Pancreatic Ductal Adenocarcinoma Metastasis Research Articles

FBXO32 Stimulates Protein Synthesis to Drive Pancreatic Cancer Progression and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related death worldwide, primarily due to its rapid progression. The current treatment options for PDAC are limited, and a better understanding of the underlying mechanisms responsible for PDAC progression is required to identify improved therapeutic strategies. Here, we identified FBXO32 as an oncogenic driver in PDAC. FBXO32 was aberrantly upregulated in PDAC, and high FBXO32 expression was significantly associated with an unfavorable prognosis in PDAC patients. FRG1 deficiency promoted FBXO32 upregulation in PDAC. FBXO32 promoted cell migration and invasion in vitro and tumor growth and metastasis in vivo. Mechanistically, FBXO32 directly interacted with eEF1A1 and promoted its polyubiquitination at the K273 site, leading to enhanced activity of eEF1A1 and increased protein synthesis in PDAC cells. Moreover, FBXO32-catalyzed eEF1A1 ubiquitination boosted the translation of ITGB5 mRNA and activated FAK signaling, thereby facilitating focal adhesion assembly and driving PDAC progression. Importantly, interfering with the FBXO32-eEF1A1 axis or pharmaceutical inhibition of FAK by defactinib, an FDA-approved FAK inhibitor, substantially inhibited PDAC growth and metastasis driven by aberrantly activated FBXO32-eEF1A1 signaling. Overall, this study uncovers a mechanism by which PDAC cells rely on FBXO32-mediated eEF1A1 activation to drive progression and metastasis. FBXO32 may serve as a promising biomarker for selecting eligible PDAC patients for treatment with defactinib.

The scRNA-sequencing landscape of pancreatic ductal adenocarcinoma revealed distinct cell populations associated with tumor initiation and progression

Pancreatic ductal adenocarcinoma (PDAC) stands as a formidable malignancy characterized by its profound lethality. The comprehensive analysis of the transcriptional landscape holds immense significance in understanding PDAC development and exploring novel treatment strategies. However, due to the firm consistency of pancreatic cancer samples, the dissociation of single cells and subsequent sequencing can be challenging. Here, we performed single-cell RNA sequencing (scRNA-seq) on 8 PDAC patients with different lymph node metastasis status. We first identified the crucial role of MMP1 in the transition from normal pancreatic cells to cancer cells. The knockdown of MMP1 in pancreatic cancer cell lines decreased the expression of ductal markers such as SOX9 while the overexpression of MMP1 in hTERT-HPNE increased the expression of ductal markers, suggesting its function of maintaining ductal identity. Secondly, we found a S100A2+ tumor subset which fueled lymph node metastasis in PDAC. The knockdown of S100A2 significantly reduced the motility of pancreatic cancer cell lines in both wound healing and transwell migration assays. While overexpression of S100A2 led to increased migratory capability. Moreover, overexpression of S100A2 in KPC1199, a mouse pancreatic cancer cell line, caused a larger tumor burden in a hemi-spleen injection model of liver metastasis. In addition, epithelial-mesenchymal transition-related genes were decreased by S100A2 knockdown revealed by bulk RNA sequencing. We also identified several pivotal contributors to the pro-tumor microenvironment, notably OMD+ fibroblast and CCL2+ macrophage. As a result, our study provides valuable insights for early detection of PDAC and promising therapeutic targets for combatting lymph node metastasis.

Investigating the influence of taurochenodeoxycholic acid (TCDCA) on pancreatic cancer cell behavior: An RNA Sequencing Approach

Pancreatic cancer (PC) poses a substantial global health challenge, ranking as the fourth leading cause of cancer-related deaths due to its high mortality rate. Late-stage diagnoses are common due to the absence of specific symptoms. Pancreatic ductal adenocarcinoma (PDAC) accounts for the majority of PC cases. Recent research has suggested a potential link between elevated serum levels of bile acids (BAs) and tumorigenesis of PDAC. This study aims to understand how taurochenodeoxycholic acid (TCDCA), a secondary BA, influences PDAC using RNA sequencing techniques on the Capan-1 cell line. We identified 2,950 differentially expressed genes (DEGs) following TCDCA treatment, with 1,597 upregulated and 1,353 downregulated genes. These DEGs were associated with critical PDAC pathways, including coagulation, angiogenesis, cell migration, and signaling regulation.Furthermore, we reviewed relevant literature highlighting genes like DKK-1, KRT80, UPLA, and SerpinB2, known for their roles in PDAC tumorigenesis and metastasis. Our study sheds light on the complex relationship between BAs and PDAC, offering insights into potential diagnostic markers and therapeutic targets. Further research is needed to unravel these findings' precise mechanisms and clinical implications, potentially improving PDAC diagnosis and treatment.

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

FAM53B promotes pancreatic ductal adenocarcinoma metastasis by regulating macrophage M2 polarization.

Our study investigated the role of FAM53B in regulating macrophage M2 polarization and its potential mechanisms in promoting pancreatic ductal adenocarcinoma (PDAC) metastasis. To further investigate the role of FAM53B in regulating macrophage M2 polarization and its potential mechanism in promoting PDAC metastasis. Our goal is to determine how FAM53B affects macrophage M2 polarization and to define its underlying mechanism in PDAC metastasis. Cell culture and various experiments, including protein analysis, immunohistochemistry, and animal model experiments, were conducted. We compared FAM53B expression between PDAC tissues and healthy tissues and assessed the correlation of FAM53B expression with clinical features. Our study analyzed the role of FAM53B in macrophage M2 polarization in vitro by examining the expression of relevant markers. Finally, we used a murine model to study the role of FAM53B in PDAC metastasis and analyzed the potential underlying mechanisms. Our research showed that there was a significant increase in FAM53B levels in PDAC tissues, which was linked to adverse tumor features. Experimental findings indicated that FAM53B can enhance macrophage M2 polarization, leading to increased anti-inflammatory factor release. The results from the mouse model further supported the role of FAM53B in PDAC metastasis, as blocking FAM53B prevented tumor cell invasion and metastasis. FAM53B promotes PDAC metastasis by regulating macrophage M2 polarization. This discovery could lead to the development of new strategies for treating PDAC. For example, interfering with the FAM53B signaling pathway may prevent cancer spread. Our research findings also provide important information for expanding our understanding of PDAC pathogenesis.

Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

The nanobody targeting PD-L1 and CXCR4 counteracts pancreatic stellate cell-mediated tumour progression by disrupting tumour microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy worldwide owing to its complex tumour microenvironment and dense physical barriers. Stromal-derived factor-1 (SDF-1), which is abundantly secreted by tumour stromal cells, plays a pivotal role in promoting PDAC growth and metastasis. In this study, we investigated the impact and molecular mechanisms of the anti-PD-L1&CXCR4 bispecific nanobody on the TME and their consequent interference with PDAC progression. We found that blocking the SDF-1/CXCR4 signalling pathway delayed the epithelial-mesenchymal transition in pancreatic cancer cells. Anti-PD-L1&CXCR4 bispecific nanobody effectively suppress the secretion of SDF-1 by pancreatic stellate cells and downregulate the expression of smooth muscle actin alpha(α-SMA), thereby preventing the activation of cancer-associated fibroblasts by downregulating the PI3K/AKT signaling pathway. This improves the pancreatic tumour microenvironment, favouring the infiltration of T cells into the tumour tissue. In conclusion, our results suggest that the anti-PD-L1&CXCR4 bispecific nanobody exerts an antitumor immune response by changing the pancreatic tumour microenvironment. Hence, the anti-PD-L1&CXCR4 bispecific nanobody is a potential candidate for pancreatic cancer treatment.

NNT-AS1 in CAFs-derived exosomes promotes progression and glucose metabolism through miR-889-3p/HIF-1α in pancreatic adenocarcinoma

It is metabolic and signaling crosstalk between stromal cells and tumors in the tumor microenvironment, which influences several aspects of tumor formation and drug resistance, including metabolic reprogramming. Despite considerable findings linking lncRNAs in HIF-1-related regulatory networks to cancer cell, little emphasis has been given to the role in communication between cancer-associated fibroblasts (CAFs) and tumor cells. Previously, we observed that NNT-AS1 was substantially expressed in CAFs cells and CAFs exosomes, and subsequently investigated the influence of CAFs exosomal NNT-AS1 on glucose metabolism, proliferation, and metastasis of pancreatic ductal adenocarcinoma (PDAC) cells. Transmission electron microscopy was used to examine exosomes secreted by PDAC patient-derived CAFs. qRT-PCR was used to evaluate the expression of NNT-AS1, miR-889-3p, and HIF-1. The role of CAFs-derived exosomal NNT-AS1 in PDAC cell progression and metabolism have been identified. Dual luciferase reporter assays examined the binding between NNT-AS1, miR-889-3p, and HIF-1. After PDAC cells co-culture exosomes secreted by CAFs, we found that they alter glucose metabolism, proliferation, and metastasis. In PDAC cells, CAF-derived exosomal lncRNA NNT-AS1 acted as a molecular sponge for miR-889-3p. Furthermore, HIF-1 could be targeted by miR-889-3p and was controlled by NNT-AS1. This study explores the mechanism by which NNT-AS1 influences the interaction of CAFs on glycolytic remodeling, proliferation, and metastasis of tumor cells through regulating miR-889-3p/HIF-1α, which also helps discover new clinical treatment targets for PDAC.

Abstract 3050: Evaluating the role of ∆Np63α in driving growth and metastasis of pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the US and is rising in incidence. PDAC tumors may be either classical or basal in transcriptomic subtype, and PDAC patients with basal tumors have worse prognosis and may have increased resistance to chemotherapy. Previous studies have shown that ∆Np63α drives basal program expression. The goal of this project is to investigate whether ∆Np63α expression is sufficient to increase pancreatic cancer cell growth, metastasis and chemoresistance and whether these cells can outcompete ∆Np63α non-expressers in mixed tumors. Non-∆Np63α expressing human PDAC cell lines AsPC1 and HPAFII were stably transduced to express either ∆Np63α or mCherry (control). For both AsPC1 and HPAFII, ∆Np63α-expressing and control cells had similar proliferation rates and susceptibility to chemotherapy agents oxaliplatin and SN38 when grown in culture. When control or ∆Np63α-expressing AsPC1 cells were implanted orthotopically into the pancreas of athymic nude mice, there was no significant difference in primary tumor mass and pathological analysis of lungs and livers did not reveal any significant difference in metastatic burden. Bulk RNA sequencing of primary tumors confirmed that tumors in mice inoculated with AsPC1-∆Np63α had significantly upregulated expression of genes associated with the basal subtype. Collectively, these data suggest that while ∆Np63α drives the basal program in PDAC, its expression is insufficient to cause resistance to these chemotherapies or a growth or metastatic advantage in an immunodeficient setting. Further work is needed to elucidate the mechanism by which ∆Np63α contributes to more aggressive PDAC tumors. Citation Format: Daniel H. Ryan, Chin-Hsien (Emily) Tai, Xianyu Zhang, Mayrel Palestino Dominguez, Christine Alewine. Evaluating the role of ∆Np63α in driving growth and metastasis of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3050.

Abstract 4130: Targeting epigenetically-driven inflammation for PDAC metastasis prevention in high-risk individuals

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer with a dire prognosis, in large part due to the late stage at which it is diagnosed in most patients. High-risk individuals (HRIs) identified through family history or germline genetic testing are candidates for imaging-based screening, yet even with intensive surveillance, metastasis often occurs between scans. Our study aims to address this critical clinical challenge by investigating the epigenetic determinants of PDAC metastasis and developing novel strategies for metastasis prevention in HRIs. Methods: Innovative transplantation models of PDAC liver and peritoneal metastasis incorporating DNA barcodes for subclonal tracking were developed to facilitate the identification of subclones with high or low metastatic potential. An in-depth epigenomic characterization and comparison of these subclones was performed utilizing assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-seq. Results: Metastasis-high subclones demonstrate widespread chromatin accessibility differences relative to their metastasis-low peers. These differences are linked to expression changes for genes involved in motility, differentiation, survival, and particularly inflammation. Top epigenetically activated genes in metastasis-high subclones include the pro-inflammatory cytokines IL-1α and IL-23α and the pro-inflammatory cytokine receptor IL-18R1. Discussion: These findings highlight the role of epigenetic changes in PDAC metastasis, especially activation of inflammatory pathways resulting from altered chromatin accessibility. These insights provide a foundation for exploring anti-inflammatory agents for PDAC metastasis prevention. Targeting inflammation, alongside imaging-based screening, could significantly improve outcomes for HRIs. Citation Format: Jesse Handler, Reza Kalhor. Targeting epigenetically-driven inflammation for PDAC metastasis prevention in high-risk individuals [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4130.

Abstract 5358: The influence of extracellular vesicles on immune cells in PDAC

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) is bad. The tumor microenvironment consists of an abundance of immunosuppressive immune cells and anti-inflammatory cytokines that contribute to local and systemic immunosuppression in PDAC. Tumor cells can influence the tumor microenvironment and, with that, the present immune cells through the secretion of cytokines or chemokines or the secretion of extracellular vesicles (EVs). EVs are a media of communication among the tumor cells themselves, but also with other cells of the body, with the goal to promote tumor expansion and metastasis and decrease any anti-tumor-directed forces of the immune system. Our lab has shown that those EVs play a significant role in tumor growth and metastasis in PDAC. We have found MUC1 to be an important marker for tumor-derived EVs (tdEVs), associated with their activity and influence on cell proliferation and tumor growth in vitro and in vivo. Determining which immune cell populations are most targeted and impacted by tdEVs is essential to improving our understanding of the role of EVs in PDAC. Methods: TdEVs were isolated from murine pancreatic cancer cell lines through differential ultracentrifugation. EVs were then labeled with a membrane dye, which allows tracking of the EVs in the mouse. We designed an in vivo study to investigate the effects that tdEVs have on different immune cell populations, as well as tumor growth and metastasis. This study has two distinct phases: a 3-week “Pre-Education Phase”, with injection of either MUC1+ or MUC1KO EVs, is followed by orthotopic implantation of tumor cells in the pancreas. Data: We hypothesized that tdEVs impact effector immune cells in PDAC, leading to phenotypic, transcriptional, and functional changes that result in a decreased anti-tumor immune response. The highest uptake of EVs was found in macrophages and dendritic cells (DCs). Furthermore, most immune cells preferably take up MUC1+ as compared to MUC1KO EVs. After three weeks of EV education, RNA sequencing of the liver showed differential gene expression in a variety of genes related to processes associated with B cells. Treatment with MUC1+ EVs downregulated B cell-related processes in the liver, B cell differentiation, proliferation and activity are reduced. Several genes coding for immunoglobulin parts were affected as well. Immune cell profiling of the blood and spleen showed that MUC1+ EVs reduce NK and macrophage numbers in the spleen as compared to MUC1KO EV treatment after three weeks. Additionally, EV-treatment increased CD8+ T cell numbers in the blood but decreased CD8+ T cells in the spleen. Conclusion: We have shown that tdEVs induce phenotypic and transcriptional changes in immune cell populations and alter immune cell frequencies in vivo. Ongoing research in our lab involves analysis of phenotypic and transcriptional changes after tumor implantation as well as functional differences in EV-treated immune cells in vitro and in vivo. Citation Format: Clara S. Mundry, Ying Huang, Paul M. Grandgenett, Michael A. Hollingsworth. The influence of extracellular vesicles on immune cells in PDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5358.

Abstract 1597: CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine

Abstract Purpose: Cancer-associated fibroblasts (CAFs) are highly heterogeneous in pancreatic ductal adenocarcinoma (PDAC). We previously reported that CAFs can acquire an endothelial phenotype under stress. Thus, we hypothesized that there are CAFs highly expressing endothelial markers and substantiated a novel subtype of CAFs promoting tumor progression. Experimental design: We performed single-cell RNA sequencing to identify the existence of CD144+CAFs. The prospective and retrospective analysis were combined to figure out the correlation between CD144+CAFs proportion and clinical outcome. The proliferation and invasion assay of cell lines, organoids and pancreatic cancer xenograft model were used to detect the effect of CD144+CAFs on tumor progression. Cytokine array assay, RNA-sequencing, IP-mass spectrum, Ch-IP and luciferase analysis were conducted to elucidate the underlying mechanism. siRNA delivery nanosystem was designed and administrated to precisely target CD144+CAFs in vivo. Results: CD144+CAFs were present in tumor microenvironment of PDAC and breast cancer and patients with a higher CD144+CAFs proportion have worse prognosis in PDAC. CD144+CAFs can promote the metastasis of PDAC through enhancing epithelial-mesenchymal transition. CD144-β-catenin-STAT3 signaling axis was activated and downstream inflammatory secreted cytokines were transcriptionally upregulated to maintain the functions and phenotype of CD144+CAFs. A novel CAF-targeting siRNA delivery nanosystem, loading FAPα and siCDH5, was designed and administrated to precisely target CD144+CAFs, which substantially inhibited the protumoral roles in vivo. Conclusion: CD144+CAFs can promote the growth and metastasis of PDAC by activating CD144-β-catenin-STAT3 signaling axis and stimulating inflammatory secretion. CAF-targeting siRNA delivery nanosystem can inhibit tumor progression by precisely targeting CD144+CAFs. Citation Format: Xugang Sun, Jun Yu, Tiansuo Zhao, Jihui Hao. CD144+cancer-associated fibroblasts drive the malignancy of pancreatic cancer via stimulating inflammatory paracrine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1597.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1392.

Abstract 4397: Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression

Abstract Most cases of Pancreatic Ductal Adenocarcinoma (PDAC) are diagnosed after metastasis has occurred, making it crucial to find effective treatments for late-stage disease. Previously, our group found widespread epigenetic reprogramming during PDAC metastasis in the absence of additional driver mutations. These cells develop dependence on glucose metabolism through the oxidative pentose phosphate pathway (oxPPP). Inhibition of oxPPP with 6-aminonicotinamide (6AN) reverses the chromatin changes and restores cells to a less malignant state (McDonald et al. Nat Gen, 2017). To identify a clinically actionable mechanism for restoration, we hypothesized that oxPPP dependence leads to downstream metabolic changes that mediate chromatin state. Using untargeted metabolomic analysis, we found that alpha-ketoglutarate (AKG), a cofactor of lysine and histone demethylases, was downregulated by oxPPP inhibition (p=0.033) in a patient-derived xenograft lung metastasis cell line, A13Lg. Treatment of cells with 6AN and a cell permeable precursor of AKG largely reversed the chromatin changes seen on a western blot. Given that AKG is mainly produced by glutamine metabolism, we inhibited glutaminolysis with glutamine deprivation (Gln(-)) or the glutamine antagonist JHU083. Both treatments decreased AKG (Gln(-) p=0.002, JHU083 p=0.038) and prevented cell growth (Gln(-) p<0.0001, JHU083 p=0.0002). Glutamine-deprived cells showed decreased ability to form colonies (p=0.004), and JHU083 treated cells showed impaired migration in a Boyden chamber assay (p=0.003). Western blots showed a decrease in H3K27 acetylation, a marker of euchromatin, and an increase in H3K9 and H3K27 methylation, markers of heterochromatin, in response to both treatments. Further, homogenized time-resolved fluorescence (HTRF) assays on glutamine-deprived cells showed that H3K9 di- (p=0.0016,) and tri- (p=0.0003) methylation was increased. Adding cell permeable AKG to media without glutamine largely negated both the phenotypic and epigenetic changes. Using CUT&RUN against H3K27Ac, we found that acetylation was depleted at promoters involved in tumor cell proliferation, invasion, and migration. Bulk RNA-sequencing also showed decreased expression of genes involved in these pathways. Epigenetic and phenotypic changes were not as apparent after treatment with CB839, a glutaminase 1 inhibitor, suggesting that broad inhibition of glutamine metabolism is necessary for chromatin restoration. Together, these data suggest that decreasing AKG in PDAC distant metastasis cells, through glutamine deprivation or JHU083, leads to chromatin changes that specify less malignant phenotypes, providing a rationale for targeting glutamine metabolism as a therapy for late-stage PDAC. *APF and MM are co-corresponding authors. Citation Format: Kenna Sherman, Jiaqi Cheng, Weiqiang Zhou, Hongkai Ji, Masahiro Maeda, Andrew P. Feinberg. Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4397.

Abstract 576: The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer

Abstract We have previously disclosed (Ahmed et al, ACS Med Chem Lett, 2020) a novel small molecule G-quadruplex binding compound, QN-302, that is highly potent in pancreatic ductal adenocarcinoma (PDAC) cells and in several in vivo models for this disease. QN-302 was invented at University College London UK and has been subsequently developed by Qualigen Therapeutics Inc. It was cleared by the FDA in August 2023 for Phase 1a clinical trials in USA and is currently under clinical evaluation. RNA-seq whole-genome transcriptome analysis revealed that QN-302 selectively targets G-quadruplexes in cancer-related genes, notably by forming stable G-quadruplex complexes with appropriate promoter sequences. This results in the transcriptional down regulation of affected genes, which map to for example, the hedgehog, axon guidance, WNT/β-catenin and P38 signal transduction pathways. In many instances individual expression changes are modest, though statistically significant: cumulatively, they affect whole pathways. We have revisited the RNA-seq data for QN-302 in PDAC MIA-PACA2 cells (GSE151741 at https://www.ncbi.nlm.nih.gov/geo/) in detail and now identify a small group of eleven down-regulated genes, all with >3-fold expression changes. Interestingly all these genes and their genes products have been previously identified as playing a role in human PDAC (they also have varying roles in other cancer types). Some have been validated as anticancer targets. 10/11 of these genes (https://www.proteinatlas.org/) have been previously reported as being over-expressed in significant fractions of large (>150) groups of human cancer patients from The Cancer Genome Atlas (TCGA) project. Cellular transcriptome data on these eleven genes is presented here, which together with earlier reports on them, supports the designation of QN-302 as a pan-G-quadruplex agent targeting a group of major genes involved in PDAC maintenance and metastasis. Data from a xenograft model for pancreatic cancer is also available for four of these genes, which concurs with the cellular data. We suggest that the identification of these 11 genes may also provide a basis for biomarker selection and possible patient stratification based on transcriptome data. Citation Format: Stephen Neidle, Ahmed A. Ahmed, Tariq Arshad. The pan-quadruplex drug QN-302 selectively down-regulates key pathway targets in pancreatic cancer cells that are up-regulated in human pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 576.

Abstract 1395: Unraveling the interplay between tumor heterogeneity and microenvironment in pancreatic ductal adenocarcinoma evolution and its clinical implications

Abstract Intratumoral heterogeneity (ITH) and tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) play important roles in tumor evolution and patient outcomes. However, the precise characterization of diverse cell populations and their crosstalk associated with PDAC progression and metastasis is still challenging. We performed single-cell RNA sequencing of primary PDAC samples with and without paired liver metastasis samples to understand the interplay between ITH and TME in the PDAC evolution and its clinical associations. We discovered that even a small proportion (~30%) of basal-like ductal cells in primary PDAC was associated with poor chemotherapy response and patient survival and that epithelial-mesenchymal transition programs were largely subtype specific. The clonal homogeneity significantly increased with more prevalent and pronounced copy number gains of oncogenes, such as KRAS and ETV1, and losses of tumor suppressor genes, such as SMAD2 and MAP2K4, along PDAC progression. Diverse immune cell populations, including naive SELLhi regulatory T cells (Tregs) and activated TIGIThi Tregs, contributed to shaping immunosuppressive TMEs of PDAC through cellular interactions with malignant ductal cells. The proportion of basal-like ductal cells negatively correlated with that of immunoreactive cell populations, such as cytotoxic T cells, but positively correlated with that of immunosuppressive cell populations, such as Tregs. Overall, our study provides a valuable resource for better understanding the roles of ITH and TME in PDAC evolution and their clinical relevance in the treatment of PDAC. Citation Format: Hyemin Kim, Hyong-oh Jeong, Se-Hoon Lee, Kwang Hyuck Lee, Kyu Taek Lee, Jong Kyun Lee, Min Woo Lee, Semin Lee, Joo Kyung Park. Unraveling the interplay between tumor heterogeneity and microenvironment in pancreatic ductal adenocarcinoma evolution and its clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1395.

Unleashing a Dual‐Warhead Nanomedicine to Precisely Sensitize Immunotherapy for Pancreatic Ductal Adenocarcinoma

AbstractDespite remarkable achievements in cancer immunotherapy, significant challenges persist in pancreatic ductal adenocarcinoma (PDAC) treatment. Particularly, there is a critical issue of inadequate intratumoral infiltration of effector T cells, primarily attributed to the physical “hard” barrier caused by extracellular matrix (ECM) and the “soft” barrier imposed by immune suppression. Here, a dual‐warhead nanomedicine is reported that effectively targets and attacks the barriers to precisely sensitize PDAC immunotherapy. The nanomedicine is achieved by designing tumor cell membrane vesicle (TMV)‐coated polyamidoamine dendrimer‐condensed siRNA complexes, with hedgehog inhibitor encapsulated in TMV bilayers. With the precision‐guided stealth feature of TMV coating, the nanomedicine achieved powerful tumor‐targeting co‐delivery of hedgehog inhibitor and siRNA, resulting in a significant decrease in collagen I secretion and efficient silencing of the target gene to disrupt the ECM and immune suppression barriers. This nanomedicine enables robust immunity against PDAC progression and metastasis, offering a potent approach to PDAC therapy.

Dual-energy computed tomography in a multiparametric regression model for diagnosing lymph node metastases in pancreatic ductal adenocarcinoma

ObjectiveTo investigate the diagnostic value of dual-energy computed tomography (DECT) quantitative parameters in the identification of regional lymph node metastasis in pancreatic ductal adenocarcinoma (PDAC).MethodsThis retrospective diagnostic study assessed 145 patients with pathologically confirmed pancreatic ductal adenocarcinoma from August 2016–October 2020. Quantitative parameters for targeted lymph nodes were measured using DECT, and all parameters were compared between benign and metastatic lymph nodes to determine their diagnostic value. A logistic regression model was constructed; the receiver operator characteristics curve was plotted; the area under the curve (AUC) was calculated to evaluate the diagnostic efficacy of each energy DECT parameter; and the DeLong test was used to compare AUC differences. Model evaluation was used for correlation analysis of each DECT parameter.ResultsStatistical differences in benign and metastatic lymph nodes were found for several parameters. Venous phase iodine density had the highest diagnostic efficacy as a single parameter, with AUC 0.949 [95% confidence interval (CI):0.915–0.972, threshold: 3.95], sensitivity 79.80%, specificity 96.00%, and accuracy 87.44%. Regression models with multiple parameters had the highest diagnostic efficacy, with AUC 0.992 (95% CI: 0.967–0.999), sensitivity 95.96%, specificity 96%, and accuracy 94.97%, which was higher than that for a single DECT parameter, and the difference was statistically significant.ConclusionAmong all DECT parameters for regional lymph node metastasis in PDAC, venous phase iodine density has the highest diagnostic efficacy as a single parameter, which is convenient for use in clinical settings, whereas a multiparametric regression model has higher diagnostic value compared with the single-parameter model.

LINC00909 up-regulates pluripotency factors and promotes cancer stemness and metastasis in pancreatic ductal adenocarcinoma by targeting SMAD4

BackgroundPancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis.MethodsThe expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People’s Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied.ResultsLINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models.ConclusionWe demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.

Correlation of laminin subunit alpha 3 expression in pancreatic ductal adenocarcinoma with tumor liver metastasis and survival.

The high mortality rate of pancreatic ductal adenocarcinoma (PDAC) is primarily attributed to metastasis. Laminin subunit alpha 3 (LAMA3) is known to modulate tumor progression. However, the influence of LAMA3 on liver metastasis in PDAC remains unclear. This study aimed to elucidate whether LAMA3 expression is increased in PDAC with liver metastasis. We extracted information related to LAMA3 expression levels and associated clinicopathological parameters from The Cancer Genome Atlas (TCGA) and four Gene Expression Omnibus (GEO) datasets. Clinicopathological analysis was performed; the Kaplan-Meier Plotter was used to evaluate LAMA3's prognostic effect in PDAC. We retrospectively collected clinicopathological data and tissue specimens from 117 surgically treated patients with PDAC at the Affiliated Hospital of Qingdao University. We assessed LAMA3 expression and investigated its correlation with the clinicopathological traits, clinical outcomes, and hepatic metastasis. Amplified expression of LAMA3 was observed in PDAC tissue compared with normal tissue in the TCGA and GEO databases. High LAMA3 expression was associated with poor overall survival (OS) and relapse-free survival (RFS) in patients with PDAC. LAMA3 expression was significantly enhanced in PDAC tissues than in adjacent tissues. Tumor tissues from patients with PDAC exhibiting liver metastasis showed higher LAMA3 expression than those without liver metastasis. High LAMA3 expression correlated with large tumor size and TNM stage. LAMA3 expression and liver metastasis were independent predictive factors for OS; the former was independently associated with liver metastasis. LAMA3 expression is elevated in patients with PDAC with liver metastasis and is a predictor of prognosis.