Pancreatic Cancer Stem Cells Markers Research Articles

Clinical significance and therapeutic implication of CD200 in pancreatic cancer

BackgroundCD200, a negative regulator of T cells as well as a marker for cancer stem cells, represents a significant prognostic factor and potential therapeutic target in certain cancers. However, its clinical significance remains unknown in pancreatic ductal adenocarcinoma (PDAC). MethodsCD200 was assessed in 220 resected PDAC patients who underwent surgery with or without neoadjuvant chemoradiotherapy (NACRT). We examined the clinicopathological outcomes associated with CD200 and further assessed its clinical implications regarding immunological and cancer stem cell properties. ResultsNACRT was associated with higher CD200 expression (66.4 % vs. 32.2 %, P < 0.001) compared to upfront surgery. CD200 was identified as an independent poor prognostic factor in NACRT (hazard ratio 1.90, 95 % confidence interval 1.12–3.23, P = 0.016), but not in upfront surgery patients. Post-recurrence survival was significantly worse in CD200+ patients compared to CD200- patients in the NACRT group, but there was no significant difference observed in the upfront surgery group. CD200 expression was correlated with significantly lower levels of CD4+, CD8+, and CD45RO+ tumor-infiltrating lymphocytes. Furthermore, the correlation of CD200 with pancreatic cancer stem cell markers CD44/CD24/ESA was stronger in irradiated human pancreatic cancer cells. ConclusionsOur data underscore novel roles for CD200 in immune evasion as well as therapy resistance in pancreatic cancer. CD200 may represent a novel poor prognostic predictive factor and potential therapeutic target for PDAC with NACRT.

Abstract B080: A porcine model of pancreatic ductal adenocarcinoma for human scale translational cancer research

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly fatal form of cancer, requiring urgent advancements in diagnostic and therapeutic strategies. Although PDAC mouse models have been crucial in advancing our comprehension of the disease, they present some limitations in replicating key human PDAC aspects. In this study, we present the development of the first large animal model of pancreatic cancer via Cre activatable mutations in the endogenous KRAS and TP53 genes 1,2. In order to activate these mutations in the pancreas, two Cre driver pig lines expressing Cre recombinase from PTF1A 3 and PDX1 were created. Crossing both Cre lines with pigs carrying latent KRAS G12D and TP53 R167H alleles produced double (KC) and triple (KPC) genetically modified animals. Pathological analyses revealed the presence of inter- and intralobular fibrosis with multifocal acinar-to-ductal metaplasia (ADM), some low-grade PanINs, dysplasia, inflammation and duct obstruction in most of the KPC pigs between 3 to 6 months of age. Interestingly, the majority of these pigs already exhibited local metastasis in the lymph nodes, suggesting that the metastatic potential is established early in the tumorigenic process. Age-matched KC pigs exhibited mainly ADM with dysplasia. Flow cytometry and RNA expression analyses of the established pancreatic tumour primary cells revealed a pronounced epithelial-to-mesenchymal transition (EMT). Moreover, both bulk tumour samples and primary tumour cells showed high expression of the pancreatic cancer stem cell marker CD44, with differential expression of its isoforms, including the major isoform and variants 6 and 9. In addition, histological and molecular analyses revealed notable similarities between pig and human PDAC, including molecular subtypes, neuronal invasion and loss of acinar identity. These findings highlight the unique advantage of the pig PDAC model, that allows longitudinal and multi-regional tumour sampling in the absence of any therapeutic interventions. This makes it a valuable tool for developing new diagnostic and therapeutic approaches that can greatly improve patient outcomes in the long run.

SNAIL2 contributes to tumorigenicity and chemotherapy resistance in pancreatic cancer by regulating IGFBP2.

Pancreatic cancer has an extremely poor prognosis because of its resistance to conventional therapies. Cancer stem cell (CSC)‐targeted therapy is considered a promising approach for this disease. Epithelial‐mesenchymal transition‐inducing transcription factors (EMT‐TFs) contribute to CSC properties in some solid tumors; however, this mechanism has not been fully elucidated in pancreatic cancer. Zinc finger protein, SNAIL2 (also known as SLUG), is a member of the SNAIL superfamily of EMT‐TFs and is commonly overexpressed in pancreatic cancer. Patients exhibiting high SNAIL2 expression have a poor prognosis. In this study, we showed that the suppression of SNAIL2 expression using RNA interference decreased tumorigenicity in vitro (sphere formation assay) and in vivo (xenograft assay) in 2 pancreatic cancer cell lines, KLM1 and KMP5. In addition, SNAIL2 suppression resulted in increased sensitivity to gemcitabine and reduced the expression of CD44, a pancreatic CSC marker. Moreover, experiments on tumor spheroids established from surgically resected pancreatic cancer tissues yielded similar results. A microarray analysis revealed that the mechanism was mediated by insulin‐like growth factor (IGF) binding protein 2. These results indicate that IGFBP2 regulated by SNAIL2 may represent an effective therapeutic target for pancreatic cancer.

Tumor-associated macrophages derived from cancer stem cells

Macrophages are the most abundant immune cells in the microenvironment of solid tumors. The present study displayed histological and immunohistochemical analyses of a malignant tumor model developed from cancer stem cells (CSCs) converted from human induced pluripotent stem cells (hiPSCs) in a cancer microenvironment prepared from the conditioned medium (CM) of a pancreatic cancer cell line. We focused on the localization and the origin of tumor-associated macrophages (TAMs), To the best of our knowledge this may be the first study to suggest the potential differentiation of CSCs to TAMs.hiPSCs were converted into CSCs in the presence of CM from PK8 cells. CSCs were then transplanted in vivo and formed primary tumors. Primary cultures for these tumors were serially transplanted again to obtain secondary tumors. Secondary tumors exhibited histopathological features of malignancy. Cells derived from tumors maintained the expression of endogenous stemness markers and pancreatic CSCs markers. Simultaneously, high immunoreactivity to anti-mouse CD68, anti-human CD68, CD206 and CD11b antibodies were detected revealing that the tumor tissue derived from CSCs was enriched for macrophages which can originate from both human and mouse cells.The model of CSCs highlighted the possibility of CSCs to differentiate into TAMs.

Abstract 2431: Identification of the underlining relationship of bivalent histone modifications with pancreatic cancer stem cells by bioinformatic analysis

Abstract Epigenetic alterations are known to involve in cancer stem cells (CSCs) reprogramming. CSCs have been reported to be more resistant to anti-tumor treatments than the non-stem cancer cells, suggesting that surviving CSCs could be responsible for tumor relapse after therapy. The most relevant epigenetic alteration, histone modification has been shown in recent scientific studies to contribute to the regulation of cancer stem cell features in tumor metastasis, progression and response to chemotherapy. The pre-transcriptional regulator, bivalent histone modifications were reported to be involved in the development of CSCs recently. The synchronous presence of functionally opposite histone marks at transcript start sequence (TSS) is known as “Bivalency”. The H3K4me3 as well as H3K27me3, the prominent histone methylations of bivalency, are concerned in transcriptional activation and transcriptional repression respectively. The balance between H3K4me3 and H3K27me3 results in diverse chromatin architectures, ensuing in different transcription states of downstream genes: “poised”, “activated” or “repressed”. The current study is aimed to explore the relationship of bivalent histone modifications, (upregulation or expression of H3K4me3, and down-regulation or repression H3K27me3) the epigenetic regulations with the expression of pancreatic cancer stem cells (CD44, CD24, CD133, Ep-CAM, ALDH1A3) in pancreatic cancer. Protein expression profiles were analyzed using biological informatics methods (string database; Cytoscape database, BioGrid, Reactome, MatrixDB, NCBI Gene and NCBI epigenomics metadata analyses) to identify the relationship of bivalent histone modifications with expressed genes of pancreatic cancer stem cells (CD44, CD24, CD133, Ep-CAM, ALDH1A3). NCBI Biosystems database showed that 197 genes are involved in the Integrated Pancreatic Cancer Pathway model using KEGG database, but the relationship of bivalent histone modifications with the expression of pancreatic cancer stem cells (CD44, CD24, CD133, Ep-CAM, ALDH1A3) in pancreatic cancer has not been showed earlier. Our analysis showed the protein-protein interaction (PPI) network was constructed for such a relationship of closely related 45 significant genes. Among these 45 genes, a total of 7 pancreatic cancer stem cell markers (CSCs) was closely associated with upregulation or expression of H3K4me3, and down-regulation or repression H3K27me3 through EZH2 and PHF19 with a high degree of interaction in the PPI network. We are also able to analyze the role of H3K27ac in regulating target genes in 18 samples of pancreatic cancer cell lines using the ChIP-sequences data set of study SRA 109996 from NCBI NLM Sequence Read Archive from Run Browser Accession number ERR2708462. Therefore, this study identified the significant relationship of bivalent histone modifications associated with pancreatic CSCs which may be the novel biomarkers and target candidates in designing drugs against pancreatic cancer. This work was part of the honors option project (HOP) of Natalia Ortega mentored by Dr. Jayanta K. Das, at MDC north campus. Citation Format: Natalia Ortega, Madhumita Das, Marco A. Ruiz, Jairo Ramos, Jayanta Kumar Das. Identification of the underlining relationship of bivalent histone modifications with pancreatic cancer stem cells by bioinformatic analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2431.

Silencing of microRNA-135b inhibits invasion, migration, and stemness of CD24+CD44+ pancreatic cancer stem cells through JADE-1-dependent AKT/mTOR pathway

BackgroundRecent studies have emphasized determining the ability of microRNAs (miRNAs) as crucial regulators in the occurrence and development of pancreatic cancer (PC), which continues to be one of the deadliest malignancies with few effective therapies. The study aimed to investigate the functional role of miR-135b and its associated mechanism to unravel the biological characteristics of tumor growth in pancreatic cancer stem cells (PCSCs).MethodsMicroarray analyses were initially performed to identify the PC-related miRNAs and genes. The expression of miR-135b and PCSC markers in PC tissues and cells was determined by RT-qPCR and western blot analysis, respectively. The potential gene (JADE-1) that could bind to miR-135b was confirmed by the dual-luciferase reporter assay. To investigate the tumorigenicity, migration, invasion, and stemness of PC cells, several gain-of-function and loss-of-function genetic experiments were conducted. Finally, tumor formation in nude mice was conducted to confirm the results in vivo.ResultsmiR-135b was highly-expressed in PC tissues and PCSCs, which was identified to specifically target JADE-1. The overexpression of miR-135b promoted proliferation, migration, and invasion of PCSC, inhibited cell apoptosis and increased the expression of stemness-related factors (Sox-2, Oct-4, Nanog, Aldh1, and Slug). Moreover, miR-135b could promote the expression of phosphorylated AKT and phosphorylated mTOR in the AKT/mTOR pathway. Additionally, miR-135b overexpression accelerated tumor growth in nude mice.ConclusionsTaken together, the silencing of miR-135b promotes the JADE-1 expression, which inactivates the AKT/mTOR pathway and ultimately results in inhibition of self-renewal and tumor growth of PCSCs. Hence, this study contributes to understanding the role of miR-135 in PCSCs and its underlying molecular mechanisms to aid in the development of effective PC therapeutics.

Combined blockade of TGf-β1 and GM-CSF improves chemotherapeutic effects for pancreatic cancer by modulating tumor microenvironment.

The interactions between tumor immune microenvironment (TIME) and pancreatic cancer cells can affect chemotherapeutic efficacy; however, the mechanisms still remain largely unknown. Thirty items in TIME were comprehensively screened by using tissue microarray from pancreatic cancer patients. Their expressions, interconnections and predictive roles for survival were analyzed. Twenty-one of 30 items could stratify the survival of the patients; however, multivariate analysis found that only 5 independent risk factors could predict worse survival (M2-polarized tumor-associated macrophages (TAMs), IgG4 positive cells, TGF-β1, GM-CSF and lymphangiogenesis). They had a much higher expression levels in tumoral tissue, compared to peritumoral tissue. The Spearman analysis showed that M2-polarized TAM, TGF-β1 and GM-CSF were positively correlated with pancreatic cancer stem cells (PCSC), angiogenesis and lymphangiogenesis. Both human and murine pancreatic cancer cells could induce M2-polarized TAM, which showed substantial roles to decease chemotherapeutic effects. After treated by gemcitabine, both human and murine pancreatic cancer cell lines expressed higher level of immune check points, PCSC markers and varieties of immunosuppressive factors; however, TGF-β1 and GM-CSF had the highest increase. Based on the above results, TGF-β1 and GM-CSF were proposed to be the optimal potential targets to improve chemotherapeutic effects. In immunocompetent murine models, we demonstrated that combined blockade of TGF-β1 and GM-CSF improved the chemotherapeutic effects by inhibition of M2-polarized TAM and induction of CD8 positive T cells. This study presents a novel promising combined strategy to improve the chemotherapeutic effects for pancreatic cancer.

Pancreatic (pro)enzymes treatment suppresses BXPC-3 pancreatic Cancer Stem Cell subpopulation and impairs tumour engrafting

Cancer stem cells (CSCs) subpopulation within the tumour is responsible for metastasis and cancer relapse. Here we investigate in vitro and in vivo the effects of a pancreatic (pro)enzyme mixture composed of Chymotrypsinogen and Trypsinogen (PRP) on CSCs derived from a human pancreatic cell line, BxPC3. Exposure of pancreatic CSCs spheres to PRP resulted in a significant decrease of ALDEFLUOR and specific pancreatic CSC markers (CD 326, CD 44 and CxCR4) signal tested by flow cytometry, further CSCs markers expression was also analyzed by western and immunofluorescence assays. PRP also inhibits primary and secondary sphere formation. Three RT2 Profiler PCR Arrays were used to study gene expression regulation after PRP treatment and resulted in, (i) epithelial-mesenchymal transition (EMT) inhibition; (ii) CSCs related genes suppression; (iii) enhanced expression of tumour suppressor genes; (iv) downregulation of migration and metastasis genes and (v) regulation of MAP Kinase Signalling Pathway. Finally, in vivo anti-tumor xenograft studies demonstrated high anti-tumour efficacy of PRP against tumours induced by BxPC3 human pancreatic CSCs. PRP impaired engrafting of pancreatic CSC’s tumours in nude mice and displayed an antigrowth effect toward initiated xenografts. We concluded that (pro)enzymes treatment is a valuable strategy to suppress the CSC population in solid pancreatic tumours.

The Anthrax Toxin Receptor 1 (ANTXR1) Is Enriched in Pancreatic Cancer Stem Cells Derived from Primary Tumor Cultures

Pancreatic ductal adenocarcinoma (PDAC) is currently the fourth leading cause of cancer-related mortality. Cancer stem cells (CSCs) have been shown to be the drivers of pancreatic tumor growth, metastasis, and chemoresistance, but our understanding of these cells is still limited by our inability to efficiently identify and isolate them. While a number of markers capable of identifying pancreatic CSCs (PaCSCs) have been discovered since 2007, there is no doubt that more markers are still needed. The anthrax toxin receptor 1 (ANTXR1) was identified as a functional biomarker of triple-negative breast CSCs, and PDAC patients stratified based on ANTXR1 expression levels showed increased mortality and enrichment of pathways known to be necessary for CSC biology, including TGF-β, NOTCH, Wnt/β-catenin, and IL-6/JAK/STAT3 signaling and epithelial to mesenchymal transition, suggesting that ANTXR1 may represent a putative PaCSC marker. In this study, we show that ANTXR1+ cells are not only detectable across a panel of 7 PDAC patient-derived xenograft primary cultures but ANTXR1 expression significantly increased in CSC-enriched 3D sphere cultures. Importantly, ANTXR1+ cells also coexpressed other known PaCSC markers such as CD44, CD133, and autofluorescence, and ANTXR1+ cells displayed enhanced CSC functional and molecular properties, including increased self-renewal and expression of pluripotency-associated genes, compared to ANTXR1− cells. Thus, this study validates ANTXR1 as a new PaCSC marker and we propose its use in identifying CSCs in this tumor type and its exploitation in the development of CSC-targeted therapies for PDAC.

Phytochemicals, withaferin A and carnosol, overcome pancreatic cancer stem cells as c-Met inhibitors

Receptor tyrosine kinases (RTKs) are pharmaceutically attractive targets due to their fundamental role in tumor formation. The hallmark of pancreatic cancer is its high mortality rate attributed to the existence of cancer stem cell (CSC) subpopulations which result in therapy resistance and recurrence. c-Met is a known pancreatic CSC marker that belongs to the family of RTKs. To surmount the hurdles related to ligand-independent c-Met activation, we aimed to elucidate the inhibitory mechanisms of withaferin A (WA) and carnosol (CA) as two hit phytochemicals against c-Met kinase domain. Both tested compounds attenuated HGF-mediated proliferation across various established c-Met+ cancer cell lines and altered cell cycle distribution accompanied by apoptosis induction. Scratch assay confirmed the anti-migratory activity of WA and CA in AsPC-1 cells. The blockade of HGF-driven cellular growth and motility was reflected by the suppression of c-Met phosphorylation and its downstream pro-survival pathway Akt. Further studies showed that the administration of WA and CA diminished the sphere-formation and clonogenic potential which was validated by down-regulation of pluripotency maintaining genes (oct-4 and nanog), demonstrating their potentiality to target pancreatic CSCs. As more than 60% of anti-cancer drugs are composed of natural product-derived inhibitors known as fourth generation inhibitors, our present data suggest that WA and CA may hold promise to eradicate CSCs in c-Met-dependent cancers.

The CD44 standard isoform contributes to radioresistance of pancreatic cancer cells.

Resistance to chemoradiotherapy is one reason for the increased recurrence rate of pancreatic cancer after these therapies. These cells change the expression levels of several proteins, such as epithelial–mesenchymal transition (EMT), while acquiring the chemo- or radio-resistance. In this study, we focused on CD44, a pancreatic cancer stem cell marker. CD44 has isoforms with different functions: standard isoform (CD44s) and several variant isoforms (CD44v). However, little is known about the roles of these isoforms after ionizing irradiation. The purpose of this study was to investigate the role of CD44 isoforms in radioresistance of pancreatic cancer cells. AsPC-1 (a human pancreatic cancer cell line) was irradiated with 4 MV X-rays. The mRNA and protein levels of CD44s were strongly upregulated, dose dependently, compared with CD44v after irradiation. Thus, we further investigated CD44s at the point of cell proliferation. We evaluated cell proliferation and survival, using CD44s knockdown cells. CD44s knockdown did not change the proliferation rate for up to 72 h after the irradiation, but decreased cell viability in the colony formation assay. As one of the reasons for these effects, we found downregulation of phosphorylated extracellular signal–regulated kinase (Erk; which is involved with cell proliferation) by CD44s knockdown, time dependently. Moreover, radiation-induced EMT-like expression changes were detected and suppressed by CD44s knockdown. In conclusion, our work demonstrated that CD44 standard isoform was especially upregulated after high-dose X-ray irradiation in several isoforms of CD44 and contributed to longer-term cell survival after the irradiation through the maintenance of Erk phosphorylation and radiation-induced EMT.

Abstract 3544: Leptin-Notch crosstalk axis: A novel target for pancreatic cancer

Abstract Background: Pancreatic cancer (PC) has consistently been the fourth cancer-related cause of death in United States for more than 10 years. Its dismal prognosis is due to the aggressive behavior, late detection, lack of reliable biomarkers and low response to chemotherapy. Obesity, characterized by high levels of leptin, correlates to low PC survival. Notch and leptin signaling have been associated with increased tumor growth and chemoresistance. We have previously shown that leptin induces proliferation of PC cell lines, increases Notch expression, PC stem cells (PCSC) and tumorsphere formation. Hypothesis: Leptin increases PC chemoresistance through its crosstalk to Notch signaling pathway that is essential for the expansion of PCSC. Methods: To test whether a leptin-Notch crosstalk axis is involved in PC-chemoresistance, human PC (MiaPaCa-2, Panc-1 and BxPC-3) cells were cultured in serum-free medium containing leptin, Gemcitabine and 5-Fluorouracil (5-FU) in combination with DAPT (γ-secretase inhibitor) and leptin signaling inhibitor bound to iron-oxide nanoparticles (IONP-LPrA2). PC cells were cultured with leptin and allow to form primary and secondary tumorspheres, enriched in PCSC. Tumorspheres were treated with the compounds described for 7-14 days. Number and size of tumorspheres were recorded, and expression of leptin receptor, OB-R and secretion of leptin were determined by Western blot (WB) and ELISA, respectively. Additionally, the levels of Notch, PCSC and EMT markers, pluripotency associated genes (Oct-4, Sox-2 and Nanog) and ATP-binding cassette transporters (ABCB1, ABCG2 and ABCC5) were determined using flow cytometry and WB. Results: PC cells expressed Leptin/OB-R system, suggesting an autocrine role in PC progression and chemoresistance. Leptin induced the formation of primary and secondary tumorspheres and increased their expression of Notch, EMT and pluripotency markers. Additionally, chemotherapeutics induced PC survival and the expression of ATP-binding cassette transporters in tumorspheres that was reinforced by leptin actions. Blockade of leptin signaling via IONP-LPrA2 and Notch activation via DAPT reduced leptin-induced molecular effects, PCSC and tumorsphere growth. Conclusion: Present data suggest that obesity, through leptin-induced Notch signaling, could increase chemoresistance in PC patients. Inhibition of leptin-Notch axis may be novel therapeutic strategy for PC, which may improve chemotherapeutic efficacy and increase survival in PC patients, particularly in obese contexts. Acknowledgements: this work has been supported by a Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386). Citation Format: Adriana Harbuzariu, Crystal C. Lipsey, Ruben R. Gonzalez-Perez. Leptin-Notch crosstalk axis: A novel target for pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3544. doi:10.1158/1538-7445.AM2017-3544

Leptin-Notch signaling axis is involved in pancreatic cancer progression

Pancreatic cancer (PC) shows a high death rate. PC incidence and prognosis are affected by obesity, a pandemic characterized by high levels of leptin. Notch is upregulated by leptin in breast cancer. Thus, leptin and Notch crosstalk could influence PC progression. Here we investigated in PC cell lines (BxPC-3, MiaPaCa-2, Panc-1, AsPC-1), derived tumorspheres and xenografts whether a functional leptin-Notch axis affects PC progression and expansion of pancreatic cancer stem cells (PCSC). PC cells and tumorspheres were treated with leptin and inhibitors of Notch (gamma-secretase inhibitor, DAPT) and leptin (iron oxide nanoparticle-leptin peptide receptor antagonist 2, IONP-LPrA2). Leptin treatment increased cell cycle progression and proliferation, and the expression of Notch receptors, ligands and targeted molecules (Notch1-4, DLL4, JAG1, Survivin and Hey2), PCSC markers (CD24/CD44/ESA, ALDH, CD133, Oct-4), ABCB1 protein, as well as tumorsphere formation. Leptin-induced effects on PC and tumorspheres were decreased by IONP-LPrA2 and DAPT. PC cells secreted leptin and expressed the leptin receptor, OB-R, which indicates a leptin autocrine/paracrine signaling loop could also affect tumor progression. IONP-LPrA2 treatment delayed the onset of MiaPaCa-2 xenografts, and decreased tumor growth and the expression of proliferation and PCSC markers. Present data suggest that leptin-Notch axis is involved in PC. PC has no targeted therapy and is mainly treated with chemotherapy, whose efficiency could be decreased by leptin and Notch activities. Thus, the leptin-Notch axis could be a novel therapeutic target, particularly for obese PC patients.

Abstract B30: Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with a propensity for metastasis and drug resistance. To overcome this and improve the outcome of patients with PDAC, there is an urgent need to elucidate the molecular mechanisms of metastasis in which cancer stem cells (CSCs) may play a role. Prrx1, a homeobox transcription factor has been identified by us, as a key molecule involved in pancreatic development, regeneration and early carcinogenesis (Reichert et al. Genes and Development 2013). Furthermore, we have reported recently that Prrx1 isoform switching governs EMT-MET plasticity to regulate PDAC invasion and metastasis (Takano et al. Genes and Development 2016). Herein, we examined that the correlation between the two Prrx1 isoforms and putative CSC markers to investigate the role of Prrx1 on pancreatic CSC functions. Materials and Methods: Mouse pancreatic ductal cell lines were isolated and established from Pdx1Cre mice (normal pancreatic ductal cells), Pdx1Cre;KrasG12D/+ mice (PanIN cells), Pdx1Cre;KrasG12D/+;p53R175H/+ mice (primary and metastatic PDAC cells). Either Prrx1a and Prrx1b was overexpressed by lentiviral transduction in each cell line with controls. Prrx1 knockdown was done in primary PDAC cells and their matched liver metastatic cells by two isoform specific siRNAs. We immunophenotyped putative pancreatic CSCs using antibodies against CSC markers (CD133, CD44, CD24 and Epcam) by flow cytometry. These subpopulations of cells were evaluated in 3D cultures, invasion assays and single-pancreatosphere formation assays. Additionally, we evaluated the expression of pancreatic CSC markers in Prrx1a and Prrx1b overexpressing and knockdown cells by qRT-PCR and flow cytometry. Results: CD133High cells and CD44HighCD24High cells are expressed highly in metastatic PDAC cells compared to primary PDAC cells. CD133High cells form spheroid cysts and CD133Low cells form spindle-shaped cells in 3D culture. CD133High cells demonstrate more invasiveness and higher self-renewal capacity compared to CD133 Low cells. The percentage of CD133High cells, CD44HighCD24High cells and EpCAMHigh cells are increased significantly in Prrx1a overexpressing primary PDAC cells. However, these cells are decreased in Prrx1a knockdown metastatic cells. Contrary to this, these subpopulation of cells are significantly decreased in Prrx1b overexpressing primary PDAC cells, and increased in Prrx1b knockdown metastatic cells. These data suggest that Prrx1a might regulate pancreatic CSC functions in metastatic PDAC cells. Conclusion: Prrx1a may be associated with a subpopulation of pancreatic cancer cells that have features of cancer stem cells. This may influence metastatic properties of pancreatic cancer cells to the liver. Citation Format: Shigetsugu Takano, Maximilian Reichert, Hideyuki Yoshitomi, Basil Bakir, Koushik K. Das, Steffen Heeg, Shingo Kagawa, Hiroaki Shimizu, Masayuki Ohtsuka, Katsunori Furukawa, Masaru Miyazaki, Anil K. Rustgi.{Authors}. Prrx1 isoforms regulate pancreatic cancer stem cell functions during pancreatic cancer progression. [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 B30.

Abstract 1034: Leptin induces early onset and aggressiveness of pancreatic cancer

Abstract Background: During the past 30 years, pancreatic cancer (PC) has constantly been the fourth leading cause of cancer death. PC is usually advanced at the time of diagnosis, with an overall five years survival rate of less than 5%. Obesity, characterized by high levels of leptin, is pandemic in United States and a risk factor for PC. Notch abnormal signaling is linked to carcinogenesis, tumor angiogenesis and self renewal capacity of PC stem cells (PCSC). We have previously shown that leptin induces proliferation of PC cell lines, increases Notch expression, PCSC and tumorsphere formation. To test leptin effects in a PC mouse model, a specific and potent signaling inhibitor bound to nanoparticles, IONP-LPrA2 was produced by us. Hypothesis: High levels of leptin induce early onset and multiplicity of PC xenografts, which is abrogated by IONP-LPrA2 treatment. Methods: Human PC cell line MiaPaCa-2 was challenged with leptin or IONP-LPrA2. The expression of pluripotency associated genes (STAT3, Oct4, Sox2 and NANOG) was evaluated. MiaPaCa-2 cells forming tumorspheres were treated with leptin or IONP-LPrA2 and inoculated into CD1 nu/nu mice. Untreated tumorspheres were inoculated as controls. A group of mice were injected twice a week with IONP-LPrA2. Tumor onset and growth were assessed. PC xenografts were analyzed for expression of Ki67 (proliferation marker), leptin targeted molecules (Notch), PCSC markers etc. Results: Leptin induced the expression of PCSC pluripotency markers. Moreover, leptin treatment of tumorspheres induced early tumor onset and multiplicity. Also, leptin increased proliferation, PCSC and oncogenic markers in PC xenografts. IONP-LPrA2 reduced leptin effects. No changes of mice food intake and body weight were observed. Conclusions: This observation might imply that obese patients are at high risk to develop PC. Inhibition of leptin signaling may be used as preventative or therapeutic strategy for PC, especially in obese patients. Acknowledgements: this work was supported by DOD W81XWH-13-1-0382; NIH/SBIR 1R41CA183399-01A1; Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP, and facilities and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR 03034 and 1C06 RR18386). Citation Format: Adriana Harbuzariu, Antonio Rampoldi, Danielle S. Daley-Brown, Tia L. Harmon, Derrick J. Beech, Gabriela Oprea-Ilies, Ruben R. Gonzalez-Perez. Leptin induces early onset and aggressiveness of pancreatic cancer. [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 1034.

Abstract 3083: Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) or pancreatic cancer is one of the most lethal malignancies in humans. The survival rate for two years is very limited in most of cases (10%) after diagnosis of this disease. This is because current therapeutic regimens have limited effectiveness. Novel therapeutic targets are needed to treat this disease. GZ17-06.12 contains a nutraceutical for its anti-cancer properties, in several cancers. Crude extracts of the plant Arum palaestinum Boiss used in traditional Israeli and Palestinian medicine, have demonstrated cytostatic and cytotoxic activities in colon, prostrate and lung carcinoma. Therefore, we hypothesize that GZ17-06.12 will inhibit tumor progression and metastasis in PDAC. Experimental Procedure: In this study, we have determined cell proliferation, pancosphere formation and apoptosis following treatment of different doses oGZ17-06.12 for 24-72 hours using MiaPaCa-2 and S2-007 human pancreatic cancer cells. Cell cycle distribution and apoptosis were measured using flow cytometic analysis. Orthotopic pancreatic cancer model in athymic mice was developed and GZ17-06.12 was given orally for 20 days to those mice. Proliferative markers, pEGFR/pAkt and apoptotic markers, Bax/Bcl-2, were monitored following treatment with GZ17-06.12 in both in vivo and in vitro models. Metastatic markers, MMP-2 and MMP-9 were measured in metastatic tissues in orthotopic models. Results: GZ17-06.12 inhibited proliferation of pancreatic cancer cell lines in a dose- and time-dependent manner. Cell cycle analysis suggested that cells were arrested in S and G2 phase following treatment. In addition, GZ17-06.12 induced apoptosis in both in vitro and in vivo pancreatic cancer. Moreover, the compound significantly inhibited epidermal growth factor receptor and Akt phosphorylation. Furthermore, GZ17-06.12 decreased the number and size of the pancospheres in S2-007 cells with concomitant inhibition of pancreatic cancer stem cell markers, DCLK1, Lgr5 and EpCam. The effect of GZ17-06.12 suppressed tumor growth and metastatic potential as indicative of MMP-2 and MMP-9 activity in primary and metastatic tumors. Conclusions: GZ17-06.12 significantly reduces tumorigenesis and metastasis in both in vitro and in vivo pancreatic cancer models, inhibiting proliferation with concomitant increase of apoptosis. Citation Format: Santanu Paul, Lisa Stehno-Bittel, Animesh Dhar. Novel inhibitor GZ17-06.12 suppresses pancreatic cancer tumorigenesis. [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 3083.

Abstract 1901: Leptin modulation of PCSC, HDAC, and microRNA in pancreatic adenocarcinoma

Abstract Background: Obesity is an important risk factor of Pancreatic Adenocarcinoma (PA) and it is characterized by the accumulation of excessive body fat and a Body Mass Index (BMI) value greater than 30. Obesity is also characterized by high levels of leptin, which has been consistently associated with the development of many different cancer including PA. Leptin can induce the proliferation of Pancreatic Cancer Stem Cells (PCSC), which are responsible for chemoresistance, invasiveness and reoccurrence of PA. PCSC express specific cell markers and can form tumorspheres in vitro. Obesity can alter the DNA acetylation and microRNA activity which are also linked to PA progression. We hypothesized that high level of leptin could modulate Histone Deacetylase (HDAC) and microRNA activity in PA cells, which induce PCSC changes and tumor progression. Methods: The PA cell lines were cultured in mammocult media that contained heparin and hydrocortisone, which will allow the proliferation of tumorspheres enriched with PCSCs. The cells were cultured with leptin, chemotherapeutic drug, and leptin signaling inhibitor IONP-LPrA2. Tumorspheres formation (number and size) was determined after 1 week of treatment. Additionally, the levels of PCSC markers, HDAC1, HDAC2, HDAC3, and HDAC8 in tumorspheres were determined using flow cytometry, western blot, and RT-PCR. In addition, the effects of treatments on miR21 and miR200a/c levels were determined using RT-PCR. Results: Leptin induced PA tumorspheres formation and size, which was accompanied by higher level of PCSC markers (CD24, CD44, ESA, and ALDH1). Moreover, leptin affected the level of HDACs, miR21 and miR200a/c in PA tumorspheres. IONP-LPrA2 abrogated leptin effects and decreased PCSC which were spared by chemotherapeutics. Conclusion: Obesity signals via leptin could be involved in the increase PA aggressiveness and chemoresistance, which may be linked to the increase of PCSC. Leptin could induce PA progression and chemoresistance via modulation of HDAC and miRNA. Acknowledgement: This work was supported by the DOD W81XWH-13-1-0382; NIH/SBIR1R41CA183399-01A1; Pilot Project Award from MSM/Tuskegee University/UAB Cancer Center Partnership grant 5U54CA118638; PC SPORE Grant from UAB to RRGP; Calvin Johnson Jr. Foundation Pancreatic Cancer Research Scholarship to CITM, and facility and support services at Morehouse School of Medicine (1G12RR026250-03; NIH RR03034 and 1C06 RR18386). Citation Format: Cynthia M. Tchio, Adriana Harbuzariu, Tia Harmon, Derrick Beech, Ruben Gonzalez-Perez. Leptin modulation of PCSC, HDAC, and microRNA in pancreatic adenocarcinoma. [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 1901.

Abstract 3955: Expression of cancer stem cell-associated proteins in exosomes derived from ascites of patients with advanced pancreatic cancer

Abstract Background: The number of patients with pancreatic cancer is rapidly growing, and the disease is the fifth leading cause of cancer-related death in Japan. At end stage of the disease, the patients are prone to suffer peritonitis carcinomatosa with chemorefractory ascites. It is known that cancer cells surviving in ascites show cancer stem cell (CSC)-like features (PLoS One 2012). The CSC-like cells robustly secrete extracellular vesicle called exosome, which plays important roles in tumorigenesis, tumor growth, metastasis, angiogenesis, pre-metastatic niche formation, immunosuppression, drug resistance, and epithelial-mesenchymal transition. The AIM of this prospective study was to assess whether exosomes taken from malignant ascites of patients with advanced pancreatic cancer included the CSC-associated proteins, that might be predictive markers for chemoresistance and prognosis. Methods: The malignant ascites was collected from the cancer patients who underwent abdominocentesis and/or cell-free and concentrated ascites reinfusion therapy (CART) in Kurume University Hospital. Ascites derived from patients with benign diseases, including decompensated liver cirrhosis (d-LC), was used as control. Informed consent was obtained from all of the patients. Exosomes in ascites were purified by using ExoQuick Exosome Precipitation Solution (System Biosciences) according to the manufacturer's protocol. Western blot analysis was performed to detect CSC-associated proteins, including CD133, CD44, CD44v9, xCT, CD24, and Dclk1. Results: Successful purification of exosomes from both the malignant ascites and the benign one was confirmed by monitoring exosome-specific proteins such as CD68, CD9, CD81, and HSP70. Among the CSC-associated proteins examined, CD133 was predominantly expressed in exosomes obtained from ascites of the pancreatic cancer patients compared with those obtained from ascites of the d-LC patients. Other molecules were faintly expressed or absent even in the malignant ascites. Conclusions: We first demonstrated abundant expression of CD133, a human pancreatic CSC marker, in exosomes derived from ascites of patients with the disease. The finding suggests that exosomal CD133 might be a potential biomarker for chemoresistance and prognosis of the patients. Citation Format: Takahiko Sakaue, Hironori Koga, Masaru Fukahori, Yasuko Imamura, Toru Nakamura, Yoshinobu Okabe, Yu Ikezono, Fumitaka Wada, Hideki Iwamoto, Atsutaka Masuda, Tomoyuki Ushijima, Keisuke Miwa, Tatsuyuki Kakuma, Osamu Tsuruta, Takuji Torimura. Expression of cancer stem cell-associated proteins in exosomes derived from ascites of patients with advanced pancreatic cancer. [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 3955.

Abstract 1727: Quinomycin A inhibits pancreatic cancer growth and affects stem cell viability by inhibiting oncogenic YAP1 function

Abstract Background: Pancreatic cancer (PCa) remains a leading cause of death in the United States. Cancer stem cells (CSC) are responsible for tumor behavior, and therapeutic resistance. Quinomycin (Qui) is an orally administered quinoxaline antibiotic that bifunctionally intercalates with double stranded DNA. As a first step for repurposing this drug, we determined whether Qui affects PCa growth, and if so whether this is by suppressing stem cells. Method: Growth and apoptosis of PCa lines (MiaPaCa-2, PanC-1, BxPC-3) and normal ductal epithelial cells (HPNE) was measured by hexosaminidase and clonogenicity, and caspase 3/7 assays, respectively. Pancosphere formation and FACS sorting were used to identify effects on stem cells. For in vivo, MiaPaCa-2 xenografts were developed in the flanks of nude mice. Immunohistochemistry was performed for stem cell markers and Hippo signaling proteins. Results: Qui demonstrated a dose- and time-dependent inhibition of proliferation and colony formation in all three PCa lines but not HPNE cells. Qui induced PCa cells to undergo apoptosis. It also significantly reduced the number and size of pancospheres, and flow cytometry and western blot analyses confirmed that Qui suppressed PCa stem cell marker proteins DCLK1, CD44, CD24 and EPCAM. We next determined whether Hippo signaling pathway is affected. For this, we tested the effect of Qui on Hippo signaling pathway, which is an active pathway in CSCs including in PCa. The key effector protein, YAP1 has been shown to be oncogenic in many cancer types. In the canonical Hippo signaling pathway, YAP1 function is inhibited. When YAP1 is phosphorylated at Ser127 by the action of upstream Mst1/2 and Lats1/2 kinases, it is sequestered in the cytoplasm, and therefore no induction of downstream gene expression. Qui significantly decreased the phosphorylation oncogenic YAP1. Furthermore, Qui inhibited the expression of YAP interacting proteins TEAD1, TEAD2, and TEAD4. On the other hand, ectopic expression of the TEAD1 partially rescued the cells from Qui-mediated growth suppression. To determine the effect of Qui on tumor growth in vivo, mice carrying MiaPaCa-2 tumor xenografts were administered the compound intraperitoneally (20 μg/kg bw) every day for 21 days. Qui treatment significantly suppressed tumor xenograft growth, with notably lower tumor volume and weight. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of CSC marker proteins, oncogenic YAP1 phosphorylation and YAP1 interacting proteins TEAD1 in the Qui-treated xenograft tissues. Conclusion: Together, these data suggest that Qui suppresses PCa growth that targets that targets stem cells by inhibiting oncogenic YAP1 in Hippo signaling pathway. Citation Format: Dharmalingam Subramaniam, Sivapriya Ponnurangam, Gaurav Fnu, Prabhu Ramamoorthy, Animesh Dhar, Ossama W. Tawfik, Shahid Umar, Scott J. Weir, Roy A. Jensen, Arun Balakrishnan, Shrikant Anant. Quinomycin A inhibits pancreatic cancer growth and affects stem cell viability by inhibiting oncogenic YAP1 function. [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 1727.

Abstract B31: A new approach to targeted therapy for obesity-related pancreatic adenocarcinoma

Abstract Background: Obesity is an important risk factor of Pancreatic Adenocarcinoma (PA) and it is characterized by the accumulation of excessive body fat and a Body Mass Index (BMI) value greater than 30. Obesity is also characterized by high levels of leptin, which has been consistently associated with the development of many different cancer including pancreatic cancer. Obesity can also alter the epigenome via epigenetics changes as DNA methylation and histones acetylation. Histone lysine residues can be acetylated by Histone Acetyltransferases or deacetylated by Histone Deacetylases (HDAC). Leptin can induce the proliferation of Pancreatic Cancer Stem Cells (PCSC), which are responsible for chemoresistance, invasiveness and reoccurrence of PA. PCSC express specific cell markers and can form tumorspheres in vitro. Our lab developed a leptin antagonist named LPrA2, which was bound to iron oxide nanoparticle (IONP-LPrA2) in order to increase its effectiveness and bioavailability. Methods: The adjuvant effects of IONP-LPrA2 in combination of chemotherapeutic drugs on PCSC and tumorspheres formation in low attachment plates were determined. Additionally, the effects of IONP-LPrA2 in combination with chemotherapeutic drugs on the differential expression of epigenetic markers HDAC-1, HDAC-2, and HDAC-8 were investigated. The PA cells were cultured in vitro and treated with leptin, IONP-LPrA2, and chemotherapeutics. Then, the expression of epigenetic and PCSC markers, cell proliferation and survival were determined via Nexcelom cellometer or Guava EasyCyte flow cytometer analyses. Results were assessed by western blot analysis. Results: Leptin affects the level of some HDACs and PCSC markers. That was abrogated by IONP-LPrA2. Chemotherapeutics showed differential effects on the epigenetic and PCSC markers and on tumorspheres formation by PA cells. Conclusion: These findings suggest that IONP-LPrA2 could be useful to design new PA therapies in combination with chemotherapeutics. Furthermore, IONP-LPrA2 may increase the efficiency of chemotherapeutic drug Gemcitabine. These results could be most relevant for obese patients that show the highest incidence of PA and lowest survival rate. Citation Format: Cynthia Ines Mantho Tchio, Adriana Harbuzariu, Tia L. Harmon, Derreck J. Beech, Ruben R. Gonzalez-Perez. A new approach to targeted therapy for obesity-related pancreatic adenocarcinoma. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B31.