Human Pancreatic Ductal Carcinoma Research Articles

Abstract C023: PTEN/STAT3 pathway in cancer-associated fibroblasts in Pancreatic Cancer

Abstract Pancreatic cancer is a devastating disease with a high mortality rate with a five-year survival rate of 12%. Recent studies have shed light on the crucial role of cancer-associated fibroblasts (CAFs) in shaping the tumor microenvironment. Our group demonstrated PTEN loss occurs in CAFs in ~25% of PDAC patient samples, which leads to increased activation of signaling pathways in CAFs, including the STAT3 pathway. Conditional deletion of STAT3 in CAFs in a dual flpO/cre recombinase PDAC model correlated with increased survival, decreased M2-like macrophages and increased T-cell infiltration. In the present study we aimed to test the hypothesis that direct interactions between PTEN and STAT3 in cancer CAFs contributes to an immuno-suppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). To address this hypothesis, we utilized CAFS isolated from the dual recombinase PDAC model. Proximity ligation assays with these CAFS demonstrated that PTEN-STAT3 are in a complex in the cell cytosol that translocate to the nucleus upon IL6 treatment. This finding was validated by co-immunoprecipitation experiments. ChIP-seq analysis of CAFs with PTEN and pSTAT3/STAT3 antibodies revealed a significant overlap in PTEN and pSTAT3/STAT3 occupation of gene promoters. Integrative analysis of PTEN-STAT3 bound genes with corresponding RNA-seq data revealed that the PTEN/STAT3 complex targets cytokines and ligands that directly affect T-cell function, contributing to an immune suppressive microenvironment. Analysis of human PDAC samples support the relationship between STAT3 activation and T-cell activity. Identifying PTEN/STAT3 targets in CAFs may provide new avenues for overcoming the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Cameron Bumbleburg, Victoria Jordan, Julia E. Lefler, Sudarshana Sharma, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C023.

Investigation of oncolytic potential of vaccine strains of yellow fever and tick-borne encephalitis viruses against glioblastoma and pancreatic carcinoma cell lines

Flaviviruses, possessing natural neurotropicity could be used in glioblastoma therapy using attenuated strains or as a delivery system for antitumor agents in an inactivated form. To investigate the sensitivity of glioblastoma and pancreatic carcinoma cell lines to vaccine strains of yellow fever and tick-borne encephalitis viruses. Cell lines: glioblastoma GL-6, T98G, LN-229, pancreatic carcinoma MIA RaCa-2 and human pancreatic ductal carcinoma PANC-1. Viral strains: 17D yellow fever virus (YF), Sofjin tick-borne encephalitis virus (TBEV). Virus concentration were determined by plaque assay and quantitative PCR. Determination of cell sensitivity to viruses by MTT assay. 17D YF was effective only against pancreatic carcinoma tumor cells MIA Paca-2 and had a limited effect against PANC-1. In glioblastoma cell lines (LN229, GL6, T98G), virus had no oncolytic effect and the viral RNA concentration fell in the culture medium. Sofjin TBEV showed CPE50 against MIA Paca-2 and a very limited cytotoxic effect against PANC-1. However, it had no oncolytic effect against glioblastoma cell lines (LN229, T98G and GL6), although virus reproduction continued in these cultures. For the GL6 glioblastoma cell line, the viral RNA concentration at the level with the infection dose was determined within 13 days, despite medium replacement, while in the case of the LN229 cell line, the virus concentration increased from 1 × 109 to 1 × 1010 copies/ml. Tumor behavior in organism is more complex and is determined by different microenvironmental factors and immune status. In the future, it is advisable to continue studying the antitumor oncolytic and immunomodulatory effects of viral strains 17D YF and Sofjin TBEV using in vivo models.

GTAD: a graph-based approach for cell spatial composition inference from integrated scRNA-seq and ST-seq data.

With the emergence of spatial transcriptome sequencing (ST-seq), research now heavily relies on the joint analysis of ST-seq and single-cell RNA sequencing (scRNA-seq) data to precisely identify cell spatial composition in tissues. However, common methods for combining these datasets often merge data from multiple cells to generate pseudo-ST data, overlooking topological relationships and failing to represent spatial arrangements accurately. We introduce GTAD, a method utilizing the Graph Attention Network for deconvolution of integrated scRNA-seq and ST-seq data. GTAD effectively captures cell spatial relationships and topological structures within tissues using a graph-based approach, enhancing cell-type identification and our understanding of complex tissue cellular landscapes. By integrating scRNA-seq and ST data into a unified graph structure, GTAD outperforms traditional 'pseudo-ST' methods, providing robust and information-rich results. GTAD performs exceptionally well with synthesized spatial data and accurately identifies cell spatial composition in tissues like the mouse cerebral cortex, cerebellum, developing human heart and pancreatic ductal carcinoma. GTAD holds the potential to enhance our understanding of tissue microenvironments and cellular diversity in complex bio-logical systems. The source code is available at https://github.com/zzhjs/GTAD.

Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape.

Necroptosis, pro-inflammatory programmed necrosis, has been reported to exert momentous roles in pancreatic cancer (PC). Herein, the objective of this study is to construct a necroptosis-related prognostic model for detecting pancreatic cancer. In this study, the intersection between necroptosis-related genes and differentially expressed genes (DEGs) of pancreatic ductal adenocarcinoma (PDAC) was obtained based on GeneCards database, GEO database (GSE28735 and GSE15471), and verified using The Cancer Genome Atlas (TCGA). Next, a prognostic model with Cox and LASSO regression analysis, and divided the patients into high-risk and low-risk groups. Subsequently, the Kaplan-Meier (KM) survival curve and the receiver operating characteristic (ROC) curves were generated to assess the predictive ability of overall survival (OS) of PC patients. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to predict the potential biofunction and possible mechanical pathways. The EMTome database and an immune analysis were applied to further explore underlying mechanism. Finally, clinical samples of PDAC patients were utilized to verify the expression of model genes via immunohistochemistry (IHC), and the normal human pancreatic ductal cell line, hTERT-HPNE as well as human pancreatic ductal carcinoma cell lines, PANC-1 and PL45, were used to identify the levels of model genes by Western blot (WB) and immunofluorescence (IF) in vitro. The results showed that 13 necroptosis-related DEGs (NRDEGs) were screened based on GEO database, and finally four of five prognostic genes, including KRT7, KRT19, IGF2BP3, CXCL5, were further identified by TCGA to successfully construct a prognostic model. Univariate and multivariate Cox analysis ultimately confirmed that this prognostic model has independent prognostic significance, KM curve suggested that the OS of low-risk group was longer than high-risk group, and the area under receiver (AUC) of ROC for 1, 3, 5 years was 0.733, 0.749 and 0.667, respectively. A GO analysis illustrated that model genes may participate in cell-cell junction, cadherin binding, cell adhesion molecule binding, and neutrophil migration and chemotaxis, while KEGG showed involvement in PI3K-Akt signaling pathway, ECMreceptor interaction, IL-17 signaling pathway, TNF signaling pathway, etc. Moreover, our results showed KRT7 and KRT19 were closely related to EMT markers, and EMTome database manifested that KRT7 and KRT19 are highly expressed in both primary and metastatic pancreatic cancer, declaring that model genes promoted invasion and metastasis potential through EMT. In addition, four model genes were positively correlated with Th2, which has been reported to take part in promoting immune escape, while model genes except CXCL5 were negatively correlated with TFH cells, indicating that model genes may participate in immunity. Additionally, IHC results showed that model genes were higher expressed in PC tissues than that in adjacent tumor tissues, and WB and IF also suggested that model genes were more highly expressed in PANC-1 and PL45 than in hTERT-HPNE. Tracing of a necroptosis-related prognostic model for pancreatic carcinoma reveals its invasion and metastasis potential through EMT and immunity. The construction of this model and the possible mechanism of necroptosis in PDAC was preliminarily explored to provide reliable new biomarkers for the early diagnosis, treatment, and prognosis for pancreatic cancer patients.

Abstract 4801: Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma

Abstract Hypoxia is a feature of the pancreatic ductal adenocarcinoma (PDA). Although gemcitabine (GEM) is widely used in chemotherapy for PDA, drug resistance restricts its clinical effectiveness. We have found that hypoxia contributes to chemoresistance, however the mechanism is still unclear. The aim of this study is to clarify the mechanism and to develop a new therapeutic strategy. First, we examined the effect of hypoxia to GEM sensitivity, stemness and energy metabolism in three human pancreatic ductal carcinoma cell lines. The inhibition rate by GEM was lower under CoCl2-induced chemical hypoxia than normoxia. Reactive oxygen species (ROS) after GEM was increased under normoxia but not under hypoxia. Flux analysis showed that both oxidative phosphorylation (OXPHOS) and glycolysis were suppressed under hypoxia, indicating a quiescent state. Next, we investigated the relationship between hypoxia and stemness. Hypoxia increased stem cell markers mRNA (C-Myc, Oct-3/4, CD24, CD44, CD133) expression and tumorsphere formation efficiency (TFE). In addition, hypoxia and GEM administration reduced sphere size but increased the number of spheres and TFE. This indicated that hypoxia and GEM suppressed proliferation, however promoted stemness. Second, to examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced OXPHOS and decreased mitochondrial ROS levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. In addition, tetramethylrhodamine-stained mitochondrial membrane potential levels decreased, suggesting mitochondrial impairment. This indicated that long hypoxia and treatment with GEM induced mitochondrial impairment and acquisition of stemness, causing chemoresistance. We further investigated the effect of lauric acid (LAA), a medium-chain fatty acid, on GEM resistance. We have previously reported that LAA causes reprogramming of energy metabolism and induces cell death in cancer cells. Treatment with LAA restored GEM sensitivity in both GEM-resistant cell lines. Flux analysis showed that both OXPHOS and glycolysis were promoted, indicating displacement from quiescent state by LAA. These results suggest that decreased energy production, decreased mitochondrial ROS levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of OXPHOS by LAA could be a tool to overcome GEM resistance. Citation Format: Tadataka Takagi, Hiroki Kuniyasu, Masayuki Sho, Rina Tani, Shiori Mori, Shingo Kishi, Yukiko Nishiguchi, Yudai Hojo. Laurine acid enhances GEM-chemosensitivity targeting hypoxia-induced mitochondrial disfunction and stemness in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4801.

Abstract 3643: PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer

Abstract This study aims to test the hypothesis that direct interactions and auto-regulatory loop between PTEN and STAT3 in cancer-associated fibroblasts (CAFs) contribute to an immunosuppressive tumor microenvironment in pancreatic ductal carcinoma (PDAC). Pancreatic cancer accounts for almost as many deaths as cases because of its poor prognosis. Many studies have led to the paradigm that stromal elements contribute to pancreatic tumor growth. Cancer-associated fibroblasts (CAFs) are a primary cell type in the desmoplastic stroma in PDAC, and targeting them to improve PDAC treatment is thought to be a promising strategy, however; studies have shown both tumor-promoting and tumor-limiting activities associated with PDAC CAFs. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is one of the most well-described tumor suppressor genes and can regulate essential transcription factors in stromal fibroblasts. On the other hand, STAT3 signaling in pancreatic CAFs contributes to the immunosuppressive and fibrotic stroma observed with disease progression. In this study, we used novel, dual recombinase mouse models of PDAC, with Kras and p53 mutations engineered in the pancreatic epithelium (Flp-based) and Stat3 deleted in a subpopulation of CAFS (Cre). CAFs isolated from these mice were used to probe PTEN-STAT3 interaction in vitro using the Proximity Ligation Assay (PLA) and by ChIP-seq using an RNA seq. Mouse and Human pancreatic tumor samples were analyzed and quantified using multiplex-immunofluorescence and multispectral imaging. In the mouse genetic model, we found PTEN downregulated in ~80% of CAFs, consistent with previous work from our group that demonstrated PTEN loss in 25% of PDAC patient samples. At the same time, pY705-STAT3 was activated in CAFs, and deletion of STAT3 in CAFs correlated with increased survival, decreased M2-like macrophages, and increased T-cell infiltration. STAT3 ablation in CAFS resulted in the re-expression of PTEN in CAFS, implying an autoregulatory loop. PLA on CAFs isolated revealed that IL6 treatment resulted in the transfer of a STAT3-PTEN protein complex into the nucleus ChIP-seq on CAFs with PTEN STAT3 antibodies revealed a significant overlap in PTEN and STAT3 occupation of gene promoters. This result is consistent with in silico analysis on the PTEN and STAT3 ChIP-Seq data in human cell lines. ChIP-Seq data is being interacted with RNA data to identify essential target genes. Human PDAC tissue microarrays are stained with pSTAT3, PTEN, CD3, and CD8.Our study suggests an autoregulatory look between PTEN and STAT3 in PDAC CAFs. PTEN may suppress specific STAT3 target genes through direct interactions with chromatin. Alternatively, PTEN/STAT3 complexes may target CAF genes that suppress tumor growth or activate anti-tumor immune responses. These investigations could identify potential targets in CAFs for restoring the anti-tumor immune responses in PDAC. Citation Format: Samaneh Saberi, Sudarshana Sharma, Cameron Bumbleburg, Julia E. Lefler, Michael C. Ostrowski. PTEN/STAT3 pathway in cancer-associated fibroblasts in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3643.

Design, synthesis and biological evaluation of a new series of arylidene indanones as small molecules for targeted therapy of non-small cell lung carcinoma and prostate cancer

In an attempt to identify small molecules for targeted therapy of non-small cell lung carcinoma (NSCLC) and prostate cancer (PCa), new arylidene indanones (1–10) were synthesized via the Claisen-Schmidt condensation of 5,6-methylenedioxy-1-indanone with p-substituted benzaldehyde. Compounds 1–10 were assessed for their cytotoxic effects on human lung adenocarcinoma (A549) and human pancreatic ductal carcinoma (PANC-1) cells as well as human normal lung fibroblast (CCD-19Lu) and human normal pancreatic ductal epithelial (hTERT-HPNE) cells. Among them, compounds 2, 4 and 10 were more effective on A549 and PANC-1 cells than cisplatin. Compounds 1 and 9 also showed more potent cytotoxic activity towards PANC-1 cells than cisplatin. In vitro assays were performed to assess their effects on DNA synthesis, apoptosis, caspase-3, mitochondrial membrane potential, intracellular calcium levels, morphological changes in cancer cells. Furthermore, all compounds were investigated for their inhibitory effects on cathepsin L (CatL) and cathepsin D (CatD). Compounds 2 and 4 exerted potent anti-NSCLC action through caspase-independent apoptosis induced by an increase in intracellular calcium level and correspondingly the disruption of the ΔΨm. These compounds also caused apoptotic morphological alterations in A549 cells. Compound 4 also inhibited both cathepsins but its inhibitory potency on CatL was more significant. Based on in vitro mechanistic assays, compound 4 was identified as a promising anticancer agent for targeted therapy of NSCLC. On the other hand, the marked anti- PCa activity of compound 1 mediated by apoptotic cell death is also noteworthy, but further enzymatic assays are required to elucidate its main mechanism of action.

The Immune Landscape of Human Pancreatic Ductal Carcinoma: Key Players, Clinical Implications, and Challenges.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancer worldwide, with an overall survival (OS) rate, all stages combined, of still <10% at 5 years. For most patients with unresectable or recurrent PDAC, few therapeutic options are available with limited efficacy (median OS ≤ 12 months). Moreover, immune checkpoint inhibitors that have been tested so far in PDAC failed to improve patient survival. This resistance of PDAC to therapies is, in part, related to the dense tumor stroma, which creates a mechanical barrier around the tumor cells and generates high interstitial pressure, preventing appropriate vascularization and thus limiting exposure to treatments. In that PDAC tumoral microenvironment, the inflammatory cell infiltrates are unbalanced toward an immunosuppressive over pro-inflammatory phenotype. Therefore, there is an urgent need to better define the composition of PDAC stroma and key immune players in order to identify new therapeutic targets and strategies.Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and deadliest cancer worldwide with an overall survival rate, all stages combined, of still <10% at 5 years. The poor prognosis is attributed to challenges in early detection, a low opportunity for radical resection, limited response to chemotherapy, radiotherapy, and resistance to immune therapy. Moreover, pancreatic tumoral cells are surrounded by an abundant desmoplastic stroma, which is responsible for creating a mechanical barrier, preventing appropriate vascularization and leading to poor immune cell infiltration. Accumulated evidence suggests that PDAC is impaired with multiple “immune defects”, including a lack of high-quality effector cells (CD4, CD8 T cells, dendritic cells), barriers to effector cell infiltration due to that desmoplastic reaction, and a dominance of immune cells such as regulatory T cells, myeloid-derived suppressor cells, and M2 macrophages, resulting in an immunosuppressive tumor microenvironment (TME). Although recent studies have brought new insights into PDAC immune TME, its understanding remains not fully elucidated. Further studies are required for a better understanding of human PDAC immune TME, which might help to develop potent new therapeutic strategies by correcting these immune defects with the hope to unlock the resistance to (immune) therapy. In this review, we describe the main effector immune cells and immunosuppressive actors involved in human PDAC TME, as well as their implications as potential biomarkers and therapeutic targets.

DPYD, down-regulated by the potentially chemopreventive agent luteolin, interacts with STAT3 in pancreatic cancer

The 5-year survival rate of pancreatic ductal carcinoma (PDAC) patients is <10% despite progress in clinical medicine. Strategies to prevent the development of PDAC are urgently required. The flavonoids Luteolin (Lut) and hesperetin (Hes) may be cancer-chemopreventive, but effects on pancreatic carcinogenesis in vivo have not been studied. Here, the chemopreventive effects of Lut and Hes on pancreatic carcinogenesis are assessed in the BOP-induced hamster PDAC model. Lut but not Hes suppressed proliferation of pancreatic intraepithelial neoplasia (PanIN) and reduced the incidence and multiplicity of PDAC in this model. Lut also inhibited the proliferation of hamster and human pancreatic cancer cells in vitro. Multi-blot and microarray assays revealed decreased phosphorylated STAT3 (pSTAT3) and dihydropyrimidine dehydrogenase (DPYD) on Lut exposure. To explore the relationship between DPYD and STAT3 activity, the former was silenced by RNAi or overexpressed using expression vectors, and the latter was inactivated by small molecule inhibitors or stimulated by IL6 in human PDAC cells. DPYD knock-down decreased, and overexpression increased, pSTAT3 and cell proliferation. DPYD expression was decreased by inactivation of STAT3 and increased by its activation. The frequency of pSTAT3-positive cells and DPYD expression was significantly correlated and was decreased in parallel by Lut in the hamster PDAC model. Finally, immunohistochemical analysis in 73 cases of human PDAC demonstrated that DPYD expression was positively correlated with the Ki-67 labeling index, and high expression was associated with poor prognosis. These results indicate that Lut is a promising chemopreventive agent for PDAC, targeting a novel STAT3-DPYD pathway.

PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis

Although induction of ferroptosis, an iron-dependent form of non-apoptotic cell death, has emerged as an anticancer strategy, the metabolic basis of ferroptotic death remains poorly elucidated. Here, we show that glucose determines the sensitivity of human pancreatic ductal carcinoma cells to ferroptosis induced by pharmacologically inhibiting system xc-. Mechanistically, SLC2A1-mediated glucose uptake promotes glycolysis and, thus, facilitates pyruvate oxidation, fuels the tricyclic acid cycle, and stimulates fatty acid synthesis, which finally facilitates lipid peroxidation-dependent ferroptotic death. Screening of a small interfering RNA (siRNA) library targeting metabolic enzymes leads to identification of pyruvate dehydrogenase kinase 4 (PDK4) as the top gene responsible for ferroptosis resistance. PDK4 inhibits ferroptosis by blocking pyruvate dehydrogenase-dependent pyruvate oxidation. Inhibiting PDK4 enhances the anticancer activity of system xc- inhibitors invitro and in suitable preclinical mouse models (e.g., a high-fat diet diabetes model). These findings reveal metabolic reprogramming as a potential target for overcoming ferroptosis resistance.

High expression of AMAP1, an ARF6 effector, is associated with elevated levels of PD-L1 and fibrosis of pancreatic cancer

BackgroundNot merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, β1-integrins, and E-cadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis.MethodsClinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed.ResultsElevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients.ConclusionsTogether with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK.2ZgVZH5hf3HfReDrMoiYXhVideo abstract

Targeting claudin-4 enhances chemosensitivity of pancreatic ductal carcinomas.

Claudin (CLDN) family comprises of protein that form a tight junction, and is involved in regulating polarity and differentiation of cells. Here, we aimed to investigate the effects of inhibiting CLDN4 in pancreatic ductal carcinomas (PDC). We first examined 91 cases of human PDC by immunohistochemistry and found that CLDN4 expression was correlated with tumor invasion, nodal metastasis, and distant metastasis. Anti‐CLDN4 extracellular domain antibody, previously established by us (4D3), inhibited the proliferation of MIA‐PaCa‐2 PDC cells and increased intracellular 5‐fluorouracil (5‐FU) concentration with lowering transepithelial electrical resistance. Concurrent treatment of 5‐FU and 4D3 resulted in synergistic inhibition of growth of MIA‐PaCa‐2 cells in nude mice. In addition, MIA‐PaCa‐2 cell tumors treated with full‐dose folfirinox (FFX) decreased tumor diameters to 50%; however, 60% of mice were dead from adverse effects. In contrast, half‐dose FFX concomitant with 4D3 treatment decreased tumors equivalent to full‐dose FFX, but without the adverse effects. These findings suggest that targeting CLDN4 might increase the effectiveness and safety of anticancer drug therapy in PDC.

MicroSPECT/CT Imaging of Cell-Line and Patient-Derived EGFR-Positive Tumor Xenografts in Mice with Panitumumab Fab Modified with Hexahistidine Peptides To Enable Labeling with 99mTc(I) Tricarbonyl Complex.

We aimed to investigate the feasibility of conjugating synthetic hexahistidine peptides (His6) peptides to panitumumab Fab (PmFab) to enable labeling with [99mTc(H2O)3(CO)3]+ complex and study these radioimmunoconjugates for imaging EGFR-overexpressing tumor xenografts in mice by microSPECT/CT. Fab were reacted with a 10-fold excess of sulfo-SMCC to introduce maleimide functional groups for reaction with the terminal thiol on peptides [CGYGGHHHHHH] that harbored the His6 motif. Modification of Fab with His6 peptides was assessed by SDS-PAGE/Western blot, and the number of His6 peptides introduced was quantified by a radiometric assay incorporating 123I-labeled peptides into the conjugation reaction. Radiolabeling was achieved by incubation of PmFab-His6 in PBS, pH 7.0, with [99mTc(H2O)3(CO)3]+ in a 1.4 MBq/μg ratio. The complex was prepared by adding [99mTcO4]- to an Isolink kit (Paul Scherrer Institute). Immunoreactivity was assessed in a direct (saturation) binding assay using MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Tumor and normal tissue uptake and imaging properties of 99mTc-PmFab-His6 (70 μg; 35-40 MBq) injected i.v. (tail vein) were compared to irrelevant 99mTc-Fab 3913 in NOD/SCID mice engrafted subcutaneously (s.c.) with EGFR-overexpressing MDA-MB-468 or PANC-1 human pancreatic ductal carcinoma (PDCa) cell-line derived xenografts (CLX) at 4 and 24 h post injection (p.i.). In addition, tumor imaging studies were performed with 99mTc-PmFab-His6 in mice with patient-derived tumor xenografts (PDX) of TNBC, PDCa, and head and neck squamous cell carcinoma (HNSCC). Biodistribution studies in nontumor bearing Balb/c mice were performed to project the radiation absorbed doses for imaging studies in humans with 99mTc-PmFab-His6. PmFab was derivatized with 0.80 ± 0.03 His6 peptides. Western blot and SDS-PAGE confirmed the presence of His6 peptides. 99mTc-PmFab-His6 was labeled to high radiochemical purity (≥95%), and the Kd for binding to EGFR on MDA-MB-468 cells was 5.5 ± 0.4 × 10-8 mol/L. Tumor uptake of 99mTc-PmFab-His6 at 24 h p.i. was significantly (P < 0.05) higher than irrelevant 99mTc-Fab 3913 in mice with MDA-MB-468 tumors (14.9 ± 3.1%ID/g vs 3.0 ± 0.9%ID/g) and in mice with PANC-1 tumors (5.6 ± 0.6 vs 0.5 ± 0.1%ID/g). In mice implanted orthotopically in the pancreas with the same PDCa PDX, tumor uptake at 24 h p.i. was 4.2 ± 0.2%ID/g. Locoregional metastases of these PDCa tumors in the peritoneum exhibited slightly and significantly lower uptake than the primary tumors (3.1 ± 0.3 vs 4.2 ± 0.3%ID/g; P = 0.02). In mice implanted with different TNBC or HNSCC PDX, tumor uptake at 24 h p.i. was variable and ranged from 3.7 to 11.4%ID/g and 3.8-14.5%ID/g, respectively. MicroSPECT/CT visualized all CLX and PDX tumor xenografts at 4 and 24 h p.i. Dosimetry estimates revealed that in humans, the whole body dose from administration of 740-1110 MBq of 99mTc-PmFab-His6 would be 2-3 mSv, which is less than for a 99mTc-medronate bone scan (4 mSv).

Evaluation of a biomarker for the diagnosis of pancreas cancer using an animal model.

Many approaches have been taken to identify new biomarkers of pancreatic ductal carcinoma (PDC). Since animal models can be sampled under controlled conditions, better standardization is possible compared with heterogeneous human studies. Transgenic rats with conditional activation of oncogenic RAS in pancreatic tissue develop PDC that closely resembles the biological and histopathological features of human PDC. Using this model, we evaluated the usefulness of leucine-rich α2-glycoprotein-1 (LRG-1) as a serum marker. In this study, we found that LRG-1 was overexpressed in rat PDC compared with normal pancreas tissue of the control rats. Serum levels of LRG-1 were also significantly higher in rats bearing PDC than in controls. Importantly, chronic pancreatitis in male Wistar Bonn/Kobori rats, which is a widely accepted as a model of chronic pancreatitis, did not cause serum levels of LRG-1 to become elevated. These results strongly support serum LRG-1 as a candidate biomarker for noninvasive diagnosis of PDC. Our models of pancreas cancer provide a useful strategy for evaluation of candidate markers applicable to human cancer.

Porphyrins to restrict progression of pancreatic cancer by stabilizing KRAS G-quadruplex: In silico, in vitro and in vivo validation of anticancer strategy

KRAS, a frequently mutated G-quadruplex forming proto-oncogene is responsible for almost every type of cancer which can form a parallel G-quadruplex structure in the promoter region. G-quadruplex structure is one of the most important drug targets for modern cancer therapy for their unique structure and specificity. Here, we have screened several synthetic porphyrin-based compounds as potential KRAS G-quadruplex stabilizing ligands, using molecular modeling and docking studies. Two novel porphyrins: Porphyrin-1(Cobalt containing) and Porphyrin-2 (Palladium containing) evidenced high affinity towards KRAS-promoter/G-quadruplex. As KRAS mutation is prevalent in pancreatic cancer, the efficacy of these ligands against human pancreatic ductal carcinoma cell line PANC-1 and MiaPaCa2 were examined. Both the Porphyrins exhibited significant cytotoxicity and block metastasis by inhibiting Epithelial to messenchymal transition. In vivo studies confirmed both porphyrin compounds to be effective against EAC tumors along with significantly low toxicity against normal Swiss albino mice. The expression of KRAS gene in porphyrin-treated PANC-1, MiaPaCa2 and tumor-derived EAC cells were drastically reduced at both protein and RNA levels. Thus interaction of porphyrin-based ligands with G-quadruplex DNA at the promoter region of KRAS, might be utilized as a target for anticancer therapeutic strategy.

Identification of the S100 fused-type protein hornerin as a regulator of tumor vascularity

Sustained angiogenesis is essential for the development of solid tumors and metastatic disease. Disruption of signaling pathways that govern tumor vascularity provide a potential avenue to thwart cancer progression. Through phage display-based functional proteomics, immunohistochemical analysis of human pancreatic ductal carcinoma (PDAC) specimens, and in vitro validation, we reveal that hornerin, an S100 fused-type protein, is highly expressed on pancreatic tumor endothelium in a vascular endothelial growth factor (VEGF)-independent manner. Murine-specific hornerin knockdown in PDAC xenografts results in tumor vessels with decreased radii and tortuosity. Hornerin knockdown tumors have significantly reduced leakiness, increased oxygenation, and greater apoptosis. Additionally, these tumors show a significant reduction in growth, a response that is further heightened when therapeutic inhibition of VEGF receptor 2 (VEGFR2) is utilized in combination with hornerin knockdown. These results indicate that hornerin is highly expressed in pancreatic tumor endothelium and alters tumor vessel parameters through a VEGF-independent mechanism.

Abstract A50: Genotype tunes PDAC tension to induce matricellular-fibrosis and tumor aggression

Abstract Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality. Nevertheless, anti-stromal therapies for pancreatic cancer patients have had mixed results, suggesting there are multifaceted, anti and pro-tumorigenic roles of fibrosis in tumor pathogenesis. We found that human PDACs lacking epithelial TGFβ activity have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis that associates with high epithelial tension and shorter patient survival. Using several Kras-driven mouse models, we found that both the loss of TGFβ signaling and elevated β1 integrin mechanosignaling engage a positive feedback loop whereby Stat3 signaling modulates pancreatic cancer malignancy by increasing matricellular fibrosis and tissue tension. By contrast, epithelial Stat3 ablation attenuated pancreatic malignancy by reducing the stromal stiffening and epithelial contractility induced by loss of TGFβ signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial actomyosin tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 as a key driver of the phenotype. These data illustrate how tumor genotype can directly tune tissue mechanics and support the development of genotype-driven, anti-stromal therapies targeting PDAC. Citation Format: Hanane Laklai, Yekaterina Miroshnikova, Michael Pickup, Eric Collisson, Kim Grace, Alex Barrett, Ryan Hill, Johnathon Lakins, David Schlaepfer, Janna Mouw, Valerie LeBleu, Sergey Novitskiy, Julie Johansen, Valeria Poli, Rahgu Kalluri, Laura Wood, Matthias Hebrok, Kirk Hansen, Harold Moses, Valerie Weaver.{Authors}. Genotype tunes PDAC tension to induce matricellular-fibrosis and tumor aggression. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A50.

Corrigendum: All trans-retinoic acid analogs promote cancer cell apoptosis through non-genomic Crabp1 mediating ERK1/2 phosphorylation.

Scientific Reports 6: Article number: 22396; published online: 03 March 2016; updated: 20 July 2016 This Article contains errors. In the last paragraph of the Results section, “Given the ability of atRA and compounds 3 and 4 to induce apoptosis in animal cancer cell line context, we surveyed severalhuman cell lines and found that CRABP1 expression was lost in human ovarian cancer cell line A2780 and human pancreatic ductal carcinoma cell line KPC”.

Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression.

Fibrosis compromises pancreatic ductal carcinoma (PDAC) treatment and contributes to patient mortality yet anti-stromal therapies are controversial. We found that human PDACs with impaired epithelial transforming growth factor β (TGF-β) signaling have elevated epithelial Stat3 activity and develop a stiffer, matricellular-enriched fibrosis associated with high epithelial tension and shorter patient survival. In several Kras-driven mouse models, both the loss of TGF-β signaling and elevated β1-integrin mechanosignaling engaged a positive feedback loop whereby Stat3 signaling promotes tumor progression by increasing matricellular fibrosis and tissue tension. In contrast, epithelial Stat3 ablation attenuated tumor progression by reducing the stromal stiffening and epithelial contractility induced by loss of TGF-β signaling. In PDAC patient biopsies, higher matricellular protein and activated Stat3 associated with SMAD4 mutation and shorter survival. The findings implicate epithelial tension and matricellular fibrosis in the aggressiveness of SMAD4 mutant pancreatic tumors, and highlight Stat3 and mechanics as key drivers of this phenotype.

The Metastatic Potential and Chemoresistance of Human Pancreatic Cancer Stem Cells.

Cancer stem cells (CSCs) typically have the capacity to evade chemotherapy and may be the principal source of metastases. CSCs for human pancreatic ductal carcinoma (PDAC) have been identified, but neither the metastatic potential nor the chemoresistance of these cells has been adequately evaluated. We have addressed these issues by examining side-population (SP) cells isolated from the Panc-1 and BxPC3 lines of human PDAC cells, the oncogenotypes of which differ. SP cells could be isolated from monolayers of Panc-1, but only from spheroids of BxPC3. Using orthotopic xenografts into the severely immunocompromised NSG mouse, we found that SP cells isolated from both cell lines produced tumors that were highly metastatic, in contrast to previous experience with PDAC cell lines. SP cells derived from both cell lines expressed the ABCG2 transporter, which was demonstrably responsible for the SP phenotype. SP cells gave rise to non-SP (NSP) cells in vitro and in vivo, a transition that was apparently due to posttranslational inhibition of the ABCG2 transporter. Twenty-two other lines of PDAC cells also expressed ABCG2. The sensitivity of PDAC SP cells to the vinca alkaloid vincristine could be greatly increased by verapamil, a general inhibitor of transporters. In contrast, verapamil had no effect on the killing of PDAC cells by gemcitabine, the current first-line therapeutic for PDAC. We conclude that the isolation of SP cells can be a convenient and effective tool for the study of PDAC CSCs; that CSCs may be the principal progenitors of metastasis by human PDAC; that the ABCG2 transporter is responsible for the SP phenotype in human PDAC cells, and may be a ubiquitous source of drug-resistance in PDAC, but does not confer resistance to gemcitabine; and that inhibition of ABCG2 might offer a useful adjunct in a therapeutic attack on the CSCs of PDAC.