Pancreatic Ductal Adenocarcinoma Specimens Research Articles

Loss of the transcriptional repressor TGIF1 results in enhanced Kras-driven development of pancreatic cancer

BackgroundThe TG-interacting factor 1 (TGIF1) gene, which encodes a nuclear transcriptional corepressor of the TGFβ1/Smad signaling pathway, has been implicated in the pathogenesis of various types of human cancer; however, its role in pancreatic ductal adenocarcinoma (PDAC) has yet to be elucidated.MethodsThe expression of TGIF1 in human and murine PDAC specimens were detected by IHC analysis. The functions of TGIF1 in in vivo PDAC growth, dissemination, and metastasis were assessed using conditional inactivation of TGIF1 in well-established autochthonous mouse models of PDAC. Primary cells from TGIF1 null or wild type PDAC mice were examined by assays for cell proliferation, migration, invasion, soft agar and xenograft tumorigenesis. Gene expression profiling, pathway analyses, epigenetic changes associated with TGIF1 loss, and in vitro and in vivo effects of 4-MU were assessed.ResultsConditional deletion of TGIF1 in the mouse pancreas had no discernible effect on pancreatic development or physiology. Notably, TGIF1 loss induced KrasG12D-driven PDAC models exhibited shorter latency and greater propensity for distant metastases. Deciphering the molecular mechanisms highlighted the TGIF1 loss-induced activation of the hyaluronan synthase 2 (HAS2)-CD44 signaling pathway and upregulation of the immune checkpoint regulator PD-L1 to facilitate the epithelial–mesenchymal transition (EMT) and tumor immune suppression. We also founded that TGIF1 might function as an epigenetic regulator and response for aberrant EMT gene expression during PDAC progression.ConclusionsOur results imply that targeting the HAS2 pathway in TGIF1 loss of PDAC could be a promising therapeutic strategy for improving the clinical efficacy against PDAC metastasis.

Clinicopathological significance of ISYNA1 expression in pancreatic ductal adenocarcinoma

Objective: To study the expression of ISYNA1 and association of ISYNA1 with clinicopathological significance in pancreatic ductal adenocarcinoma (PDAC). Methods: Collecting clinical data and specimens of 68 PDAC patients at Department of General Surgery, the First Hospital of China Medical University from March 2008 to December 2017.There were 39 males and 29 females, aged 33 to 81 years(median 59 years).The expression of ISYNA1 in 68 paraffin embedded PDAC specimens was detected by immunohistochemistry,in which 34 had paired non-cancerous pancreatic tissues,the relationship between ISYNA1 expression and clinicopathological parameters was analyzed; and the correlation between ISYNA1 and p53 in 48 PDAC specimens were estimated.qRT-PCR and Western blot were used to examine the expression of ISYNA1 mRNA and protein level in 17 paired fresh PDAC specimens and adjacent non-cancerous pancreatic tissues,respectively.siRNA interference was used to knockdown the expression of p53 in Capan-2,SW1990 and Miapaca-2 cells,and association of p53 with ISYNA1 expression was explored. Statistical methods included Student's test,χ(2) test, Kaplan-Meier curve, Log-rank test and Pearson analysis, respectively. Results: Immunohistochemistry results showed that the expression of ISYNA1 in PDAC(3.681±2.198)was significantly lower than that in normal pancreatic tissues(6.012±3.428)(t=-3.611,P=0.001).In 17 paired fresh PDAC specimens,ISYNA1 mRNA expression in non-cancerous pancreatic tissues(()ΔC(T): (3.721±2.234)was obviously higher than that in PDAC tissues ()ΔC(T): (5).889±1.607) (t=-4.636,P<0.01), and ISYNA1 protein level in non-cancerous pancreatic tissues(0.815±0.418)was similarly higher than that in PDAC tissues(0.517±0.240)(t=2.948,P=0.009).χ(2) test showed the expression of ISYNA1 was negatively associated with tumor invasion depth(χ(2)=7.534,P=0.030)and vascular invasion(χ(2)=5.048,P=0.043);Pearson analysis showed there was no relationship between ISYNA1 and mutant p53(χ(2)=1.377,P=0.359).In p53 wild-type Capan-2 and SW1990 cells,Knockdown of p53 significantly down regulated ISYNA1 expression, whereas had no effect on ISYNA1 expression in p53 mutant Miapaca-2 cells. Kaplan-Meier survival analysis and Log-Rank test indicated patients with negative ISYNA1 expression had a shorter median survival time and poorer prognosis(χ(2)=4.953, P=0.026). Conclusions: The expression of ISYNA1 in PDAC tissues is significantly decreased,which is associated with the prognosis of PDAC patients,it is only related to wild type p53,and has no relationship with mutant p53.Abnormal expression of ISYNA1 may play an important role in the progression of PDAC.

Dissecting the single cell profile of pancreatic ductal adenocarcinoma

Background: The interplay of the tumor, stroma and immune system is becoming increasingly recognized as crucial in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Traditional bulk RNA sequencing relies on utilizing RNA from a tumor specimen, and subjecting it to modern RNA sequencing techniques. Although this technique has allowed us to make great strides in understanding the heterogeneity and profile of pancreatic adenocarcinoma, we are unable to track RNA expression on the single cell level—which would allow us to pinpoint how treatments or lack thereof are contributing to phenotype and resultant survival. Methods: Immediately following resection of a pancreatic cancer specimen or alternatively after biopsy of a metastatic lesion, a tumor suspension is made. The tumor is broken down with a combination of mechanical and enzymatic digestion. This is followed by isolation of live single cells, and removal of RBCs and debris (Figure 1A). We immediately run a cell suspension of 10,000 live cells on the 10× Genomics single cell platform (Figure 1B), whereby each individual cell receives a unique barcode that it carries throughout the sequencing process. We then perform RNA sequencing. After sequencing, all cells with mitochondrial DNA content >10%, consistent with a dead cell, are removed from further analysis. We are left with a single cell RNA profile of live cells from the tumor. Results: Thus far we have processed 4 primary pancreatic ductal adenocarcinoma specimens, and 1 metastatic lesion, from a total of 5 patients, with 3 samples analyzed thus far. On average, we are left with 2470 live cells per patient sample. For each patient, we were able to identify 12 unique cell populations. Patient 1174, underwent a Whipple without preoperative chemoradiotherapy for a resectable PDAC head mass (T1cN2 on final path). Single cell analysis of the tumor specimen, identified 42.7% of the tumor mass to be PDAC cells (Figure 1C). In this case 4 distinct tumor subpopulations can be identified (Figure 1C). T and B lymphocytes comprised 35.6% of infiltrating cells, macrophages comprised 13.2% of the tumor population, with mast cells, pancreatic stellate cells, acinar cells and endothelial cells comprising the remainder of the tumor mass. Each cell population is identified by multiple characteristic markers, with 1 identifying marker displayed in Figure 1C. Example characteristic marker being KRT19 for pancreatic ductal cancer cells. In contrast to patient 1174, patient 1105, who received upfront neoadjuvant chemotherapy, but was found to be unresectable in the operating room, had a liver tumor with only 23.9% PDAC cells, and ∼50% of infiltrating cells comprising T and B lymphocytes, as well as NK cells. Finally, patient 1160, who had a resectable pancreatic head mass (T2N1 on final path) and did not receive preoperative therapy, had 20.6% PDAC cells, ∼33% infiltrating lymphocytes, and 20% macrophages. Conclusion: Single cell RNA sequencing is a viable strategy to better dissect the makeup of PDACs. It allows for RNA profiling on a single cell level, and provides a landscape of the tumor and tumor microenvironment in a non-biased fashion. Our group is enrolling multiple patients treated and not treated prior to pancreatic cancer resection, as well as consecutive specimens (pre treatment and post surgical) from several patients in order to better assess both the PDAC landscape and how it changes based on different factors such as chemotherapy, stage or location.

Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.

Abstract A156: PDA tumor cell death as following combination anti-PD-1 blockade and CXCR4 blockade is a direct effect of CD8+ T-cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the fourth most common cause of cancer death in the United States. Despite the fact that the human PDA microenvironment contains a mixture of infiltrating immune cells, including activated effector CD8+ T-cells, attempts to apply immune checkpoint inhibitor therapy have not yet been successful in achieving a clinical response (1). Combining blockade of the immune checkpoint receptor PD-1 with blockade of the chemokine receptor CXCR4 in a mouse model of PDA produced T-cell-mediated tumor cell killing (2). We therefore hypothesized that, in a human PDA slice culture model system that maintains the intact tumor microenvironment (3), combination of CXCR4 blockade with PD-1 blockade would lead to anti-tumor T-cell activation. Methods: Cores (6 mm) were taken from freshly resected, sterile human PDA specimens and cut into 250 µm thick slices using a vibratome. The slices were treated with either IgG antibody isotype control, AMD3100 (a CXCR4 blocking small molecule drug) plus antibody isotype control, anti-PD-1 antibody, or a combination of anti-PD-1 antibody and AMD3100 for two days. The slices were then stained with fluorescently labelled antibodies for CD8 and EpCAM, as well as with SR-FLICA, a reagent that produces a fluorescent signal when bound to activated Caspase 3 and 7 enzymes. Live slices were maintained in a humidified, temperature-controlled CO2 chamber for time-lapse confocal imaging for 1 hour. Similarly, untreated slices were imaged for 90 minutes prior to and following combined anti-PD-1 and AMD3100 drug treatment. Each slice was imaged at 3 different positions to observe CD8+ T-cells, EpCAM+ tumor cells, and SR-FLICA+ apoptotic cells throughout the slice. Each cell type was counted at each position over the entire time course imaged. Results: Compared to antibody isotype control or monotherapy, slices treated with combined PD-1 and CXCR4 blockade contained a greater fraction of EpCAM+ tumor cells that were undergoing apoptosis, indicated by SR-FLICA positivity. This treatment also increased the proportion of EpCAM+ tumor cells with a CD8+ T-cell within 20 µm, and EpCAM+ cells had increased evidence of apoptosis when a CD8+ cell was nearby. When individual slices were imaged before and after combined PD-1 and CXCR4 blockade, a greater proportion of EpCAM+ cells were found to have a CD8+ cell nearby after treatment compared to before treatment, and the tumor cells with a CD8+ cell nearby were more like to be undergoing apoptosis compared to before treatment. Conclusion: Direct visualization of live pancreatic cancer slice cultures demonstrates that combined PD-1 and CXCR4 blockade enhances both the migration of CD8+ T-cells towards epithelial tumor cells and their anti-tumor cytotoxic activity.

RET, a targetable driver of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) remains a deadly disease, affecting about 40,000 individuals in the United States annually. We aimed to characterize the role of RET as a co-driver of pancreas tumorigenesis. To assess the role of RET as a co-driver of PDA, we generated a novel triple mutant transgenic mouse based on the cre-activated p53R172H gene and a constitutively active RET M919T mutant (PRC). Survival analysis was performed using Kaplan-Meier analysis. Study of human PDA specimens and Pdx-1-Cre/KrasG12D /p53R172H (KPC) mice revealed that RET is upregulated during pancreas tumorigenesis, from inception through precursor lesions, to invasive cancer. We demonstrated that activation of RET is capable of inducing invasive pancreatic carcinomas in the background of the P53 inactivation mutation. Compared to KPC mice, PRC animals had distinct phenotypes, including longer latency to tumor progression, longer survival, and the presence of multiple macrometastases. Enhanced activation of the MAPK pathway was observed as early as the PanIN 2 stage. Sequencing of the exonic regions of KRAS in PRC-derived PDA cells revealed no evidence of KRAS mutations. RET can be an essential co-driver of pancreatic tumorigenesis in conjugation with KRAS activity. These data suggest that RET may be a potential target in the treatment of PDA.

Long noncoding RNA DLEU1 aggravates pancreatic ductal adenocarcinoma carcinogenesis via the miR-381/CXCR4 axis.

Recent evidence has highlighted that long noncoding RNAs (lncRNA) are associated with many diseases, particularly cancer. However, current understanding of the lncRNA deleted in lymphocytic leukemia 1 (DLEU1) in pancreatic ductal adenocarcinoma (PDAC) remains limited. Our studies indicated that the DLEU1 expression level was upregulated in PDAC tissue samples compared with adjacent normal tissue. Moreover, the aberrant overexpression of DLEU1 indicated poor prognosis of patients with PDAC. Loss-of-function experiments revealed that DLEU1 knockdown inhibited the proliferation, migration, and invasion of PDAC cells in vitro and decreased tumor growth in vivo. Bioinformatics analysis predicted that miR-381 potentially targeted the DLEU1 3'-untranslated region (UTR), suggesting an interaction between miR-381 and DLEU1. Furthermore, miR-381 also targeted the chemokine receptor-4 (CXCR4) messenger RNA 3'-UTR, which was validated by luciferase reporter assay. Taken together, our study demonstrated the oncogenic role of DLEU1 in clinical PDAC specimens and cellular experiments, showing the potential involvement of DLEU1/miR-381/CXCR4 pathway. These results provide novel insight into PDAC tumorigenesis.

High cytoplasm HABP1 expression as a predictor of poor survival and late tumor stage in pancreatic ductal adenocarcinoma patients

BackgroundHyaluronan-binding protein 1 (HABP1) overexpression has been confirmed in different malignancies and found to be strongly associated with tumor development and progression. The aim of the present study was to explore the impact of HABP1 in pancreatic ductal adenocarcinoma (PDAC) patients. MethodHABP1 expression was evaluated in 89 PDAC specimens. ResultsThe expression of HABP1 was significantly higher in tumor tissues than that in adjacent normal tissues. High nucleus HABP1 expression and high cytoplasm HABP1 expression were both detected in PDAC tissues. Overall survival analysis by optical density showed that the mean survival was similar between patients with low and high optical density values of HABP1 expression (P = 0.312). The similar result was also found between patients with low-moderate or high nucleus HABP1 expression (P = 0.275). However, the mean survival was significantly poorer in patients with cytoplasm HABP1 overexpression (P < 0.001). High cytoplasm HABP1 expression was strongly correlated with late tumor stages, arterial involvement, lymph node metastasis and carbohydrate antigen 19-9 levels. ConclusionHigh cytoplasm HABP1 expression may prove to be a predictor of poor survival and late tumor stage in PDAC patients. HABP1 could serve as a promising biomarker to identify subsets of PDAC patients with high malignant clinical behavior.

The proton preparation NSC-631570 (Ukrain) and its selective effect

First indications on the selective effect of NSC-631570 on the cancer cells were provided in an early study when different oxygen consumption by normal liver cells and Ehrlich's tumor ascitic cells after the incubation with NSC-631570 was revealed.In the tests on the Jurkat lymphoma model, NSC-631570 has been proven to be a strong apoptosis inducer. Profound research showed NSC-631570 brought about the depolarization of mitochondrial membranes and consequently the activation of caspases.NSC-631570 induced apoptosis in a panel of cancer cell lines (cervical cancer HeLa, HeKB, HeKS32, HeBcl3, HeNFR and HeIKK, human colon cancer SW480, human renal carcinoma HEK293, human osteosarcoma MG-63) by activating the caspases of the intrinsic cell death pathway. Interestingly, non-transformed fibroblasts (hTERT) cell line was insensitive to the drug.In the tests on human cervix carcinoma cells HeLa, squamous carcinoma cells WHCO5, normal kidney cell line Graham 293, and transformed kidney cell line Vero from African green monkey, NSC-631570 inhibited the tubulin polymerization and caused a metaphase block in cancer cells which is characterized by abnormal chromosomal distribution, and results in the formation of micronuclei and in apoptosis.The effects of NSC-631570 on cell survival, alteration of the cell cycle and induction of apoptosis without and in combination with ionizing radiation (IR) were investigated on the exponentially growing human tumor cells MDA-MB-231 (breast), PA-TU-8902 (pancreas), CCL-221 (colon), U-138MG (glioblastoma), and human skin and lung fibroblasts HSF1, HSF2 and CCD32-LU.Without IR, NSC-631570 exerted a time- and dose-dependent cytotoxic effect, more pronounced against the cancer cells. The combination of NSC-631570 plus IR enhanced toxicity in CCL-221 and U-138MG cells with their accumulation in the G2/M phase, but not in MDA-MB-231 and PA-TU-8902 cells. A radio protective effect was found in normal human fibroblasts.NSC-631570 caused the accumulation of prostate cancer cells as well as epidermoid carcinoma cells in the G2/M phase, however, not of normal cells. The cytotoxic effects of NSC-631570 were evaluated in primary pancreatic cancer cell lines (PPTCC), fibroblasts derived from pancreatic ductal adenocarcinoma specimens (F-PDAC), and an immortalized epithelial ductal pancreatic cell line HPNE. Cytotoxic effects of NSC-631570 in PPTCCs were significantly higher than those observed in F-PDAC and HPNE cells. Furthermore, it was revealed that PPTCCs cells consumed more drug than F-PDAC and HPNE cells. This selective effect of NSC-631570 in PPTCCs may be related to a different transport system or higher metabolism of the drug in PDAC.Altogether, in comparative studies NSC-631570 has been tested on 18 cancer and 12 benign cell lines at identical conditions so far. In all these experiments the selective effect of NSC-631570 against cancer cells was confirmed. This selective effect of NSC-631570 against cancer cells explains its good tolerability in clinical use.

Inositol polyphosphate-4-phosphatase type II plays critical roles in the modulation of cadherin-mediated adhesion dynamics of pancreatic ductal adenocarcinomas

ABSTRACTThe inositol polyphosphate-4-phosphatase type II (INPP4B) has been mostly proposed to act as a tumor suppressor whose expression is frequently dysregulated in numerous human cancers. To date, little is unveiled about whether and how INPP4B will exert its tumor suppressive function on the turnover of cadherin-based cell-cell adhesion system in pancreatic ductal adenocarcinomas (PDACs) in vitro. Here we provide the evidence that INPP4B manipulates cadherin switch in certain PDAC cell lines through a phosphorylated AKT-inactivation manner. The knockdown of INPP4B in AsPC-1 results in a more invasive phenotype, and overexpression of it in PANC-1 leads to partial reversion of mesenchymal status and impediment of in vitro invasion but not migration. More importantly, E-cadherin (Ecad) is enriched in the early and sorting endosomes containing INPP4B by which its recycling rather than degradation is enabled. Immunohistochemical analysis of 39 operatively resected PDAC specimens reveals it is poorly differentiated, non-cohesive ones in which the INPP4B and Ecad are partially or completely compromised in expression. We therefore identify INPP4B as an tumor suppressor in PDAC which attenuates AKT activation and participates in preservation of Ecad in endocytic pool and cellular membrane.

KRAS mutation and epithelial-macrophage interplay in pancreatic neoplastic transformation.

Pancreatic ductal adenocarcinoma (PDA) is characterized by epithelial mutations in KRAS and prominent tumor-associated inflammation, including macrophage infiltration. But knowledge of early interactions between neoplastic epithelium and macrophages in PDA carcinogenesis is limited. Using a pancreatic organoid model, we found that the expression of mutant KRAS in organoids increased (i) ductal to acinar gene expression ratios, (ii) epithelial cells proliferation and (iii) colony formation capacity in vitro, and endowed pancreatic cells with the ability to generate neoplastic tumors in vivo. KRAS mutations induced a protumorigenic phenotype in macrophages. Altered macrophages decreased epithelial pigment epithelial derived factor (PEDF) expression and induced a cancerous phenotype. We validated our findings using annotated patient samples from The Cancer Genome Atlas (TCGA) and in our human PDA specimens. Epithelium-macrophage cross-talk occurs early in pancreatic carcinogenesis where KRAS directly induces cancer-related phenotypes in epithelium, and also promotes a protumorigenic phenotype in macrophages, in turn augmenting neoplastic growth.

Disruption of C1galt1 Gene Promotes Development and Metastasis of Pancreatic Adenocarcinomas in Mice

Pancreatic ductal adenocarcinomas (PDACs) produce higher levels of truncated O-glycan structures (such as Tn and sTn) than normal pancreata. Dysregulated activity of core 1 synthase glycoprotein-N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) leads to increased expression of these truncated O-glycans. We investigated whether and how truncated O-glycans contributes to the development and progression of PDAC using mice with disruption of C1galt1. We crossed C1galt1 floxed mice (C1galt1loxP/loxP) with KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) mice to create KPCC mice. Growth and progression of pancreatic tumors were compared between KPC and KPCC mice; pancreatic tissues were collected and analyzed by immunohistochemistry; immunofluorescence; and Sirius red, alcian blue, and lectin staining. We used the CRISPR/Cas9 system to disrupt C1GALT1 in human PDAC cells (T3M4 and CD18/HPAF) and levels of O-glycans were analyzed by lectin blotting, mass spectrometry, and lectin pulldown assay. Orthotopic studies and RNA sequencing analyses were performed with control and C1GALT1 knockout PDAC cells. C1GALT1 expression was analyzed in well-differentiated (n= 36) and poorly differentiated (n= 23) PDAC samples by immunohistochemistry. KPCC mice had significantly shorter survival times (median 102 days) than KPC mice (median 200 days) and developed early pancreatic intraepithelial neoplasias at 3 weeks, PDAC at 5 weeks, and metastasis at 10 weeks compared with KPC mice. Pancreatic tumors that developed in KPCC mice were more aggressive (more invasive and metastases) than those in KPC mice, had a decreased amount of stroma, and had increased production of Tn. Poorly differentiated PDAC specimens had significantly lower levels of C1GALT1 than well-differentiated PDACs. Human PDAC cells with knockout of C1GALT1 had aberrant glycosylation of MUC16 compared with control cells and increased expression of genes that regulate tumorigenesis and metastasis. In studies of KPC mice with disruption of C1galt1, we found that loss of C1galt1 promotes development of aggressive PDACs and increased metastasis. Knockout of C1galt1 leads to increased tumorigenicity and truncation of O-glycosylation on MUC16, which could contribute to increased aggressiveness.

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.

MicroRNA-29b-2-5p inhibits cell proliferation by directly targeting Cbl-b in pancreatic ductal adenocarcinoma

BackgroundMicroRNAs can be used in the prognosis of malignancies; however, their regulatory mechanisms are unknown, especially in pancreatic ductal adenocarcinoma (PDAC).MethodsIn 120 PDAC specimens, miRNA levels were assessed by quantitative real time polymerase chain reaction (qRT-PCR). Then, the role of miR-29b-2-5p in cell proliferation was evaluated both in vitro (Trypan blue staining and cell cycle analysis in the two PDAC cell lines SW1990 and Capan-2) and in vivo using a xenograft mouse model. Next, bioinformatics methods, a luciferase reporter assay, Western blot, and immunohistochemistry (IHC) were applied to assess the biological effects of Cbl-b inhibition by miR-29b-2-5p. Moreover, the relationship between Cbl-b and p53 was evaluated by immunoprecipitation (IP), Western blot, and immunofluorescence.ResultsFrom the 120 PDAC patients who underwent surgical resection, ten patients with longest survival and ten with shortest survival were selected. We found that high miR-29b-2-5p expression was associated with good prognosis (p = 0.02). The validation cohort confirmed miR-29b-2-5p as an independent prognostic factor in PDAC (n = 100, 95% CI = 0.305–0.756, p = 0.002). Furthermore, miR-29b-2-5p inhibited cell proliferation, induced cell cycle arrest, and promoted apoptosis both in vivo and in vitro. Interestingly, miR-29b-2-5p directly bound the Cbl-b gene, down-regulating its expression and reducing Cbl-b-mediated degradation of p53. Meanwhile, miR-29b-2-5p expression was negatively correlated with Cbl-b in PDAC tissues (r = − 0.33, p = 0.001).ConclusionsTaken together, these findings indicated that miR-29b-2-5p improves prognosis in PDAC by targeting Cbl-b to promote p53 expression, and would constitute an important prognostic factor in PDAC.

Intestinal microbiota enhances pancreatic carcinogenesis in preclinical models.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States yet data are scant regarding host factors influencing pancreatic carcinogenesis. Increasing evidence support the role of the host microbiota in carcinogenesis but its role in PDAC is not well established. Herein, we report that antibiotic-mediated microbial depletion of KrasG12D/PTENlox/+ mice showed a decreased proportion of poorly differentiated tumors compared to microbiota-intact KrasG12D/PTENlox/+ mice. Subsequent 16S rRNA PCR showed that ~50% of KrasG12D/PTENlox/+ mice with PDAC harbored intrapancreatic bacteria. To determine if a similar observation in humans correlates with presence of PDAC, benign and malignant human pancreatic surgical specimens demonstrated a microbiota by 16S bacterial sequencing and culture confirmation. However, the microbial composition did not differentiate PDAC from non-PDAC tissue. Furthermore, murine pancreas did not naturally acquire a pancreatic microbiota, as germ-free mice transferred to specific pathogen-free housing failed to acquire intrapancreatic bacteria over time, which was not augmented by a murine model of colitis. Finally, antibiotic-mediated microbial depletion of Nod-SCID mice, compared to microbiota-intact, showed increased time to PDAC xenograft formation, smaller tumors, and attenuated growth. Interestingly, both xenograft cohorts were devoid of intratumoral bacteria by 16S rRNA PCR, suggesting that intrapancreatic/intratumoral microbiota is not the sole driver of PDAC acceleration. Xenografts from microbiota-intact mice demonstrated innate immune suppression by immunohistochemistry and differential regulation of oncogenic pathways as determined by RNA sequencing. Our work supports a long-distance role of the intestinal microbiota on PDAC progression and opens new research avenues regarding pancreatic carcinogenesis.

Stromal heterogeneity in pancreatic cancer and chronic pancreatitis

Background/objectivesAn abundant stromal reaction is a hallmark of pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis (CP). The cells mainly responsible for the stromal reaction are activated pancreatic stellate cells (PSCs). Despite their crucial role, PSCs are not well characterized. PSCs share characteristics with the better-known hepatic stellate cells (HSCs). The aim of this study was a detailed analysis of PSCs in PDAC and CP. MethodsWhole-slide specimens of CP (n = 12) and PDAC (n = 10) were studied by histochemistry and immunohistochemistry. The stroma was evaluated using Movat's pentachrome stain. PSCs were tested by immunohistochemistry for PSC markers (α-SMA, CD34, desmin, NGFR, SPARC and tenascin C) and HSC markers (α-crystallin B, CD56, NGF, NT-3, synaptophysin and TrkC). Alpha-SMA, tenascin C, SPARC and NT-3 staining were verified on tissue micro arrays (TMAs) from a well-characterized cohort of 223 PDAC patients. PSCs isolated from human PDAC and CP tissue samples as well as HSCs were evaluated by immunofluorescence. ResultsWhile the stroma of CP cases was characterized by a collagen-rich fibrosis, PDAC stroma displayed higher mucin content (p = 0.0002). PSCs showed variable expression of tested markers. In PDAC samples, staining of most markers was found around tumor complexes, while CP samples showed a greater variety of localizations. Alpha-SMA staining correlated with collagen-rich fibrosis (p = 0.012), while NT-3 staining correlated with mucin-rich stroma (p = 0.008). A peritumoral staining was confirmed for α-SMA, tenascin C, SPARC and NT-3 in the PDAC TMA cohort (n = 223). In a subgroup of patients with pancreatic head tumors and UICC 2009 IIB (n = 144), α-SMA staining intensity was a prognostic factor for overall survival at uni- and multivariate analysis (p = 0.036 and p = 0.002). ConclusionsThe close similarities between PSCs and HSCs were confirmed. Heterogeneous expression patterns of the tested markers might reflect different levels of activation or differentiation, or even multiple subpopulations of PSCs. Survival analysis suggests an impact of stromal composition on survival.

Vitamin D Supplementation is a Promising Therapy for Pancreatic Ductal Adenocarcinoma in Conjunction with Current Chemoradiation Therapy.

The cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC) are well known to play a dominant role in distant metastasis. Nevertheless, the effect on CAFs with current chemoradiation therapies remains uncertain. This study aimed to reveal the role of CAFs under current chemoradiation therapy (CRT) and investigate the factors regulating CAFs. α-SMA-positive cells in 86 resected PDAC specimens with/without preoperative CRT were evaluated by immunohistochemistry. Various factors, including the plasma levels of vitamin D, were investigated for association with the number of CAFs or distant metastasis-free survival (DMFS). Human pancreatic satellite cells (hPSCs) extracted from clinical specimens were used to validate the factors. All PDAC samples contained CAFs but the number varied widely. Multivariate analysis for DMFS indicated a larger number of CAFs was a significant risk factor. Univariate analysis for the number of CAFs identified two clinical factors: preoperative CRT and lower plasma levels of vitamin D. In subgroup analysis, the higher plasma level of vitamin D was a dominant factor for longer DMFS in PDAC patients after preoperative CRT. These results were validated by using extracted hPSCs. Irradiation activated stromal cells into CAFs facilitating malignant characteristics of PDAC and the change was inhibited by vitamin D supplementation in vitro. In conjunction with established current therapies, vitamin D supplementation may be an effective treatment for PDAC patients by inactivating CAFs.

Tumor-associated macrophages promote progression and the Warburg effect via CCL18/NF-kB/VCAM-1 pathway in pancreatic ductal adenocarcinoma

Tumor-associated macrophages (TAMs) are frequently found near pancreatic cancer cells, but it is uncertain whether they are involved in pancreatic cancer progression and the Warburg effect. Here, we show that CCL18 secreted by TAMs facilitates malignant progression and induced a glycolytic phenotype in pancreatic cancer, partially owing to paracrine induction of VCAM-1 in pancreatic cancer cells. Reciprocally, VCAM-1-induced lactate production from pancreatic cancer cells with enhanced aerobic glycolysis activates macrophages to a TAM-like phenotype, forming a positive feedback loop. VCAM-1 was found to be highly expressed in human pancreatic ductal adenocarcinoma (PDAC) tissues and cell lines, and is associated with disease progression and predicts clinical outcome in PDAC patients. Flow cytometry analysis further demonstrated that VCAM-1 downregulation induced an accumulation of PDAC cells in G0/G1 phase, accompanied by a significant decrease in S phase. Downregulation of VCAM-1 significantly inhibited proliferation, colony formation, migration, and invasion of PDAC cells in vitro, whereas the ectopic expression of VCAM-1 had the opposite effect. VCAM-1 on pancreatic cancer cells might tethers THP-1 monocytes to cancer cells via counter–receptor interaction, providing a survival advantage to pancreatic cancer cells that infiltrate leukocyte-rich microenvironments. Furthermore, downregulation of VCAM-1 could repress tumor growth in mouse xenograft models. In particular, our results highlighted the contribution of VCAM-1 to the maintenance of the Warburg effect in PDAC cells. Finally, we investigated the clinical correlations of CCL18 and VCAM-1 in human PDAC specimens. In summary, these findings indicate that the CCL18/PITPNM3/NF-kB/VCAM-1 regulatory network might provide a potential new therapeutic strategy for PDAC.

Decreased expression of USP9X is associated with poor prognosis in Chinese pancreatic ductal adenocarcinoma patients.

The present study aimed to investigate the expression level of ubiquitin specific peptidase 9X (USP9X) and its clinical significance in Chinese patients with pancreatic ductal adenocarcinoma (PDAC). The mRNA expression levels of USP9X in 30 paired PDAC tissue samples were examined by reverse transcription-quantitative polymerase chain reaction. The protein expression of USP9X was analyzed by immunohistochemistry (IHC) in a tissue microarray containing 205 PDAC specimens. All analyses were performed by SPSS 20.0 and GraphPad Prism 5.0 software. The USP9X mRNA level was significantly decreased in 18/30 (60.0%) PDAC tissue samples compared with matched surrounding non-tumor tissue samples. The results of IHC revealed that decreased expression of USP9X was inversely associated with liver metastasis (P=0.032). Kaplan-Meier survival curves indicated that patients with high expression of USP9X presented a longer clinical overall survival time (P<0.001). Univariate and multivariate COX regression analysis revealed that USP9X protein expression level was a significant, and independent prognostic factor for the overall survival rate of patients with PDAC. The results of the present study indicate that USP9X may serve as a candidate tumor suppressor and prognostic biomarker in PDAC.

Tumor-Stroma IL1β-IRAK4 Feedforward Circuitry Drives Tumor Fibrosis, Chemoresistance, and Poor Prognosis in Pancreatic Cancer.

Targeting the desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC) holds promise to augment the effect of chemotherapy, but success in the clinic has thus far been limited. Preclinical mouse models suggest that near-depletion of cancer-associated fibroblasts (CAF) carries a risk of accelerating PDAC progression, underscoring the need to concurrently target key signaling mechanisms that drive the malignant attributes of both CAF and PDAC cells. We previously reported that inhibition of IL1 receptor-associated kinase 4 (IRAK4) suppresses NFκB activity and promotes response to chemotherapy in PDAC cells. In this study, we report that CAF in PDAC tumors robustly express activated IRAK4 and NFκB. IRAK4 expression in CAF promoted NFκB activity, drove tumor fibrosis, and supported PDAC cell proliferation, survival, and chemoresistance. Cytokine array analysis of CAF and microarray analysis of PDAC cells identified IL1β as a key cytokine that activated IRAK4 in CAF. Targeting IRAK4 or IL1β rendered PDAC tumors less fibrotic and more sensitive to gemcitabine. In clinical specimens of human PDAC, high stromal IL1β expression associated strongly with poor overall survival. Together, our studies establish a tumor-stroma IL1β-IRAK4 feedforward signal that can be therapeutically disrupted to increase chemotherapeutic efficacy in PDAC.Significance: Targeting the IL1β-IRAK4 signaling pathway potentiates the effect of chemotherapy in pancreatic cancer. Cancer Res; 78(7); 1700-12. ©2018 AACR.