Pancreatic Intraepithelial Neoplasia Cells Research Articles

Abstract C056: Characterization of chromatin accessibility patterns in the human pancreas and early pancreatic neoplastic lesions using snATAC-seq

Abstract Study of early pancreas neoplasia in humans had previously been limited by lack of available tissue. Recent work by our group on deceased donor pancreata identified pancreatic intraepithelial neoplasia (PanIN) lesions in non-diseased organs and defined a transcriptomic signature for the microenvironment of these pre-cancerous lesions. Prevalence of PanINs in healthy human tissue was higher than expected and established a novel model to expand understanding of the complex biology of these precursor lesions. Epigenetic changes in chromatin structure and the subsequent effects on gene expression are essential processes in neoplasia that have yet to be described at the single cell level in the human pancreas. We hypothesized that previously defined gene signatures could identify acinar, ductal, and PanIN cells in donor pancreata based on chromatin accessibility profiling by single nuclear transposase-accessible chromatin preparation followed by high-throughput sequencing (snATAC-seq). Comparison between the accessibility patterns, transcription factor motifs, and differences in accessibility versus expression profiles can then be used to expand prior knowledge of early pancreatic neoplasia and identify novel targets for further study and therapy selection. To test this hypothesis, single nuclei from seventeen donor pancreata (twenty total samples) were isolated for snATAC-seq using the 10x Genomics platform. Data analysis was conducted using CellRanger, Seurat, Signac, AUCell, and other programs to identify and label chromatin peaks in individual nuclei corresponding to acinar, ductal, and PanIN origin according to gene signatures identified by scRNA-seq and special transcriptomics on matched donor pancreata. Using this method, cells corresponding to these gene signatures, including PanIN- like cells, were indeed identifiable with differentially accessible regions of the genome then analyzed for transcription factor motif enrichment between these cell types. Motifs enriched in cells identified as PanINs compared to either ductal or acinar cells included several transcription factors implicated in tumorigenesis. These results are similar to previously demonstrated essential factors in pancreatic neoplasia, suggesting this strategy is a relevant method for data analysis and discovery of novel factors implicated in tumorigenesis. Ongoing studies include validation of these findings in vitro using human pancreatic normal epithelial cells, human tumor cell lines, and organoid models derived from human normal and tumor samples. Together, this study demonstrates that PanINs can be identified in human pancreas tissue by snATAC-seq and that differentially expressed motifs can be identified, providing opportunity for further integration with transcriptomic information to elucidate detailed understanding of early neoplasia in the pancreas for future drug development and targeted therapeutics studies. Citation Format: Jamie N Mills, Aaron Dendekker, Joyce K Thompson, Hannah Watkoske, Simone Benitz, Padma Kadiyala, Ahmed Elhossiny, Howard Crawford, Eileen Carpenter, Marina Pasca di Magliano, Filip Bednar. Characterization of chromatin accessibility patterns in the human pancreas and early pancreatic neoplastic lesions using snATAC-seq [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C056.

Abstract C069: A conserved reepithelialization program driven by FOSL1 underlies malignancy in pancreatic ductal adenocarcinoma

Abstract Benign lesions named pancreatic intraepithelial neoplasia (PanIN) are frequent in the healthy population but are precursors to deadly pancreatic ductal adenocarcinoma (PDAC). Conversion of PanINs to PDAC is poorly understood. Here we harness high resolution spatial transcriptomics to identify a core transcriptional program associated with invasive growth across human PDAC and required for PanIN-to-PDAC progression in mice. The malignancy program is lineage restricted, and predominantly comprised of transcripts activated to drive reepithelialization during skin wound healing. This wound-like program appears in rare PanIN cells that also activate tumor suppressor genes (TSGs), which keep the incipient malignant cells in check. TSG KO in PanINs leads to rapid outgrowth of the wound-like population, unleashing invasive growth, driven by the wound-induced TF FOSL1. Bidirectional interactions between malignant epithelial cells and wound-like CTHRC1-expressing CAFs enforce the malignant state. Our results suggest that wound recapitulation drives the highly invasive properties of PDAC. Citation Format: Charles David, Melian Zhuo, Yong Li, Yifeng Zhang, Chenlu Yang, Ziqing Deng, Yupei Zhao, Wennming Wu, Junya Peng, Mo Chen. A conserved reepithelialization program driven by FOSL1 underlies malignancy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C069.

Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.

Immunoprevention is an emerging consideration for solid tumors, including pancreatic ductal adenocarcinoma (PDAC). We and others have shown that Kras mutations in genetic models of spontaneous pancreatic intraepithelial neoplasia (PanIN), which is a precursor to PDAC, results in CD73 expression in the neoplastic epithelium and some populations of infiltrating immune cells, including macrophages and CD8 T cells. CD73 is an ecto-enzyme that converts extracellular adenosine monophosphate to adenosine, a critical immune inhibitory molecule in PDAC. We hypothesized inhibition of CD73 would reduce the incidence of PanIN formation and alter the immune microenvironment. To test our hypothesis, we used the KrasG12D; PdxCre1 (KC) genetically engineered mouse model and tested the utility of AB-680, a small molecule inhibitor targeting CD73, to inhibit PanIN progression. AB-680, or vehicle control, was administered using oral gavage delivery 3 days/week at 10 mg/kg, beginning when the mice were 2 months old and lasting 3 months. We euthanized the mice at 5 months old. In the KC model, we quantified significantly less pancreatitis, early and advanced PanIN, and quantified a significant increase in M1 macrophages in AB-680-treated mice. Single-cell RNA sequencing (scRNA-seq) of pancreata of AB-680-treated mice revealed increased infiltration of CD4+ T cells, CD8+ T cells, and mature B cells. The scRNA-seq analysis showed that CD73 inhibition reduced M2 macrophages, acinar, and PanIN cell populations. CD73 inhibition enhanced immune surveillance and expanded unique clonotypes of TCR and BCR, indicating that inhibition of CD73 augments adaptive immunity early in the neoplastic microenvironment. Prevention Relevance: Previous studies found PanIN lesions in healthy pancreata. Not all progress to PDAC, suggesting a window for enhanced antitumor immunity through immunoprevention therapy. CD73 inhibition in our study prevents PanIN progression, reduces immune-suppressive macrophages and expands TCR and BCR unique clonotypes, highlighting an encouraging therapeutic avenue for high-risk individuals.

Abstract 1206: Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity

Abstract Immunotherapies are generally ineffective in pancreatic ductal adenocarcinoma (PDAC). Paradoxically, inflammation is believed to play a key role in PDAC development and invasion: patients suffering from chronic pancreatitis have a 13-fold increase in risk of developing PDAC. PDAC cells are surrounded by a dense network of fibrotic tissue containing immunosuppressive cells such as regulatory T cells, tumor-associated macrophages, and cancer-associated fibroblasts. Better understanding of the role of inflammation in PDAC could lead to the design of effective immunotherapies. Pancreatic intraepithelial neoplasia (PanIN) is a precursor to PDAC. While most of us will develop PanINs, few of these lesions will progress to invasive cancer. Efforts to determine which PanINs are likely to progress have shown that the size, incidence, and genetic variation of these lesions is high. Here, we add to these efforts through in-depth quantitative analysis of the pancreatic immune microenvironment. CODA is a novel 3D imaging technique used extensively to study the structure, transcriptomic signatures, and genetic heterogeneity of PanINs. In serial histological samples, CODA integrates image registration, cell detection, and segmentation of nine pancreatic structures: PanIN, normal ducts, islets, acini, nerves, vasculature, fat, stroma, and immune aggregates. Through integration with CD45, CD3, and FoxP3 labelled images, we explored immune infiltration in 48 large (cm3) samples of human pancreas tissues containing >1,000 PanIN lesions to describe spatial correlations between local immune hotspots, 3D tumor morphology, and the surrounding pancreas structures. We quantified inter- and intra-sample heterogeneity in immune response to pancreatic cancer precursor lesions. We determined that this cohort varied greatly in both the number of PanIN (range 3 - 92 per cm3) and overall immune cell density (range 3,000 - 40,000 immune cells/mm3). We determined that, while PanIN size is not related to local immune cell density, overall PanIN burden is highly correlated to global immune cell density. This suggests that PanIN content does not have a short-range relationship to inflammation, but does have a long-range relationship with pancreatic structures and immune infiltration. As chronic inflammation in the pancreas shows correlation with development of pancreatic cancer, increased knowledge of the heterogeneous, local immune cell environment in precancerous samples will help us determine the role of inflammation in reacting to and controlling malignant progression. Citation Format: Ashley L. Kiemen, Cristina Almagro-Pérez, Valentina Matos-Romero, Alicia M. Braxton, Jaanvi Mahesh-Babu, Elizabeth D. Thompson, Toby C. Cornish, PeiHsun Wu, Laura D. Wood, Arrate Muñoz-Barrutia, Ralph H. Hruban, Denis Wirtz. Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1206.

Sodium butyrate induces cell senescence in murine pancreatic intraepithelial neoplasia cells

Sodium butyrate induces cell senescence in murine pancreatic intraepithelial neoplasia cells

Abstract PR007: Three-dimensional genomic analysis of human pancreatic intraepithelial neoplasia (PanIN) reveals striking multifocality and genetic heterogeneity

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease with a 5 year survival rate of 11%. PDAC arises from precursor lesions, the most common of which is pancreatic intraepithelial neoplasia (PanIN). Molecular analysis of human PanINs is critical to understand early pancreatic tumorigenesis, which could inform risk stratification, early detection, and cancer prevention approaches. Because PanINs are microscopic, it is challenging to determine their full extent and anatomic relationships using two dimensional histological sections. To determine the density and connectivity of PanINs, we utilized a novel machine learning algorithm (CODA) for three-dimensional (3D) reconstruction and cellular quantification. Large blocks of grossly normal pancreas were harvested from 39 surgical pancreatectomy specimens, followed by formalin fixation, paraffin-embedding, and complete serial sectioning. 3D reconstruction using CODA revealed striking multifocality of PanINs within the pancreata of most analyzed patients, with more than 600 spatially PanINs being modeled. Next, to determine whether these multifocal PanINs arose as independent neoplasms or via intraductal spread of a single PanIN, we assessed their clonal relationships using somatic multi-region DNA next generation sequencing (NGS). To do so, 8 additional blocks of grossly normal pancreatic tissue were harvested and 3D modeled, yielding 37 anatomically distinct PanINs for genomic analysis. Each spatially unconnected PanIN was separately microdissected in five different regions to assess both intra-PanIN and inter-PanIN genetic heterogeneity. 99 samples from the 37 PanIN lesions underwent targeted NGS using a panel covering major drivers of pancreatic ductal neoplasia. For PanINs with sufficient DNA, whole exome sequencing and deep sequencing of KRAS was also performed. Across the 8 blocks, 10 PanINs shared no mutations with other PanINs in the same block, indicating independent clonal origin. In addition, 8 PanINs shared only KRAS hotspot mutations with numerous other unshared mutations, likely indicating independent PanINs that shared hotspot mutations by chance. Six spatially separate low grade PanINs shared both driver and passenger mutations with at least one other spatially unconnected PanIN, demonstrating the ability of PanIN cells to dissociate from one lesion and establish unconnected, genetically related PanINs nearby. Furthermore, 5 PanINs harbored multiple KRAS mutations within a single PanIN, suggesting a polyclonal origin. One PanIN lacked any driver gene mutations. The genetic origins of the remaining 7 spatially separate PanINs could not be resolved due to a lack of discrete mutations found by targeted sequencing. Our 3D genomic analysis of anatomically distinct PanINs demonstrates that the unexpectedly large number of PanINs in normal pancreas most often arise independently, providing new foundations for our understanding of early pancreatic tumorigenesis. Determining the mechanisms for this multifocal neoplasia is an important direction for future research. Citation Format: Alicia M. Braxton, Ashley Kiemen, Rachel Karchin, Yuchen Jiao, Pei-Hsun Wu, Ralph H Hruban, Denis Wirtz, Laura D. Wood. Three-dimensional genomic analysis of human pancreatic intraepithelial neoplasia (PanIN) reveals striking multifocality and genetic heterogeneity [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr PR007.

Complement factor B regulates cellular senescence and is associated with poor prognosis in pancreatic cancer

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.

A Fucoxanthinol Induces Apoptosis in a Pancreatic Intraepithelial Neoplasia Cell.

Fucoxanthinol (FxOH), a predominant metabolite from fucoxanthin (Fx), can exert potential anti-cancer effects in various cancers. However, limited data are available on the effect of FxOH or Fx on pancreatic cancer. The present study investigated the effect of FxOH on a cell line derived from pancreatic cancer tissue developed in Ptf1aCre/+; LSL-k-rasG12D/+ mice. Using flow-cytometric, microarrays, and western blotting analyses, alterations in FxOH-induced apoptosis-related gene expression and protein levels were evaluated in a mice pancreatic cancer cell line, KMPC44. FxOH significantly arrested the cells at S phase along with suppression of many gene sets, such as cytokine- cytokine receptor interaction and cell adhesion molecule CAMS. Moreover, attenuated protein levels for cytokine receptors, adhesion, phosphatidylinositol-3 kinase/protein kinase B, and mitogen-activated protein kinase were observed. FxOH may prevent pancreatic cancer development in a murine cancer model.

Promoter-Level Transcriptome Identifies Stemness Associated With Relatively High Proliferation in Pancreatic Cancer Cells.

Both pancreatic intraepithelial neoplasia (PanIN), a frequent precursor of pancreatic cancer, and intraductal papillary mucinous neoplasm (IPMN), a less common precursor, undergo several phases of molecular conversions and finally develop into highly malignant solid tumors with negative effects on the quality of life. We approached this long-standing issue by examining the following PanIN/IPMN cell lines derived from mouse models of pancreatic cancer: Ptf1a-Cre; KrasG12D; p53f/+ and Ptf1a-Cre; KrasG12D; and Brg1f/f pancreatic ductal adenocarcinomas (PDAs). The mRNA from these cells was subjected to a cap analysis of gene expression (CAGE) to map the transcription starting sites and quantify the expression of promoters across the genome. Two RNA samples extracted from three individual subcutaneous tumors generated by the transplantation of PanIN or IPMN cancer cell lines were used to generate libraries and Illumina Seq, with four RNA samples in total, to depict discrete transcriptional network between IPMN and PanIN. Moreover, in IPMN cells, the transcriptome tended to be enriched for suppressive and inhibitory biological processes. In contrast, the transcriptome of PanIN cells exhibited properties of stemness. Notably, the proliferation capacity of the latter cells in culture was only minimally constrained by well-known chemotherapy drugs such as GSK690693 and gemcitabine. The various transcriptional factor network systems detected in PanIN and IPMN cells reflect the distinct molecular profiles of these cell types. Further, we hope that these findings will enhance our mechanistic understanding of the characteristic molecular alterations underlying pancreatic cancer precursors. These data may provide a promising direction for therapeutic research.

Immune Cell Production of Interleukin 17 Induces Stem Cell Features of Pancreatic Intraepithelial Neoplasia Cells

Little is known about how the immune system affects stem cell features of pancreatic cancer cells. Immune cells that produce interleukin 17A (IL17A) in the chronically inflamed pancreas (chronic pancreatitis) contribute to pancreatic interepithelial neoplasia (PanIN) initiation and progression. We investigated the effects that IL17A signaling exerts on pancreatic cancer progenitor cells and the clinical relevance of this phenomena. We performed studies with Mist1Cre;LSLKras;Rosa26mTmG (KCiMist;G) and Kras(G12D);Trp53(R172H);Pdx1-Cre (KPC) mice (which upon tamoxifen induction spontaneously develop PanINs) and control littermates. Some mice were injected with neutralizing antibodies against IL17A or control antibody. Pancreata were collected, PanIN epithelial cells were isolated by flow cytometry based on lineage tracing, and gene expression profiles were compared. We collected cells from pancreatic tumors of KPC mice, incubated them with IL17 or control media, measured expression of genes regulated by IL17 signaling, injected the cancer cells into immune competent mice, and measured tumor growth. IL17A was overexpressed in pancreata of KCiMist mice from an adenoviral vector. Pancreata were collected from all mice and analyzed by histology and immunohistochemistry. Levels of DCLK1 and other proteins were knocked down in KPC pancreatic cancer cells using small interfering or short hairpin RNAs; cells were analyzed by immunoblotting. We obtained 65 pancreatic tumor specimens from patients, analyzed protein levels by immunohistochemistry, and compared results with patient survival times. We also analyzed gene expression levels and patient outcome using The Cancer Genome Atlas database. PanIN cells from KCiMist;G mice had a gene expression pattern associated with embryonic stem cells. Mice given injections of IL17-neutralizing antibodies, or with immune cells that did not secrete IL17, lost this expression pattern and had significantly decreased expression of DCLK1 and POU2F3, which regulate tuft cell development. KCiMist mice that overexpressed IL17 formed more PanINs, with more DCLK1-positive cells, than control mice. Pancreatic tumor cells from KPC mice and human Capan-2 cells exposed to IL17A had increased activation of NF-κB and mitogen-activated protein kinase signaling and increased expression of DCLK1 and ALDH1A1 (a marker of embryonic stem cells) compared with cells in control media. These cells also formed tumors faster that cells not exposed to IL17 when they were injected into immunocompetent mice. KPC cells with knockdown of DCLK1 expressed lower levels of ALDH1A1 after incubation with IL17 than cells without knockdown. Expression of the IL17 receptor C was higher in DCLK1-positive PanIN cells from mice compared with DCLK1-negative PanIN cells. In human pancreatic tumor tissues, high levels of DCLK1 associated with a shorter median survival time of patients (17.7 months, compared with 26.6 months of patients whose tumors had low levels of DCLK1). Tumor levels of POU2F3 and LAMC2 were also associated with patient survival time. In studies of mouse and human pancreatic tumors and precursors, we found that immune cell-derived IL17 regulated development of tuft cells and stem cell features of pancreatic cancer cells via increased expression of DCLK1, POU2F3, ALDH1A1, and IL17RC. Strategies to disrupt this pathway might be developed to prevent pancreatic tumor growth and progression.

HMGA1 expression levels are elevated in pancreatic intraepithelial neoplasia cells in the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer

Background:Pancreatic cancer is currently the third leading cause of cancer deaths in the United States and it is predicted to become the second by the year 2030. High-mobility group A1 protein (HMGA1) is an oncogenic transcription factor, localised and active in cell nuclei, that is linked to tumour progression in many human cancers, including pancreatic cancer. Overexpression of HMGA1 renders cancer cells resistant to chemotherapy. Although the Ptf1a-Cre; LSL-KrasG12D transgenic mouse is perhaps the most widely utilised animal model for human pancreatic cancer, expression levels of HMGA1 in pancreata from this mouse model have not been characterised.Methods:Quantitative immunohistochemical analysis was used to determine nuclear HMGA1 levels in pancreatic tissue sections from Ptf1a-Cre; LSL-KrasG12D mice aged 5, 11, and 15 months. The H Score method was used for quantitative analysis.Results:The HMGA1 levels were significantly elevated in pancreatic intraepithelial neoplasia (PanIN) epithelia compared with untransformed acinar tissues or fibroinflammatory stroma.Conclusions:The PanINs have long been regarded as precancerous precursors to pancreatic adenocarcinoma. Significantly elevated HMGA1 levels observed in the nuclei of PanINs in Ptf1a-Cre; LSL-KrasG12D mice validate this animal model for investigating the role that HMGA1 plays in cancer progression and testing therapeutic approaches targeting HMGA1 in human cancers.

Quantitative assessment of pancreatic cancer precursor lesions in IHC-stained tissue with a tissue image analysis platform

Tissue image analysis (tIA) is emerging as a powerful tool for quantifying biomarker expression and distribution in complex diseases and tissues. Pancreatic ductal adenocarcinoma (PDAC) develops in a highly complex and heterogeneous tissue environment and, generally, has a very poor prognosis. Early detection of PDAC is confounded by limited knowledge of the pre-neoplastic disease stages and limited methods to quantitatively assess disease heterogeneity. We sought to develop a tIA approach to assess the most common PDAC precursor lesions, pancreatic intraepithelial neoplasia (PanIN), in tissues from KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre (KPC) mice, a validated model of PDAC development. tIA profiling of training regions of PanIN and tumor microenvironment (TME) cells was utilized to guide identification of PanIN/TME tissue compartment stratification criteria. A custom CellMap algorithm implementing these criteria was applied to whole-slide images of KPC mice pancreata sections to quantify p53 and Ki-67 biomarker staining in each tissue compartment as a proof-of-concept for the algorithm platform. The algorithm robustly identified a higher percentage of p53-positive cells in PanIN lesions relative to the TME, whereas no difference was observed for Ki-67. Ki-67 expression was also quantified in a human pancreatic tissue sample available to demonstrate the translatability of the CellMap algorithm to human samples. Together, our data demonstrated the utility of CellMap to enable objective and quantitative assessments, across entire tissue sections, of PDAC precursor lesions in preclinical and clinical models of this disease to support efforts leading to novel insights into disease progression, diagnostic markers, and potential therapeutic targets.

Pigment Epithelium-derived Factor (PEDF) Blocks Wnt3a Protein-induced Autophagy in Pancreatic Intraepithelial Neoplasms

An increase in autophagy characterizes pancreatic carcinogenesis, but the signals that regulate this process are incompletely understood. Because canonical Wnt/β-catenin signaling is necessary for the transition from early to advanced pancreatic intraepithelial neoplasia (PanIN) lesions, we assessed whether Wnt ligands and endogenous inhibitors of Wnt signaling modulate autophagy. In this study, canonical Wnt3a ligand induced autophagy markers and vacuoles in murine PanIN cells. Furthermore, pigment epithelium-derived factor (PEDF), a secreted glycoprotein known for its anti-tumor properties, blocked Wnt3a-directed induction of autophagy proteins. Autophagy inhibition was complemented by reciprocal regulation of the oxidative stress enzymes, superoxide dismutase 2 (SOD2) and catalase. Transcriptional control of Sod2 expression was mediated by PEDF-induced NFκB nuclear translocation. PEDF-dependent SOD2 expression in PanIN lesions was recapitulated in a murine model of PanIN formation where PEDF was deleted. In human PanIN lesions, co-expression of PEDF and SOD2 was observed in the majority of early PanIN lesions (47/50, 94%), whereas PEDF and SOD2 immunolocalization in high-grade human PanIN-2/3 was uncommon (7/50, 14%). These results indicate that PEDF regulates autophagy through coordinate Wnt signaling blockade and NFκB activation.

Abstract 4941: Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancer types, with a 5-year survival rate <5%. Tumor specific biomarkers that can aid in early detection and treatment monitoring are urgently needed in order to improve the outcomes. Human Reg family proteins (Reg1A, Reg1B, Reg3A, Reg3G and Reg4) are a group of calcium-dependent (C-type) lectins that promote cell growth and respond to inflammatory stimulants. We examined their associations with pancreatic ductal adenocarcinoma (PDAC). In patients with PDAC, the serum levels of Reg1A and 1B were significantly elevated (p = 0.0003 and 0.0018), which correlated with TNM stages of the patients. By using immunohistochemistry (IHC), the expression of Reg1A, Reg1B and Reg4 was observed in the malignant glandular epithelium of pancreatic intraepithelial neoplasia (PanIN) and invasive PDAC. The intensity of Reg1A and Reg1B staining positively correlated with the histological grades of PanIN. The serum levels and tissue expression of Reg1A and Reg1B were negatively correlated with the differentiation grades of invasive PDAC. Additionally, patchy expression of Reg1A andReg3A/G was also observed in areas with acinar-to-ductal metaplasia (ADM) adjacent to cancer cells. Our data suggests that the changes in Reg proteins were closely associated with the carcinogenesis of PDAC. Reg1A and 1B proteins showed specificity for pancreatic ductal differentiation and could be used as clinical markers for early detection of PDAC, monitoring therapeutic response and predicting tumor recurrence after therapy and tumor metastasis. The expression of Reg family proteins in both ADM and PanIN cells supports the notion that ADM could be an alternative mechanism of PDAC carcinogenesis. Citation Format: Qing Li, Hao Wang, George Zogopoulos, Qin Shao, Jun-Li Liu, Zu-Hua Gao. Overexpression of Reg proteins in human pancreatic ductal adenocarcinoma and acinar-to-ductal metaplasia. [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 4941.

Adipocytes promote pancreatic cancer cell proliferation via glutamine transfer

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.

Pancreatic preneoplastic lesions plasma signatures and biomarkers based on proteome profiling of mouse models.

Background:Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a mortality that is almost identical to incidence. Because early detected PDAC is potentially curable, blood-based biomarkers that could detect currently developing neoplasia would improve patient survival and management. PDAC develops from pancreatic intraepithelial neoplasia (PanIN) lesions, graded from low grade (PanIN1) to high grade (PanIN3). We made the hypothesis that specific proteomic signatures from each precancerous stage exist and are detectable in plasma.Methods:We explored the peptide profiles of microdissected PanIN cells and of plasma samples corresponding to the different PanIN grade from genetically engineered mouse models of PDAC using capillary electrophoresis coupled to mass spectrometry (CE-MS) and Chip-MS/MS.Results:We successfully characterised differential peptides profiles from PanIN microdissected cells. We found that plasma from tumor-bearing mice and age-matched controls exhibit discriminative peptide signatures. We also determined plasma peptide signatures corresponding to low- and high-grade precancerous step present in the mice pancreas using the two mass spectrometry technologies. Importantly, we identified biomarkers specific of PanIN3.Conclusions:We demonstrate that benign and advanced PanIN lesions display distinct plasma peptide patterns. This strongly supports the perspectives of developing a non-invasive screening test for prediction and early detection of PDAC.

Abstract 924: GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis

Abstract Introduction: Nicotine and nitrosamine exposure from smoking causes pancreatic cell injury and contributes to a cascade of oncogenic events that may be contributing to the rising rate of pancreatic cancer (PDAC). Cytokines activate kinases and transcription factors including cyclic AMP response element binding (CREB) protein. CREB activation through phosphorylation regulates diverse cellular responses. We studied whether granulocyte-macrophage colony stimulating factor (GM-CSF)-dependent phosphorylated CREB plays a role in smoking-induced pathogenesis of PDAC. Experimental procedure: Human tissue microarray analysis was performed to determine the significance of pCREB expression amongst smokers and non-smokers. Total RNA extracted from immortalized human pancreatic ductal cell (H6c7) and pancreatic intraepithelial neoplasia (PanIN) mouse cell lines (LSL-KrasG12D/+; Pdx1Cre/+) were treated with tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and RNA sequencing (RNA-seq) was performed. Network, pathway and functional analyses of the transcriptome were conducted. To analyze the signaling pathway involved in NNK-induced tumorigenicity, we performed a phospho-kinase antibody array and cytokine antibody array on NNK treated H6c7 and PanIN cells or conditioned media (CM) protein. Western blot and ELISA were used to validate the array data findings. NNK-induced activation of GM-CSF, G-CSF and IL-6 were blocked using monoclonal antibodies. PKT (Ptf1acre/+;LSL-KrasG12D/+;Tgfbr2flox/flox) mice and nude mice with MiaPaCa2 flank xenografts were treated with MEK inhibition (AZD6244) with or without NNK/in vivo smoking. Tumors from these mice were immunoblotted for phosphorylation of MEK1/2, MSK1/2, RSK and CREB. PDAC cells with CREB siRNA or human rGM-CSF were treated with NNK and analyzed for in vitro functional assays and EMT characteristics. Results: Expression of pCREB was significantly higher (p<0.001) in smokers when compared to non-smokers. Overall survival of smokers with high pCREB expression in their primary tumor was associated with significantly decreased median survival when compared to non-smokers with low pCREB expression. Exposure of cells to NNK resulted in phosphorylation of CREB, c-Jun and β-catenin, and release of GM-CSF, G-CSF and IL-6. RNA-seq analysis confirmed activation of MEK/ERK signaling. Studies were performed to elucidate the possible regulatory mechanism behind NNK mediated induction of GM-CSF and its downstream signaling. Importantly, blocking GM-CSF inhibited NNK-induced phosphorylation of CREB and it was mediated through MEK signaling. The in vivo and in vitro tumorigenicity assays showed an increase in the tumorigenic potential and EMT of NNK treated PDAC cells. Conclusions: Our study demonstrates that NNK induces pancreatic tumorigenesis through GM-CSF mediated activation of CREB. Citation Format: Jason Castellanos, Kumaraswamy Honnenahally, Chanjuan Shi, Nipun Merchant, Nagaraj Nagathihalli. GM-CSF induces CREB signaling pathways and modulates tobacco carcinogen-induced pancreatic tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 924. doi:10.1158/1538-7445.AM2015-924

Abstract B116: Modeling of early to invasive stages of pancreatic cancer progression with an iPSC-like line from human pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the worst prognoses of any human malignancy. When human PDAC cells are injected into immunodeficient animals, they create tumors of the late stage from which they were derived. We hypothesized that if human PDAC cells were converted to pluripotency and then allowed to differentiate back into pancreatic tissue, they might undergo early stages of cancer. Although most induced pluripotent stem cell (iPSC) lines that we generated from PDAC were not of the expected cancer genotype, one PDAC iPS-like line, 10–22 cells, conferred the highly disrupted cancer genome of the starting tumor epithelial population. When 10-22 iPS-like cells are injected into immunodeficient mice, they generate pancreatic intraepithelial neoplasia (PanIN) precursors that progress to the invasive stage PDAC. Using this unique system, we established serum-free cultures of human PanIN lesions from immunodeficient mice injected with 10-22 cells and analyzed the proteome of secreted and released proteins. The live human PanIN explants secrete or release proteins from many genes that are known to be expressed in human pancreatic cancer progression and that predicted an HNF4a network achieved in intermediate-stage lesions from new clinical samples. Recent studies show that the PanIN cells progress to metastases at longer time. The 10-22 cell line is being engineered with near-infrared fluorescent protein under the control of the ductal K19 promoter so that populations can be retrieved by FACS for transcriptome analysis as well as tumor progression and regression in response to drugs can be monitored in vivo. We are now assessing the secreted or released proteins from early lesions grown in mice as diagnostic markers for human PDAC prognosis. Thus, rare events allow iPSC technology to provide a live human cell model of early pancreatic cancer and insights into disease progression. Citation Format: Jungsun Kim, Kenneth S. Zaret. Modeling of early to invasive stages of pancreatic cancer progression with an iPSC-like line from human pancreatic ductal adenocarcinoma. [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 B116.

ATDC induces an invasive switch in KRAS-induced pancreatic tumorigenesis

The initiation of pancreatic ductal adenocarcinoma (PDA) is linked to activating mutations in KRAS. However, in PDA mouse models, expression of oncogenic mutant KRAS during development gives rise to tumors only after a prolonged latency or following induction of pancreatitis. Here we describe a novel mouse model expressing ataxia telangiectasia group D complementing gene (ATDC, also known as TRIM29 [tripartite motif 29]) that, in the presence of oncogenic KRAS, accelerates pancreatic intraepithelial neoplasia (PanIN) formation and the development of invasive and metastatic cancers. We found that ATDC up-regulates CD44 in mouse and human PanIN lesions via activation of β-catenin signaling, leading to the induction of an epithelial-to-mesenchymal transition (EMT) phenotype characterized by expression of Zeb1 and Snail1. We show that ATDC is up-regulated by oncogenic Kras in a subset of PanIN cells that are capable of invading the surrounding stroma. These results delineate a novel molecular pathway for EMT in pancreatic tumorigenesis, showing that ATDC is a proximal regulator of EMT.

Abstract B02: Modeling of early to invasive stages of pancreatic cancer progression with an iPSC-like line from human pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) has one of the worst prognoses of any human malignancy. When human PDAC cells are injected into immunodeficient animals, they create tumors of the late stage from which they were derived. We hypothesized that if human PDAC cells were converted to pluripotency and then allowed to differentiate back into pancreatic tissue, they might undergo early stages of cancer. Although most induced pluripotent stem cell (iPSC) lines were not of the expected cancer genotype, one PDAC line, 10–22 cells, when injected into immunodeficient mice, generates pancreatic intraepithelial neoplasia (PanIN) precursors to PDAC that progress to the invasive stage. The PanIN-like cells secrete or release proteins from many genes that are known to be expressed in human pancreatic cancer progression and that predicted an HNF4a network in intermediate-stage lesions. Recent studies show that the PanIN cells progress to metastases at longer time. The cells are being engineered so that tumor progression, and regression in response to drugs, can be monitored in vivo. Thus, rare events allow iPSC technology to provide a live human cell model of early pancreatic cancer and insights into disease progression. Citation Format: Jungsun Kim, John P. Hoffman, Maximilian Reichert, Andrew Rhim, Ben Z. Stanger, Andrew Gumbs, Kenneth S. Zaret. Modeling of early to invasive stages of pancreatic cancer progression with an iPSC-like line from human pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B02.