Metastatic Pancreatic Ductal Adenocarcinoma Cells Research Articles

ZKSCAN5 activates LAPTM5 expression by recruiting SETD7 to promote metastasis in pancreatic ductal adenocarcinoma.

Lysosomal-associated transmembrane protein 5 (LAPTM5) has been associated with poor prognosis in cancer patients. Its role in regulating metastasis in pancreatic ductal adenocarcinoma (PDAC), however, remains vague. The study here aimed to expound the metastasis-promoting properties of LAPTM5 in PDAC and the detailed mechanism. LAPTM5 was overexpressed in metastatic PDAC cells and was related to the dismal prognosis of patients in GEO datasets. By using lentiviral vectors harboring short hairpin RNA, we found that LAPTM5 downregulation reduced PDAC cell viability, proliferation, and aggressiveness in vitro and liver metastasis in vivo. Zinc finger with KRAB and SCAN domains 5 (ZKSCAN5) was predicted and verified to mediate LAPTM5 transcription in PDAC cells. Both ZKSCAN5 and SET domains, containing lysine methyltransferase 7 (SETD7) bound to the LAPTM5 promoter, and ZKSCAN5 recruited SETD7 to form a complex promoting LAPTM5 transcription. LAPTM5 knockdown reversed the promoting effect of ZKSCAN5 on the metastasis of PDAC cells. Thus, our findings on the ZKSCAN5/SETD7/LAPTM5 axis provide insights into the underlying mechanism of liver metastasis dissemination in PDAC.

Abstract B081: SOAT1 as a targetable KRAS dependency in PDAC

Abstract Metabolic rewiring is an emerging hallmark of neoplasia. In pancreatic ductal adenocarcinoma (PDAC), KRAS alters myriad metabolic programs to promote cellular transformation. Among them, the cholesterol synthesis pathway has emerged as a vulnerability of cancer cells due to the importance of cholesterol in membrane fluidity and the production of isoprenoid intermediates that enable KRAS membrane localization and activation in pancreatic cancer. We recently reported that the non-essential gene SOAT1 (Sterol-O-Acyl transferase 1), encoding an enzyme that esterifies free cholesterol into an insoluble form for storage and transport, is markedly upregulated in metastatic PDAC cells. SOAT1 promotes the hyperactivation of the mevalonate synthesis pathway due to loss of negative feedback by free cholesterol, thereby leading to increased production of isoprenoids such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are required for RAS and RHO membrane localization and activity, respectively. Genetic ablation of Soat1 reduced RAS and RHO membrane localization and inhibited cellular proliferation and mitochondrial function in vitro, and these effects were largely rescued by exogenous FPP and GGPP. Therefore, our study nominated SOAT1 as a new candidate gene for therapeutic targeting. Based on SuFEx Click Chemistry (ASCC) technology, we have designed and synthesized a panel of drug-like candidates. The first series of Soat1 probes selectively impair the proliferation of PDAC cells that express SOAT1, reduce SOAT1 protein content in cells, and covalently bind to SOAT1. In mice, the most active SOAT1 probes were tolerated in a 28-day daily dosing study up to 120 mg/kg. Marked tumor growth inhibition was noted in mice bearing PDAC xenografts and GEMM mice treated as monotherapy with 15 mg/kg/d of the most active SOAT1 probe, and tissue analysis demonstrated tumor necrosis, decreased proliferation, and increased apoptosis. Mechanistically, SOAT1 loss or inhibition targets multiple important components of cellular transformation in PDAC including KRAS, RHO, and mitochondrial biology, and our work suggests that it is an attractive target to pursue for further drug development in this cancer. Citation Format: Wenjun Lan, Shoujun Sun, Jeremy Nigri, Josh Homer, Young Park, Astrid Deschenes, Paolo Cifani, Justin Cross, Victoria Gaeth, Sunny Kim, Shawn Ting, Rachel Rubino, John Moses, David Tuveson. SOAT1 as a targetable KRAS dependency in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B081.

Abstract B100: Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression

Abstract Background: Nerves are a major component of the tumor microenvironment, with active crosstalk between cancer cells and nerves that may contribute to cancer progression. Netrin1 (Ntn1) is an axon guidance molecule that is important during early neural development. Recent studies suggest that Ntn1 may also play a role in tumorigenesis but its role in pancreatic cancer is not well understood. We hypothesized that Ntn1 plays a pro-tumorigenic role in pancreatic ductal adenocarcinoma (PDAC) progression and metastasis in part through modulation of nerves in the microenvironment. Methods: Expression of Ntn1 and its receptor Neogenin1(Neo1) were investigated in mouse models of spontaneous PDAC (LSL-Kras+/G12D/Pdx1-Cre; KC, LSL-Kras+/G12D/LSL-Trp53+/R172H/Pdx1-Cre; KPC). The influence of Ntn1 on pancreatic cancer progression and innervation was evaluated by KC mice with or without conditional knockout of Ntn1 (LSL-Kras+/G12D/Pdx1-Cre/Netrin1fl/fl; KCN). Murine PDAC cell lines and organoids were used to examine the functional role of Ntn1 and Neo1 through genetic and pharmacological modulations. The capacity of these cell lines to generate metastasis and the effect on neurite outgrowth was studied in a co-culture system with Dorsal Root Ganglia (DRG). Results: Expression of Ntn1 was not observed in normal acinar cells or ductal cells, but was found in both PanIN lesions and PDAC. Neo1 expression was similar to Ntn1 and there was a positive correlation between the expression of Ntn1 and Neo1. A lower rate of PanIN progression was observed in KCN mice compared to KC mice. In the metastatic model, overexpression of Ntn1 increased tumor burden and decreased survival while injections of a neutralizing Ntn1 antibody or genetic knockdown of Neo1 improved both endpoints. Ntn1 and Neo1 were upregulated in pancreatic organoids upon KRAS activation. Recombinant Ntn1 (rNTN1) promoted organoid forming capacity and upregulate Sox2 and Sox9 gene expression, which was inhibited by genetic knockdown of Neo1 or an anti-Neo1 antibody, supporting a direct autocrine role in modulating stemness. Nerve fibers were significantly increased at the site of PDAC liver metastasis, with Ntn1 showing the highest expression level among neurotrophins, as it was significantly increased in metastatic PDAC cells. Consistent with this observation, we observed less innervation in KCN mice, which correlated with reduced PanIN progression. Co-culture of DRGs with PDAC cells that overexpressed Ntn1 resulted in much greater axonal elongation consistent with a role of Ntn1 in promoting innervation. Conclusion: The Netrin1/Neogenin1 interaction is an important regulator of PDAC progression, acting both directly on PDAC cells to promote stemness, and indirectly via modulating tumor innervation. Blockade of Ntn1/Neo1 interaction reduces the capacity of PDAC to metastasize to the liver and improves survival. Overall, targeting the Ntn1/Neo1 interaction may represent a potential new therapeutic approach for the treatment of pancreatic cancer. Citation Format: Yosuke Ochiai, Sunagawa Masaki, Ermanno Malagola, Hiroki Kobayashi, Feijing Wu, Ruth A. White, Leah B. Zamechek, Timothy C. Wang. Netrin-1/Neogenin-1 interaction modulates pancreatic innervation to promote tumorigenesis and accelerates cancer progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B100.

Lipopolysaccharide (LPS) stimulation of Pancreatic Ductal Adenocarcinoma (PDAC) and macrophages activates the NLRP3 inflammasome that influences the levels of pro-inflammatory cytokines in a co-culture model

ABSTRACT Modified macrophages, tumor-associated macrophages (TAMs), are key contributors to the survival, growth, and metastatic behavior of pancreatic ductal adenocarcinoma (PDAC) cells. Central to the role of inflammation and TAMs lies the NLRP3 inflammasome. This study investigated the effects of LPS-stimulated inflammation on cell proliferation, levels of pro-inflammatory cytokines, and the NLRP3 inflammasome pathway in a co-culture model using PDAC cells and macrophages in the presence or absence of MCC950, a NLRP3-specific inhibitor. The effects of LPS-stimulated inflammation were tested on two PDAC cell lines (Panc 10.05 and SW 1990) co-cultured with RAW 264.7 macrophages. Cell proliferation was determined using the MTT assay. Levels of pro-inflammatory cytokines, IL-1β, and TNF-α were determined by ELISA. Western blot analyses were used to examine the expression of NLRP3 in both PDAC cells and macrophages. The co-culture and interaction between PDAC cell lines and macrophages led to pro-inflammatory microenvironment under LPS stimulation as evidenced by high levels of secreted IL-1β and TNF-α. Inhibition of the NLRP3 inflammasome by MCC950 counteracted the effects of LPS stimulation on the regulation of the NLRP3 inflammasome and pro-inflammatory cytokines in PDAC and macrophages. However, MCC950 differentially modified the viability of the metastatic vs primary PDAC cell lines. LPS stimulation increased PDAC cell viability by regulating the NLRP3 inflammasome and pro-inflammatory cytokines in the tumor microenvironment of PDAC cells/macrophages co-cultures. The specific inhibition of the NLRP inflammasome by MCC950 effectively counteracted the LPS-stimulated inflammation.

Abstract 378: Ferritin regulates oxaliplatin sensitivity and immunosuppression in pancreatic ductal adenocarcinoma

Abstract Oxaliplatin, a third-generation of platinum analog, has been used for the treatment of colorectal, gastric, and advanced pancreatic ductal adenocarcinoma (PDAC); however, its relatively high toxicity reduced its efficacy in clinical use. Recently, we found that organic cation transporter 2 (OCT2) expression is positively correlated to Oxaliplatin sensitivity in PDAC cells and better survival outcomes in PDAC patients with chemotherapy, suggesting that OCT2 level is important not only in predicting sensitivity to oxaliplatin but also in guiding dose adjustments to minimize toxicity. Here, we observed the significance of serum Ferritin as a prognostic marker for pancreatic cancer, and further identified Ferritin heavy chain (FTH1) down-regulated OCT2 level and reduced Oxaliplatin sensitivity of advanced PDAC cells in vitro and in vivo. Our study reveals the negative correlation between FTH1 and OCT2 levels in PDAC database analysis and reducing FTH1 expression with Deferasirox (DFX), which is an agent that has been widely used for patients with iron overload, reversed OCT2 expression and Oxaliplatin sensitization in metastatic PDAC cells. Additionally, we identified that exosomal Ferritin/FTH1 from PDAC cells may enhance M1-type to M2-type macrophage polarization to build up immunosuppression in the tumor microenvironment. Citation Format: Ching-Feng Chiu. Ferritin regulates oxaliplatin sensitivity and immunosuppression 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 378.

Tissue-Specific Human Extracellular Matrix Scaffolds Promote Pancreatic Tumour Progression and Chemotherapy Resistance.

Over 80% of patients with pancreatic ductal adenocarcinoma (PDAC) are diagnosed at a late stage and are locally advanced or with concurrent metastases. The aggressive phenotype and relative chemo- and radiotherapeutic resistance of PDAC is thought to be mediated largely by its prominent stroma, which is supported by an extracellular matrix (ECM). Therefore, we investigated the impact of tissue-matched human ECM in driving PDAC and the role of the ECM in promoting chemotherapy resistance. Decellularized human pancreata and livers were recellularized with PANC-1 and MIA PaCa-2 (PDAC cell lines), as well as PK-1 cells (liver-derived metastatic PDAC cell line). PANC-1 cells migrated into the pancreatic scaffolds, MIA PaCa-2 cells were able to migrate into both scaffolds, whereas PK-1 cells were able to migrate into the liver scaffolds only. These differences were supported by significant deregulations in gene and protein expression between the pancreas scaffolds, liver scaffolds, and 2D culture. Moreover, these cell lines were significantly more resistant to gemcitabine and doxorubicin chemotherapy treatments in the 3D models compared to 2D cultures, even after confirmed uptake by confocal microscopy. These results suggest that tissue-specific ECM provides the preserved native cues for primary and metastatic PDAC cells necessary for a more reliable in vitro cell culture.

Cystathionine β-synthase overexpression drives metastatic dissemination in pancreatic ductal adenocarcinoma via inducing epithelial-to-mesenchymal transformation of cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancer types with a constant rise in global incidence. Therefore, better understanding of PDAC biology, in order to design more efficient diagnostic and treatment modalities, is a priority.Here we found that the expression levels of cystathionine β-synthase (CBS), a transsulfuration enzyme, is markedly elevated in metastatic PDAC cells compared to cell lines isolated from non-metastatic primary tumors. On human immunohistochemical samples from PDAC patients we also found higher CBS staining in cancerous ductal cells compared to in non-tumor tissue, which was further elevated in the lymph node metastasis of the same patients. In mice, orthotopically injected CBS-silenced T3M4 cells induced fewer liver metastases compared to control cells indicating important roles for CBS in PDAC cancer cell invasion and malignant transformation. Wound healing and colony formation assays in cell culture confirmed that CBS-deficient metastatic T3M4 and non-metastatic BxPC3 primary tumor cells migrate slower and have impaired anchorage-independent growth capacities compared to control T3M4 cells. CBS silencing in T3M4 cells lowered WNT5a and SNAI1 gene expression down to levels that were observed in BxPC3 cells as well as resulted in an increase in E-cadherin and a decrease in Vimentin signals in mouse tumors when injected orthotopically. These observations suggested a primary role for the epithelial to mesenchymal transformation of cancer cells in CBS-mediated tumor aggressiveness. Under normal conditions, STAT3, an upstream regulator of Wnt signaling pathways, was less phosphorylated and more oxidized in shCBS T3M4 and BxPC3 compared to control T3M4 cells, which is consistent with decreased transcriptional activity at lower CBS levels due to less protection against oxidation. Sulfur metabolome analyses suggested that this CBS-mediated protection against oxidative modifications is likely to be related to persulfide/sulfide producing activities of the enzyme rather than its canonical function to produce cystathionine for cysteine synthesis. Taken together, CBS overexpression through regulation of the EMT plays a significant role in PDAC cancer cell invasion and metastasis.

The role of S100A9 in the interaction between pancreatic ductal adenocarcinoma cells and stromal cells

BackgroundA major feature of the microenvironment in pancreatic ductal adenocarcinoma (PDAC) is the significant amount of extracellular matrix produced by pancreatic stellate cells (PSCs), which have been reported to enhance the invasiveness of pancreatic cancer cells and negatively impact the prognosis.MethodsWe analyzed the data from two publicly available microarray datasets deposited in the Gene Expression Omnibus and found candidate genes that were differentially expressed in PDAC cells with metastatic potential and PDAC cells cocultured with PSCs. We studied the interaction between PDAC cells and PSCs in vitro and verified our finding with the survival data of patients with PDAC from the website of The Human Protein Atlas.ResultsWe found that PSCs stimulated PDAC cells to secrete S100A9, which attracted circulatory monocytes into cancer tissue and enhanced the expression of programmed death-ligand 1 (PD-L1) on macrophages. When analyzing the correlation of S100A9 and PD-L1 expression with the clinical outcomes of patients with PDAC, we ascertained that high expression of S100A9 and PD-L1 was associated with poor survival in patients with PDAC.ConclusionsPSCs stimulated PDAC cells to secrete S100A9, which acts as a chemoattractant to attract circulatory monocytes into cancer microenvironment and induces expression of PD-L1 on macrophages. High expression of S100A9 and PD-L1 was associated with worse overall survival in a cohort of patients with PDAC.

Riproximin Exhibits Diversity in Sugar Binding, and Modulates some Metastasis-Related Proteins with Lectin like Properties in Pancreatic Ductal Adenocarcinoma.

Riproximin (Rpx) is a type II ribosome-inactivating protein with specific anti-proliferative activity. It was purified from Ximenia americana by affinity chromatography using a resin coupled with lactosyl residues. The same technique facilitated isolation of proteins with lectin-like properties from human Suit2-007 and rat ASML pancreatic cancer cells, which were termed lactosyl-sepharose binding proteins (LSBPs). The role of these proteins in cancer progression was investigated at mRNA level using chip array data of Suit2-007 and ASML cells re-isolated from nude rats. These data compared significant mRNA expression changes when relating primary (pancreas) and metastatic (liver) sites following orthotopic and intraportal implantation of Pancreatic Ductal Adenocarcinoma (PDAC) cells, respectively. The affinity of Rpx to 13 simple sugar structures was modeled by docking experiments, the ranking of which was principally confirmed by NMR-spectroscopy. In addition, Rpx and LSBPs were evaluated for anti-proliferative activity and their cellular uptake was assessed by fluorescence microscopy. From 13 monosaccharides evaluated, open-chain rhamnose, β-d-galactose, and α-l-galactopyranose showed the highest affinities for site 1 of Rpx’s B-chain. NMR evaluation yielded a similar ranking, as galactose was among the best binders. Both, Rpx and LSBPs reduced cell proliferation in vitro, but their anti-proliferative effects were decreased by 15–20% in the presence of galactose. The program “Ingenuity Pathway Analysis” identified 2,415 genes showing significantly modulated mRNA expression following exposure of Suit2-007 cells to Rpx in vitro. These genes were then matched to those 1,639 genes, which were significantly modulated in the rat model when comparing primary and metastatic growth of Suit2-007 cells. In this overlap analysis, LSBP genes were considered separately. The potential suitability of Rpx for treating metastatic Suit2-007 PDAC cells was reflected by those genes, which were modulated by Rpx in a way opposite to that observed in cancer progression. Remarkably, these were 14% of all genes modulated during cancer progression, but 71% of the respective LSBP gene subgroup. Based on these findings, we predict that Rpx has the potential to treat PDAC metastasis by modulating genes involved in metastatic progression, especially by targeting LSBPs.

E-cadherin might be a stage-dependent modulator in aggressiveness in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) pathology is known for its uncontrollable progress due to highly invasive characteristics and refractory behavior against existing chemotherapies. The aberrant expression of CDH1 (expresses the protein E-cadherin) is associated with increased overall survival in various cancers, however, E-cadherin expression in PDAC progression has remained elusive. We investigated the impact of exogenously elevated E-cadherin levels on the tumorigenicity of transduced low grade and metastatic PDAC cell lines, Panc-1 and AsPC-1, respectively. Constitutive expression of E-cadherin promoted a more hybrid E/M state in AsPC-1 cells, while it was associated with the acquisition of a more epithelial-like state in Panc1 cells. Our study suggests that E-cadherin may play differential roles in determining the metastatic characteristics of primary and metastatic pancreatic cancer cells.

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5

BackgroundPreviously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC.MethodsWe expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC.ResultsSecretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases.ConclusionsMetastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.

Primary and Metastatic Pancreatic Cancer Cells Exhibit Differential Migratory Potentials.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastatic spread in more than 50% of patients. In this study, we sought to understand the migratory properties of (non)metastatic PDAC cells and determine whether the migration of cancer stem cell (CSC) populations accounts for the aggressive nature of this disease. The migratory abilities of primary and metastatic PDAC cell lines were investigated using a microfluidic device and time-lapse photography. The velocity, time of delay of mobilization, and number of migratory cells were analyzed. Cancer stem cell subpopulations were isolated by fluorescence-activated cell sorting and their migratory properties compared with their non-CSC counterparts. Primary cancer cells exhibited higher velocities, greater number of migratory cells, and a shorter time of delay of mobilization in comparison to metastatic cell lines. Characterization of CSC populations revealed primary PDAC cell lines were composed of fewer CD133 and CD24CD44 CSC subpopulations than metastatic cells. Moreover, migratory analysis of CSC subpopulations revealed lower velocities, fewer migratory cells, and a greater time of delay of mobilization than non-CSC. Primary cancer cells demonstrate enhanced migratory abilities in comparison to metastatic PDAC cells. Those differences may result from lower CSC subpopulations in primary cells because CSC populations demonstrated impaired migratory abilities in contrast to non-CSC.

Inflammatory IFIT3 renders chemotherapy resistance by regulating post-translational modification of VDAC2 in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide and effective therapy remains a challenge. IFIT3 is an interferon-stimulated gene with antiviral and pro-inflammatory functions. Our previous work has shown that high expression of IFIT3 is correlated with poor survival in PDAC patients who receive chemotherapy suggesting a link between IFIT3 and chemotherapy resistance in PDAC. However, the exact role and molecular mechanism of IFIT3 in chemotherapy resistance in PDAC has been unclear.Methods: A group of transcriptome datasets were downloaded and analyzed for the characterization of IFIT3 in PDAC. Highly metastatic PDAC cell line L3.6pl and patient-derived primary cell TBO368 were used and IFIT3 knockdown and the corresponding knockin cells were established for in vitro studies. Chemotherapy-induced apoptosis, ROS production, confocal immunofluorescence, subcellular fractionation, chromatin-immunoprecipitation, co-immunoprecipitation and mass spectrometry analysis were determined to further explore the biological role of IFIT3 in chemotherapy resistance of PDAC.Results: Based on PDAC transcriptome data, we show that IFIT3 expression is associated with the squamous molecular subtype of PDAC and an increase in inflammatory response and apoptosis pathways. We further identify a crucial role for IFIT3 in the regulation of mitochondria-associated apoptosis during chemotherapy. Knockdown of IFIT3 attenuates the chemotherapy resistance of PDAC cells to gemcitabine, paclitaxel, and FOLFIRINOX regimen treatments, independent of individual chemotherapy regimens. While IFIT3 overexpression was found to promote drug resistance. Co-immunoprecipitation identified a direct interaction between IFIT3 and the mitochondrial channel protein VDAC2, an important regulator of mitochondria-associated apoptosis. It was subsequently found that IFIT3 regulates the post-translational modification-O-GlcNAcylation of VDAC2 by stabilizing the interaction of VDAC2 with O-GlcNAc transferase. Increased O-GlcNAcylation of VDAC2 protected PDAC cells from chemotherapy induced apoptosis.Conclusions: These results effectively demonstrate a central mechanism by which IFIT3 expression can affect chemotherapy resistance in PDAC. Targeting IFIT3/VDAC2 may represent a novel strategy to sensitize aggressive forms of pancreatic cancer to conventional chemotherapy regimens.

KMT2D deficiency enhances the anti-cancer activity of L48H37 in pancreatic ductal adenocarcinoma.

BACKGROUNDNovel therapeutic strategies are urgently needed for patients with a delayed diagnosis of pancreatic ductal adenocarcinoma (PDAC) in order to improve their chances of survival. Recent studies have shown potent anti-neoplastic effects of curcumin and its analogues. In addition, the role of histone methyltransferases on cancer therapeutics has also been elucidated. However, the relationship between these two factors in the treatment of pancreatic cancer remains unknown. Our working hypothesis was that L48H37, a novel curcumin analog, has better efficacy in pancreatic cancer cell growth inhibition in the absence of histone-lysine N-methyltransferase 2D (KMT2D).AIMTo determine the anti-cancer effects of L48H37 in PDAC, and the role of KMT2D on its therapeutic efficacy.METHODSThe viability and proliferation of primary (PANC-1 and MIA PaCa-2) and metastatic (SW1990 and ASPC-1) PDAC cell lines treated with L48H37 was determined by CCK8 and colony formation assay. Apoptosis, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) levels, and cell cycle profile were determined by staining the cells with Annexin-V/7-AAD, JC-1, DCFH-DA, and PI respectively, as well as flow cytometric acquisition. In vitro migration was assessed by the wound healing assay. The protein and mRNA levels of relevant factors were analyzed using Western blotting, immunofluorescence and real time-quantitative PCR. The in situ expression of KMT2D in both human PDAC and paired adjacent normal tissues was determined by immunohistochemistry. In vivo tumor xenografts were established by injecting nude mice with PDAC cells. Bioinformatics analyses were also conducted using gene expression databases and TCGA.RESULTSL48H37 inhibited the proliferation and induced apoptosis in SW1990 and ASPC-1 cells in a dose- and time-dependent manner, while also reducing MMP, increasing ROS levels, arresting cell cycle at the G2/M stages and activating the endoplasmic reticulum (ER) stress-associated protein kinase RNA-like endoplasmic reticulum kinase/eukaryotic initiation factor 2α/activating transcription factor 4 (ATF4)/CHOP signaling pathway. Knocking down ATF4 significantly upregulated KMT2D in PDAC cells, and also decreased L48H37-induced apoptosis. Furthermore, silencing KMT2D in L48H37-treated cells significantly augmented apoptosis and the ER stress pathway, indicating that KMT2D depletion is essential for the anti-neoplastic effects of L48H37. Administering L48H37 to mice bearing tumors derived from control or KMT2D-knockdown PDAC cells significantly decreased the tumor burden. We also identified several differentially expressed genes in PDAC cell lines expressing very low levels of KMT2D that were functionally categorized into the extrinsic apoptotic signaling pathway. The KMT2D high- and low-expressing PDAC patients from the TCGA database showed similar survival rates,but higher KMT2D expression was associated with poor tumor grade in clinical and pathological analyses.CONCLUSIONL48H37 exerts a potent anti-cancer effect in PDAC, which is augmented by KMT2D deficiency.

Super-enhancers: novel target for pancreatic ductal adenocarcinoma.

Super-enhancers (SEs) are unique areas of the genome which drive high-level of transcription and play a pivotal role in the cell physiology. Previous studies have established several important genes in cancer as SE-driven oncogenes. It is likely that oncogenes may hack the resident tissue regenerative program and interfere with SE-driven repair networks, leading to the specific pancreatic ductal adenocarcinoma (PDAC) phenotype. Here, we used ChIP-Seq to identify the presence of SE in PDAC cell lines. Differential H3K27AC marks were identified at enhancer regions of genes including c-MYC, MED1, OCT-4, NANOG, and SOX2 that can act as SE in non-cancerous, cancerous and metastatic PDAC cell lines. GZ17-6.02 affects acetylation of the genes, reduces transcription of major transcription factors, sonic hedgehog pathway proteins, and stem cell markers. In accordance with the decrease in Oct-4 expression, ChIP-Seq revealed a significant decrease in the occupancy of OCT-4 in the entire genome after GZ17-6.02 treatment suggesting the possible inhibitory effect of GZ17-6.02 on PDAC. Hence, SE genes are associated with PDAC and targeting their regulation with GZ17-6.02 offers a novel approach for treatment.

Expression of annexinII and stromal tenascinC promotes epithelial to mesenchymal transition and correlates with distant metastasis in pancreatic cancer.

The interaction between cancer cells and stromal components contributes to cancer invasion and metastasis in pancreatic ductal adenocarcinoma (PDAC). The present study investigated the role of the correlation between annexin II (ANX2) and stromal tenascin C (TNC) with the progression of PDAC. The functions of the expression ANX2 and TNC were assessed in in vitro experiments using mouse and human PDAC cells, and the clinical effect was analyzed using immunohistochemistry with surgically resected PDAC tissues. The effects on epithelial to mesenchymal transition (EMT), invasion, putative cancer stemness, and anoikis resistance were examined in vitro using murine precancerous pancreatic intraepithelial neoplasia (PanIN) cells and murine and human invasive PDAC cells with ANX2 knockdown using specific small interfering RNA (siRNA)s and recombinant TNC (rTNC). ANX2 was expressed at a high level in primary PanIN cells and invasive PDAC cells, compared with the levels in liver metastatic PDAC cells. In the ANX2-knockdown cells, there were fewer cells with a morphological mesenchymal appearance in three-dimensional culture and invasion was reduced compared with that in the control cells. Morphological change into the mesenchymal phenotype and invasion were enhanced by rTNC treatment in the control PDAC cells but not in the ANX2-knockdown cells. Pancreatosphere formation assays showed that ANX2 and TNC facilitated the maintenance of stem-like characters in PDAC cells. Furthermore, anoikis assays indicated that the interaction of ANX2-TNC contributed to anoikis resistance in PDAC cells. In the immunohistochemistry analyses, the group with a high expression of ANX2 and high stromal TNC was significantly correlated with distant metastasis, and was associated with hematogenous/peritoneal recurrence and poor outcomes following surgery in resected human primary PDAC tissues. In conclusion, the results demonstrated that ANX2 and stromal TNC regulated invasion in addition to stemness and anoikis resistance, which are crucial for metastasis in the progression of PDAC. These results indicate the potential of the ANX2-TNC axis as a therapeutic target for PDAC metastasis.

Effect of macrophages on pancreatic cancer.

324 Background: Pancreatic Ductal Adenocarcinoma (PDAC) is expected to be the second leading cause of cancer related deaths by 2030. TGF-β is a well-studied PDAC mediator with a context dependent role as initially a tumor suppressor with potential to convert to a tumor promoter in later stages. Tumor associated macrophages and interleukins, such as the pro-inflammatory interleukin, IL23, are not well studied regarding PDAC. We hypothesized PDAC treated with TGF-β and macrophages would induce a more aggressive phenotype. Methods: We investigated aggressive behavior with a primary PDAC cell line in vivo and a metastatic PDAC cell line in vitro. A primary pancreatic cell line, Panc-1 cells, were pre-treated with PBS, IL23, macrophages (10:1 ratio of Panc-1 cells to macrophages), IL23 + macrophages, TGF-ß, TGF-ß + macrophages, or TGF-ß + macrophages + IL23. After treatment, cells were orthotopically implanted into the pancreas of NOD SCID gamma mice with 5 mice per group. Mice weights were recorded twice weekly for 4-weeks. Primary lesions and metastasis were investigated with ANOVA. AsPC-1 cells, a metastatic pancreatic cell line, were pre-treated with the same seven treatments. We investigated pSTAT3 expression and the streak closure in vitro. Results: Panc-1 cells treated with macrophages had the largest pancreatic tumor weight and diameter compared to PBS control, IL23 alone, and TGF-β alone (P < 0.001). When macrophages treatment included TGF-β, pancreatic tumor weights and diameters decreased as compared to macrophages alone and macrophages + IL23 (P < 0.001). Macrophage treatment induced higher liver weights and higher number of surface liver metastatic lesions suggesting higher metastatic disease burden (P < 0.03). AsPC-1 cells treated with combinations of macrophages and TGF-β increased pSTAT3 expression compared to PBS control. AsPC-1 cells treated with macrophages closed the gap in the scratch assay faster than PBS control 24 hours after treatment (P < 0.001). Conclusions: We demonstrated macrophages have a key role in converting primary pancreatic cancer into a more aggressive phenotype in vivo whereas they have less effect on metastatic pancreatic cancer in vitro.

PTEN-Dependent Stabilization of MTSS1 Inhibits Metastatic Phenotype in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) presents at metastatic stage in over 50% of patients. With a survival rate of just 2.7% for patients presenting with distant disease, it is imperative to uncover novel mechanisms capable of suppressing metastasis in PDAC. Previously, we reported that the loss of metastasis suppressor protein 1 (MTSS1) in PDAC cells results in significant increase in cellular migration and invasion. Conversely, we also found that overexpressing MTSS1 in metastatic PDAC cell lines corresponds with not only decreased metastatic phenotype, but also greater overall survival. While it is known that MTSS1 is downregulated in late-stage PDAC, the mechanism behind that loss has not yet been elucidated. Here, we build off our previous findings to present a novel regulatory mechanism for the stabilization of MTSS1 via the tumor suppressor protein phosphatase and tensin homolog (PTEN). We show that PTEN loss in PDAC cells results in a decrease in MTSS1 expression and increased metastatic potential. Additionally, we demonstrate that PTEN forms a complex with MTSS1 in order to stabilize and protect it from proteasomal degradation. Finally, we show that the inflammatory tumor microenvironment, which makes up over 90% of PDAC tumor bulk, is capable of downregulating PTEN expression through secretion of miRNA-23b, potentially uncovering a novel extrinsic mechanism of MTSS1 regulation. Collectively, these data offer new insight into the role and regulation of MTSS1in suppressing tumor cell invasion and migration and help shed light as to what molecular mechanisms could be leading to early cell dissemination in PDAC.

Grainyhead-like 2 (GRHL2) regulates epithelial plasticity in pancreatic cancer progression.

The epithelial‐mesenchymal transition (EMT) and mesenchymal‐epithelial transition (MET) contribute to cancer metastasis of pancreatic ductal adenocarcinoma (PDAC). We explored the role of grainyhead‐like 2 (GRHL2), a suppressor of EMT, in the progression of PDAC. Expressions of GRHL2 were assessed using surgically resected PDAC tissues by immunohistochemistry analysis, and in vitro using human and mouse PDAC cells. Effects on epithelial plasticity and stemness of GRHL2 were examined in vitro using liver metastatic PDAC cells (CFPAC‐1) with GRHL2 knockdown by specific siRNAs. GRHL2 has a significantly positive correlation with E‐cadherin and CD133 in 155 resected human primary PDAC tissues. GRHL2 is highly expressed in liver metastatic cells than in primary invasive cells of both human and mouse PDAC, accompanied by a positive correlation with E‐cadherin expression. GRHL2 knockdown CFPAC‐1 cells demonstrated morphological changes into mesenchymal appearances and reduced proliferation through EMT. Notably, knockdown studies followed by flow cytometry analysis for a subpopulation of CD133+ showed that GRHL2 facilitates CFPAC‐1 cells to maintain stem‐like characters including self‐renewal capacity and anoikis resistance. GRHL2 regulates epithelial plasticity along with stemness in PDAC, both of which are crucial for metastasis, implicating the possibility of GRHL2 as a therapeutic target for PDAC liver metastasis.

Metadherin promotes metastasis by supporting putative cancer stem cell properties and epithelial plasticity in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a high metastatic potential. However, the mechanism of metastatic colonization in PDAC remains poorly understood. Metadherin (MTDH) has emerged in recent years as a crucial mediator of metastasis in several cancer types, although the biological role of MTDH in PDAC has not been investigated. Here, we demonstrated the functional roles of MTDH in PDAC progression, especially focusing on the metastatic cascade. In vitro studies showed that MTDH provides cancer stem cell (CSC) properties in metastatic PDAC cells and contributes to anoikis resistance with epithelial characteristics in PDAC cells. We also performed in vivo studies using both orthotopic transplantation and intra-portal vein injection as experimental models of liver metastasis to examine the function of MTDH at the metastatic site. MTDH knockdown dramatically reduced the incidence of liver metastases along with epithelial features in both experimental mouse models. Collectively, MTDH facilitates metastatic colonization with putative CSC and epithelial properties in PDAC cells. PDAC cells were transiently treated with TGF-β1 to investigate the roles of MTDH on epithelial plasticity. Intriguingly, MTDH expression was negatively correlated with Twist1 expression during the Mesenchymal-Epithelial transition (MET) induction in metastatic PDAC cells. These results suggest that MTDH may contribute to MET induction via downregulation of Twsit1. Lastly, immunohistochemistry indicated that MTDH overexpression is closely associated with hematogenous metastasis and predicts poor prognosis in patients with PDAC. This is the first demonstration of MTDH function in PDAC metastatic colonization. Our data suggest that MTDH targeting therapy could be applied to control PDAC metastasis.