Inhibition of C5a-C5aR1 axis suppresses tumour progression by enhancing antitumour immunity and chemotherapeutic effect in pancreatic ductal adenocarcinoma.

Background: The clinical standard of care for patients with pancreatic ductal adenocarcinoma (PDAC) is treatment with Gemcitabine (Gem) despite Gem's limited benefits. To date, no surrogate marker has been identified to indicate whether Gem treatment directly affects PDAC tumor cells. In order to identify such a marker, we analyzed Gem treated cell lines and clinical samples. Methods: 12 PDAC cell lines were treated with 5μM Gem for three hours and then allowed to grow in the absence of Gem for 24hrs before mRNA expression analysis (Illumina Gene Expression Array). IHC and WB were used to quantify IL-8 expression. ShRNA mediated silencing was utilized to study the therapeutic value of IL-8. Results: mRNA expression and WB confirmed the induction of IL-8 after Gem treatment. Gem induced IL-8 expression was transcriptionally controlled by NFκB and C/EBP. PDAC cells (MPanc96 and BxPc-3-CXCR1), HPSC (Human Pancreatic Stellate Cells-CXCR1) and HUVEC (Human Umbilical Vein Endothelial cells-CXCR1 & 2) expressed the receptor of IL-8. Conditioned media from Gem treated cells and rIL-8 treated cells, stimulated cell growth (PDAC, HPSC and HUVEC), polygon formation in HUVEC and α-SMA expression in HPSC. Stable silencing of IL-8 in Capan2 cells, which develop an abundant stroma, led to a significant reduction in tumor volume. Histologically, IL-8 silenced tumors showed reduced micro-vessel density (CD37), as well as highly reduced desmoplasia (Trichrome stain & α-SMA) associated with necrosis. Further, IHC revealed strong IL-8 expression in neo-adjuvant Gem-treated human PDAC tissues (strong expression 42.1%, weak expression 57.9%, negative 0%) in comparison to tissue from patients that underwent surgery alone (strong expression 0%, weak expression 77.8%, negative 22.2%).Conclusion: Gem induced IL-8 functions as a growth factor for cancer cells and its microenvironment. Thus, IL-8 can serve as a surrogate marker to indicate Gem effects in PDAC. Citation Format: Sobeyda Gomez, Takahiko Fujii, Haojie Huang, Rosa Hwang, Viji Ramachandran, Craig Logsdon, Thiru Arumugam. Dynamics of Gemcitabine induced interleukin-8 (IL-8) expression in pancreatic tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1094. doi:10.1158/1538-7445.AM2014-1094

BackgroundThe typical pathological feature of pancreatic ductal adenocarcinoma (PDAC) is a significant increase in stromal reaction, leading to a hypoxic and poorly vascularized tumor microenvironment. Tumor cells undergo metabolic reprogramming, such as the Warburg effect, yet the underlying mechanisms are not fully understood.MethodsInterference and overexpression experiments were conducted to analyze the in vivo and in vitro effects of USP7 on the growth and glycolysis of tumor cells. Small-molecule inhibitors of USP7 and transgenic mouse models of PDAC were employed to assess the consequences of targeting USP7 in PDAC. The molecular mechanism underlying USP7-induced c-Myc stabilization was determined by RNA sequencing, co-IP and western blot analyses.ResultsUSP7 is abnormally overexpressed in PDAC and predicts a poor prognosis. Hypoxia and extracellular matrix stiffness can induce USP7 expression in PDAC cells. Genetic silencing of USP7 inhibits the glycolytic phenotypes in PDAC cells, while its overexpression has the opposite effect, as demonstrated by glucose uptake, lactate production, and extracellular acidification rate. Importantly, USP7 promotes PDAC tumor growth in a glycolysis-dependent manner. The small-molecule inhibitor P5091 targeting USP7 effectively suppresses the Warburg effect and cell growth in PDAC. In a transgenic mouse model of PDAC, named KPC, P5091 effectively blocks tumor progression. Mechanistically, USP7 interacts with c-Myc, enhancing its stability and expression, which in turn upregulates expression of glycolysis-related genes.ConclusionsThis study sheds light on the molecular mechanisms underlying the Warburg effect in PDAC and unveils USP7 as a potential therapeutic target for improving PDAC treatment.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers. At present, surgical resection is the only curative treatment for PDAC, but the majority of PDAC patients show early recurrence even after curative resection. There is a possibility of having been distant micrometastasis at early stage of carcinogenesis in PDAC. The control of PDAC metastatic progression leads to improve the prognosis of PDAC patients. Recently, it is reported that a verified mediator of distant metastasis, Metadherin (MTDH), promotes metastasis in experimental models of breast cancer, and its high expression is associated with poor prognosis in a large spectrum of cancer types. Recent evidences imply that the epithelial-mesenchymal transition (EMT) and putative cancer stem cell (CSC) functions orchestrates to play crucial roles in cancer progression. In this study, we aim to investigate the function of MTDH in PDAC progression concomitant with undergoing EMT/MET plasticity or involving putative CSC functions. Experimental methods: We investigated MTDH expression in 7 human and 6 mouse PDAC cell lines and elucidated the roles of MTDH in PDAC progression by in vitro experiments for the loss of function using siMTDH and shMTDH. In immunohistochemical (IHC) staining of MTDH in resected PDAC tissues, we analyzed the correlation between MTDH expression and the clinico-pathological parameters as well as clinical outcome of the 82 PDAC patients. Results: Western blot analyses showed higher MTDH protein expression in metastatic PDAC compared to primary PDAC cell lines. In metastatic cell lines, MTDH expression has a positive correlation with E-cadherin expression. These data suggest that MTDH might be related to mesenchymal-epithelial transition (MET) and play a crucial role in metastatic cascade of PDAC. Conversely, MTDH has a negative correlation with E-cadherin in primary PDAC cell lines. It is implicated that MTDH might play inverse functions in primary site and metastatic site. Next, to examine what is the role of MTDH in the EMT/MET process, we evaluated the expression pattern of EMT markers and MTDH in response to transient TGF-β1 treatment. We found that E-cadherin expression decreases at 8 to 24 hours after treatment (EMT initiation), and then comes back to increase of the expression at 4 days after treatment (MET induction). While MTDH and E-cadherin expression shows an inverse relation during EMT initiation, these two expressions have a positive correlation during MET period (from 7 days after treatment). Additionally, we found EMT induction by TGF-β1 is attenuated by knockdown of MTDH, These results suggest MTDH might contribute to the phenotypic switch of PDAC cells via the EMT/MET process. MTDH knockdown significantly impaired the ability of pancreatosphere formation in PDAC cells and sensitized PDAC cells to gemcitabine. In FACS analyses, we also found that the percentage of CD133-positive cancer stem cell subpopulation was significantly reduced in MTDH knockdown metastatic PDAC cells. In anoikis assay, (this assay evaluate the resistance for apoptosis after losing contact from extracelluar matrix), MTDH knockdown reduced anoikis resistance in PDAC cells. These data indicate that MTDH might foster putative CSC functions in PDAC cells. In IHC staining, high MTDH expression indicated significantly higher incidence of hematogenous metastasis (p=0.02). Furthermore, High expression group showed poorer prognosis of PDAC patients compared to Low expression group in Kaplan Meier analysis (p=0.029). Conclusions: MTDH might promote PDAC metastasis to undergo EMT in primary PDAC and MET in metastatic PDAC along with.putative CSC functions. The regulation of MTDH might develop the new strategy for PDAC treatment, and lead to improve the outcome of patients. Citation Format: Kensuke Suzuki, Shigetsugu Takano, Hideyuki Yoshitomi, Shingo Kagawa, Hiroaki Shimizu, Masayuki Otsuka, Katunori Furukawa, Tukasa Takayashiki, Satoshi Kuboki, Daisuke Suzuki, Nozomu Sakai, Hiroyuki Nojima, Masaru Miyazaki.{Authors}. The elucidation for functional roles of Metadherin in metastatic cascade of pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B29.

Introduction: We have previously identified C4b-binding protein α-chain (C4BPA) as a serum biomarker for pancreatic ductal adenocarcinoma (PDAC). Serum C4BPA level was significantly higher, especially in the early stage of PDAC patients, than in healthy controls as well as in patients with pancreatitis and other malignancies (Br J Cancer 2016). C4BPA is known as a potent soluble inhibitor of the complement system; however, the functional role of C4BPA remains unknown during tumor progression. In this study, we investigated the clinical significance of C4BPA expression and explored the possibility of correlation between C4BPA and antitumoral immunity in PDAC progression. Materials and Methods: Immunohistochemistry (IHC) for C4BPA was performed in resected PDAC tissues. PDAC tissues were obtained from 172 consecutive patients who underwent pancreatectomy at the Department of General Surgery, Chiba University Hospital, Japan from January 2011 to December 2014. In in vitro experiments, we evaluated C4BPA expression in human pancreatic duct epithelial (HPDE) cell, 7 human PDAC cell lines, resected PDAC sample, and the adjacent normal pancreatic tissues. C4BPA is known as a soluble inhibitor of the complement system and an activating ligand for CD40, which alters tumor microenvironment. The binding of CD40 on B cells or macrophages activates antitumoral T cells and antigen-presenting cells and induces a variety of downstream effects. To elucidate the molecular mechanism, we have addressed the association between C4BPA and CD40 expression in PDAC cells. Results: In IHC analysis, we found that C4BPA was mainly expressed in the stroma surrounding PDAC cells. Based on the staining intensity, PDAC samples were categorized into two groups: 75 cases as a high-expression group and 97 cases as a low-expression group. The Kaplan-Meier analysis showed that patients with low C4BPA expression presented a significantly shorter disease-free (P=0.011) and overall survival time than those with high C4BPA expression (P=0.0016, log-rank test). On multivariate analyses, high venous invasion and low C4BPA expression were observed as an independent prognostic factor in patients with PDAC after curative surgery. In in vitro experiment, C4BPA was strongly expressed in human pancreatic duct epithelial (HPDE) cell line but not in PDAC cell lines by Western blotting. Now, we have confirmed C4BPA expression in frozen section of PDAC tissues and the adjacent normal pancreas tissues, and CD40 expression in various PDAC cell lines. Discussion: Our results suggested that low C4BPA expression in the stroma of PDAC is associated with unfavorable prognosis of PDAC patients. These results also suggested that C4BPA might play a role in the process of elimination and equilibrium phase based on the immunoediting theory. Citation Format: Kosuke Sasaki, Shigetsugu Takano, Hideyuki Yoshitomi, Kazuyuki Sogawa, Shingo Kagawa, Katsunori Furukawa, Tsukasa Takayashiki, Satoshi Kuboki, Daisuke Suzuki, Nozomu Sakai, Takashi Mishima, Eri Nakadai, Masayuki Ohtsuka. C4BPA identified as a novel biomarker is associated with favorable outcome of patients with pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C64.

Introduction: Despite our deep understanding of genetic drivers of the disease, pancreatic ductal adenocarcinoma (PDA) continues to be associated with dismal survival rates. Targeting the DNA repair machinery has emerged as a promising therapeutic strategy to treat pancreatic cancer patients carrying DNA damage repair (DDR) mutations. Such mutations promote tumorigenesis, but also paradoxically render tumor cells particularly susceptible to platinum-based agents and PARP inhibitors (PARPi). However, despite promising preclinical and clinical results, early data demonstrate that eventually most tumors, regardless of DDR status, become resistant to PARPi-therapies. The mRNA-binding protein HuR, predominantly expressed in the nucleus, translocates to the cytoplasm upon tumor-associated stress where it post-transcriptionally regulates select mRNA cargo, resulting in resistance to DNA damaging agents in a harsh tumor microenvironment. Here, we sought to evaluate the role of HuR in regulating PARPi efficacy. Results: In response to cellular stress induced by IC50 dosages of a panel of PARPis such as Veliparib, Olaparib, Rucaparib, Talazoparib and Niraparib, nuclear localized HuR undergoes cytoplasmic translocation. Silencing of HuR via siRNA, CRISPR and a DOX-inducible system resulted in significant decrease in long and short- term PDA cell survival, irrespective of DDR status. To complement and validate in vitro findings, we employed a heterotropic mouse xenograft model using Mia.sh290 cells wherein DOX induction significantly reduced HuR expression. Olaparib mediated PARP inhibition (50mg/kg, 5 days a week) combined with DOX-induced HuR silencing resulted in significant reduction in tumor volumes, compared to Olaparib alone or DOX alone. Mechanistically, we demonstrate that the pro-survival protein HuR facilitates PDA cells to recover from PARPi insult by, in part, regulating poly ADP ribose glycohydrolase (PARG), the major enzyme responsible for hydrolyzing poly-ADP ribose (PAR) polymers, on chromatin and associated proteins. HuR binds to two 41- 43bp long sites in the 3’ untranslated region (3’UTR) of PARG, increasing its mRNA stability and protein expression. Increased PARG activity, further validated via exogenous overexpression, promotes DNA repair efficiency and increases PDA cell survival. Functional analysis indicate that such inhibition of HuR and/or PARG significantly enhances PARPi sensitivity in PDA cells, via increased accumulation of DNA damage γH2AX foci, preventing efficient removal of PAR polymers, and enhancing detrimental trapping of PARP1 on chromatin. Conclusions: Taken together, our results indicate that HuR- mediated upregulation of PARG acts as a universal pro-survival mechanism and HuR inhibition could significantly potentiate PARPi therapy in PDA, irrespective of DNA repair status. Citation Format: Saswati N. Chand, Mahsa Zarei, Akshay R. Kamath, Matthew J. Schiewer, Carmella Romeo, Joseph A. Cozzitorto, Nicole Meisner- Kober, Eric Londin, Isidore Rigoutsos, Karen Knudsen, John M. Pascal, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. HuR dependent inhibition of PARG enhances PARP inhibitor therapy for DNA repair proficient and deficient pancreatic cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1995.

Targeting B cells in pancreatic adenocarcinoma: does RESOLVE resolve the question?

Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

Purpose: Most patients with pancreatic ductal adenocarcinoma (PDAC) have vascular invasion and metastasis, leading to low surgical resection rate and dismal prognosis. Tumor angiogenesis is related to vascular invasion and metastasis. However, anti-angiogenesis therapeutic effects in PDAC are limited. Therefore, it is imperative to explore molecular mechanism of angiogenesis in PDAC.Experimental Design: scRNA-seq data were utilized to delineatetranscriptional profiles of endothelial cells in PDAC. The in vitro and vivo angiogenesis models were used to explore the role of PDAC derived exosomes under hypoxic condition in tumor angiogenesis.Results: Endothelial cells in PDAC had distinct gene expression profiles compared with normal pancreas. The marker genes of endothelial cells in PDAC were enriched for hypoxia and angiogenesis. MiR-30b-5p were significantly enriched in hypoxic PDAC cells derived exosomes, which could be transferred to HUVEC, resulting in the upregulation of miR-30b-5p. Hypoxic PDAC cells derived exosomes could promote tube formation and endothelial cells migration via miR-30b-5p mediated downregulation of gap junction protein GJA1. Moreover, hypoxic PDAC cells derived exosomes increased new microvascular density in vivo. Patients with PDAC had higher levels of total miR-30b-5p and exosomal miR-30b-5p in peripheral blood plasma than healthy subjects. In addition, there were significant correlations for the levels of total miR-30b-5p or exosomal miR-30b-5p between peripheral blood plasma and portal vein plasma.Conclusions: Hypoxic PDAC cells derived exosomal miR-30b-5p promoted angiogenesis by inhibiting GJA1, and miR-30b-5p was a potential diagnostic marker for PDAC.

Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells.

Hedgehog (Hh) signaling is deregulated in multiple human cancers including pancreatic ductal adenocarcinoma (PDA). Because KRAS mutation represents one of the earliest genetic alterations and occurs almost universally in PDA, we hypothesized that oncogenic KRAS promotes pancreatic tumorigenesis in part through activation of the Hh pathway. Here, we report that oncogenic KRAS activates hedgehog signaling in PDA cells, utilizing a downstream effector pathway mediated by RAF/MEK/MAPK but not phosphatidylinositol 3-kinase (PI3K)/AKT. Oncogenic KRAS transformation of human pancreatic ductal epithelial cells increases GLI transcriptional activity, an effect that is inhibited by the MEK-specific inhibitors U0126 and PD98059, but not by the PI3K-specific inhibitor wortmannin. Inactivation of KRAS activity by a small interfering RNA specific for oncogenic KRAS inhibits GLI activity and GLI1 expression in PDA cell lines with activating KRAS mutation; the MEK inhibitors U0126 and PD98059 elicit a similar response. In addition, expression of the constitutively active form of BRAF(E600), but not myr-AKT, blocks the inhibitory effects of KRAS knockdown on Hh signaling. Finally, suppressing GLI activity leads to a selective attenuation of the oncogenic transformation activity of mutant KRAS-expressing PDA cells. These results demonstrate that oncogenic KRAS, through RAF/MEK/MAPK signaling, is directly involved in the activation of the hedgehog pathway in PDA cells and that collaboration between these two signaling pathways may play an important role in PDA progression.

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.

Background: Super-enhancers (SEs) are implicated in regulating genes that establish cell identity, state, and fate. Thus, therapeutic targeting of SEs has emerged as a promising approach for cancer treatment. To understand SE-regulation of gene expression in the tumor microenvironment (TME), we mapped the SE landscape in pancreatic ductal adenocarcinoma (PDAC) cell lines and cancer-associated fibroblasts (CAF) derived from different PDAC patients. Given the dismal 5-year survival rate of just 9% for patients with PDAC, novel treatments are desperately needed. Agents that can inhibit oncogenic SE networks may have potential in the treatment of PDAC. Methods: SE landscapes in 9 PDAC and 9 CAF cell lines were mapped using chromatin immunoprecipitation followed by sequencing (ChIP-seq) for genome-wide binding of H3K27ac. SE ranking and calling were performed using ROSE. RNAseq was carried out to establish gene expression profiles. The effect of epigenetic modulators on SE-associated gene expression was evaluated using Nanostring technology. CRISPR interference (CRISPRi) was used to functionally validate the effect of SE disruption on the expression of a critical SE-associated oncogene. Results: While total enhancers and SEs mapped were higher in CAFs versus PDAC cells, the fraction of enhancers constituting SEs was similar (~5% of all enhancers) in both cell types. PDAC and CAF cells had distinct SE landscapes. Among the 4,192 SEs identified in both cell types, 31% of SEs were shared by at least one or more PDAC or CAF cells, while 20 SEs were recurrent in all eighteen samples. More than 150 SEs were common in most CAFs, while around 100 SEs were present in majority of PDAC cell lines. Strikingly, the presence of SE was almost always associated with upregulation of the gene closest to the SE element. Gene ontology analysis of SE-associated genes highlighted significant enrichment in biologic pathways of tissue morphogenesis, vasculature development, and epithelial cell differentiation in both PDAC and CAFs. Epigenetic inhibitors that target either the bromo-domain proteins or those that inhibit the transcription factor complex II had the most prominent effects on transcriptional repression of SE-associated genes. CRISPRi mediated disruption of an SE element regulating the MYC oncogene resulted in its transcriptional repression, suggesting that SEs positively exert transcriptional pressure on its associated target gene(s). Conclusions: Overall, potentially critical biologic pathways that may be under SE control in PDAC and CAF cells were elucidated. The cell-type specific distinct nature of SE landscape suggests that SEs may be evolutionarily conserved and may be exploited for therapeutic potential. These results offer a means to identify agents that disrupt SEs and further study the consequences of perturbing SE networks in PDAC and its TME. (This work was supported partly by a BSWRI/TGen Collaborative Grant in Oncology Research and the SU2C-CRUK-Lustgarten Foundation Pancreatic Cancer Dream Team grant.) Citation Format: Pawan Noel, Shaimaa Hussein, Kenian Chen, Daniel D. Von Hoff, Yin Lin, Haiyong Han. Mapping super-enhancer signatures in pancreatic ductal adenocarcinoma, cancer-associated fibroblasts and their targeting by epigenetic inhibitors [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A37.

Background: Glucocorticoids are among the most potent anti-inflammatory agents that act by inhibiting the synthesis of almost all known cytokines and influencing multiple transduction pathways. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only potential curative therapeutic. In the present work we investigated the influence of glucocorticoids on PDAC cells in vitro as well as in vivo in a pancreatic carcinoma resection mouse model. Methods: The influence of dexamethasone (DEX), a synthetic glucocorticoid, on proliferation and IL8 secretion in pancreatic cells (BxPC3, Colo357, PancTuI) was analyzed by cell counting and ELISA. NFkB-activity of PancTuI cells treated with DEX was determined by electrophoretic mobility shift assay (EMSA). Furthermore, effects of DEX on the invasiveness were studied by a fibroblast-based invasion assay. In the mouse resection model subtotal pancreatectomy was performed after orthotopic inoculation of human PDAC cells. DEX was administered after resection as an adjuvant treatment regime and 4 weeks later, local recurrent tumor sizes as well as number of liver and spleen metastases were analyzed. Results: In vitro, DEX did not have an anti-proliferative effect on PDAC cells, but strongly reduced the invasiveness well as the activation of NF-κB. The secretion of IL-8 into the supernatant of the tumor cells correlated inversely with the reduced activation of NF-κB. In vivo, we observed a significant reduction of the local recurrent tumor volume and the number of liver and spleen metastases.Conclusions: DEX has a profound influence on the malignant phenotype of PDAC tumor cells in vitro in terms of inhibition of invasiveness and pro-inflammatory signaling. This was approved in vivo by reduced metastasizing capacity and reduced size of local tumor recurrence. Therefore, DEX-treatment appears to be an interesting therapeutical option in an adjuvant setting after pancreatic cancer resection.

BackgroundSqualene epoxidase (SQLE), originally identified as a rate-limiting enzyme for cholesterol biosynthesis, is associated with multiple malignancies. We aim to investigate the regulatory role, molecular mechanisms and clinical implications of...

BackgroundThe interplay between cancer cells and stromal components, including soluble mediators released from cancer cells, contributes to the progression of pancreatic ductal adenocarcinoma (PDAC). Here, we set out to identify key secreted proteins involved in PDAC progression.MethodsWe performed secretome analyses of culture media of mouse pancreatic intraepithelial neoplasia (PanIN) and PDAC cells using Stable Isotope Labeling by Amino acid in Cell culture (SILAC) with click chemistry and liquid chromatography-mass spectrometry (LC-MS/MS). The results obtained were verified in primary PDAC tissue samples and cell line models.ResultsComplement factor B (CFB) was identified as one of the robustly upregulated proteins, and found to exhibit elevated expression in PDAC cells compared to PanIN cells. Endogenous CFB knockdown by a specific siRNA dramatically decreased the proliferation of PDAC cells, PANC-1 and MIA PaCa-II. CFB knockdown induced increases in the number of senescence-associated-β-galactosidase (SA-β-gal) positive cells exhibiting p21 expression upregulation, which promotes cellular senescence with cyclinD1 accumulation. Furthermore, CFB knockdown facilitated downregulation of proliferating cell nuclear antigen and led to cell cycle arrest in the G1 phase in PDAC cells. Using immunohistochemistry, we found that high stromal CFB expression was associated with unfavorable clinical outcomes with hematogenous dissemination after surgery in human PDAC patients. Despite the presence of enriched CD8+ tumor infiltrating lymphocytes in the PDAC tumor microenvironments, patients with a high stromal CFB expression exhibited a significantly poorer prognosis compared to those with a low stromal CFB expression. Immunofluorescence staining revealed a correlation between stromal CFB expression in the tumor microenvironment and an enrichment of immunosuppressive regulatory T-cells (Tregs), myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). We also found that high stromal CFB expression showed a positive correlation with high CD8+/Foxp3+ Tregs populations in PDAC tissues.ConclusionsOur data indicate that CFB, a key secreted protein, promotes proliferation by preventing cellular senescence and is associated with immunological tumor promotion in PDAC. These findings suggest that CFB may be a potential target for the treatment of PDAC.