Invasive Pancreatic Ductal Adenocarcinoma Research Articles

ADAM9 contributes to vascular invasion in pancreatic ductal adenocarcinoma.

A disintegrin and a metalloprotease (ADAM)‐9 is a metzincin cell‐surface protease with strongly elevated expression in solid tumors, including pancreatic ductal adenocarcinoma (PDAC). In this study, we performed immunohistochemistry (IHC) of a tissue microarray (TMA) to examine the expression of ADAM9 in a cohort of >100 clinically annotated PDAC cases. We report that ADAM9 is prominently expressed by PDAC tumor cells, and increased ADAM9 expression levels correlate with poor tumor grading (P = 0.027) and the presence of vasculature invasion (P = 0.017). We employed gene expression silencing to generate a loss‐of‐function system for ADAM9 in two established PDAC cell lines. In vitro analysis showed that loss of ADAM9 does not impede cellular proliferation and invasiveness in basement membrane. However, ADAM9 plays a crucial role in mediating cell migration and adhesion to extracellular matrix substrates such as fibronectin, tenascin, and vitronectin. This effect appears to depend on its catalytic activity. In addition, ADAM9 facilitates anchorage‐independent growth. In AsPC1 cells, but not in MiaPaCa‐2 cells, we noted a pronounced yet heterogeneous impact of ADAM9 on the abundance of various integrins, a process that we characterized as post‐translational regulation. Sprout formation of human umbilical vein endothelial cells (HUVECs) is promoted by ADAM9, as examined by transfer of cancer cell conditioned medium; this finding further supports a pro‐angiogenic role of ADAM9 expressed by PDAC cancer cells. Immunoblotting analysis of cancer cell conditioned medium highlighted that ADAM9 regulates the levels of angiogenic factors, including shed heparin‐binding EGF‐like growth factor (HB‐EGF). Finally, we carried out orthotopic seeding of either wild‐type AsPC‐1 cells or AsPC‐1 cells with silenced ADAM9 expression into murine pancreas. In this in vivo setting, ADAM9 was also found to foster angiogenesis without an impact on tumor cell proliferation. In summary, our results characterize ADAM9 as an important regulator in PDAC tumor biology with a strong pro‐angiogenic impact.

RAMPS – Surgical Treatment for a Pancreatic Tumor

Background: Radical antegrade modular pancreatosplenectomy (RAMPS) is not the standard procedure for cancers of the body and tail of the pancreas but this approach provides improvement in the establishment of an operation with oncologic rationales both for the dissection planes used to achieve negative margins and the extent of lymph node dissection. The authors present a clinical case report. Methods: The authors present a clinical case report. Results: 55-year-old lady, without comorbidities, felt epigastric pain and noted weight loss (10Kg) for a month. Had a CT scan that showed a pancreatic body mass (4x2cm) with splenic vessels encasement and segmental involvement of splenoportal confluent. The case was discussed in a multidisciplinary meeting and decided with surgery. We performed a radical antegrade modular pancreatosplenectomy with side wall portal ressection. This procedure has complicated with the development of diabetes and a type b pancreatic fistula (conservative treatment). Pathology showed an invasive pancreatic ductal adenocarcinoma (6.5cm) with perineural invasion and metastases in 9 of 27 identified lymph nodes (pT4N2). Conclusion: RAMPS procedure for lesions of the body and tail of the pancreas can retrieve significantly more lymph nodes than standard distal pancreatectomy and splenectomy and, therefore, improving tumor staging and outcome.

Significance of Expression of Cancer Stem Cell Markers CD133 and Nestin in Pancreatic Intraepithelial Carcinoma-invasive Adenocarcinoma Sequence.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and one of the most leading causes of cancer related deaths. It has a very poor prognosis with high recurrence rate. We investigated the expression of CSC markers (CD133 and nestin) in 100 patients [30 pancreatic intraepithelial tumor cases (PanIN) and 70 PDAC cases] and correlate the expression levels of these markers with clinicopathological data with the aid of Ki67 expression. Our findings showed that both cancer stem markers are related to the grade, stage, metastasis of PDAC and to the grade of PanIN cases and revealed that both markers are associated with PanIN-PDAC sequence with inverse relation between them. Both markers may contribute to proliferation, differentiation, invasiveness, and histologic types of PDAC. Sothey may also be useful for developing new therapeutic modalities for PDAC.

S100A8 and S100A9 proteins form part of a paracrine feedback loop between pancreatic cancer cells and monocytes

BackgroundThe secretion of soluble factors enables communication between tumour cells and the surrounding microenvironment and plays an important role in oncogenesis. Pancreatic ductal adenocarcinoma (PDAC) is characterised by a highly reactive microenvironment, harbouring a variety of cell types, including S100A8/S100A9-expressing monocytes. S100A8/S100A9 proteins regulate the behaviour of cancer cells by inducing pre-metastatic cascades associated with cancer spread. The aim of this study was to examine how S100A8/A9 proteins mediate tumour-stroma crosstalk in PDAC.MethodsCytokine profiling of pancreatic cancer cell-derived conditioned media was performed using Bio-Plex Pro 27 Plex Human Cytokine assays. Protein expression and activation of downstream signalling effectors and NF-κB were assessed by western blotting analysis and reporter assays respectively.ResultsStimulation of cultured pancreatic cancer cells with S100A8 and S100A9 increased the secretion of the pro-inflammatory cytokines IL-8, TNF-α, and FGF. S100A8, but not S100A9 induced PDGF secretion. Conversely, pancreatic cancer cell-derived conditioned media and the individual cytokines, TNF-α and TGF-β induced the expression of S100A8 and S100A9 proteins in the HL-60 monocytic cell line and primary human monocytes, while FGF and IL-8 induced the expression of S100A9 only. S100A8 and S100A9 activated MAPK and NF-κB signalling in pancreatic cancer. This was partially mediated via activation of the receptor of advanced glycosylation end-product (RAGE).ConclusionS100A8 and S100A9 proteins induce specific cytokine secretion from PDAC cells, which in turn enhances the expression of S100A8/A9. This paracrine crosstalk could have implications for PDAC invasiveness and metastatic potential.

A novel feedback loop between high MALAT-1 and low miR-200c-3p promotes cell migration and invasion in pancreatic ductal adenocarcinoma and is predictive of poor prognosis

BackgroundIt was demonstrated that long non-coding RNAs occupied an important position in tumor pathogenesis and progression. We have previously found that the metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1) promotes cell proliferation and metastases in pancreatic ductal adenocarcinoma (PDAC). The present study was aimed to discuss the underlying mechanisms.MethodsBioinformatics method was used to identify the miRNA target of MALAT-1. Expressions of relative genes were assessed by quantitative real-time PCR and western blotting, respectively. Sulforhodamine B assay and Transwell assay were employed to detect cell proliferation, migration and invasion, respectively. Moreover, RNA immunoprecipitation was performed to determine whether RNA-induced silencing complex contained MALAT-1 and its potential binding miRNA. Luciferase assays was used to confirm potential binding site.ResultsBioinformatics search predicted that miR-200c-3p was a direct target of MALAT-1. Further, we found a reciprocal suppression between MALAT-1 and miR-200c-3p expression. In terms of mechanisms, high MALAT-1 and low miR-200c-3p may form a novel feedback loop. On the one hand, MALAT-1 functioned as a competing endogenous RNA to suppress miR-200c-3p expression, leading to upregulation of ZEB1 expression. On the other hand, miR-200c-3p inhibited the level of MALAT-1 expression was in a way similar to miRNA-mediated downregulation of target genes. Clinical data further indicated that MALAT-1 and ZEB1 expression was negatively correlated with miR-200c-3p transcript level of PDAC tissues. There was a positive correlation between MALAT-1 and ZEB1 level. MALAT-1 (high)/miR-200c-3p (low) correlated with shorter overall survival of PDAC patients. Multivariate analysis revealed that both MALAT-1 and miR-200c-3p levels were independent prognostic factors.ConclusionOur findings firstly revealed a novel feedback loop between high MALAT-1 and low miR-200c-3p. Targeting the feedback loop between high MALAT-1 and low miR-200c-3p will be a therapeutic strategy for PDAC.

Cancerization of the Pancreatic Ducts: Demonstration of a Common and Under-recognized Process Using Immunolabeling of Paired Duct Lesions and Invasive Pancreatic Ductal Adenocarcinoma for p53 and Smad4 Expression.

Invasive pancreatic ductal adenocarcinoma (PDAC) can infiltrate back into and spread along preexisting pancreatic ducts and ductules in a process known as cancerization of ducts (COD). Histologically COD can mimic high-grade pancreatic intraepithelial neoplasia (HG-PanIN). We reviewed pancreatic resections from 100 patients with PDAC for the presence or absence of ducts with histologic features of COD. Features supporting COD included adjacent histologically similar invasive PDAC and an abrupt transition between markedly atypical intraductal epithelium and normal duct epithelium or circumferential involvement of a duct. As the TP53 and SMAD4 genes are frequently targeted in invasive PDAC but not HG-PanIN, paired PDAC and histologically suspected COD lesions were immunolabeled with antibodies to the p53 and Smad4 proteins. Suspected COD was identified on hematoxylin and eosin sections in 89 (89%) of the cases. Immunolabeling for p53 and Smad4 was performed in 68 (76%) of 89 cases. p53 was interpretable in 55 cases and all 55 (100%) cases showed concordant labeling between COD and invasive PDAC. There was matched aberrant p53 immunolabeling in 37 (67%) cases including overexpression in 30 (55%) cases and lack of expression in 7 (13%) cases. Smad4 immunolabeling was interpretable in 61 cases and 59 (97%) cases showed concordant labeling between COD and invasive PDAC. Matched loss of Smad4 was seen in 28 (46%) cases. The immunolabeling of invasive PDAC and COD for p53 and Smad4 supports the high prevalence of COD observed on hematoxylin and eosin and highlights the utility of p53 and Smad4 immunolabeling in differentiating COD and HG-PanIN.

Tenascin C is upregulated around perineural invasion of pancreatic ductal adenocarcinoma and correlates with locoregional recurrence

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by stromal desmoplasia and perineural invasion. However, mechanisms which drive stromal change and neural tropism in cancer progression remain unclear. Tenascin C (TNC), an extracellular matrix glycoprotein, has been reported to be expressed mainly in cancer stroma and associated with metastasis and poor prognosis in PDAC. The aim of this study is to evaluate relationships between TNC expressions around nerves and clinicopathological features in resected PDAC. Method: A total of 79 PDAC patients without receiving any preoperative treatment who underwent surgical resection between January 2000 and May 2017 were enrolled in this study. TNC expression was examined immunohistochemically on paraffin-embedded sections from resected specimen. The perineural staining intensity of TNC in invasive front of pancreatic cancer was defined as high or low (Image), by comparing with that in adjacent non-cancerous tissue in the same section. The relationships between TNC expression and clinicopathological features were retrospectively analyzed. Results: High TNC perineural expression in invasive front was seen in 30 (38%) patients, and associated with perineural invasion (p=0.006), pT3 ≤ (p=0.01), lymph node metastasis (p=0.038) and postoperative locoregional recurrence (p=0.039). High perineural TNC expression was significantly associated with worse recurrence free survival rates by univariate analysis. Conclusions: Perineural TNC expression is a potential predictable marker for locoregional recurrence.

Impact of histological tumor positive resection after pancreatoduodenectomy in patients with ductal adenocarcinoma and lymph node invasion

Introduction: In patients with pancreatic ductal adenocarcinoma (PDAC), complete tumor resection (R0) has been proven to improve overall survival (OS). The present study aimed to compare OS after R0 and R1 resections among patients with PDAC and lymph node invasion who underwent pancreatoduodenectomy. Methods: All consecutive PDAC patients of our prospectively-maintained pancreas database who underwent pancreatoduodenectomy were retrospectively analyzed (2000-2015). Only patients without neoadjuvant treatment were included. R0 resection was defined as absence of microscopic cancer cells at resection margin (1-mm clearance requirement). OS was calculated using Kaplan-Meier method. Results: During the study period, 192 consecutive patients underwent upfront pancreatoduodenectomy. On histopathology, 165 patients had lymph node invasion (86%), while 27 had no lymph node invasion (14%). Among patients with lymph node invasion (n=165), 92 had R0 resection (56%), 61 R1 resection (37%), and 12 R2 resection (7%). The 12 R2 resections were excluded (median OS: 14 months). Preoperative characteristics and demographics of R0 and R1 groups were similar. Overall complication rates (Clavien II-V) were similar in both groups (53/92=58% vs. 41/61=67%, p=0.232). Median OS was 24 months for the R0 and R1 groups (p=0.725). Recurrence rate was 59% for the R0 group and 64% for the R1 group (p=0.533). Multivariate Cox proportional hazards model did not identify the R status or any other item as predictive factor for OS in patients with lymph node invasion. Conclusion: In PDAC patients who underwent upfront pancreatoduodenectomy without neoadjuvant treatment, the R status did not influence OS in case of lymph node invasion.

Successful surgical treatment of invasive pancreatic ductal adenocarcinoma during pregnancy

The treatment of pancreatic cancer is demanding. Surgical resection is the sine qua non for prolonged survival. Pregnancy can sometimes be an obstacle to offer the patient the required treatment options who is suffering from a concomitant malignancy. We are presenting a successful management of a pregnant women with pancreatic cancer. A 40-year-old woman suspected of pancreatic mass during the 20th week of her pregnancy was admitted to our clinics. Detailed work-up revealed pancreatic cystic mass of 9cm with 3.5cm solid component which was interpreted to be a malignant pancreatic tumor by radiology. No distant metastasis was detected. The tumor was determined resectable. Surgical intervention was allowed by the gynecology and obstetrics department without need of a medical abortion according to the decision of the patient who was in the second trimester of her pregnancy. Distal pancreatectomy with splenectomy and omentectomy was performed. She was discharged in the 6th day after an uneventful postoperative period with her healthy fetus. The histopathologic examination revealed invasive ductal adenocarcinoma originating from mucinous cystic neoplasia of high grade dysplasia without any lymphatic metastasis. Medical oncology decided to delay the adjuvant chemotheraphy just after forthcoming birth. She gave a birth to a healthy baby afterwards. The patient is in a good condition without any recurrence after app. 2 years of follow-up. Pregnancy with concomitant malignancy can be a challenging clinical situation. Prompt decision-making, good timing and, meticulous surgery followed by adequate adjuvant therapy can be live-saving for the patient and the fetus as well.

Influence of recurrence's locations of pancreatic adenocarcinoma on patient survival

Introduction: The recurrence of invasive pancreatic ductal adenocarcinomas (PDAC) is related to a poor prognosis. The aim of this study is to compare patient survival rates with different recurrence's locations. Methods: A review was carried out of a prospectively maintained database with consecutive patients who underwent radical surgery for PDAC from January 2007 to December 2016. We analysed variables related to the appearance of recurrence and prognosis. Univariate analysis of interval from recurrence to death (IRD) was performed for each location. Results: 67 patients underwent pancreatectomy, with a median follow-up of 24.7 months. Recurrence appeared in 48 patients (71.6%) with a mean disease-free survival and overall survival rate of 21.5 and 10.5 months, respectively. The mean IRD was 9.2 months. A Whipple procedure was performed in 91.6% of patients, with venous resection in 37.5%. Adjuvant chemotherapy was given to 72.9% of patients. A positive microscopic margin was present in 43.8% and poor differentiation in 27.1%. There was lymphovascular and perineural invasion in 47.9% and 70.8%, respectively. The recurrence locations were: liver (39.6%), local (31.3%), lung (10.4%), bone (6.3%) and peritoneum (4.2%); 8.3% of recurrences occurred as disseminated disease (≥2 locations). IRD related to bone recurrences was significantly shorter when compared to the other locations (9.6 vs. 3.7 months; p = 0.004), while hepatic recurrences showed a longer IRD (7.6 vs. 12.0 months; p = 0.045). Conclusion: Bone recurrences in PDAC present shorter IRD, while hepatic recurrences show a longer interval.

BAG3 regulates stability of IL-8 mRNA via interplay between HuR and miR-4312 in PDACs

Bcl-2 associated athanogene 3 (BAG3) is highly expressed in pancreatic ductal adenocarcinoma (PDAC), and its high expression appears to be a poor prognostic factor for patients with PDAC. In this study, we show that BAG3 knockdown significantly decreases migration and invasion of PDACs via reduction of interleukine-8 (IL-8) production. BAG3 knockdown regulates IL-8 expression at the posttranscriptional levels via interplay between recruitment of RNA-binding protein HuR and miR-4312. HuR binds to the cis-elements located in the 3′-untranslational region (UTR) of the IL-8 transcript to stabilize it, whereas miR-4312-containing miRNA-induced silencing complex (miRISC) is recruited to the adjacent seed element to destabilize it. The binding of HuR prevents the recruitment of Argonaute (Ago2), overriding miR-4312-mediated translation inhibition of IL-8. BAG3 knockdown decreases cytoplasmic distribution of HuR via increasing its phosphorylation at Ser202, therefore compromising its recruitment while promoting recruitment of miR-4312 containing miRISC to IL-8 transcript. Furthermore, our data indicate that only phosphorylated Ago2 at Ser387 interacts with IL-8 transcript. BAG3 knockdown increases phosphorylation of Ago2 at Ser387, thereby further promoting loading of miR-4312 containing miRISC to IL-8 transcript. Taken together, we propose that BAG3 promotes invasion by stabilizing IL-8 transcript via HuR recruitment, and subsequently suppressing the loading of miR-4312 containing miRISC in PDACs. Our results reveal a novel pathway linking BAG3 expression to enhanced PDAC metastasis, thus making BAG3 a potential target for intervention in pancreatic cancer.

Aberrant expression of PDZ-binding kinase/T-LAK cell-originated protein kinase modulates the invasive ability of human pancreatic cancer cells via the stabilization of oncoprotein c-MYC.

High frequency of mortality in patients with pancreatic ductal adenocarcinoma (PDAC) is vastly associated with the invasive and metastatic nature of these cancer cells. Little is known about the factors involved in this invasive/metastatic process. The current challenge in the treatment of these patients is the lack of viable options besides gemcitabine. The aim of this study was to evaluate the role of PDZ-binding kinase (PBK)/T-LAK cell-originated protein kinase (TOPK) in invasive PDAC cells and to determine whether PBK/TOPK expression drives invasiveness in PDAC. Using gain-of-function and loss-of-function studies in established and patient-derived xenograft-PDAC cell lines, and examining patient-derived archival tissue samples, we demonstrate for the first time that PBK/TOPK is upregulated in pancreatic cancer and expression levels are closely associated with the invasive property of pancreatic cancer cells. Modulation of PBK/TOPK causally regulates the invasive ability of PDAC cells. We also demonstrate that two key players in metastatic invasion, matrix metalloproteinases-2 (MMP-2) and MMP-9 gelatinase activity and gene promoter activities, are regulated by PBK/TOPK. Moreover, we demonstrate for the first time that PBK/TOPK provides stability of an oncoprotein, c-MYC, which transcriptionally regulates MMP-2 and MMP-9 in these invasive PDAC cells. Our in vitro and in situ data corroborate that PBK/TOPK is closely associated with the invasive nature of PDAC and reveal a novel mechanism by which the metastatic behavior of human pancreatic cancer cells is regulated. These findings provide a rationale for targeting PBK/TOPK for the therapeutic intervention of invasive/metastatic pancreatic cancer in human.

Downregulated miR-98-5p promotes PDAC proliferation and metastasis by reversely regulating MAP4K4

BackgroundThe aberrant expression of microRNAs (miRNAs) has emerged as important hallmarks of cancer. However, the molecular mechanisms underlying the differences of miRNA expression remain unclear. Many studies have reported that miR-98-5p plays vital functions in the development and progression of multiple cancers. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unknown.MethodsThe expression of miR-98-5p and its specific target gene were determined in human PDAC specimens and cell lines by miRNA qRT-PCR, qRT-PCR and western blot. The effects of miR-98-5p depletion or ectopic expression on PDAC proliferation, migration and invasion were evaluated in vitro using CCK-8 proliferation assays, colony formation assays, wound healing assays and transwell assays. Furthermore, the in vivo effects were investigated using the mouse subcutaneous xenotransplantation and pancreatic tail xenotransplantation models. Luciferase reporter assays were employed to identify interactions between miR-98-5p and its specific target gene.ResultsMiR-98-5p expression was significantly lower in cancerous tissues and associated with tumor size, TNM stage, lymph node metastasis and survival. Notably, a series of gain- and loss-of-function assays elucidated that miR-98-5p suppressed PDAC cell proliferation, migration and invasion both in vitro and in vivo. Luciferase reporter assays, western blot and qRT-PCR revealed MAP4K4 to be a direct target of miR-98-5p. The effects of ectopic miR-98-5p were rescued by MAP4K4 overexpression. In contrast, the effects of miR-98-5p depletion were impaired by MAP4K4 knockdown. Furthermore, miR-98-5p suppressed the MAPK/ERK signaling pathway through downregulation of MAP4K4. In addition, the expression level of miR-98-5p was negatively correlated with MAP4K4 expression in PDAC tissues and cell lines.ConclusionsThese results suggest that downregulation of miR-98-5p promotes tumor development by downregulation of MAP4K4 and inhibition of the downstream MAPK/ERK signaling, thus, highlighting the potential of miR-98-5p as a therapeutic target for PDAC.

Abstract 1113: PODXL1 as a critical accelerator for pancreatic cancer metastasis

Abstract Podocalyxin-like 1 (PODXL1) is an anti-adhesive transmembrane protein belonging to the CD34 family and also stem cell marker that has been associated with an aggressive tumor phenotype and poor prognosis in several forms of cancer. In this study, we report the biological role of PODXL1 in progression of pancreatic invasive ductal adenocarcinoma (PDAC), especially focusing to invasion and metastasis of PDAC cells. Knockout of PODXL1 gene in three different human PDAC lines, MiaPaCa-2, AsPC1 and Panc-1, almost completely abrogated metastatic lesion in liver-metastatic mouse model in vivo. Molecular analysis revealed that PODXL1 functioned as a critical activator to multiple cytokine receptors expressed on cancer cells, and its interaction facilitated PDAC cell invasion. Endogenous expression of PODXL1 was strongly detected on cancer cells at the invasive front of the PDAC tissues, where coincidently expressed specific cytokine receptor that critically mediates cancer metastasis in vivo. Thus, we identify the novel functional aspect of PODXL1 and its biological role on PDAC as one of the most intractable malignancies. Citation Format: Eisaku Kondo, Ken Saito. PODXL1 as a critical accelerator for pancreatic cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1113.

Abstract 5741: Dexamethasone suppresses cytokine-induced dual oxidase 2 and VEGF-A expression in human pancreatic cancer cells in vitro and pancreatic cancer growth in xenografts

Abstract Chronic pancreatic inflammation is strongly associated with pancreatic cancer. We previously demonstrated that inflammatory cytokines interact to produce a DUOX2 -dependent, reactive oxygen species (ROS)-related, pro-oxidant milieu that could increase the pathologic potential of pancreatic inflammation, enhancing malignant transformation. Furthermore, we have shown that DUOXuox2 expression is upregulated in patients with chronic pancreatitis as well as pancreatic ductal adenocarcinoma (PDAC). Dexamethasone (Dex) has been reported to inhibit PDAC invasiveness, the formation of pancreatic intraepithelial neoplasia in genetically-engineered mouse models of pancreatic cancer, as well as epithelial to mesenchymal transition in PDACs, and local tumor recurrence and metastasis in vivo. Using cultured human pancreatic cancer cell lines, we found that in a dose- and time-dependent fashion Dex inhibited cytokine (IFN-γ/LPS, IL-4, and IL-17)-mediated up-regulation of DUOXuox2 and VEGF-A expression in several human pancreatic cancer cell lines, including BxPC-3, ASPC-1, and CFPAC-1. Dex pretreatment also blocked DUOX2-related DNA double strand breaks (detected as activated γH2AX) and the expression of HIF-1α in PDAC cells treated with pro-inflammatory cytokines. The effects of Dex were abolished by pre-treatment with the Dex antagonist RU-486. As expected, we did not observe anti-proliferative effects of Dex on PDAC cells in vitro. However, Dex strongly repressed DUOXuox2 mRNA and protein expression as well as the growth of xenografts initiated from BxPC-3 cells; in contrast, for the MIA-Paca line that is unresponsive to cytokines in culture, Dex produced no effect on DUOXuox2 expression and tumor growth when these cells were grown as xenografts. Examination of the human DUOXuox2 promotor in silico revealed a putative negative glucocorticoid receptor (GR) binding element. Western analysis, using nuclear extracts from pancreatic cancer cells treated with Dex, revealed that both activated glucocorticoid receptor and certain co-repressors, such as NCOR-1/2 and histone deacetylases (HDAC1, 2, and 3) exist in human pancreatic cancer cell nuclei. Our ongoing experiments are focused on understanding the molecular mechanism of Dex-mediated DUOXuox2 repression both in vitro and in vivo. In summary, these studies suggest that cytokine-related oxidant stress, generated by Duox2, could play a role in the progression of pancreatic cancer. Citation Format: Yongzhong Wu, James H. Doroshow. Dexamethasone suppresses cytokine-induced dual oxidase 2 and VEGF-A expression in human pancreatic cancer cells in vitro and pancreatic cancer growth in xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5741.

Rho Kinase Inhibition by AT13148 Blocks Pancreatic Ductal Adenocarcinoma Invasion and Tumor Growth.

The high mortality of pancreatic cancer demands that new therapeutic avenues be developed. The orally available small-molecule inhibitor AT13148 potently inhibits ROCK1 and ROCK2 kinases that regulate the actomyosin cytoskeleton. We previously reported that ROCK kinase expression increases with human and mouse pancreatic cancer progression and that conditional ROCK activation accelerates mortality in a genetically modified LSL-KrasG12D; LSL-p53R172H; Pdx1-Cre; (KPC) mouse pancreatic cancer model. In this study, we show that treatment of KPC mouse and human TKCC5 patient-derived pancreatic tumor cells with AT13148, as well as the ROCK-selective inhibitors Y27632 and H1152, act comparably in blocking ROCK substrate phosphorylation. AT13148, Y27632, and H1152 induced morphologic changes and reduced cellular contractile force generation, motility on pliable discontinuous substrates, and three-dimensional collagen matrix invasion. AT13148 treatment reduced subcutaneous tumor growth and blocked invasion of healthy pancreatic tissue by KPC tumor cells in vivo without affecting proliferation, suggesting a role for local tissue invasion as a contributor to primary tumor growth. These results suggest that AT13148 has antitumor properties that may be beneficial in combination therapies or in the adjuvant setting to reduce pancreatic cancer cell invasion and slow primary tumor growth. AT13148 might also have the additional benefit of enabling tumor resection by maintaining separation between tumor and healthy tissue boundaries.Significance: Preclinical evaluation of a small-molecule ROCK inhibitor reveals significant effects on PDAC invasion and tumor growth, further validating ROCK kinases as viable therapeutic targets in pancreatic cancer. Cancer Res; 78(12); 3321-36. ©2018 AACR.

Perineural Invasion in Pancreatic Ductal Adenocarcinoma

Perineural Invasion in Pancreatic Ductal Adenocarcinoma

MicroRNA‑539 inhibits cell proliferation, colony formation and invasion in pancreatic ductal adenocarcinoma by directly targeting IGF‑1R.

MicroRNAs (miRNAs) possess oncogenic and tumour‑suppressive roles in the carcinogenesis and progression of pancreatic ductal adenocarcinoma (PDAC) by regulating the expression of numerous cancer‑related genes. Thus, the investigation on the expression and roles of miRNAs in PDAC may facilitate the identification of novel and effective targets for the clinical diagnosis and treatment of patients with PDAC. miRNA‑539 (miR‑539) has been studied in multiple types of human cancer. However, its expression and potential biological function in PDAC remain unclear. In the current study, the expression level, clinical significance, roles and underlying molecular mechanism of miR‑539 in PDAC. The present results demonstrated that miR‑539 expression was downregulated in PDAC tissues and cell lines. A low miR‑539 level was associated with TNM stage and lymph node metastasis of patients with PDAC. miR‑539 overexpression induced a significant reduction in the proliferation, colony formation and invasion of PDAC cells. Insulin‑like growth factor 1 receptor (IGF‑1R) was confirmed as a direct target gene of miR‑539 in PDAC. Further analysis indicated that IGF‑1R was overexpressed in PDAC tissues. Notably, the mRNA expression of IGF‑1R was negatively correlated with miR‑539 levels in PDAC tissues. In addition, the recovered IGF‑1R expression also partially counteracted the suppressive roles of miR‑539 overexpression in PDAC cells. Overall, miR‑539 may inhibit the aggressive behaviour of PDAC by directly targeting IGF‑1R and may serve as a novel therapeutic target for patients with this disease.

MicroRNA‑874 inhibits proliferation and invasion of pancreatic ductal adenocarcinoma cells by directly targeting paired box 6.

Studies have demonstrated that a number of microRNAs (miRNAs) are dysregulated in pancreatic ductal adenocarcinoma (PDAC), and alterations in their expression may affect the onset and progression of PDAC. Therefore, the expression patterns, biological functions and associated molecular mechanisms of miRNAs in PDAC should be elucidated for the development of novel therapeutic methods. Previous studies reported significant miRNA‑874 (miR‑874) dysregulation in multiple types of human cancer. However, the expression pattern, possible roles and underlying mechanisms of miR‑874 in PDAC remain to be elucidated. This study evaluated miR‑874 expression in PDAC and examined its biological functions and underlying mechanism of action in PDAC progression. miR‑874 expression was downregulated in PDAC tissues and cell lines. Functional experiments demonstrated that upregulation of miR‑874 inhibited cell proliferation and invasion in PDAC. Additionally, paired box 6 (PAX6) was predicted as a putative target of miR‑874 using bioinformatics analysis. Further experiments demonstrated that PAX6 may be the direct target gene of miR‑874 in PDAC. PAX6 knockdown exhibited similar inhibitory effects to miR‑874 overexpression in PDAC cells. In addition, restored PAX6 expression may reverse the suppressive roles of miR‑874 overexpression in PDAC cells. The results demonstrated that miR‑874 may serve tumor suppressive roles in PDAC by directly targeting PAX6. Therefore, miR‑874 may exhibit potential applications for treatment of patients with PDAC.

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

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