Non-cancerous Pancreatic Tissues Research Articles

A prognostic model for anoikis-related genes in pancreatic cancer

Anoikis, a distinct form of programmed cell death, is crucial for both organismal development and maintaining tissue equilibrium. Its role extends to the proliferation and progression of cancer cells. This study aimed to establish an anoikis-related prognostic model to predict the prognosis of pancreatic cancer (PC) patients. Gene expression data and patient clinical profiles were sourced from The Cancer Genome Atlas (TCGA-PAAD: Pancreatic Adenocarcinoma) and the International Cancer Genome Consortium (ICGC-PACA: Pancreatic Ductal Adenocarcinoma). Non-cancerous pancreatic tissue gene expression data were obtained from the Genotype-Tissue Expression (GTEx) project. The R package was used to construct anoikis-related PC prognostic models, which were later validated with the ICGC-PACA database. Survival analyses demonstrated a poorer prognosis for patients in the high-risk group, consistent across both TCGA-PAAD and ICGC-PACA datasets. A nomogram was designed as a predictive tool to estimate patient mortality. The study also analyzed tumor mutations and immune infiltration across various risk groups, uncovering notable differences in tumor mutation patterns and immune landscapes between high- and low-risk groups. In conclusion, this research successfully developed a prognostic model centered on anoikis-related genes, offering a novel tool for predicting the clinical trajectory of PC patients.

MLPH regulates EMT in pancreatic adenocarcinoma through the PI3K-AKT signaling pathway.

Pancreatic adenocarcinoma (PAAD) is an extremely malignant tumor, and most patients develop postoperative metastases. Melanophilin (MLPH) is involved in the progression of various tumors, but its molecular mechanisms and role in pancreatic cancer progression are unknown. In this study, differential MLPH expression in cancer tissues and the adjacent tissues was evaluated using the Gene Expression Profiling Interaction Analysis 2 (GEPIA 2) and Human Protein Atlas (HPA) databases. The role of MLPH in PAAD proliferation, invasion, and migration in vitro was explored via clone formation, Cell Counting Kit-8 assay, Transwell assay, and western blot. The in vivo validation of function was performed using a metastatic nude mouse model. The result showed that the pancreatic cancer tissues had significantly higher MLPH expression levels than the noncancerous pancreatic tissues. MLPH expression changes were related to PAAD cell proliferation, invasion, and migration. The western blotting demonstrated that PAAD cells had reduced Epithelial-mesenchymal transition (EMT)-related marker expression. Furthermore, overexpressing MLPH enhanced cell proliferation, migration, and invasion, and increased EMT-related marker expression. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the molecular mechanism underlying the effect of MLPH on PAAD was significantly related to the PI3K-AKT pathway. LY294002 blocked the MLPH overexpression-mediated enhanced cell invasion and migration and inhibited EMT-associated marker expression. Conversely, 740Y-P reversed the inhibitory effects of MLPH downregulation and led to cell migration, invasion, and EMT. MLPH regulated EMT to mediate PAAD cell invasive migration through the PI3K-AKT pathway. The results indicated that MLPH is a possible target for blocking PAAD metastasis.

Disease-related protein co-expression networks are associated with the prognosis of resectable node-positive pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a multifactorial disease, the molecular profile of which remains unclear. This study aimed at unveiling the disease-related protein networks associated with different outcomes of resectable, node-positive PDAC cases. We assessed laser-microdissected cancerous cells from PDAC tissues of a poor outcome group (POG; n = 4) and a better outcome group (BOG; n = 4). Noncancerous pancreatic duct tissues (n = 5) were used as the reference. We identified four representative network modules by applying a weighted network correlation analysis to the obtained quantitative PDAC proteome datasets. Two network modules that were significant for POG were associated with the heat shock response to hypoxia-related stress; in the latter, a large involvement of the non-canonical Hedgehog pathway (regulated by GLI1), the internal ribosome entry site-mediated cap-independent translation, the inositol requiring enzyme 1-alpha (IRE1α)/X-box binding protein 1 pathway of the unfolding protein response (UPR), and the aerobic glycolysis was observed. By contrast, the BOG characteristic module was involved in the inactivation of the UPR pathway via the synoviolin 1-dependent proteasomal degradation of IRE1α, the activation of SOX2, and the loss of PALB2 (partner and localizer of BRCA2) function, all potentially suppressing malignant tumor development. Our findings might facilitate future therapeutic strategies for PDAC.

Abstract 6285: MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and an inherently chemoresistant tumor. Dense fibrotic stroma is a major obstacle for drug delivery to the tumor cells in PDAC. Pancreatic stellate cells (PSCs) are the major stromal cells responsible for fibrotic microenvironment. MicroRNA-217 (miR-217) is known mainly as a tumor suppressor. However, the detailed roles of miR-217 on pathophysiology of PDAC remain unclarified. Methods: Expressions levels of miR-217 were measured in surgically resected human PDAC samples. Using Lipofectamine RNAiMAX, miR-217 or negative control miR (NC) were transfected in human pancreatic cancer cells, Panc-1, MIApaca-2, BxPC-3 and murine PAN02 cells, and chemosensitivities to gemcitabine and paclitaxel were investigated by MTS assay. Human hPSC-1 (obtained from Riken cell bank, Japan) was cultured in 20%FBS+DMEM and the changes of phenotypes and expression levels of miR-217 after the stimulation with TGF-β1 was measured by fluorescein microscope and digital PCR, respectively. Results: The expression level of miR-217 was markedly downregulated in PDAC tissues than in noncancerous pancreatic tissues (P<0.001, n=9). Overexpression of miR-217 significantly inhibited the proliferation of Panc-1, MIApaca-2 and BxPC-3 (P<0.001) and migration of Panc-1, MIApaca-2 and PAN02 (P<0.05). Lipofection of miR-217 further reduced the number of PAN02 exposed to 1-5nM gemcitabine (P<0.0001) and 0.01-0.1uM paclitaxel (P=0.0085) which resulted in the reduced the IC-50 as compared with NC. Human PSC-1 stimulated with TGF-β1 (10ng/ml) significantly reduced miR-217 expression compared to nascent cells. The stimulation with TGF-β1 upregulated the expression of cancer associated fibroblast (CAF) markers, α-smooth muscle actin (SMA) and vimentin. However, the expression levels of these markers were significantly reduced by the kipofection of miR-217. Conclusion: MiR-217 affects both cancer and stromal cells in PDAC which may regulate chemosensitivity in PDAC. Restoring miR-217 levels in tumor cells and/or PSCs could be a future therapeutic strategy. Citation Format: Yuki Kaneko, HIdeyuki Ohzawa, Yuki Kimura, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6285.

Clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma: prediction of early recurrence based on genome-wide DNA methylation profiling

PurposeThe present study was conducted to clarify the clinicopathological impacts of DNA methylation alterations on pancreatic ductal adenocarcinoma (PDAC).MethodsGenome-wide DNA methylation screening was performed using the Infinium HumanMethylation450 BeadChip, and DNA methylation quantification was verified using pyrosequencing. We analyzed fresh-frozen tissues from an initial cohort (17 samples of normal control pancreatic tissue [C] from 17 patients without PDAC, and 34 samples of non-cancerous pancreatic tissue [N] and 82 samples of cancerous tissue [T] both obtained from 82 PDAC patients) and formalin-fixed paraffin-embedded T samples from 34 patients in a validation cohort.ResultsThe DNA methylation profiles of N samples tended to differ from those of C samples, and 91,907 probes showed significant differences in DNA methylation levels between C and T samples. Epigenetic clustering of T samples was significantly correlated with a larger tumor diameter and early recurrence (ER), defined as relapse within 6 months after surgery. Three marker CpG sites, applicable to formalin-fixed paraffin-embedded surgically resected materials regardless of their tumor cell content, were identified for prediction of ER. The sensitivity and specificity for detection of patients belonging to the ER group using a panel combining these three marker CpG sites, including a CpG site in the CDK14 gene, were 81.8% and 71.7% and 88.9% and 70.4% in the initial and validation cohorts, respectively.ConclusionThese findings indicate that DNA methylation alterations may have a clinicopathological impact on PDAC. Application of our criteria will ultimately allow prediction of ER after surgery to improve the outcome of PDAC patients.

LACTB mRNA expression is increased in pancreatic adenocarcinoma and high expression indicates a poor prognosis.

This study aimed to find the prognostic value of Beta-lactamase-like (LACTB) in pancreatic adenocarcinoma (PAAD) patients. The mRNA expression of LACTB was upregulated in PAAD and was correlated with vital status (P = 0.0199). The immunoreactive scores of LACTB protein in human PAAD tissues were significantly higher than those in adjacent noncancerous pancreatic tissues. Receiver operating characteristic (ROC) curve assessment showed that LACTB mRNA expression has high diagnostic value in PAAD. Kaplan-Meier curve and Cox analyses suggested that patients with high LACTB mRNA expression have a poor prognosis, indicating that LACTB mRNA is an independent prognostic factor for overall survival [hazard ratio (HR) = 1.72, P = 0.015, 95% confidence interval (CI) = 1.106-2.253] and disease-specific survival (HR = 1.97, P = 0.004, 95% CI = 1.238-3.152) of PAAD patients. Gene set enrichment analysis (GSEA) revealed that hallmark_g2m_checkpoint, hallmark_myc_targets_v1, hallmark_e2f_targets, and kegg_cell_cycle were differentially enriched in phenotypes with high LACTB expression. In addition, CDC20, CDK4, MCM6, MAD2L1, MCM2 and MCM5 were leading genes intersecting in these four pathways, and a positive correlation between mRNA expression and LACTB was observed in most normal and cancer tissues. Finally, elevated LACTB mRNA expression was significantly related to multiple immune marker sets. Our results elucidate that LACTB is involved in the development of cancer, and that high LACTB expression in patients with PAAD can predict a poor prognosis. High LACTB expression was significantly correlated with cell cycle-related genes and multiple immune marker sets.

Membrane expression and significance of TRAIL death receptors DR4 and DR5 in Pancreatic cancer

Introduction: Tumor necrosis factor related-apoptosis-inducing ligand (TRAIL) is a powerful and selective activator of apoptosis in many cancer cells. We aim to investigate the expression and significance of TRAIL death receptor DR4 and DR5 in pancreatic cancer (PC) tissues.
 Method: Twenty-eight histologically verified samples of PC tissue were collected between 2018 and 2019. TRAIL death receptor expression profiles were determined by immunohistochemistry.
 Result: Death receptor DR4 and DR5 were expressed in the PC tissue and the adjacent non-cancerous pancreatic tissues, the expression of DR4 and DR5 in the PC tissue was significantly higher than that of the adjacent non-cancerous pancreatic tissues (p<0.05). Additionally, in both the tissue group, the expression of DR4 was significantly stronger than the DR5 (p<0.05). To assess the relationship between DR4 and DR5 expression, differentiation, and tumor staging of PC, the result reveals that the expression of DR4 and DR5 was significantly higher in stage I tumors than the stage II, III, IV tumors (p<0.05). In contrast, the expression of DR4 and DR5 was decreased with a decrease in the degree of differentiation of tumors. However, the difference was not statistically significant.
 Conclusion: The membrane expression of TRAIL death receptor DR4 and DR5 is greater in PC than in the adjacent non-cancerous pancreatic tissues. Furthermore, increased membrane expression of TRAIL death receptor DR4 and DR5 in stage I PC and well-differentiated PC may predict the prognosis and feasibility of using TRAIL gene therapy as a treatment option for early PC.

GOLPH3 Promotes Cancer Growth by Interacting With STIP1 and Regulating Telomerase Activity in Pancreatic Ductal Adenocarcinoma.

Overexpression of Golgi phosphoprotein 3 (GOLPH3) predicts poor prognosis and is a potential therapeutic target in pancreatic ductal adenocarcinoma (PDAC). However, its role and underlying molecular mechanisms in the progression of PDAC remain unknown. In the present study, using high-throughput bimolecular fluorescence complementation (BiFC) analysis, we identified that stress-inducible protein-1 (STIP1) interacts with GOLPH3 and confirmed the interaction using co-localization and co-immunoprecipitation. The levels of GOLPH3 and STIP1 in PDAC tissues and adjacent non-cancerous pancreatic tissues were determined using immunohistochemistry (IHC) and quantitative real-time reverse transcription PCR. Real-time Quantitative-telomere repeat amplification (Q-TRAP) was applied to detect relative telomerase activity, and cell proliferation was measured when small interfering RNAs targeting GOLPH3 or STIP1 were transfected into PDAC cell lines. BALB/c nude mice were used to assess tumor growth inhibition of BXPC3 cells stably transfected with GOLPH3 short hairpin RNA. In summary, GOLPH3 was found to interact with STIP1 and both proteins were overexpressed and co-localized in PDAC tissues and cell lines. Moreover, suppression of GOLPH3 expression using shRNAs in PANC1 and BXPC3 cells inhibited tumor cell proliferation both in vitro and in vivo. Mechanistically, GOLPH3 interacts with STIP1 to activate telomerase reverse transcriptase (hTERT) and telomerase activity by c-Myc, and then upregulates cell cycle-related signaling proteins, including cyclin D1, to promote tumor cell growth, suggesting that disrupting the interaction between STIP1 and GOLPH3 would be a promising new strategy to treat PDAC.

Deep learning to distinguish pancreatic cancer tissue from non-cancerous pancreatic tissue: a retrospective study with cross-racial external validation

The diagnostic performance of CT for pancreatic cancer is interpreter-dependent, and approximately 40% of tumours smaller than 2 cm evade detection. Convolutional neural networks (CNNs) have shown promise in image analysis, but the networks' potential for pancreatic cancer detection and diagnosis is unclear. We aimed to investigate whether CNN could distinguish individuals with and without pancreatic cancer on CT, compared with radiologist interpretation. In this retrospective, diagnostic study, contrast-enhanced CT images of 370 patients with pancreatic cancer and 320 controls from a Taiwanese centre were manually labelled and randomly divided for training and validation (295 patients with pancreatic cancer and 256 controls) and testing (75 patients with pancreatic cancer and 64 controls; local test set 1). Images were preprocessed into patches, and a CNN was trained to classify patches as cancerous or non-cancerous. Individuals were classified as with or without pancreatic cancer on the basis of the proportion of patches diagnosed as cancerous by the CNN, using a cutoff determined using the training and validation set. The CNN was further tested with another local test set (101 patients with pancreatic cancers and 88 controls; local test set 2) and a US dataset (281 pancreatic cancers and 82 controls). Radiologist reports of pancreatic cancer images in the local test sets were retrieved for comparison. Between Jan 1, 2006, and Dec 31, 2018, we obtained CT images. In local test set 1, CNN-based analysis had a sensitivity of 0·973, specificity of 1·000, and accuracy of 0·986 (area under the curve [AUC] 0·997 (95% CI 0·992-1·000). In local test set 2, CNN-based analysis had a sensitivity of 0·990, specificity of 0·989, and accuracy of 0·989 (AUC 0·999 [0·998-1·000]). In the US test set, CNN-based analysis had a sensitivity of 0·790, specificity of 0·976, and accuracy of 0·832 (AUC 0·920 [0·891-0·948)]. CNN-based analysis achieved higher sensitivity than radiologists did (0·983 vs 0·929, difference 0·054 [95% CI 0·011-0·098]; p=0·014) in the two local test sets combined. CNN missed three (1·7%) of 176 pancreatic cancers (1·1-1·2 cm). Radiologists missed 12 (7%) of 168 pancreatic cancers (1·0-3·3 cm), of which 11 (92%) were correctly classified using CNN. The sensitivity of CNN for tumours smaller than 2 cm was 92·1% in the local test sets and 63·1% in the US test set. CNN could accurately distinguish pancreatic cancer on CT, with acceptable generalisability to images of patients from various races and ethnicities. CNN could supplement radiologist interpretation. Taiwan Ministry of Science and Technology.

Analysis of the methylation of CpG islands in the CDO1, TAC1 and CHFR genes in pancreatic ductal cancer.

No difference in the gene methylation status of tumor-suppression genes between pancreatic cancer tissues and adjacent non-cancer tissues is observed. The present study investigated whether the promoter CpG islands of the cysteine dioxygenase 1 (CDO1), tachykinin precursor 1 (TAC1) and checkpoint with forkhead and ring finger domains (CHFR) genes were methylated in pancreatic cancer and adjacent non-cancerous pancreatic tissue in order to determine if they could be considered as markers for the detection of pancreatic cancer. A total of 38 Formalin-fixed and paraffin-embedded pancreatic adenocarcinoma tissues and their adjacent non-cancerous specimens from patients with pancreatic cancer, as well as 9 non-cancerous pancreatic samples from patients without pancreatic adenocarcinoma were obtained following surgical resection. The hypermethylation of CpG islands was detected using a methylation-specific quantitative PCR. The methylation values were calculated using the ∆Cq method and were expressed as 2−ΔCq. The 2−ΔCq value of the CDO1 promoter from pancreatic adenocarcinoma specimens was significantly higher compared with that of adjacent non-cancerous and tumor-free pancreatic tissues (P<0.0001 and P=0.0008, respectively). The 2−ΔCq value of the TAC1 promoter of pancreatic adenocarcinoma was also significantly higher compared with that of adjacent non-cancerous tissues and tumor-free pancreatic samples (both P<0.0001). However, there was no significant difference in the 2−ΔCq value of the CHFR promoter among the pancreatic cancer, adjacent non-cancer tissue and tumor-free pancreatic samples. Furthermore, 12 out of the 38 pancreatic adenocarcinoma cases (31.6%) presented some methylation in the CHFR promoter. The results from Kaplan-Meier analysis between CHFR promoter methylation values and the clinicopathological characteristics of patients with pancreatic adenocarcinoma demonstrated that CHFR promoter methylation was significantly associated with lymph node metastasis. The methylation values of CDO1 and TAC1 promoters in cancer tissues were higher compared with adjacent tissues. However, whether hypermethylation of CDO1 and TAC1 promoters may serve as a biomarker in the diagnosis of pancreatic adenocarcinoma remains unclear.

Expression of programmed cell death 4 and pok erythroid myeloid oncogenic factor in pancreatic cancer and its relationship with clinicopathological characteristics

Objective To explore the relationship and the biological behaviour of pancreatic cancer between the expression of programmed cell death 4 (PDCD4) and pok erythroid myeloid oncogenic factor (Pokemon). Methods Pancreatic cancer tissues pathologically confirmed From January 2012 to April 2019 on Affiliated hospital of guangdong medical university were collected, Immunohistochemical method were used to assess the expression of PDCD4 and Pokemon in 42 cases of pancreatic cancer tissues and 21 cases of non-cancerous adjacent pancreatic tissues. Results The positive rate of PDCD4 protein expression in colorectal carcinoma tissues 54.76% (23/42) was significantly lower than that in non-cancerous adjacent colorectal tissues 80.95% (17/21) (χ2=4.143, P<0.05), the positive rate of Pokemon protein expression in colorectal carcinoma tissues 57.14% (24/42) was significantly higher than that in non-cancerous adjacent colorectal tissues 14.29% (3/21) (χ2=10.500, P<0.01); The expressions of PDCD4 protein were related with pancreatic cancer to tumor size, TNM stage, lymph node metastasis (Respectively, χ2=5.999, χ2=5.015, 6.313, P<0.05), the expressions of Pokemon protein were related with pancreatic cancer to TNM stage, lymph node metastasis (Respectively, χ2=5.839, 4.582, P<0.05). Conclusion Combined detection of PDCD4 and Pokemon to judge the invasion and metastasis of pancreatic cancer may have guiding significance. Key words: Pancreatic cancer; Immunohistochemical method; Programmed cell death 4; Pok erythroid myeloid oncogenic factor

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.

Different Roles of Sphingosine Kinase 1 and 2 in Pancreatic Cancer Progression

BackgroundPancreatic cancer is a disease with poor prognosis, and development of new treatments is necessary. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays a critical role in progression of many types of cancer. However, little is known about the role of sphingosine kinases in pancreatic cancer. This study investigated the roles of sphingosine kinases in pancreatic cancer progression. Materials and methodsS1P levels in pancreatic cancer and noncancerous pancreatic tissue were measured in 10 patients. We generated PAN02 murine pancreatic cancer cell lines with a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system genes 9 (Cas9)-mediated deletion of SphK1 or SphK2 and assessed cell growth and migration. In an animal model, we assessed the survival of mice injected with PAN02 cells intraperitoneally. ResultsS1P levels in the pancreatic cancer tissue were significantly higher than those in noncancerous tissue. SphK1 knockout (KO) cells showed greater proliferation and migration than wild type (WT) cells, and SphK2 KO cells showed less proliferation and migration than WT cells. Animal experiments showed that the survival of mice injected with SphK1 KO cells was significantly shorter than those injected with WT cells, and the survival of mice injected with SphK2 KO cells was longer than those injected with WT cells. Surprisingly, cytotoxic assay using gemcitabine showed that SphK1 KO cells survived less than WT cells, and SphK2 KO cells survived more than WT cells. ConclusionsS1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cells. Targeting both SphK1 and SphK2 may be a potential strategy for pancreatic cancer treatment.

Abstract 1928: Identification of interleukin 13 receptor alpha 2 (IL-13Ra2) overexpression in LSL-KrasG12D/+, LSL-Try53R172H/+, and PDx-1-Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma as a target for receptor directed anticancer therapy

Abstract Pancreatic ductal adenocarcinoma (PDA) is a uniformly lethal disease because it escapes early diagnosis and at an advanced stage there are no effective therapies available. Previously, we have demonstrated that IL-13R α2 is overexpressed in 71% of human PDA specimens. We have targeted IL-13Rα2 in an orthotopic mouse model of human PDA and shown effectiveness of the receptor targeting agent, IL-13-PE, a recombinant immunotoxin comprised of IL-13 and Pseudomonas exotoxin. To determine the effectiveness of IL-13-PE and develop receptor targeting approaches, we used a LSL-KrasG12D/+, LSL-Try53R172H/+ and PDx-1-Cre (KPC) genetically engineered mouse model that spontaneously develops PDA in C57BL/6J immunocompetent mice and simulates human PDA disease. These spontaneous PDA tumors showed significant overexpression of IL-13R α2 mRNA compared to normal mouse pancreas and surrounding non-cancerous pancreatic tissues as determined by RT-qPCR. By in situ hybridization (ISH) and immunohistochemistry (IHC) analysis, we confirmed these results at mRNA and protein levels of tumor and non-tumor tissue specimens by hybridizing the tumor sections with biotinylated anti-sense riboprobe and immunostaining with anti-murine IL-13R α2 antibody. We established two tumor cell lines from KPC mice PDA, which maintained abundant expression of IL-13R α2 mRNA and protein levels. The overexpressed IL-13R α2 on PDA tumor cells is found biologically active, as it binds to IL-13-PE in a concentration dependent manner killing tumors cells at IC50 of ~25ng/ml (concentration of IL-13-PE that killed 50% of tumor cells). In a clonogenic assay, PDA tumor cells formed colonies, which were again sensitive to IL-13-PE in a concentration dependent manner with IC50 of ~28ng/ml. Based on these data, we have initiated assessment of anti-cancer activity of IL-13-PE against spontaneously developed PDA tumors developed in mice between the ages of 14-16 weeks. These mice were randomized to either receive excipient control i.p. or 50 µg/Kg IL-13-PE every alternate day for two weeks and then evaluated for general health and tumor growth by ultrasound technique. Five week follow up data reveal that mice treated with excipient alone developed larger tumors compared to treated mice and the general health of IL-13-PE treated mice was better compared to excipient-treated mice. These animals are being followed for their overall survival. Our studies indicate that IL-13R α2 is overexpressed in genetically engineered mouse model of PDA suggesting that IL-13R α 2 is a useful drug target not only in orthotopic mouse model but also in spontaneously developed PDA in mice, which may mimic its biological function more closely to human PDA. Citation Format: Akiko Suzuki, Pamela Leland, Bharat H. Joshi, Raj K. Puri. Identification of interleukin 13 receptor alpha 2 (IL-13Ra2) overexpression in LSL-KrasG12D/+, LSL-Try53R172H/+, and PDx-1-Cre (KPC) genetically engineered mouse model of pancreatic ductal adenocarcinoma as a target for receptor directed anticancer therapy [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 1928.

Mechanisms by which CXCR4/CXCL12 cause metastatic behavior in pancreatic cancer.

C-X-C motif chemokine receptor (CXCR) 4/CXCL12 is associated with tumor invasion and metastasis in pancreatic cancer. The present study aimed to investigate the possible mechanisms behind this process by studying the association between the expression of CXCR4 and numerous molecular markers. A total of 60 patients with pancreatic cancer who had been treated with radical surgery between July 2012 and February 2016 were included in the present study. The expression of CXCR4/CXCL12 in primary pancreatic cancer lesions, tissues adjacent to cancerous tissue, non-cancerous pancreatic tissues and in the surrounding lymph nodes was evaluated by immunohistochemistry. Expression levels of four candidate biomarkers [vascular endothelial growth factor-C (VEGF-C), Ki-67, matrix metalloproteinase 2 (MMP-2) and β-catenin] were also evaluated. The correlation between CXCR4 and these four biomarkers was assessed. CXCR4 (CXCL12) expression levels were higher in pancreatic cancer 56.7% (86.7%), paracancerous tissue 50.0% (85.0%) and surrounding lymph nodes 53.3% (80.0%), compared with in normal tissues 18.3% (45.0%). CXCR4 expression was significantly associated with the lymph node metastasis of tumors (P=0.001), pathological type (P=0.037) and tumor-node-metastasis stage (P=0.031). CXCR4 expression exhibited a positive correlation with VEGF-C (r=0.417; P=0.001), Ki-67 (r=0.316; P=0.014), MMP-2 (r=0.284; P=0.028) and β-catenin (r=0.368; P=0.04). Furthermore, logistic regression analysis revealed VEGF-C (β=1.722; P=0.005) and Ki-67 (β=1.196; P=0.047) to be two biomarkers that cause metastasis via CXCR4. CXCR4/CXCL12 is closely associated with tumor grade and lymphatic metastasis. VEGF-C and Ki-67 are two important biomarkers, through which CXCR4 initiates metastatic behavior in pancreatic cancer. Therefore, angiogenesis inhibitors will continue to be effective agents in treating pancreatic cancer.

MiR-92b-3p acts as a tumor suppressor by targeting Gabra3 in pancreatic cancer

BackgroundMicroRNAs (miRNAs) can act as oncogenes or tumor suppressors by controlling cell proliferation, differentiation, metastasis and apoptosis, and miRNA dysregulation is involved in the development of pancreatic cancer (PC). Our previous study demonstrated that Gabra3 plays critical roles in cancer progression. However, whether Gabra3 is regulated by miRNAs in PC remains unknown.MethodsThe expression levels of miR-92b-3p and Gabra3 were measured by quantitative PCR (qPCR), immunoblotting, in situ hybridization (ISH) and immunohistochemistry (IHC). The proliferation rate of PC cells was detected by MTS assay. Wound-healing and transwell assays were used to examine the invasive abilities of PC cells. Dual-luciferase reporter assays were used to determine how miR-92b-3p regulates Gabra3. Xenograft mouse models were used to assess the role of miR-92b-3p in PC tumor formation in vivo.ResultsHere, we provide evidence that miR-92b-3p acted as a tumor suppressor in PC by regulating Gabra3 expression. MiR-92b-3p expression levels were lower in PC tissues than corresponding noncancerous pancreatic (CNP) tissues and were associated with a poor prognosis in PC patients. MiR-92b-3p overexpression suppressed the proliferation and invasion of PC cells in both in vivo and in vitro models. Conversely, miR-92b-3p knockdown induced an aggressive phenotype in PC cells. Mechanistically, miR-92b-3p overexpression suppressed Gabra3 expression, which then led to the inactivation of important oncogenic pathways, including the AKT/mTOR and JNK pathways.ConclusionOur results suggest that miR-92b-3p acted as a tumor suppressor by targeting Gabra3-associated oncogenic pathways; these results provide novel insight into future treatments for PC patients.

MiR-3656 expression enhances the chemosensitivity of pancreatic cancer to gemcitabine through modulation of the RHOF/EMT axis

The highly refractory nature of pancreatic cancer (PC) to chemotherapeutic drugs is one of the key reasons contributing to the poor prognosis of this disease. MicroRNAs (miRNAs) are key regulators of gene expression and have been implicated in a variety of processes from cancer development through to drug resistance. Herein, through miRNA profiling of gemcitabine-resistant (GR) and parental PANC-1 cell lines, we found a consistent reduction of miR-3656 in GR PANC-1 cells. miR-3656 overexpression enhanced the antitumor effect of gemcitabine, whereas silencing of miR-3656 resulted in the opposite effect. By performing mechanistic studies using both in vitro and in vivo models, we found that miR-3656 could target RHOF, a member of the Rho subfamily of small GTPases, and regulate the EMT process. Moreover, enforced EMT progression via TWIST1 overexpression compromised the chemotherapy-enhancing effects of miR-3656. Finally, we found significantly lower levels of miR-3656 and higher levels of RHOF in PC tissues compared with adjacent noncancerous pancreatic tissues, and this was also associated with poor PC patients’ prognosis. Taken together, our results suggest that the miR-3656/RHOF/EMT axis is an important factor involved in regulating GR in PC, and highlights the potential of novel miR-3656-based clinical modalities as a therapeutic approach in PC patients.

LINE-1 methylation level and prognosis in pancreas cancer: pyrosequencing technology and literature review

Global DNA hypomethylation plays an important role in genomic instability and carcinogenesis. The long interspersed nucleotide element-1 (LINE-1) methylation level is a good surrogate marker of the global DNA methylation level. Previously, we demonstrated a strong relationship between LINE-1 hypomethylation and poor prognosis in certain cancers. However, the relationship between the LINE-1 methylation level and the clinical outcome of pancreatic cancer (PC) remains unclear. We used a pyrosequencing assay to measure LINE-1 methylation levels in 126 samples of resected PC and evaluated the prognostic value of the LINE-1 methylation level. LINE-1 methylation levels were significantly lower in PC tissues than in matched noncancerous pancreatic tissues (p=0.039, n=36). The tumoral LINE-1 methylation range was 41.3-92.8 (n=126, mean 77.7, median 78.5, standard deviation 5.7). The LINE-1 methylation level was unrelated to clinical and pathological features. Moreover, LINE-1 hypomethylation was not significantly associated with overall survival, cancer specific survival, or disease-free survival (log-rank p=0.30, p=0.18 and p=0.50, respectively). The LINE-1 methylation level appears not to be associated with poor prognosis in PC. The effect of the LINE-1 methylation level on the survival of PC patients needs to be confirmed in a larger-cohort study.

Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors.

Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis. In particular, macropinocytosis can be a nutrient source for pancreatic cancer cells, but it is not fully understood how the tumor environment influences metabolic phenotypes and whether macropinocytosis supports the maintenance of amino acid levels within pancreatic tumors. Here we utilize miniaturized plasma exchange to deliver labeled albumin to tissues in live mice, and we demonstrate that breakdown of albumin contributes to the supply of free amino acids in pancreatic tumors. We also deliver albumin directly into tumors using an implantable microdevice, which was adapted and modified from ref. 9. Following implantation, we directly observe protein catabolism and macropinocytosis in situ by pancreatic cancer cells, but not by adjacent, non-cancerous pancreatic tissue. In addition, we find that intratumoral inhibition of macropinocytosis decreases amino acid levels. Taken together, these data suggest that pancreatic cancer cells consume extracellular protein, including albumin, and that this consumption serves as an important source of amino acids for pancreatic cancer cells in vivo.

Reduced miRNA-218 expression in pancreatic cancer patients as a predictor of poor prognosis.

microRNA-218 (miR-218) is a vertebrate-specific miRNA that plays a crucial role in tumorigenesis and tumor progression. This study analyzed the miR-218 expression level and clinical significance in pancreatic cancer. One hundred and seven pairs of pancreatic cancer and adjacent normal tissues were analyzed by quantitative reverse-transcriptase polymerase chain reaction. The correlation between miR-218 expression and clinicopathological characters was determined by the two-sample Student t-test. The survival correlations were analyzed by the Kaplan-Meier method and Cox proportional hazards model. The relative expression of miR-218 in pancreatic cancer tissues (2.63 ± 1.59) was significantly lower than that in matched noncancerous pancreatic tissues (6.52 ± 2.50, P < 0.001). The low expression of miR-218 in the pancreatic cancer tissues were strongly correlated with the TNM classification (P = 0.02), distant metastasis (P = 0.001), and tumor differentiation (P = 0.003). The low level of miR-218 expression was significantly correlated with the shorter overall survival time of pancreatic cancer patients (5-year overall survival rate: 7.5 vs 34.9%; log-rank test: P < 0.001). Multivariate analyses confirmed that a low level of miR-218 expression was an independent predictor of poor prognosis in pancreatic cancer patients (Hazard ratio: 7.24; 95% confidence interval: 2.01-18.28; P = 0.007). Our findings suggested a significant downregulation in the expression of miR-218; this might have considerable potential value in the prognosis for pancreatic cancer.