STAT1 Mediates FAK-Driven Choline Metabolism and Metastasis in Pancreatic Ductal Adenocarcinoma

BackgroundThis study investigated the molecular mechanism of long intergenic non-protein coding RNA 1605 (LINC01605) in the process of tumor growth and liver metastasis of pancreatic ductal adenocarcinoma (PDAC).MethodsLINC01605 was filtered out with specificity through TCGA datasets (related to DFS) and our RNA-sequencing data of PDAC tissue samples from Renji Hospital. The expression level and clinical relevance of LINC01605 were then verified in clinical cohorts and samples by immunohistochemical staining assay and survival analysis. Loss- and gain-of-function experiments were performed to estimate the regulatory effects of LINC01605 in vitro. RNA-seq of LINC01605-knockdown PDAC cells and subsequent inhibitor-based cellular function, western blotting, immunofluorescence and rescue experiments were conducted to explore the mechanisms by which LINC01605 regulates the behaviors of PDAC tumor cells. Subcutaneous xenograft models and intrasplenic liver metastasis models were employed to study its role in PDAC tumor growth and liver metastasis in vivo.ResultsLINC01605 expression is upregulated in both PDAC primary tumor and liver metastasis tissues and correlates with poor clinical prognosis. Loss and gain of function experiments in cells demonstrated that LINC01605 promotes the proliferation and migration of PDAC cells in vitro. In subsequent verification experiments, we found that LINC01605 contributes to PDAC progression through cholesterol metabolism regulation in a LIN28B-interacting manner by activating the mTOR signaling pathway. Furthermore, the animal models showed that LINC01605 facilitates the proliferation and metastatic invasion of PDAC cells in vivo.ConclusionsOur results indicate that the upregulated lncRNA LINC01605 promotes PDAC tumor cell proliferation and migration by regulating cholesterol metabolism via activation of the mTOR signaling pathway in a LIN28B-interacting manner. These findings provide new insight into the role of LINC01605 in PDAC tumor growth and liver metastasis as well as its value for clinical approaches as a metabolic therapeutic target in PDAC.

SummaryMetastasis is the leading cause of death for nearly 90% of patients with cancer. In the digestive system, malignancies preferentially metastasize to the liver, which occurs in 76–80% of patients with pancreatic ductal adenocarcinoma (PDAC). Given the shared endoderm origin of embryonic pancreas and liver, this study investigated whether genes highly expressed in liver progenitors drive PDAC cells metastasize to the liver. Using an in vitro liver differentiation model, genes highly expressed in liver progenitors were identified. Among them, TFAP2A was highly expressed in PDAC and closely related to PDAC liver metastasis. Cancer associated fibroblasts (CAFs) upregulated TFAP2A expression by bone morphogenetic protein 4 (BMP4). Functional experiments demonstrated that TFAP2A overexpression promoted PDAC cell stemness and liver metastasis in vitro and in vivo. Mechanistically, TFAP2A could promote epithelial mesenchymal transition (EMT) and recruit macrophage by upregulating MYC, facilitating PDAC cell intravasation. Collectively, these findings unveil molecular mechanisms for PDAC liver metastasis and potential therapeutic targets.

<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, as it commonly metastasizes to the liver resulting in an overall poor prognosis. However, the molecular mechanism involved in liver metastasis remains poorly understood. Here, we aimed to identify the MUC16-mediated molecular mechanism of PDAC-liver metastasis. Previous studies demonstrated that MUC16 and its C-terminal (Cter) domain are involved in the aggressiveness of PDAC. In this study, we observed MUC16 and its Cter expression significantly high in human PDAC tissues, PDAC organoids, and metastatic liver tissues, while no expression was observed in normal pancreatic tissues using IHC and immunofluorescence (IFC) analyses. MUC16 knockdown in SW1990 and CD18/HPAF PDAC cells significantly decreased the colony formation, migration, and endothelial/p-selectin binding. In contrast, MUC16-Cter ectopic overexpression showed significantly increased colony formation and motility in MiaPaCa2 pancreatic cancer cells. Interestingly, MUC16 promoted cell survival and colonization in the liver, mimicking an <i>ex vivo</i> environment. Furthermore, MUC16 enhanced liver metastasis in the <i>in vivo</i> mouse model. Our integrated analyses of RNA-sequencing suggested that MUC16 alters Neuropilin-2 (NRP2) and cell adhesion molecules in pancreatic cancer cells. Furthermore, we identified that MUC16 regulated NRP2 via JAK2/STAT1 signaling in PDAC. NRP2 knockdown in MUC16-overexpressed PDAC cells showed significantly decreased cell adhesion and migration. Overall, the findings indicate that MUC16 regulates NRP2 and induces metastasis in PDAC.</p>Implications:<p>This study shows that MUC16 plays a critical role in PDAC liver metastasis by mediating NRP2 regulation by JAK2/STAT1 axis, thereby paving the way for future therapy efforts for metastatic PDAC.</p></div>

<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, as it commonly metastasizes to the liver resulting in an overall poor prognosis. However, the molecular mechanism involved in liver metastasis remains poorly understood. Here, we aimed to identify the MUC16-mediated molecular mechanism of PDAC-liver metastasis. Previous studies demonstrated that MUC16 and its C-terminal (Cter) domain are involved in the aggressiveness of PDAC. In this study, we observed MUC16 and its Cter expression significantly high in human PDAC tissues, PDAC organoids, and metastatic liver tissues, while no expression was observed in normal pancreatic tissues using IHC and immunofluorescence (IFC) analyses. MUC16 knockdown in SW1990 and CD18/HPAF PDAC cells significantly decreased the colony formation, migration, and endothelial/p-selectin binding. In contrast, MUC16-Cter ectopic overexpression showed significantly increased colony formation and motility in MiaPaCa2 pancreatic cancer cells. Interestingly, MUC16 promoted cell survival and colonization in the liver, mimicking an <i>ex vivo</i> environment. Furthermore, MUC16 enhanced liver metastasis in the <i>in vivo</i> mouse model. Our integrated analyses of RNA-sequencing suggested that MUC16 alters Neuropilin-2 (NRP2) and cell adhesion molecules in pancreatic cancer cells. Furthermore, we identified that MUC16 regulated NRP2 via JAK2/STAT1 signaling in PDAC. NRP2 knockdown in MUC16-overexpressed PDAC cells showed significantly decreased cell adhesion and migration. Overall, the findings indicate that MUC16 regulates NRP2 and induces metastasis in PDAC.</p>Implications:<p>This study shows that MUC16 plays a critical role in PDAC liver metastasis by mediating NRP2 regulation by JAK2/STAT1 axis, thereby paving the way for future therapy efforts for metastatic PDAC.</p></div>

Liver metastasis (LM) is the predominant distant metastatic target of pancreatic ductal adenocarcinoma (PDAC) and a major contributor to poor patient outcomes, with pre-metastatic niche (PMN) formation as its critical prerequisite. However, its mechanisms remain incompletely elucidated. RNA sequencing was performed on primary tumor tissues and paired adjacent non-tumor tissues from PDAC patients, combined with the analysis of a GEO dataset related to PDAC liver metastasis (PDAC-LM), followed by validation using in vitro assays and mouse orthotopic liver metastasis models. Transcription factor database screening coupled with dual-luciferase reporter assays identified downstream molecules. Mass spectrometry was conducted on plasma extracellular vesicles (EVs) from PDAC patients and healthy controls, with validation by ELISA. The hepatic effects of EVs were assessed using mouse models, and in vitro EV uptake assays were performed to dissect the underlying molecular mechanisms. Transcriptomic sequencing of PDAC patients showed that elevated primary-tumor runt-related transcription factor 1 (RUNX1) expression correlates with PDAC-LM. In orthotopic murine models, RUNX1 knockdown significantly reduced liver metastasis rate from 61.54% to 8.33%. Mechanistically, RUNX1 transcriptionally upregulated integrin beta 1 (ITGB1), which was enriched in PDAC cell-secreted EVs and internalized by hepatic stellate cells (HSCs), activating the NF-κB pathway to induce HSC activation and secretion of ECM proteins, inflammatory factors and chemokines. This EV-induced HSC activation thereby promoted fibrotic hepatic PMN formation and facilitated LM, as validated in murine models. Clinically, ITGB1 in plasma EVs correlated with PDAC-LM and poor prognosis, with favorable diagnostic efficacy in distinguishing PDAC-LM patients. Here, we first demonstrate that ITGB1 upregulated by RUNX1 in primary PDAC cells is enriched in tumor-derived EVs and contributes to fibrotic PMN formation, thereby accelerating PDAC-LM. These pivotal findings not only unravel the intricate molecular circuitry governing PDAC-LM but also pinpoint promising biomarkers to facilitate the diagnosis and therapy of this lethal metastatic cascade.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, and the patients are generally diagnosed with distant metastasis. Liver is one of the preferred organs of distant metastasis, and liver metastasis is the leading cause of death in PDAC. Diet-induced obesity (DIO) is a risk factor for PDAC, and it remains unclear whether and how DIO contributes to liver metastasis of PDAC. In our study, we found that DIO significantly promoted PDAC liver metastasis compared with normal diet (ND) in intrasplenic injection mouse model. RNA-seq analysis for liver metastasis nodules showed that the various chemokines and several chemokine receptors were altered between ND and DIO samples. The expression levels of CX3CL1 and CX3CR1 were significantly upregulated in DIO-induced liver metastasis of PDAC compared to ND. Increased CX3CL1 promoted the recruitment of CX3CR1-expressing pancreatic tumor cells. Taken together, our data demonstrated that DIO promoted PDAC liver metastasis via CX3CL1/CX3CR1 axis.

Advancing age is the strongest independent risk factor for developing pancreatic ductal adenocarcinoma (PDAC). Older patients are more likely to present with advanced stage disease and have inferior overall survival when liver metastases are present. There is a critical need to understand the molecular factors that promote PDAC metastasis in order to guide development of more effective therapies. Despite the strong association of PDAC incidence and liver metastasis with aging, mechanisms through which aging impacts this process are not understood. Our prior work identified age-related changes in the PDAC tumor microenvironment (TME) that promote orthotopic PDAC tumor growth in aged (>12-month-old) compared to young (8-week-old) mice. GDF-15, a divergent TGFβ superfamily member, is a critical factor released by aged fibroblasts in the TME that promotes PDAC growth in an age-dependent fashion. We have recently identified that liver metastases are significantly more frequent in aged hosts, consistent with clinical observations. To determine how aging promotes PDAC metastases, we performed studies using novel human and mouse models that incorporate age as a variable. Primary tumors from aged mice have increased angiogenesis and decreased CD8+ T cell infiltration, findings associated with metastasis. To determine if GDF-15, released at high levels by aged but not young pancreatic fibroblasts, is involved in priming tumors for metastasis, we performed recombinant GDF-15 treatment of young mice harboring orthotopic KPC tumors. We found that GDF-15 significantly increases angiogenesis and decreases CD8+ T cell infiltration, changes associated with increased likelihood of metastasis. Tumors in aged GDF-15 knock out mice give rise to fewer metastases and have decreased angiogenesis and CD8+ T cell infiltration compared to aged wild type mice. Treatment of both human and mouse pancreatic cancer cells in vitro with aged fibroblast conditioned media or recombinant GDF-15 results in increased migration and invasion driven by GDF-15 specific activation of AKT signaling relative to controls. We next tested if the aged liver, independently of effects in a primary tumor, contributes to metastasis. Portal vein injection of KPC and KPCY clones into aged mice resulted in increased and larger metastases compared to young mice. Using imaging mass cytometry, we identified that liver metastases in aged mice were immune excluded with low CD8+ T cell infiltration and increased spatial interactions between immune cells and fibroblast populations with immunosuppressive phenotypes. Together, these data show that aging alters the primary tumor and metastatic liver microenvironments to promote more efficient metastatic spread of PDAC cells. We will present this and other ongoing work targeting the aging-driven GDF-15 signaling for the treatment and prevention of PDAC metastasis. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Sara E. Young, Elizabeth M. Jaffee, Won Jin Ho, Daniel J. Zabransk. Aged microenvironments promote liver metastasis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2551.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal tumors. Because most PDAC patients are diagnosed with an already advanced and metastatic stage of the disease, only 15% are suitable for surgical intervention. Thus, an improved understanding of the metastatic mechanism is critical to improve the outcome of these patients. The most common target for PDAC metastasis is the liver. Outgrowth of metastases requires acquisition of tumor genetic alterations in conjunction with alterations in the pancreas and liver microenvironment. The aim of the study was to investigate the role of the tumor microenvironment and the molecular alterations in tumor and stroma cells leading to PDAC liver metastasis formation. To this purpose a metastatic cell variant was established from the FC1199 pancreatic cancer cell line, derived from tumors arisen in LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice and growing orthotopically in C57BL/6 mice. The FC199LV metastatic variant was obtained by successive rounds of in vivo selection of cells implanted in the pancreas, isolated from liver metastases and re-implanted in the pancreas for a total of 5 rounds. IHC and in vivo MicroCT analysis of livers at different time points after tumor cell injection highlighted the presence of metastases only in mice transplanted with FC1199LV. Single cell RNA-sequencing analysis of dissociated primary tumors and livers of mice transplanted with FC1199 and FC1199LV highlighted differences in the microenvironmental transcription profile between the two tumor variants. The spontaneous metastatic model of PDAC is an important tool to study the interplay between tumor and stroma in PDAC metastatic process and to identify a tumor and tumor microenvironment gene expression signature associated with PDAC liver metastasis. Supported by AIRC IG 2019 ID23443 Citation Format: Claudia Fioravanti, Arianna Vallerga, Giulia Garattini, Ilaria Craparotta, Federica Di Leva, Chiara Grasselli, Fabio Sangalli, Andrea Resovi, Raffaella Giavazzi, Marco Bolis, Giulia Taraboletti, Dorina Belotti. Dissecting PDAC cell-microenvironment interplay in liver metastasis by single cell analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2566.

Pancreatic ductal adenocarcinoma (PDAC) requires innovative therapeutic strategies to counteract its progression and metastatic potential. Since the majority of patients are diagnosed with advanced metastatic disease, treatment strategies targeting not only the primary tumor but also metastatic lesions are needed. Tumor-Associated Macrophages (TAMs) have emerged as central players, significantly influencing PDAC progression and metastasis. Our objective was to validate an innovative therapeutic strategy involving the reprogramming of TAMs using lipid nanosystems to prevent the formation of a pro-metastatic microenvironment in the liver. In vitro results demonstrate that M2-polarized macrophages lose their M2-phenotype following treatment with lipid nanoemulsions composed of vitamin E and sphingomyelin (VitE:SM), transitioning to an M0/M1 state. Specifically, VitE:SM nanoemulsiontreatment decreased the expression of macrophage M2 markers such as Arg1 and Egr2, while M1 markers such as Cd86, Il-1b and Il-12b increased. Additionally, the TGF-βR1 inhibitor Galunisertib (LY2157299) was loaded into VitE:SMnanoemulsions and delivered to C57BL/6 mice orthotopically injected with KPC PDAC tumor cells. Treated mice showed diminished primary tumor growth and reduced TAM infiltration in the liver. Moreover, we observed a decrease in liver metastasis with the nanoemulsion treatment in an intrasplenic model of PDAC liver metastasis. Finally, we validated the translatability of our VitE:SM nanosystem therapy in a human cell-based 3D co-culture model in vivo, underscoring the pivotal role of macrophages in the nanosystem's therapeutic effect in the context of human PDAC metastasis. The demonstrated effectiveness and safety of our nanosystem therapy highlights a promising therapeutic approach for PDAC, showcasing its potential in reprogramming TAMs and mitigating the occurrence of liver metastasis.

Pancreatic ductal adenocarcinoma (PDAC) presents a major therapeutic challenge due to its aggressive metastatic behavior. In the liver metastasis microenvironment of PDAC, macrophages are key drivers of tumor progression. Targeting these pro-tumorigenic macrophages requires a deep understanding of the various macrophage populations and the factors influencing their phenotypes. Our study has revealed a distinct subset of macrophages with a pro-tumorigenic phenotype that emerges early in PDAC liver metastasis. We found that the establishment of hepatic metastases triggers local tissue damage, which, through efferocytosis, reprograms macrophages into an immunosuppressive state, thereby facilitating metastasis. Inhibiting efferocytosis pharmacologically prevented the conversion of macrophages, enhanced CD8+ T cell responses, and reduced liver metastasis. These results highlight the role of macrophages in tissue repair processes that promote liver metastasis in PDAC and suggest potential therapeutic targets to hinder its progression. Citation Format: Yuliana Astuti, Meirion Raymant, Valeria Quaranta, Kim Clarke, Maidinaimu Abudula, Olivia Smith, Gaia Bellomo, Vatshala Chandran-Gorner, Craig Nourse, Christopher Halloran, Paula Ghaneh, Daniel Palmer, Robert Jones, Fiona Campbell, Jeffrey Pollard, Jennifer Morton, Ainhoa Mielgo, Michael Schmid. Efferocytic macrophage promotes pancreatic cancer liver metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR011.

Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients, with most patients receiving palliative chemotherapy. We identified neuronal pentraxin 1 (NPTX1) as a cancer-secreted protein that becomes overexpressed in human and murine PDAC cells during metastatic progression and identified adhesion molecule with Ig-like domain 2 (AMIGO2) as its receptor. Molecular, genetic, biochemical, and pharmacologic experiments revealed that secreted NPTX1 acts cell-autonomously on the AMIGO2 receptor to drive PDAC metastatic colonization of the liver-the primary site of PDAC metastasis. NPTX1-AMIGO2 signaling enhanced hypoxic growth and was critically required for hypoxia-inducible factor-1α (HIF1α) nuclear retention and function. NPTX1 is overexpressed in human PDAC tumors and upregulated in liver metastases. Therapeutic targeting of NPTX1 with a high-affinity monoclonal antibody substantially reduced PDAC liver metastatic colonization. We thus identify NPTX1-AMIGO2 as druggable critical upstream regulators of the HIF1α hypoxic response in PDAC. Significance: We identified the NPTX1-AMIGO2 axis as a regulatory mechanism upstream of HIF1α-driven hypoxia response that promotes PDAC liver metastasis. Therapeutic NPTX1 targeting outperformed a common chemotherapy regimen in inhibiting liver metastasis and suppressed primary tumor growth in preclinical models, revealing a novel therapeutic strategy targeting hypoxic response in PDAC.

<div>Abstract<p>Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients, with most patients receiving palliative chemotherapy. We identified neuronal pentraxin 1 (NPTX1) as a cancer-secreted protein that becomes overexpressed in human and murine PDAC cells during metastatic progression and identified adhesion molecule with Ig-like domain 2 (AMIGO2) as its receptor. Molecular, genetic, biochemical, and pharmacologic experiments revealed that secreted NPTX1 acts cell-autonomously on the AMIGO2 receptor to drive PDAC metastatic colonization of the liver—the primary site of PDAC metastasis. NPTX1–AMIGO2 signaling enhanced hypoxic growth and was critically required for hypoxia-inducible factor-1α (HIF1α) nuclear retention and function. NPTX1 is overexpressed in human PDAC tumors and upregulated in liver metastases. Therapeutic targeting of NPTX1 with a high-affinity monoclonal antibody substantially reduced PDAC liver metastatic colonization. We thus identify NPTX1–AMIGO2 as druggable critical upstream regulators of the HIF1α hypoxic response in PDAC.</p><p><b>Significance:</b> We identified the NPTX1–AMIGO2 axis as a regulatory mechanism upstream of HIF1α-driven hypoxia response that promotes PDAC liver metastasis. Therapeutic NPTX1 targeting outperformed a common chemotherapy regimen in inhibiting liver metastasis and suppressed primary tumor growth in preclinical models, revealing a novel therapeutic strategy targeting hypoxic response in PDAC.</p></div>

Background & Aims: Circular RNAs (circRNAs) play important roles in many biological processes. However, the detailed mechanism underlying the critical roles of circRNAs in cancer remains largely unexplored. We aim to explore the molecular mechanisms of circRTN4 with critical roles in pancreatic ductal adenocarcinoma (PDAC). Methods: CircRTN4 expression level was examined in PDAC primary tumors. The oncogenic roles of circRTN4 in PDAC tumor growth and metastasis were studied in mouse tumor models. Bioinformatics analysis, luciferase assay and miRNA pulldown assay were performed to study the novel circRTN4-miRNA-lncRNA pathway. To identify circRTN4-interacting proteins, we performed circRNA-pulldown and mass spectrometry in PDAC cells. Protein stability assay and 3-Dimensional structure modeling were performed to reveal the role of circRTN4 in stabilizing RAB11FIP1. Results: circRTN4 was significantly upregulated in primary tumors from PDAC patients. In vitro and in vivo functional studies revealed that circRTN4 promoted PDAC tumor growth and liver metastasis. Mechanistically, circRTN4 interacted with tumor suppressor miR-497-5p in PDAC cells. CircRTN4 knockdown upregulated miR-497-5p to inhibit the oncogenic lncRNA HOTTIP expression. Furthermore, we identified critical circRTN4-intercting proteins by circRNA-pulldown in PDAC cells. CircRTN4 interacted with important epithelial-mesenchymal transition (EMT)- driver RAB11FIP1 to block its ubiquitination site. We found that circRTN4 knockdown promoted the degradation of RAB11FIP1 by increasing its ubiquitination. Also, circRTN4 knockdown inhibited the expression of RAB11FIP1-regulating EMT-markers Slug, Snai1, Twist, Zeb1 and N-cadherin in PDAC. Conclusion: The upregulated circRTN4 promotes tumor growth and liver metastasis in PDAC through the novel circRTN4-miR-497-5p-HOTTIP pathway. Also, circRTN4 stabilizes RAB11FIP1 to contribute EMT. Citation Format: Chi Hin Wong, Ut Kei Lou, Frederic Khe-Cheong Fung, Joanna H. M. Tong, Ka-Fai To, Stephen Lam Chan, Yangchao Chen. CircRTN4 promotes pancreatic cancer progression through a novel circRNA-miRNA-lncRNA pathway and stabilizing epithelial-mesenchymal transition protein [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-006.

Targeted inhibition of CHKα and mTOR in models of pancreatic ductal adenocarcinoma: A novel regimen for metastasis

RIOK3 promotes pancreatic ductal adenocarcinoma cell invasion and metastasis by stabilizing FAK