SRC, STAT1, PTPN11, PKM, and PAK1 with high expression were identified as hub DEPs in the PPI network, and these DEPs were associated with poor overall survival and STAT 1, MAPK/ERK, and PI3K/Akt/mTOR signaling pathways in PDAC patients. QYHJ significantly promoted tumor death in nude mice. Moreover, quercetin inhibited the proliferation, migration, and invasion of PDAC cells. Additionally, Que induced apoptosis and autophagy in PDAC cells. Mechanistically, QYHJ and Que significantly activated STAT 1 and remarkably inhibited the MAPK/ERK and PI3K/Akt/mTOR signaling pathways in vivo and in vitro, respectively. Importantly, ERK1/2 inactivation contributes to que-induced apoptosis in SW1990 and PANC-1cells. These results suggest that QYHJ and Que are promising anti-PDAC avenues that benefit from their multiform mechanisms.

CircPTPRA promotes the progression of pancreatic ductal adenocarcinoma via the miR-140-5p/LMNB1 axis.

Growing evidences suggest that circular RNAs (circRNAs) are important factors in cancer progression. Nevertheless, the role of circRNAs in the progression of pancreatic ductal adenocarcinoma (PDAC) remains unclear. CircPTPRA was identified based on our previous circRNA array data analysis. Wound healing, transwell, and EdU assays were performed to investigate the effect of circPTPRA on the migration, invasion, and proliferation of PDAC cells in vitro. RNA pull-down, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP), and dual-luciferase reporter assays were conducted to verify the binding of circPTPRA with miR-140-5p. Subcutaneous xenograft model was constructed for in vivo experiment. CircPTPRA was significantly upregulated in PDAC tissues and cells compared to normal controls. Moreover, circPTPRA overexpression was positively correlated with lymph node invasion and worse prognosis in PDAC patients. In addition, overexpression of circPTPRA promoted PDAC migration, invasion, proliferation, and epithelial-mesenchymal transition (EMT) in vitro and in vivo. Mechanistically, circPTPRA upregulates LaminB1 (LMNB1) expression by sponging miR-140-5p and ultimately promotes the progression of PDAC. This study revealed that circPTPRA plays an important role in the progression of PDAC by sponging miR-140-5p. It can be explored as a potential prognostic marker and therapeutic target for PDAC.

Abstract 2218: The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma

Abstract KRAS is frequently mutated in pancreatic ductal adenocarcinoma (PDAC). However, targeting mutant KRAS is still challenging. Kinase inhibitors are ideal targeted therapeutics for mutant KRAS-driven cancer. In our previous study, an esiRNA screening was prepared to identify some vital kinases that took part in KRAS mutant-driven pancreatic cancer. The STK31 was identified as a potential therapeutic target in KRAS mutant PDAC. This study aimed to investigate the underlying mechanism of STK31 in KRAS mutant PDAC as well as its therapeutic potential. The results demonstrated that STK31 was upregulated in KRAS mutant PDAC patients with poor survival. STK31 was also highly expressed in PDAC cell lines with KRAS mutant background. Inhibition of STK31 in KRAS mutant cell lines significantly reduced PDAC cell proliferation, colony formation, and migration. Gain and loss of function experiments revealed that STK31 was a downstream target of KRAS in PDAC. Pharmacological inhibition of several signaling pathways involved in KRAS showed MAPK/ERK signaling involved in STK31 regulation. Then, further mechanistic study validated that c-Jun, regulated by KRAS/MAPK/ERK signaling, directly regulated the transcription level of STK31 by binding to its promoter region. In summary, our results show that STK31 promotes PDAC cell proliferation under the control of the KRAS/MAPK/ERK/c-Jun transcriptional axis in PDAC. STK31 may act as a potential target in PDAC treatment. Citation Format: Yuting Liu, Shing Chun Tang, Yangchao Chen. The molecular mechanism underlying KRAS regulation on STK31 expression in pancreatic ductal adenocarcinoma [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 2218.

Abstract 5493: Novel strategy to make KRAS targeted therapies more effective for PDAC treatment

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) patients exhibit extremely poor prognosis. KRAS mutation on codon-12 is present in 70-95% of PDAC cases and it drives PDAC growth and progression. Galectin-1 (Gal-1) is present in both PDAC and stromal cells, being involved in tumor microenvironment, immune cell activation and metastasis. Therefore, this study discusses the efficiency of combined inhibition of mutated KRASG12D and Gal-1 inhibition to effectively suppress PDAC growth and progression. For this we have delivered KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: SPION nano-formulation was used to deliver siKRASG12D and investigate in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a KrasG12D;LSL-Trp53R172H syngeneic mouse model. Results: Our results demonstrate that SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells and tumor spheroid growth in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. We have used -KrasG12D;LSL-Trp53R172H syngeneic mouse model of PDAC for investigating efficacy of combined SP-siKRAS formulation and galectin-1 inhibitor. Our results showed that the combination treatment inhibited the fibrotic tumor growth and increased survival rate. The combined treatment increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 50% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 57% (FoxP3+CD25+CD45+CD3+) and increased memory T cells by 34% in mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability. Citation Format: Ana I. Bulnes, Orlando Garcia, Poornima Shaji, Swathi Holla, Nirnoy Dan, Anupam Dhasmana, Shabnam Malik, Murali Yallapu, Stephen Behrman, Subhash Chauhan, Sheema S. Khan. Novel strategy to make KRAS targeted therapies more effective for PDAC treatment. [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 5493.

Abstract 1337: Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression

Abstract Advancing age is a major independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cells that eventually comprise the PDAC tumor microenvironment (TME) are incompletely understood. Fibroblasts comprise a large part of PDAC tumors, can alter the behavior of cancer cells, and impact treatment responses. Our studies investigate how normal pancreatic fibroblasts acquire tumor-promoting properties during aging and alter the tumorigenic properties of PDAC both in vitro and in vivo. To determine if PDAC tumor growth is altered by an aged microenvironment, we performed orthotopic injection of KPC and Panc02 PDAC cells into the pancreata of aged (>64-week-old) and young (6-8-week-old) C57Bl/6J mice. Tumors in aged mice are significantly larger compared to those in young mice, and there is increased incidence of liver metastases in the aged mouse cohort. We next queried whether aged pancreatic fibroblasts may contribute to this increase in tumor growth. We generated a panel of aged (donors >55 years old) and young (donors <35 years old) normal human pancreatic fibroblasts to determine the effects of healthy aging fibroblasts on PDAC cell behavior. Conditioned media from or co-culture with aged human pancreatic fibroblasts increases the proliferation, migration, and invasion of PDAC cells compared to young fibroblasts. Aged fibroblast conditioned media activated AKT signaling and enhances features of EMT and stemness. Proteomic analysis of conditioned media reveals a significantly altered secretome in aged fibroblasts, with a number of known tumor-promoting proteins released at higher levels from aged compared to young fibroblasts. We examined the contribution of GDF-15, secreted by aged fibroblasts at relatively high levels, as a paracrine modulator of PDAC growth and progression. Addition of recombinant GDF-15 to young fibroblast conditioned media increases proliferation and invasion of PDAC cells to levels similar to aged fibroblast conditioned media. Treatment of young mice with recombinant GDF-15 increases tumor growth and AKT phosphorylation in vivo. Additionally, inhibition of AKT reduces tumor size in aged but not young mice, suggesting that the aged microenvironment at least in part exerts its pro-tumor effects through AKT. In conclusion, we show that aged, non-cancer-associated pancreatic fibroblasts have the potential to promote growth, migration, and invasiveness in multiple models of pancreatic ductal adenocarcinoma. Further studies examining fibroblast-secreted factors and other changes in the aged TME are ongoing. Citation Format: Daniel J. Zabransky, Yash Chhabra, Mitchell Fane, Emma Kartalia, James Leatherman, Jacquelyn Zimmerman, Elizabeth Jaffee, Ashani Weeraratna. Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression [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 1337.

Knocking down GALNT6 promotes pyroptosis of pancreatic ductal adenocarcinoma cells through NF-κB/NLRP3/GSDMD and GSDME signaling pathway.

Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly lethal malignancy with poor prognosis. Polypeptide N-acetylgalactosaminyltransferase-6 (GALNT6) is frequently overexpressed in PDAC. However, the role of GALNT6 in the PDAC remains unclear. The expression of GALNT6 in pancreatic cancer and normal tissues were analyzed by bioinformatic analyses and immunohistochemistry. CCK8 and colony formation were used to detect cell proliferation. Flow cytometry was applied to detect cell cycle.The pyroptosis was detected by scanning electron microscopy. The mRNA expression was detected by qRT-PCR. The protein expression and localization were detected by western blot and immunofluorescence assay. ELISA was used to detect the levels of inflammatory factors. The expression of GALNT6 was associated with advanced tumor stage, and had an area under curve (AUC) value of 0.919 in pancreatic cancer based on the cancer genome atlas (TCGA) dataset. Knockdown of GALNT6 inhibited cell proliferation, migration, invasion and cell cycle arrest of PDAC cells. Meanwhile, knockdown of GALNT6 increased the expression levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-18 (IL-18), the release of inflammasome and an increasing of Gasdermin D (GSDMD), N-terminal of GSDMD (GSDMD-N), Gasdermin E (GSDME) and N-terminal of GSDME (GSDME-N) in PDAC cells. GALNT6 suppressed the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) and GSDMD by glycosylation of NF-κB and inhibiting the nucleus localization of NF-κB. Additionally, GALNT6 promotes the degradation of GSDME by O-glycosylation. We found that GALNT6 is highly expressed in pancreatic cancer and plays a carcinogenic role. The results suggested that GALNT6 regulates the pyroptosis of PDAC cells through NF-κB/NLRP3/GSDMD and GSDME signaling. Our study might provides novel insights into the roles of GALNT6 in PDAC progression.

M6A-modified circRNA MYO1C participates in the tumor immune surveillance of pancreatic ductal adenocarcinoma through m6A/PD-L1 manner

Emerging evidence indicates the critical roles of N6-methyladenosine (m6A) modification in human cancers. Herein, our work reported that a novel m6A-modified circRNA from the MYO1C gene, circMYO1C, upregulated in the pancreatic ductal adenocarcinoma (PDAC). Our findings demonstrated that circMYO1C is highly expressed in PDAC tissues. Functionally, circMYO1C promoted the proliferation and migration of PDAC cells in vitro and its silencing reduced the tumor growth in vivo. Mechanistically, circMYO1C cyclization was mediated by m6A methyltransferase METTL3. Moreover, methylated RNA immunoprecipitation sequencing (MeRIP-seq) unveiled the remarkable m6A modification on PD-L1 mRNA. Moreover, circMYO1C targeted the m6A site of PD-L1 mRNA to enhance its stability by cooperating with IGF2BP2, thereby accelerating PDAC immune escape. In conclusion, these findings highlight the oncogenic role of METTL3-induced circMYO1C in PDAC tumorigenesis via an m6A-dependent manner, inspiring a novel strategy to explore PDAC epigenetic therapy.

LHPP Inhibits the Viability, Migration, and Proliferation of PDAC Cells and Significantly Affects the Expression of SDC1 and S100p.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy with a poor response to chemotherapy and an extremely poor prognosis. Recent studies have revealed that phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) can inhibit the growth of various cancers. Therefore, the current study was conducted to investigate the antitumor effects of LHPP in PDAC and to explore its mechanism using proteomics analysis. Immunohistochemical analysis of clinical samples demonstrated that LHPP expression levels were lower in tumor tissues compared to adjacent nontumor tissues. Moreover, multivariate COX regression analysis showed that LHPP expression level was an independent prognostic factor for the patients with PDAC. Patients with high LHPP expression had a better prognosis. The lentiviral vectors for normal control (NC), LHPP knockdown (KD), and LHPP overexpression (OE) were infected with BxPC-3 and PANC-1 cell lines. Cell counting kit-8 assay, Transwell assay, and flow cytometry analyses showed that LHPP overexpression significantly inhibited the cell viability, migration, and proliferation of BxPC-3 and PANC-1 cells. Moreover, xenograft tumor model demonstrated that LHPP overexpression inhibited xenograft tumor growth in vivo. Subsequently, proteins with significantly altered expression in BxPC-3 cells after lentivirus infection were detected using proteomics analyses. Interestingly, compared to the NC group, the expression of Syndecan 1 (SDC1) was significantly upregulated in the KD group, while that of S100P was significantly downregulated in the OE group. LHPP might emerge as an important target for delaying the advancement of PDAC, thereby providing a novel therapeutic approach for the treatment of PDAC.

Inhibition of the SLC35B2–TPST2 Axis of Tyrosine Sulfation Attenuates the Growth and Metastasis of Pancreatic Ductal Adenocarcinom

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States. Tyrosine sulfation, catalyzed by the tyrosylprotein sulfotransferase 2 (TPST2), is a post-translational modification essential for protein-protein interactions and cellular functions. Solute carrier family 35 member B (SLC35B2) is a key transporter that transports the universal sulfate donor 3'-phosphoadenosine 5'-phosphosulfate into the Golgi apparatus where the protein sulfation occurs. The goal of this study was to determine whether and how the SLC35B2-TPST2 axis of tyrosine sulfation plays a role in PDAC. Gene expression was analyzed in PDAC patients and mice. Human PDAC MIA PaCa-2 and PANC-1 cells were used for invitro studies. TPST2-deficient MIA PaCa-2 cells were generated to assess xenograft tumor growth invivo. Mouse PDAC cells derived from the KrasLSL-G12D/+;Tp53L/+;Pdx1-Cre (KPC) mice were used to generate Tpst2 knockout KPC cells to evaluate tumor growth and metastasis invivo. High expressions of SLC35B2 and TPST2 were correlated with poor PDAC patient survival. Knocking down SLC35B2 or TPST2, or pharmacologicically inhibiting sulfation, resulted in the inhibition of PDAC cell proliferation and migration invitro. TPST2-deficient MIA PaCa-2 cells showed inhibited xenograft tumor growth. Orthotopic inoculation of Tpst2 knockout KPC cells in mice showed inhibition of primary tumor growth, local invasion, and metastasis. Mechanistically, the integrin β4 was found to be a novel substrate of TPST2. Inhibition of sulfation destabilizes integrin β4 protein, which may have accounted for the suppression of metastasis. Targeting the SLC35B2-TPST2 axis of tyrosine sulfation may represent a novel approach for therapeutic intervention of PDAC.

S100A10 Promotes Pancreatic Ductal Adenocarcinoma Cells Proliferation, Migration and Adhesion through JNK/LAMB3-LAMC2 Axis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive tumors, characterized by diagnosis at an advanced stage and a poor prognosis. As a member of the S100 protein family, S100A10 regulates multiple biological functions related to cancer progression and metastasis. However, the role of S100A10 in PDAC is still not completely elucidated. In this study, we reported that S100A10 was significantly up-regulated in PDAC tissue and associated with a poor prognosis by integrated bioinformatic analysis and human PDAC tissue samples. In vitro, down-regulation of S100A10 reduced the proliferation, migration, and adhesion of PDAC cell lines, whereas up-regulation of S100A10 showed the opposite effect. Furthermore, LAMB3 was proved to be activated by S100A10 using RNA-sequencing and western blotting. The effect of LAMB3 on the proliferation, migration, and adhesion of PDAC cells was similar to that of S100A10. Up-regulation or down-regulation of LAMB3 could reverse the corresponding effect of S100A10. Moreover, we validated S100A10 activates LAMB3 through the JNK pathway, and LAMB3 was further proved to interact with LAMC2. Mice-bearing orthotopic pancreatic tumors showed that S100A10 knocked-down PANC-1 cells had a smaller tumor size than the control group. In conclusion, S100A10 promotes PDAC cells proliferation, migration, and adhesion through JNK/LAMB3-LAMC2 axis.

COL10A1-DDR2 axis promotes the progression of pancreatic cancer by regulating MEK/ERK signal transduction.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant tumors with a poor prognosis. Type X collagen α 1 chain (COL10A1), a member of the collagen family, is a gene associated with the progression of a variety of human tumors, but the specific function and molecular mechanism of COL10A1 in pancreatic cancer remain unclear. Our study found that COL10A1 is highly expressed in pancreatic cancer cells and tissues, and its high expression is related to poor prognosis and some clinicopathological features, such as tumor size and differentiation. Biological functional experiments showed that overexpression of COL10A1 enhanced the proliferation and migration of PDAC cells. Interestingly, discoid protein domain receptor 2 (DDR2), the receptor of COL10A1, is regulated by COL10A1. We found that the COL10A1-DDR2 axis activates the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, which leads to epithelial-mesenchymal transformation (EMT) and accelerates the progression of pancreatic cancer. In summary, COL10A1 regulates PDAC cell proliferation and MEK/ERK signaling pathways by binding to DDR2 to promote migration, invasion and EMT. Our study suggested that COL10A1 might be a critical factor in promoting PDAC progression. More research is needed to confirm COL10A1 as a potential biomarker and therapeutic target for PDAC.

Clinical significance and functional role of adhesion G-protein-coupled receptors in human pancreatic ductal adenocarcinoma.

The adhesion G-protein-coupled receptors (GPCRs) play crucial roles in tumour pathogenesis, however, their clinical significance in pancreatic ductal adenocarcinoma (PDAC) remains unclear. We analysed 796 PDAC patients, including 331 from public data sets (TCGA, ICGC and GSE57495) and 465 from independent cohorts (training: n = 321, validation: n = 144). Using in-vitro studies, we confirmed the biological function of the candidate GPCRs. Analysis of all 33 adhesion GPCRs, led to identify GPR115, as the only significant prognostic factor in all public data sets. The patients with high GPR115 expression exhibited significantly poorer prognosis for OS and RFS, in training (P < 0.01, P < 0.01) and validation cohort (P < 0.01, P = 0.04). Multivariate analysis indicated that GPR115 high expression was an independent prognostic factor in both cohorts (HR = 1.43; P = 0.01, HR = 2.55; P < 0.01). A risk-prediction model using Cox regression by incorporating GPR115 and clinicopathological factors accurately predicted 5-year survival following surgery. In addition, GPR115 silencing inhibited cell proliferation and migration in PDAC cells. We demonstrated that GPR115 has important prognostic significance and functional role in tumour progression; providing a rationale that this may be a potential therapeutic target in patients with PDAC.

Identification of a novel target of SETD1A histone methyltransferase and the clinical significance in pancreaticcancer.

Although histone H3K4 methyltransferase SETD1A is overexpressed in various cancer types, the molecular mechanism underlying its overexpression and its target genes in pancreatic ductal adenocarcinoma (PDAC) remain unclarified. We conducted immunohistochemical staining for SETD1A in 105 human PDAC specimens to assess the relationship between SETD1A overexpression and clinicopathological features. The function and target genes of SETD1A were investigated using human pancreatic cancer cell lines. SETD1A expression was upregulated in 51.4% of patients with PDAC and was an independent prognostic factor associated with shorter disease-free survival after resection (p< 0.05). Knockdown and overexpression of SETD1A showed that SETD1A plays a crucial role in increasing the proliferation and motility of PDAC cells. SETD1A overexpression increased tumorigenicity. RNA sequencing of SETD1A-knockdown cells revealed downregulation of RUVBL1, an oncogenic protein ATP-dependent DNA helicase gene. ChIP analysis revealed that SETD1A binds to the RUVBL1 promoter region, resulting in increased H3K4me3 levels. Knockdown of RUVBL1 showed inhibition of cell proliferation, migration, and invasion of PDAC cells, which are similar biological effects to SETD1A knockdown. High expression of both SETD1A and RUVBL1 was an independent prognostic factor not only for disease-free survival but also for overall survival (p< 0.05). In conclusion, we identified RUVBL1 as a novel downstream target gene of the SETD1A-H3K4me3 pathway. Co-expression of SETD1A and RUVBL1 is an important factor for predicting the prognosis of patients with PDAC.

C/EBPδ Suppresses Motility-Associated Gene Signatures and Reduces PDAC Cell Migration.

Pancreatic Ductal Adenocarcinoma (PDAC) is among the most aggressive human cancers and occurs globally at an increasing incidence. Metastases are the primary cause of cancer-related death and, in the majority of cases, PDAC is accompanied by metastatic disease at the time of diagnosis, making it a particularly lethal cancer. Regrettably, to date, no curative treatment has been developed for patients with metastatic disease, resulting in a 5-year survival rate of only 11%. We previously found that the protein expression of the transcription factor CCAAT/Enhancer-Binding Protein Delta (C/EBPδ) negatively correlates with lymph node involvement in PDAC patients. To better comprehend the etiology of metastatic PDAC, we explored the role of C/EBPδ at different steps of the metastatic cascade, using established in vitro models. We found that C/EBPδ has a major impact on cell motility, an important prerequisite for tumor cells to leave the primary tumor and to reach distant sites. Our data suggest that C/EBPδ induces downstream pathways that modulate actin cytoskeleton dynamics to reduce cell migration and to induce a more epithelial-like cellular phenotype. Understanding the mechanisms dictating epithelial and mesenchymal features holds great promise for improving the treatment of PDAC.

Fusobacterium nucleatum induces proliferation and migration in pancreatic cancer cells through host autocrine and paracrine signaling.

The tumor microbiome is increasingly implicated in cancer progression and resistance to chemotherapy. In pancreatic ductal adenocarcinoma (PDAC), high intratumoral loads of Fusobacterium nucleatum correlate with shorter survival in patients. Here, we investigated the potential mechanisms underlying this association. We found that F. nucleatum infection induced both normal pancreatic epithelial cells and PDAC cells to secrete increased amounts of the cytokines GM-CSF, CXCL1, IL-8, and MIP-3α. These cytokines increased proliferation, migration, and invasive cell motility in both infected and noninfected PDAC cells but not in noncancerous pancreatic epithelial cells, suggesting autocrine and paracrine signaling to PDAC cells. This phenomenon occurred in response to Fusobacterium infection regardless of the strain and in the absence of immune and other stromal cells. Blocking GM-CSF signaling markedly limited proliferative gains after infection. Thus, F. nucleatum infection in the pancreas elicits cytokine secretion from both normal and cancerous cells that promotes phenotypes in PDAC cells associated with tumor progression. The findings support the importance of exploring host-microbe interactions in pancreatic cancer to guide future therapeutic interventions.