Promotes Pancreatic Ductal Adenocarcinoma Progression Research Articles

Fibulin-5 Blocks Microenvironmental ROS in Pancreatic Cancer.

Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anticancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity, and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We used genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDAC) and found that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDAC therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5-mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDAC progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production, and thus tip the balance in favor of tumor cell survival and treatment-refractory disease.

Abstract A114: Alternatively spliced tissue factor: A new therapeutic target in pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF), the trigger of blood coagulation. Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. We recently reported that asTF is abundant in PDAC lesions and, when overexpressed in PDAC cells, it promotes tumor growth, metastatic spread, monocyte recruitment, and elevates procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we sought to determine whether i) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; ii) asTF mechanistically contributes to PDAC cell migration; and iii) antibody-based targeting of asTF slows PDAC progression and/or asTF release into systemic circulation. Methods: Cohorts of nude mice (n=5/cohort) were orthotopically transplanted with grade III human PDAC cells Pt45P1 harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi, 5x105 cells/mouse); mice began receiving Dox (2µg/mL) in sucrose at day 1 of the study (“Dox”), day 25 of the study (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo via SapC-DOPS imaging over 7 wks. PDAC cell migration toward serum was assessed using laminin-coated transwell inserts seeded with Pt45P1/asTFi cells. Plasma samples from mice bearing PDAC tumors were assayed for asTF using a custom sandwich ELISA. asTF-specific rabbit monoclonal antibody Rb1 was co-implanted with Pt45P1 cells, and tumor progression was monitored as above over 5 wks. Tumor specimens were assessed for vessel density (isolectin B4) and monocyte/macrophage infiltration (anti-F4/80 antibody). Results: “Late Dox” mice developed tumors that were comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); interestingly, tumor spread was also significantly less pronounced in “late Dox” mice compared to “Dox” mice ([7.52e+7] vs [1.22e+8]P/s/mm2, p=0.01). “Dox” mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p<0.001). When cells were co-treated with Dox and inhibitory anti-β1 antibody, migration was inhibited to the levels observed with untreated cells; anti-α6 antibody had a partial effect. Strikingly, migration of Pt45P1/asTFi cells co-treated with Dox and Rb1 was inhibited to the levels observed with untreated cells and/or Dox/anti-β1 antibody co-treated cells; rabbit isotype control IgG had no effect. Pt45P1 cells, when co-implanted with just 100μg of Rb1, grew significantly smaller tumors that were less vascularized and had ~3.5 fold fewer stromal macrophages compared to the cells co-implanted with vehicle (PBS) and/or isotype control IgG (Rb1: avg. wt. 0.835g, 20±9 cells/LPF; PBS: avg. wt. 1.624g, 73±30 cells/LPF; IgG: avg. wt. 1.665g, 66±47 cells/LPF; p<0.005 Rb1 vs PBS/IgG). Mice in the Pt45P1/Rb1 cohort had ~2 fold decrease in the levels of circulating asTF compared to mice in the Pt45P1/PBS and/or isotype IgG cohorts (0.27 ng/mL vs 0.60/0.54 ng/mL; p<0.001). Conclusion: asTF can promote PDAC progression during earlier as well as later stages of the disease. asTF-β1 integrin interactions render PDAC cells more motile, likely via competing with laminin for β1 integrin binding; Rb1 fully inhibits asTF-potentiated PDAC cell migration. When co-implanted with Rb1, Pt45P1 cells grow much smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel therapeutic strategy to stem PDAC progression. Citation Format: Dusten Unruh, Xiaoyang Qi, Zhengtao Chu, Robert Sturm, Ryan Keil, Syed Ahmad, Henri H. Versteeg, Vladimir Y. Bogdanov. Alternatively spliced tissue factor: A new therapeutic target in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A114.

Kindlin-2 induced by TGF-β signaling promotes pancreatic ductal adenocarcinoma progression through downregulation of transcriptional factor HOXB9

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related deaths with no effective therapeutics. Invasion and metastasis are the major characteristics of PDAC. However, mechanisms underlying PDAC invasion and metastasis are elusive. In this report, we found that Kindlin-2 is a target protein of transforming growth factor β (TGF-β) signaling and is upregulated by TGF-β1 in PDAC cells. TGF-β1-upregulated Kindlin-2 promotes PDAC cell growth, migration and invasion, whereas Kindlin-2 upregulates transforming growth factor receptor I (TβRI), a key component of TGF-β signaling. Thereby Kindlin-2 and TGF-β signaling constitute a positive feedback loop. Mechanistically, Kindlin-2 promotes PDAC progression by downregulation of HOXB9 and E-cadherin. For clinical relevance, enhanced expression of Kindlin-2 predicts a poor overall survival for PDAC patients. Gene expression levels of Kindlin-2, TGF-β, TβRI and HOXB9 are all correlated with the overall survival of PDAC patients in an Oncomine dataset. Taken together, our findings demonstrated that TGF-β1-induced Kindlin-2 expression promotes PDAC progression by downregulation of HOXB9 and E-cadherin.

Alternatively Spliced Tissue Factor As a New Therapeutic Target in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is marked by high expression of Tissue Factor (TF). Alternatively spliced TF (asTF) is a secreted TF form that signals non-proteolytically via β1/β3 integrins. asTF is abundant in PDAC lesions and its constitutive overexpression promotes tumor growth, metastatic spread, monocyte recruitment, and elevated procoagulant potential of PDAC cells and cell-derived microparticles. In this study, we determined if 1) delayed-onset overexpression of asTF yields a phenotype distinct from that obtained with constitutive overexpression; 2) asTF contributes to PDAC cell migration; and 3) monoclonal antibody-based targeting of tumor-derived asTF slows PDAC progression and/or asTF release into circulation in an orthotopic setting.Nude mice were orthotopically transplanted with human PDAC cells harboring a doxycycline (Dox)-inducible asTF transgene system (line Pt45P1/asTFi); mice received Dox (2µg/mL) in sucrose at day 1 (“Dox”), day 25 (“late Dox”), or sucrose alone (“no Dox”), and tumor progression was monitored in vivo over 7 weeks via SapC-DOPS imaging. Migration of Pt45P1/asTFi cells toward serum was assessed using laminin-coated transwell inserts. Plasma of PDAC tumor-bearing mice was assayed for asTF using a custom ELISA. Pt45P1 cells were resuspended in PBS containing anti-asTF rabbit monoclonal antibody Rb1 and implanted in a) nude mice; b) SCID mice, and c) SCID mice with TF levels reduced by 99% (SCID/TF-low) and tumor progression was monitored as above over 5 weeks. Paraffin-embedded tumor specimens were assessed for vessel density (isolectin B4 and/or anti-CD31 staining) and monocyte/macrophage infiltration (anti-F4/80 and/or anti-CD206 staining).“Late Dox” mice developed tumors comparable in size and vessel density to those in “Dox” mice, yet less infiltrated with macrophages (Dox: 70±22 cells/LPF, late Dox: 51±17 cells/LPF, p=0.009); tumor spread was significantly reduced in “late Dox” mice compared to “Dox” mice (7.52e+7 vs 1.22e+8 P/s/mm2, p=0.01). “Dox” mice also had high levels of asTF in circulation (~1 ng/mL), which did not differ significantly from those in “late Dox” mice. The PDAC cell migration assay revealed a ~4 fold increase in the migration of Pt45P1/asTFi cells treated with Dox compared to untreated cells (p<0.001). When cells were co-treated with Dox and inhibitory anti-β1 antibody, migration was inhibited to the levels observed with untreated cells. Migration of Pt45P1/asTFi cells co-treated with Dox and Rb1 was inhibited to the levels observed with Dox/anti-β1 antibody co-treated cells; rabbit isotype control IgG had no effect. Pt45P1 cells, when implanted together with just 100 μg of Rb1, grew significantly smaller tumors that were less vascularized and had ~3.5 fold fewer stromal macrophages compared to the cells implanted together with vehicle (PBS) and/or isotype control IgG (Rb1: avg. wt. 0.835g, 20±9 cells/low power field (LPF); PBS: avg. wt. 1.624g, 73±30 cells/LPF; IgG: avg. wt. 1.665g, 66±47 cells/LPF; p<0.005 Rb1 vs PBS/IgG). Mice in the Pt45P1/Rb1 cohort had ~2 fold decrease in the levels of circulating asTF compared to mice in the Pt45P1/PBS and/or isotype IgG cohorts (0.27 ng/mL vs 0.60/0.54 ng/mL; p<0.001). While Pt45P1 tumors grown in SCID and SCID/TF-low mice were comparable in size, tumor spread was significantly diminished in SCID/TF-low mice (7.74e+8 P/s/mm2 vs 1.09e+9 P/s/mm2, p=0.007); tumors in SCID/TF-low mice also had fewer M2 polarized macrophages (p=0.011) and lower vessel density (4,576µm2/LPF vs 1,834µm2/LPF, p=0.017). In the presence of Rb1, Pt45P1 cells implanted in SCID mice grew tumors significantly smaller compared to IgG control (p=0.005); the effects of Rb1 on primary tumor growth in SCID/low-TF mice were similar to those in SCID mice.We here report that asTF can promote PDAC progression during early and late stages of the disease. asTF-β1 integrin interaction renders PDAC cells more motile, while Rb1 fully inhibits asTF-potentiated PDAC cell migration. Host-derived TF has no impact on the size of primary PDAC tumor, yet it contributes to tumor vascularization and, consequently, metastatic spread. In the presence of Rb1 in nude and/or SCID mice, Pt45P1 cells grow significantly smaller tumors with fewer monocytes and blood vessels that release less asTF in the circulation. Thus, antibody-based targeting of asTF may comprise a novel strategy to stem PDAC progression. DisclosuresNo relevant conflicts of interest to declare.

An Increased Abundance of Tumor-Infiltrating Regulatory T Cells Is Correlated with the Progression and Prognosis of Pancreatic Ductal Adenocarcinoma

CD4+CD25+Foxp3+ regulatory T cells (Tregs) can inhibit cytotoxic responses. Though several studies have analyzed Treg frequency in the peripheral blood mononuclear cells (PBMCs) of pancreatic ductal adenocarcinoma (PDA) patients using flow cytometry (FCM), few studies have examined how intratumoral Tregs might contribute to immunosuppression in the tumor microenvironment. Thus, the potential role of intratumoral Tregs in PDA patients remains to be elucidated. In this study, we found that the percentages of Tregs, CD4+ T cells and CD8+ T cells were all increased significantly in tumor tissue compared to control pancreatic tissue, as assessed via FCM, whereas the percentages of these cell types in PBMCs did not differ between PDA patients and healthy volunteers. The percentages of CD8+ T cells in tumors were significantly lower than in PDA patient PBMCs. In addition, the relative numbers of CD4+CD25+Foxp3+ Tregs and CD8+ T cells were negatively correlated in the tissue of PDA patients, and the abundance of Tregs was significantly correlated with tumor differentiation. Additionally, Foxp3+ T cells were observed more frequently in juxtatumoral stroma (immediately adjacent to the tumor epithelial cells). Patients showing an increased prevalence of Foxp3+ T cells had a poorer prognosis, which was an independent factor for patient survival. These results suggest that Tregs may promote PDA progression by inhibiting the antitumor immunity of CD8+ T cells at local intratumoral sites. Moreover, a high proportion of Tregs in tumor tissues may reflect suppressed antitumor immunity.

Celecoxib and GABA Cooperatively Prevent the Progression of Pancreatic Cancer In Vitro and in Xenograft Models of Stress-Free and Stress-Exposed Mice

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is associated with high levels of psychological distress. We have shown that beta-adrenergic receptors (β-ARs), which are activated by stress neurotransmitters, regulate PDAC cells via cyclic AMP (cAMP)-dependent signaling in vitro, that social stress promotes PDAC progression in mouse xenografts and that γ-aminobutyric acid (GABA) inhibits these responses in vitro and in vivo. The targeted inhibition of stress-induced regulatory pathways may abolish the potentially negative impact of psychological stress on clinical outcomes. Our current data show that chronic exposure of PDAC cell lines Panc-1 (activating point mutations in K-ras) and BXPC-3 (no mutations in K-ras) in vitro to the stress neurotransmitter epinephrine at the concentration (15 nM) previously measured in the serum of mice exposed to social stress significantly increased proliferation and migration. These responses were inhibited in a concentration-dependent manner by celecoxib. The effects of celecoxib alone and in combination with γ-aminobutyric acid (GABA) on the progression of subcutaneous mouse xenografts from the cell line (BXPC-3) most responsive to epinephrine were then investigated in the presence and absence of social stress. Cancer-stimulating factors (VEGF & prostaglandin E2 [PGE2]) and levels of cAMP were measured by immunoassays in blood and xenograft tissue. Phosphorylation of the signaling proteins ERK, CREB, Src, and AKT was assessed by ELISA assays and Western blotting. Expression of COX-2, 5-lipoxygenase, and p-5-LOX were determined by semi-quantitative Western blotting. Celecoxib alone significantly inhibited xenograft progression and decreased systemic and tumor VEGF, PGE2, and cAMP as well as phosphorylated signaling proteins in stress-exposed and stress-free mice. These responses were significantly enhanced by co-treatment with GABA. The celecoxib-induced downregulation of COX-2 protein and p-5-LOX was also significantly enhanced by GABA under both experimental conditions. Our findings identify the targeted inhibition of stress-induced pathways as a promising area for more effective cancer intervention in pancreatic cancer.

P-0098 Aggravated Hypoxic Microenvironment in Pancreatic Cancer with Hyperglycemia

IntroductionHyperglycemia is one of the clinical features of pancreatic ductal adenocarcinoma (PDAC). It is considered to be the prognostic factor of PDAC and could directly contribute to tumor progression. Previous studies indicated that the PDAC cell lines subcutaneous xenotransplants in streptozotocin (STZ)-induced diabetes BALB/c-nu/nu mice showed more aggressive progression than those in mice with euglycemia. Hypoxia in tumor microenvironment is another characteristic of PDAC, and promotes cancer progression via a variety of signaling pathways modulated by hypoxia-inducible factor 1 (HIF-1). The purpose of this study was to investigate the underlying mechanism by which hyperglycemia combined hypoxia microenvironment promotes PDAC. MethodsWe assess 100 patients who suffered from PDAC and underwent pancreatoduodenectomy, and divided them into two groups according to the serum glucose level (euglycemia and hyperglycemia). Additionally, we created STZ-induced diabetes nude mice with orthotopic xenografts of PDAC cell BxPC-3. Immunohistochemical stainings were performed to detect the expression of CD31, alpha-smooth muscle actin (&agr;-SMA), HIF-1&agr;, and carbonic anhydrase IX (CA IX) in PDAC tissues. ResultsOf all 100 cases, 41 had hyperglycemia. The HIF-1&agr; staining in the nucleus and CA IX staining in the cytoplasm of the cancer cells were significantly stronger (P<0.005) in the hyperglycemia group than those in the euglycemia group, in both cancer region and normal pancreas region, indicating that there was an aggravated hypoxia condition in the hyperglycemia group. In cancer stroma, expression level of &agr;-SMA was higher in the hyperglycemia group, coordinated with the higher hypoxia level. But the microvessel density (MVD) of CD31 staining in cancer region was lower in the hyperglycemia group than that in the euglycemia group. In situ tumors showed more aggressive phenotype in diabetic nude mice than those in nondiabetic mice. That is, more serious ascites, cachexia and liver metastasis were observed in mice with hyperglycemia. ConclusionThere was aggravated hypoxia condition in pancreas of patients and mice with diabetes. Hyperglycemia might promote PDAC progression via upregulated hypoxic stress in tumor microenvironment.

Abstract LB-504: Mast cell cytokines stimulate cancer-associated fibroblasts proliferation and promote pancreatic cancer progression

Abstract Objectives: Pancreatic ductal adenocarcinoma (PDAC) is a uniformly lethal disease and is notoriously resistant to most therapies. In patients, cancer cells exist in a complex microenvironment containing cancer-associated fibroblasts (pancreatic stellate cells, PSCs) and immune cells. These components provide a fibrotic niche that is an impediment to successful therapy. In our preliminary studies, we found that mast cells infiltrated at the tumor margin. This high infiltration of human mast cells into the tumor microenvironment was associated with poor survival. Mast cells were found to be essential to PDAC tumorigenesis in a mast cell-deficient Kitw-sh/w-sh mouse model compared to control mice. In addition, when we treated tumor-bearing mice with cromolyn, a mast cell stabilizer, tumor growth was depressed. How mast cells regulate the tumor microenvironment and contribute to PDAC tumorigenesis are unknown. We hypothesized that mast cell cytokines mediate cancer-associated fibroblasts proliferation that promotes PDAC progression. Methods: We measured mast cell infiltration in 16 normal human pancreas (NP), 26 pancreatic intraepithelial lesions (PanINs), and 25 PDAC patients’ tissue samples. In an in vitro co-culture system, we measured the crosstalk of human mast cells, PDAC cells, and PSCs. The effect of mast cell cytokine release on PDAC and PSC proliferation and migration was measured using conditioned medium. To determine the role of IL-1β, IL-13, and tryptase on PDAC and PSC proliferations, neutralizing antibodies were used. To determine whether inhibiting mast cell degranulation and cytokine production has a therapeutic effect in vivo, we treated tumor-bearing mice with masitinib, which is a tyrosine kinase inhibitor targeting c-KIT. Tumor sizes were measured by bioluminescence imaging. Results: Mast cell infiltration was associated with human malignancy progression (NP → PanIN, P &amp;gt; 0.05; PanIN → PDAC, P &amp;lt; 0.001), which was consistent with tryptase expression pattern in a spontaneous PDAC mouse model. In vitro, tumor cells promoted mast cell migration (P &amp;lt; 0.05). PDAC cells and PSCs stimulated mast cell activation, as measured by degranulation and tryptase release (P &amp;lt; 0.05). Conversely, mast cell-derived IL-13 stimulated PDAC proliferation (P &amp;lt; 0.001). Strikingly, IL-1β, tryptase, or IL-13 from mast cells promoted PSC proliferation. In vivo, PDAC growth was significantly reduced by masitinib (P &amp;lt; 0.01 vs saline control) in tumor-bearing mice. Conclusions: Mast cells promote PDAC progression. Tumor cells promote mast cell activation and migration to the tumor site. Once there, mast cell cytokines activate proliferation of both tumor cells and fibroblasts, potentially contributing to the fibrotic microenvironment found in PDAC. Therefore, therapy targeting mast cells cytokines, IL-1β, tryptase, or IL-13, may overcome stromal formation and improve PDAC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-504. doi:1538-7445.AM2012-LB-504

Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-β signaling in cooperation with active Kras expression

Pancreatic ductal adenocarcinoma (PDAC) is an almost uniformly lethal disease in humans. Transforming growth factor-beta (TGF-beta) signaling plays an important role in PDAC progression, as indicated by the fact that Smad4, which encodes a central signal mediator downstream from TGF-beta, is deleted or mutated in 55% and the type II TGF-beta receptor (Tgfbr2) gene is altered in a smaller subset of human PDAC. Pancreas-specific Tgfbr2 knockout mice have been generated, alone or in the context of active Kras (Kras(G12D)) expression, using the Cre-loxP system driven by the endogenous Ptf1a (pancreatic transcription factor-1a) locus. Pancreas-selective Tgfbr2 knockout alone gave no discernable phenotype in 1.5 yr. Pancreas-specific Kras(G12D) activation alone essentially generated only intraepithelial neoplasia within 1 yr. In contrast, the Tgfbr2 knockout combined with Kras(G12D) expression developed well-differentiated PDAC with 100% penetrance and a median survival of 59 d. Heterozygous deletion of Tgfbr2 with Kras(G12D) expression also developed PDAC, which indicated a haploinsufficiency of TGF-beta signaling in this genetic context. The clinical and histopathological manifestations of the combined Kras(G12D) expression and Tgfbr2 knockout mice recapitulated human PDAC. The data show that blockade of TGF-beta signaling and activated Ras signaling cooperate to promote PDAC progression.