Pancreatic Cancer Progression Research Articles

MicroRNA-130b Suppresses Malignant Behaviours and Inhibits the Activation of the PI3K/Akt Signaling Pathway by Targeting MET in Pancreatic Cancer.

There has been interested in the microRNAs' roles in pancreatic cancer (PC) cell biology, particularly in regulating pathways related to tumorigenesis. The study aimed to explore the hub miRNAs in PC and underlying mechanisms by bioinformatics and fundamental experiments. RNA datasets collected from the Gene Expression Omnibus were analysed to find out differentially expressed RNAs (DERNAs). The miRNA-mRNA and protein-protein interaction (PPI) networks were built. The clinicopathological features and expressions of hub miRNAs and hub mRNAs were explored. Dual-luciferase reporter gene assay was performed to assess the interaction between microRNA and target gene. RT-qPCR and western blot were employed to explore RNA expression. The roles of RNA were detected by CCK-8 test, wound healing, transwell, and flow cytometry experiment. We verified 40 DEmiRNAs and 1613 DEmRNAs, then detected a total of 69 final functional mRNAs (FmRNAs) and 23 DEmiRNAs. In the miRNA-mRNA networks, microRNA-130b (miR-130b) was the hub RNA with highest degrees. Clinical analysis revealed that miR-130b was considerably lower expressed in cancerous tissues than in healthy ones, and patients with higher-expressed miR-130b had a better prognosis. Mechanically, miR-130b directly targeted MET in PC cells. Cell functional experiments verified that miR-130b suppressed cell proliferation, migration, promoted apoptosis, and inhibited the PI3K/Akt pathway by targeting MET in PC cells. Our findings illustrated the specific molecular mechanism of miR-130b regulating PC progress. The miR-130b/MET axis may be an alternative target in the therapeutic intervention of PC and provide an opportunity to deepen our understanding of the pathogenesis of PC.

VDR promotes pancreatic cancer progression in vivo by activating CCL20-mediated M2 polarization of tumor associated macrophage.

Activation of VDR pathway was a promising anti-tumor therapy strategy. However, numerous clinical studies have demonstrated the effect of activating VDR is limited, which indicates that VDR plays a complex role in vivos. We analyzed the TCGA database to examine the association between VDR expression and immune cell infiltration in pancreatic adenocarcinoma (PAAD). Western blot, ELISA, ChIP, and dual-luciferase reporter assays were performed to determine the mechanism of VDR regulating CCL20. Migration assay and immunofluorescence were used to investigate the role of CCL20 in M2 macrophage polarization and recruitment. We employed multiplexed immunohistochemical staining and mouse models to validate the correlation of VDR on macrophages infiltration in PAAD. Flow cytometry analysis of M2/M1 ratio in subcutaneous graft tumors. VDR is extensively expressed in PAAD, and patients with elevated VDR levels exhibited a significantly reduced overall survival. VDR expression in PAAD tissues was associated with increased M2 macrophages infiltration. PAAD cells overexpressing VDR promote macrophages polarization towards M2 phenotype and recruitment in vitro and vivo. Mechanistically, VDR binds to the CCL20 promoter and up-regulates its transcription. The effects of polarization and recruitment on macrophages can be rescued by blocking CCL20. Finally, the relationship between VDR and M2 macrophages infiltration was evaluated using clinical cohort and subcutaneous graft tumors. A positive correlation was demonstrated between VDR/CCL20/CD163 in PAAD tissues and mouse models. High expression of VDR in PAAD promotes M2 macrophage polarization and recruitment through the secretion of CCL20, which activates tumor progression. This finding suggests that the combination of anti-macrophage therapy may improve the efficacy of VDR activation therapy in PAAD.

The Expression Analysis of Long Non-coding RNAs Related to Wnt/β-Catenin Signaling in Pancreatic Cancer Patients.

Background The oncogenic Wnt/β-catenin signaling plays a critical role in carcinogenesis, prognosis, and resistance to therapy. Pancreatic cancer (PC) has high mortality because of its poor prognosis. Several studies have suggested that lncRNAs are directly involved in the development and progression of PC as well as in Wnt/β-catenin signaling. In this study, we investigated and compared the expression of Wnt/β-catenin signaling-related ZFAS1 and HCG11 lncRNAs, and their targets, CTNNB1 and IGF2BP1 genes in the blood of patients with PC and healthy individuals. A total of 47 PC patients and 50 healthy individuals participated in this study. RNA was extracted from the peripheral blood samples of participants, and cDNA was synthesized. The expression level of the selected genes was quantified by real-time PCR. The expression of HCG11 lncRNA and CTNNB1 genes in patients with PC was significantly upregulated compared to healthy individuals, and the expression of the ZFAS1 lncRNA was significantly downregulated. According to the analysis of the ROC curve, the diagnostic powers of ZFAS1 and CTNNB1 in PC were 0.67 and 0.69, respectively. Altogether, the present study suggests a role for ZFAS1 and HCG11 lncRNAs and CTNNB1 and IGF2BP1 in the pathogenesis of pancreatic cancer. Moreover, the peripheral expression of these lncRNAs may be useful as potential biomarkers for PC.

Identification of fibronectin type III domain containing 3B as a potential prognostic and therapeutic target for pancreatic cancer: a preliminary analysis

BackgroundFibronectin type III domain containing 3B (FNDC3B), a member of the fibronectin type III domain-containing protein family, has been indicated in various malignancies. However, the precise role of FNDC3B in the progression of pancreatic cancer (PC) still remains to be elucidated.MethodsIn this study, we integrated data from the National Center for Biotechnology Information, the Cancer Genome Atlas, Genotype-Tissue Expression database, and Gene Expression Omnibus datasets to analyze FNDC3B expression and its association with various clinicopathological parameters. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, along with Gene Set Enrichment Analysis (GSEA), single sample Gene Set Enrichment Analysis (ssGSEA) and estimate analysis were recruited to delve into the biological function and immune infiltration based on FNDC3B expression. Additionally, the prognostic estimation was conducted using Cox analysis and Kaplan–Meier analysis. Subsequently, a nomogram was constructed according to the result of Cox analysis to enhance the prognostic ability of FNDC3B. Finally, the preliminary biological function of FNDC3B in PC cells was explored.ResultsThe study demonstrated a significantly higher expression of FNDC3B in tumor tissues compared to normal pancreatic tissues, and this expression was significantly associated with various clinicopathological parameters. GSEA revealed the involvement of FNDC3B in biological processes and signaling pathways related to integrin signaling pathway and cell adhesion. Additionally, ssGSEA analysis indicated a positive correlation between FNDC3B expression and infiltration of Th2 cells and neutrophils, while showing a negative correlation with plasmacytoid dendritic cells and Th17 cells infiltration. Kaplan–Meier analysis further supported that high FNDC3B expression in PC patients was linked to shorter overall survival, disease-specific survival, and progression-free interval. However, although univariate analysis demonstrated a significant correlation between FNDC3B expression and prognosis in PC patients, this association did not hold true in multivariate analysis. Finally, our findings highlight the crucial role of FNDC3B expression in regulating proliferation, migration, and invasion abilities of PC cells.ConclusionDespite limitations, the findings of this study underscored the potential of FNDC3B as a prognostic biomarker and its pivotal role in driving the progression of PC, particularly in orchestrating immune responses.

Abstract LB190: Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer

Abstract In the context of the tumor microenvironment (TME), a substantial increased secreted protein, chitinase-3 like-protein-1 (CHI3L1) has been observed, emanating from various cell types such as cancer cells, macrophages, and T cells. The development of an immunosuppressive and fibrotic TME has been implicated in CHI3L1-mediated cancer progression. Despite recognizing the potential for CHI3L1 blockade to facilitate cancer regression, there remains a limited understanding of the regulatory mechanisms governing the expression of this tumor-promoting protein within cancer cells. Our investigations, along with others, have identified low levels of CHI3L1 protein in normal epithelial cells, in stark contrast to its abundant presence in pancreatic cancer tumor specimens. We propose that the interaction between cancer and stromal cells in the TME induces CHI3L1 expression within cancer cells. To test this hypothesis, we established a co-culture system involving cancer and stromal cells including pancreatic stellate cells, macrophages and T cells to emulate the TME of pancreatic cancer. Subsequently, we observed a ten-fold increase in CHI3L1 expression in cancer cells upon exposure to co-culture media. Through secretomic analysis, C-X-C motif chemokine ligand 10 (CXCL10) emerged as a potential inducer of CHI3L1 expression in response to co-culture media. CXCL10/CXCR3 signaling has been reported to correlate with poor prognosis in pancreatic cancer, while their role in disease development is still unclear. Concurrently, Gene Set Enrichment Analysis (GSEA) identified the activation of TNFα signaling via NF-κB and TGF-β pathways in pancreatic cancer patients with high CHI3L1 expression. Notably, chromatin-immunoprecipitation and promoter activity assays validated the direct binding and transcriptional regulatory role of NF-κB, a downstream transcription factor of CXCL10, on CHI3L1 gene in cancer cells. In addition, epigenetic reprogramming occurred during co-culture media education. Our observations indicated demethylation of the CHI3L1 promoter during co-culture media exposure, a phenomenon confirmed to be mediated by STAT3 activation. In summary, this study elucidates, for the first time, the intricate crosstalk between cancer and stromal cells within the TME of pancreatic cancer as a pivotal factor for the transcriptional and epigenetic dysregulation of CHI3L1, which then contributes to TME remodeling and malignant pancreatic cancer progression. Citation Format: Pei-Chia (Peggy) Su, Yi-Ching Wang. Cancer-stromal cells interaction dominates the expression of pro-tumor decreted protein chitinase-3-like-1 in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB190.

Revolutionizing Pancreatic Cancer Diagnosis and Prognosis through Machine Learning Technology

Abstract: Pancreatic cancer presents significant challenges in terms of early detection and treatment, resulting in poor patient outcomes. This research article explores the application of random forest, a powerful machine learning algorithm, to enhance predictive modeling in pancreatic cancer research. Leveraging a dataset comprising clinical and molecular features, we trained and evaluated random forest models to predict key outcomes such as tumor progression, treatment response, and overall survival. Our findings demonstrate the effectiveness of random forest in accurately stratifying patients based on their prognosis and treatment outcomes. Furthermore, we identified key biomarkers and clinical variables contributing to predictive accuracy, providing valuable insights into the underlying biological mechanisms of pancreatic cancer progression. The integration of random forest into pancreatic cancer research holds promise for improving patient stratification, guiding treatment decisions, and ultimately, advancing personalized medicine approaches in the management of this challenging disease

IFIT1 modulates the proliferation, migration and invasion of pancreatic cancer cells via Wnt/β-catenin signaling.

Previously, Interferon-induced Protein with Tetratricopeptide Repeats 1 (IFIT1) has been shown to promote cancer development. Here, we aimed to explore the role of IFIT1 in the development and progression of pancreatic cancer, including the underlying mechanisms. We explored IFIT1 expression in pancreatic cancer samples using The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. Cell Counting Kit-8 (CCK8), colony formation, scratch wound-healing and Transwell assays were performed to assess the proliferation, migration and invasion abilities of pancreatic cancer cells. Gene Set Enrichment Analysis (GSEA) and Western blotting were performed to assess the regulatory effect of IFIT1 on the Wnt/β-catenin pathway. We found that upregulation of IFIT1 expression is common in pancreatic cancer and is negatively associated with overall patient survival. Knockdown of IFIT1 expression led to decreased proliferation, migration and invasion of pancreatic cancer cells. We also found that IFIT1 could regulate Wnt/β-catenin signaling, and that a Wnt/β-catenin agonist could reverse this effect. In addition, we found that IFIT1 can promote epithelial-mesenchymal transition (EMT) of pancreatic cancer cells. Our data indicate that IFIT1 increases pancreatic cancer cell proliferation, migration and invasion by activating the Wnt/β-catenin pathway. In addition, we found that EMT could be regulated by IFIT1. IFIT1 may serve as a potential therapeutic target for pancreatic cancer.

Abstract 6220: TRIP13 augments pancreatic cancer progression- An integrated systems biology study

Abstract Background- Pancreatic cancer (PanCa) is one of the most aggressive forms of cancer and its incidence rate is continuously increasing every year. It is expected that by 2030, PanCa will become the 2nd leading cause of cancer related deaths in the United States due to the lack of understanding of the complexity of disease, early diagnostic methods, and extremely poor survival. Despite great advancements in biomedical research, there are very limited modalities available for the early detection of PanCa and its treatment. Thus, understanding of disease biology and identification of newer diagnostic and therapeutic modalities are high priority research in the field. Our group has identified Thyroid Receptor Interacting Protein -13 (TRIP13) as an oncogenic protein which contributes to early events of PanCa. Methodology- High dimensional omics and structural elucidation of TRIP13 was conducted using publicly available databases like Consurf, Phosphosite, GTEx and GEPIA2. Molecular biology techniques were applied to cross validate all our bioinformatics data. qPCR, immunoblotting and IHC analysis was performed in progressive PDAC cell lines and human tissues to decipher the expression level of TRIP13 in various pathological staging including functional enrichment analysis using Linkdomics. Results- The structural elucidation analysis revealed that most of the functionally exposed residues, phosphorylation and ubiquitylation sites situated at AAA domain of TRIP13. As compared to normal, TRIP13 is significantly over expressed in pancreatic tumor samples. Pathological staging 2 and 1 (early stages) showed relatively higher expression of TRIP13 as compared advanced stage. Of the total 7 isoforms of TRIP13, only one isoform (TRIP13-001) has shown the over expression in pancreatic tumor samples and shown association with poor survival. In correlation studies, TRIP13 has shown positive association with other pancreatic cancer biomarkers (CEACAM5, S100A4, MUC1, MSLN, CA125). The functional enrichment analyses suggest that TRIP13 is involved in important patho-physiological pathways like DNA repair, viral carcinogenesis, cellular senescence, and cell cycle. Our wet lab experiments also support our computational biology observation. Conclusion- This integrated computational biology study suggests a potential role of TRIP13 in pancreatic cancer progression via modulation of important oncological pathways. Wet lab experimentations also validated the involvement of TRIP13 in pancreatic cancer progression. TRIP13 expression may be associated with patient prognosis. Thus, its inhibitory small molecules can be useful for boosting pancreatic cancer therapies in clinical settings. Key words- TRIP13, Pancreatic cancer, Early events of cancer, Integrative Biology, Transcriptomics, Computational biology Citation Format: Swati Dhasmana, Anupam Dhasmana, Stella Rios, Iris A. Perez, Sheema Khan, Farrukh Afaq, Upender Manne, Murali M. Yallapu, Subhash C. Chauhan. TRIP13 augments pancreatic cancer progression- An integrated systems biology study [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 6220.

Abstract 7304: Modulation of cytokine and chemokine profiles in pancreatic tumors and plasma via stage-specific intervention with Momordica Charantia juice in the LSL-KrasG12D/+, p48Cre/+ mouse model

Abstract According to the American Cancer Society ≈64,050 people will be newly diagnosed with pancreatic cancer in the United States, while ≈55, 550 (men and women numbers combined) will die of this malignancy in 2023 alone. Epidemiological studies have indicated that a diet rich in natural agents might reduce pancreatic ductal adenocarcinoma (PDAC) risk, prompting investigations into a natural agent with health benefits in pancreatic diseases [such as the fruits of Momordica Charantia (Bitter Melon)] as a potential natural and non-toxic anti-cancer approach against PDAC. Previously we have reported the protective effects of oral Bitter Melon Juice (BMJ-standardized-lyophilized powder) against growth and progression of PDAC in preclinical animal models (xenograft and patient-derived tumor models). In a recent study we reported the efficacy of BMJ against pancreatic cancer in the LSL-KrasG12D/+, p48Cre/+ transgenic mouse model [this model mimics the stage-specific progression of intraepithelial neoplasms to PDAC as it occurs in humans]. Both male and female LSL-KrasG12D/+, p48Cre/+ transgenic mice were subjected to BMJ intervention (daily oral gavage, dose: 200mg/kg body wt.) during different stages in disease progression. Intervention was started at 4 or 15 weeks of mice age and continued till study end (10 and 30 weeks of mice age respectively). Additionally, in one group of mice, intervention was initiated at 6 weeks of mice age and continued till 41 weeks of mice age (study end). Outcomes were compared with wild-type and disease-positive untreated control groups. Pancreatic tissue/tumors were harvested at study end and blood plasma was also collected. In the present study, we performed a stage specific evaluation of tissue and plasma cytokine/chemokine profiling after BMJ intervention to gain detailed mechanistic insights into the potential of BMJ to mediate it is anti-pancreatic cancer effect via modulation of inflammatory pathways associated with PDAC growth and progression. BMJ (15 weeks of treatment) demonstrated a significant response in modulating cytokine/chemokine profiles in LSL KrasG12D/+, p48Cre/+ mice pancreatic cancerous tissues (30 weeks old mice). BMJ intervention specifically downregulated the levels of monocyte chemoattractant protein (MCP-1), macrophage inflammatory protein-1 (MIP-1-gamma), tumor necrosis factor (TNF) receptor (TNFR1), Vascular cell adhesion molecule 1 (VCAM-1/CD106) and L-selectin (CD62L). Furthermore, Ingenuity Pathway Analysis indicated significant inhibitory predictions on TNF-α, NF-κB and MCP-1 (CCL-2) associated inflammatory pathways. Overall, these outcomes signify that the anti- cancer effects of BMJ in KRAS harboring PDAC tumors is associated with down-regulation of inflammatory pathways associated with growth and progression of pancreatic cancer. Citation Format: Lakshmi Sai Pratyusha Bugata, Robin Kumar, Md. Imtiazul Kabir, Rama Kant, Chapla Agarwal, Rajesh Agarwal, Komal Raina. Modulation of cytokine and chemokine profiles in pancreatic tumors and plasma via stage-specific intervention with Momordica Charantia juice in the LSL-KrasG12D/+, p48Cre/+ mouse model [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 7304.

Abstract 6984: Investigating the function of the OSM-OSMR axis in pancreatic ductal adenocarcinoma (PDAC)

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer, characterized by a low survival rate and a significant global mortality impact. The interaction between PDAC and the immune system is complex and plays a crucial role in the progression of the disease. PDAC creates an immunosuppressive tumor microenvironment, which hampers the ability of immune cells to effectively infiltrate the tumor and mount an anti-cancer immune response. Additionally, there is an imbalance of cytokines, with an overabundance of immunosuppressive factors and a deficiency in pro-inflammatory cytokines. This hostile immunological landscape contributes to the aggressive nature of PDAC and hinders the success of immunotherapies and adjuvant treatments. One cytokine of particular interest is oncostatin M (OSM), a member of the interleukin-6 (IL-6) family. OSM has been implicated in the regulation of cancer and has been associated with poor overall survival in pancreatic cancer and other malignancies. Similar to other cytokines, OSM is secreted by immune cells and acts as a regulator of the inflammatory microenvironment surrounding the tumor. The levels of OSM in this milieu can potentially influence cancer development and regulation through multiple mechanisms. Despite the growing understanding of cytokines in cancer, research on the role of OSM in PDAC is still limited and requires further investigation to identify potential therapeutic targets. Therefore, there is a critical need to delve deeper into the functions of OSM and its receptor, oncostatin M receptor (OSMR), in the initiation and progression of pancreatic cancer. As such, our goal is to examine the functions of OSM and its receptor (OSMR) in initiating and advancing pancreatic cancer to better comprehend the role of the OSM-OSMR axis in PDAC, both in vivo and in vitro. Citation Format: Yuan Sui, Tony Hunter. Investigating the function of the OSM-OSMR axis in pancreatic ductal adenocarcinoma (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 6984.

Abstract 2802: Enterococcus faecalis-derived X facilitates pancreatic tumor by regulating immune microenvironment

Abstract Previous research in human samples has found a link between the intratumoral microbiome and the development and progression of pancreatic cancer. Nevertheless, the bacterial strains that may have an effect, and the underlying mechanisms, are largely unknown. To identify potential bacterial species, microbiome sequencing was performed on fecal and tumor samples obtained from KPC mice and patients with pancreatic tumors in this study. To identify metabolites associated with bacteria, untargeted metabolomics were analyzed on serum and tumor samples. Furthermore, advanced and high-throughput techniques, such as single-cell RNA sequencing, tumor organoids, engineered bacteria and germ-free mouse models, were used to elucidate the specific influence on the immune microenvironment of pancreatic tumors. Our findings reveal an increased abundance of Enterococcus faecalis (E. faecalis) in the gut during the development of pancreatic cancer, accompanied by its accumulation in the tumor. Patient-derived E. faecalis strain and its metabolite X, facilitated pancreatic tumor growth, reduced the recruitment of CD8+T cells, and caused their dysfunction in the tumor, relying heavily on E. faecalis intratumoral colonization. We found that X inhibits the production of anti-tumor chemokines like CCL21 and CCL8 in tumor cells, reducing peripheral CD8+ T cell infiltration into the tumor. Furthermore, X was found to inhibit glycolysis and compromise the function of tumor-infiltrating CD8+ T cells by decreasing the activity of lactate dehydrogenase (LDH) in CD8+ T cells. Additionally, oral doxycycline, an antibiotic that targets intratumoral E. faecalis, effectively enhanced the therapeutic effect of gemcitabine in pancreatic cancer. Finally, we present evidence that X may predicts efficacy and survival in pancreatic cancer patients. Together, our findings provide new insights into the role of E. faecalis in mediating the immune environment of pancreatic tumors, highlighting E. faecalis and X as potential therapeutic targets for pancreatic cancer patients. Citation Format: Bin Wang, Chao Yang, Yongjie Xie, Tianxing Zhou, Jun Yu, Jihui Hao. Enterococcus faecalis-derived X facilitates pancreatic tumor by regulating immune microenvironment [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 2802.

Abstract 880: Identifying ANKRD22 and fostamatinib as a theragnostic target and drug candidate for metastatic pancreatic cancer through in silico analysis

Abstract Introduction: Pancreatic cancer (PC) presents significant clinical challenges, marked by a low 5-year survival rate and frequent distant metastasis. When PC metastasizes to other organs, it leads to a notable decrease in the 5-year survival rate for PC in metastasis, dropping from 11% to 3%. Despite the effectiveness demonstrated by advanced anti-cancer therapies targeting these mutated genes, the improvement in survival rates remains modest. Another critical consideration is the rising resistance to Gemcitabine, a standard drug used for treating PC. Hence, it is imperative to gain a more profound understanding of the comprehensive mechanisms underlying metastatic pancreatic cancer (mPC) and actively explore novel drugs for its treatment. Methods: First, we reanalyzed the RNA-seq of three of PC's most common metastatic sites, including liver, lung, and peritoneum. Using the Galaxy platform, we identified DEGs (differentially expressed genes) between metastatic sites. After that, we further utilized other web-based tools and databases, such as GEPIA, TIMER 2.0, Alphafold, PubChem, Drugbank, and Chembl, to identify ANKRD22 and perform molecular docking of Fostamatinib binds to this gene. Results: Our research focused on exploring the role of ANKRD22, which contains four copies of L-shaped ankyrin motifs, an emerging oncogenic molecule associated with various cancers. In the current study, we identified that ANKRD22 exhibited significant expression in the pancreatic adenocarcinoma (PAAD) cohort, correlating with deteriorating OS rates in PC patients. Moreover, it displayed distinct expression patterns in advanced stages of the disease compared to early stages, Furthermore, we uncovered a noteworthy correlation between ANKRD22 and the Kras, TP53, CDKN2A, and SMAD4 genetic anomaly, linked to diminished survival rates, as widely recognized, in the initiation and progression of PC, these four genes stand out as the most frequently mutated. Mechanistically, ANKRD22 exhibited associations with KRAS dependency signature, U12 type spliceosome assembly, multivesicular body routing, cell invasion, tumor cell line invasion, and tumor cell line migration, all indicative of PC progression and dissemination. Expanding our investigation through the DrugBank and ChEMBL databases, we delved into fostamatinib, an FDA-approved drug known as a spleen tyrosine kinase (SYK) inhibitor. Molecular docking experiments revealed the potential interaction between ANKRD22 and fostamatinib, suggesting its candidacy to treat mPC. Conclusion: Our findings identify ANKRD22 as a pivotal oncogenic factor promoting metastasis in PC and propose fostamatinib as a potential inhibitory treatment. Citation Format: Huong Thi Luu Kim Huynh, Hendrick Gao-Min Lim, Yuan-Chii Gladys Lee, Chien-Hsin Chen, Alexander T.H. Wu. Identifying ANKRD22 and fostamatinib as a theragnostic target and drug candidate for metastatic pancreatic cancer through in silico analysis [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 880.

Abstract 1589: Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is primarily characterized by a desmoplastic reaction, mainly consisting of cancer-associated fibroblasts (CAFs). In order to investigate the heterogeneity of CAFs, we initially conducted single-cell Assay for Transposase-Accessible Chromatin with high-throughput sequencing (scATAC-seq) using pancreas tissue from 12-week-old KPC (K-rasLSL.G12D/+; Trp53R172H/+; Pdx-1-Cre) PDAC mouse models. We identified three clusters enriched with myofibroblast CAF (myCAF) gene markers and one cluster enriched with inflammatory CAF (iCAF) gene markers. Next, we performed single-cell RNA sequencing (scRNA-seq) on pancreas tissues from KPC mice. scRNA-seq revealed four distinct fibroblast clusters, each exhibiting characteristic gene expression profiles of normal fibroblasts, iCAF, myCAF and antigen-presenting CAF (apCAF). These findings suggest that CAFs in the myCAF cluster identified in scATAC-seq may have potential to differentiate into either myCAFs or other CAF subtypes based on certain cues. Next, we focus on the expression of myCAF marker genes, particularly two commonly used markers, Tagln and Acta2. Among myCAFs, two subtypes were identified: Tagln-positive and Acta2-positive CAFs, and Tagln-negative and Acta2-positive CAFs. To confirm our findings, we conducted Immunohistochemical analysis (IHC) on KPC pancreas samples. We observed CAFs that were positive for both Talng and αSMA exclusively in close proximity to tumor cells. Fibroblast expressing only αSMA mostly located in normal area. Tagln is known as an actin-binding protein and plays an important role in activated fibroblasts. Orthotopic PDAC mouse models using Tagln homozygous knockout mice showed a significant reduction in tumor weight compared to the wild-type mice (P = 0.0076). Indeed, we found that high TAGLN expression in PDAC samples was associated with poor survival, as demonstrated by the analysis of the TCGA dataset. These data suggest that myCAFs expressing TAGLN contribute to the promotion of PDAC growth. Targeting stromal TAGLN might be a potential therapeutic strategy to counteract PDAC progression. Citation Format: Keiko Shinjo, Xingxing Wang, Kohei Kumegawa, Reo Maruyama, Shinji Mii, Yoshiteru Murofushi, Miho Suzuki, Atsushi Enomoto, Yutaka Kondo. Integrated multiomics analysis revealed the significance of Transgelin in driving pancreatic cancer-associated fibroblast-mediated cancer 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 1589.

Abstract 5692: Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers

Abstract Aberrant expression of mutant KRAS and miRNA-21 (miR-21) are commonly attribute to malignancy and tumorigenic progression of pancreatic cancers. However, few studies have explored the molecular mechanism linking these two prognostic factors. In this study, we explored the underlying molecular bridge between KRAS and miR-21 regulation in pancreatic cancer. Functional integrative analysis of KRAS, an essential oncogenic driver in pancreatic cancer progression, and together with miR-21 revealed XRN1, a 5′-3′ exoribonuclease, as RNA-linked top-scored molecule with a strong survival correlation and malignancy in pancreatic cancer patients. Depletion of XRN1 led to severe reduction in mature miR-21 expression, while unaltered precursor miR-21 levels; indicating that the XRN1 is critical in miR-21 maturation at post-Drosha step in conjunction with Dicer. Moreover, miR-21 sensor-based experiment also confirmed the critical role of XRN1 on miR-21 biogenesis. Depletion experiments showed that XRN1 positively regulates proliferation and self-renewal capacity of pancreatic cancer cells via its 5′-3′ exoribonuclease activity and concomitantly elevated the expression of PTEN and PDCD4 which are the key miR-21 cellular downstream targets. shRNA-mediated XRN1 depletion also yielded smaller tumor volume and longer survival in pancreatic cancer xenografts and syngeneic model demonstrating the therapeutic potential of targeting XRN1. Subsequent silencing of KRAS reduced XRN1 expression in pancreatic cancer cells, suggesting that KRAS plays a key role in this XRN1-miR-21 axis. Furthermore, drug-resistant pancreatic cells showed elevated expression of both KRAS and XRN1-miR-21 indicating that this axis is also associated with drug sensitivity. In sum, these results suggest that a novel KRAS-XRN1 posttranscriptional regulatory axis plays a pivotal role in tumor progression by mediating miR-21-directed signaling, and thus may suggest a novel targeting strategy for pancreatic cancer therapeutics. Significance: This study identifies molecular regulatory interplay between KRAS and XRN1-miR-21 signaling in pancreatic cancers, elucidates how KRAS-XRN1 modulates tumorigenesis via miR-21, and demonstrates that targeting this signaling can contribute to therapeutic strategies for pancreatic cancers. [Y. Seo and S. E. Oh contributed equally to this work.] Citation Format: Yoona Seo, Seung Eon Oh, Jong Heon Kim. Posttranscriptional molecular network facilitates KRAS-linked tumorigenesis via XRN1-miR-21 signaling in pancreatic cancers [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 5692.

Abstract 1598: Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models

Abstract Background & Aim: Progression and therapeutic resistance in pancreatic cancer depends on the ability of cancer cells to adopt specific cell states. These phenotypes largely depend on environmental tissue niches. Here, we investigated therapy-induced tumor dynamics in vivo at single-cell spatial and temporal resolution in different subtypes of PDAC using several genetically engineered mouse models, which reflect distinct human PDAC ecosystems. Methods & Results: We established orthotopic tumor models of pancreatic cancer with distinct tumor microenvironment (TME) formation (reactive vs deserted stroma), which were monitored for tumor growth to define tumor response patterns under MAPK inhibition. Serial ultrasound-guided biopsy sampling was performed to collect tissues from the same tumors at pre-treatment, responsive and recurrence periods.Transcriptomic analysis of biopsies using the Mfuzz algorithm delineated highly dynamic gene and pathway activities during treatment. Reactive, but not deserted, TME showed an upregulation of immune-related pathways (antigen presentation, T cell receptor, NK cytotoxicity) immediately after MAPK inhibition in regressed tumors that dropped dramatically during recurrence of tumors, in which upregulation of TGF-b signaling was observed. In the deserted stroma model, on the other hand, an enrichment in drug metabolic pathways during recurrence was observed.CAF cluster deconvolution analysis revealed an increase in a LRRC15+ CAF cluster, which was TGF-b-driven and immunosuppressive, during recurrence period only in the reactive model. The dynamic changes of immune cells and CAFs were validated at protein level by multiplex immunofluorescent imaging. Cmputational spatial analysis revealed a tumor-encapsulating and CD8+ cytotoxic T cell-repelling pattern exhibited by LRRC15+FAP+ CAFs, implying a potential role of this CAF subset in T cell suppression during recurrence in reactive subtype. Using spectral cell sorting with CellView image technology, specific CAF subsets, tumor cells as well as immune cell subsets based on 30+ markers and cell morphology were isolated, characterized and assessed by ex vivo functional assays to confirm the immunosuppressive and tumor protective mechanism mediated by CAFs. Conclusion: Longitudinal in vivo assessment of distinct PDAC subtypes provides deep mechanistic insights to accurately unlock specific response patterns of tumors with heterogeneous TME dynamics upon therapeutic perturbation, leveraging identification of key nodes for intervention. Citation Format: Jens T. Siveke, Rui Fang, Phyllis Fung-Yi Cheung, Jiajin Yang, Juanfei Peng, Kristina Althoff, Konstantinos Savvatakis. Individualized longitudinal assessment reveals therapy-induced tumor and microenvironment dynamics in preclinical pancreatic cancer models [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 1598.

Abstract 4112: Identification of Th1-specific epitopes from non-mutated KRAS lesions for off-the-shelf cancer vaccines

Abstract Background KRAS is crucial for cell growth, differentiation, and signaling. Mutations in the KRAS gene (i.e., codon 12 or 13) are frequently associated with the development and progression of cancer, particularly lung, colorectal, and pancreatic cancers. Recently, it has been captured that KRAS targeting cancer vaccine research and clinical trials have been conducted in certain tumor types and vaccine approaches are encouraged to move forward. It has been well known that the tumor-eradicating efficacy of cancer vaccines arises from Th1 epitope selection. While tumor growth may persist after immunization with non-selected epitopes (including full-length), immunization with a highly selected Th1-specific epitope vaccine can lead to tumor regression by suppressing the activation of immune inhibitory cells. The Th-Vac® discovery platform uses a combination of in-silico, in-vitro, and in-vivo studies to precisely identify Th1-specific epitopes. It consists of two modules: module 1 (in-silico) and module 2 (in-vitro and efficacy evaluation). The aim is to identify MHC class II epitopes that are specific to antigens for CD4+ T cells, displaying the most favorable binding affinity across diverse alleles. Th1-specific epitope vaccines aim to boost and sustain T cell immunity against tumor antigens, overcoming the lack of a precise T cell response or pre-existing tolerance in the immunosuppressive TME. This study aimed to explore Th1-specific epitopes for KRAS vaccine to be potentially effective in numerous KRAS mutation-driven cancers. Methods Peptide sequence candidates that have highly potential of MHC class II binding affinity were comprehensively predicted via a module 1 (ASEP program). All of peptides selected in ASEP were synthesized as 15 mers. Each peptide was evaluated for immunogenicity using ELISpot and/or FACS analysis in module 2 (2a for in vitro and 2b for in vivo immunologic evaluation) of the Th-Vac® discovery platform. Specifically, in Module 2b, immunogenicity was evaluated using a 38-mer peptide formed by combining five overlapping peptides selected in 2a. These kinds of immunogenicity evaluations were conducted using type 1 and/or type 2 cytokine(s) produced by CD4+ T cells. Results In ASEP prediction, twelve peptides were selected as potential candidates demonstrating a high affinity to MHC class II, which were in different lesions of common mutation of KRAS. In module 2 using ELISpot and FACS analysis, two epitopes (non-mutated sequences) were finally selected as Th1-specific epitopes based on the type 1 T cell response. Conclusion Unlike previous vaccines using mutant lesions, in this study, two non-mutated Th1-specific epitopes are under the in-vivo efficacy studies as mono and combining regimen(s) in certain tumor types. Additionally, the Th-Vac® platform was fully validated in terms of the performance and its application would be expanded beyond a cancer vaccine. Citation Format: Youngki Choi, Jinback Lim, Hyo-Hyun Park, Jinho Kang, Hun Jung. Identification of Th1-specific epitopes from non-mutated KRAS lesions for off-the-shelf cancer vaccines [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 4112.

Abstract 6792: Investigating differential expression of Palladin isoform 3 in 2D and 3D pancreatic cancer cell cultures

Abstract Palladin (PALLD) is an actin-binding protein widely expressed in mammalian tissues that modulates cytoskeletal dynamics. Palladin is linked to the progression of breast, pancreatic, and renal cancers. There are at least 9 known isoforms of PALLD but isoforms 3 (iso3) and 4 (iso4) appear most significant in cancer cells and associated fibroblasts. Our goal is to understand iso3 in pancreatic cancer (PC) cell lines grown in various culture environments to shed light on the intricate interplay between culture conditions and gene regulation. Cell models were developed as 3D spheroids from hanging drops after optimizing culture conditions by utilizing a combination of inexpensive methylcellulose (MC) and rat tail collagen viscosity ranges across cell seeding densities. The quality of the developed spheroids was assessed using multiple parameters, like diameter, integrity, and circularity to determine ideal spheroid characteristics. Spheroids that formed at a lower MC viscosity range (350-550 mPa·s) led to peripheral loose cells and a higher viscosity range (7,000-10,000 mPa·s) resulted in elongated shapes. Moderate viscosity ranges (1,000-1,800 mPa·s and 3,500-5,600 mPa·s) consistently produced circular and compact spheroids. Optimal incubation times varied from 72 to 96 hr depending on the cell line. Invasion dynamics within collagen-MC gels revealed that PANC-1 had significantly higher invasion potential compared to BxPC-3 and a control cell line C2C12. RT-qPCR results show that PALLD iso3 was particularly overexpressed in the 3D culture environment of PANC-1, suggesting that elevated PALLD isoform 3 expression may be a characteristic of 3D culture, particularly in invasive PC types. Taken together, the study validates the importance of extracellular viscosity for influencing spheroid characteristics toward generating reproducible 3D spheroids. The investigation also highlights PALLD iso3 as a potentially valuable biomarker for aggressive forms of pancreatic cancer. Citation Format: Sonali Biswas, Jason A. Bush. Investigating differential expression of Palladin isoform 3 in 2D and 3D pancreatic cancer cell cultures [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 6792.

Abstract 5904: Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways

Abstract Pancreatic cancer (PaCa) ranks as the fourth leading cause of cancer-related deaths in the United States and is among the top five worldwide. Its hallmark characteristics include early invasion, metastasis, resistance to chemotherapy and radiotherapy, and aggressive tumor progression. Evaluating new generation treatments with significant potential in this ongoing battle against cancer is crucial. Voltage-gated sodium channels (VGSC), long detected in various metastatic cell lines and tissue samples beyond breast, lung, and prostate cancer, are of particular interest. This study aimed to explore the therapeutic effects of inhibiting NaV1.7 and its neonatal variant, nNaV1.7, in pancreatic cancer cells. The study utilized human normal keratinocyte cell line HaCaT and pancreatic cancer cell lines PANC-1 and MiaPaCa-2. It investigated the roles and effects of adult and neonatal isoforms of the NaV1.7 channel in pancreatic cancer proliferation, invasion, and metastasis. The findings revealed significantly higher adult and neonatal NaV1.7 mRNA expressions in PANC-1 and MiaPaCa-2 cell lines compared to HaCaT cells. Specific siRNAs effectively downregulated channel mRNA and protein expressions. Notably, NaV1.7 siRNA treatments significantly inhibited cell proliferation and colony-forming capacity in PANC-1 and MiaPaCa-2 cells, without cytotoxic effects on HaCaT cells. Combining gemcitabine with specific NaV1.7 siRNAs enhanced drug efficacy in the PANC-1 cell line. Furthermore, NaV1.7 siRNAs significantly reduced cell invasion, migration, and wound healing in both cell lines. These siRNAs also induced apoptosis and G1 arrest in the cell cycle. They inhibited protein expressions involved in key signaling pathways, including P-Src/P-Fak/Integrin β1, EF2K, P-Akt, P-mTOR, PARP, Caspase-3/9, Cyclin D1/E1. This study is pioneering, as literature lacks research on the expression and function of NaV1.7 and nNaV1.7 in pancreatic cancer cells or tissue samples. In conclusion, NaV1.7 channel isoforms play a significant role in the development, progression, and metastasis of pancreatic cancer and present as a potential therapeutic target in PaCa. Citation Format: Mumin Alper Erdogan, Ayse Caner. Targeting NaV1.7 channels in pancreatic cancer: Implications for EF2K and P-Src/P-Fak/integrin β1 signaling pathways [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 5904.

Abstract 5161: Integrating patient’s derived organoids and liquid biopsy: An exploratory study of early stage pancreatic cancer

Abstract Background: Pancreatic cancer (PC) has a 5-year survival of 12% despite recent therapeutic advancements. There is a desperate need to discover novel ways of treating PC since a large proportion of patients do not benefit from therapy and experience significant toxicity. A major cause of therapy failure is therapy resistance due to tumor heterogeneity. Nowadays, a deeper understanding of tumor heterogeneity could be assisted by the use of patients-derived organoids (PDOs). Furthermore, the use of liquid biopsy may allow for the investigation of circulating factors with predictive value and for disease monitoring over time. Methods: Patients were enrolled in the comprehensive, multiomic study of pancreatic cancer CONTAINER. Patients with early stage PC treated with preoperative chemotherapy or undergoing surgery first were selected for the present study. When possible, a surgical specimen was collected to obtain PDOs. Tissue from resected tumors was dissociated by GentleMACSTM dissociator (Miltenyi Biotec), fragments were embedded in MatrigelTMand cultured in PancreaCultTM human Organoid Media (stemcell technologies). PDO histological features were investigated by H&E staining and IHC. Blood samples were collected before and one month after surgery. From blood samples circulating tumor cells (CTCs) were enriched and isolated, extracellular vesicles (EVs) were isolated from plasma with size-exclusion chromatography and their concentration and size distribution was evaluated with NanoSight NS300. Then, EV surface antigens were characterized by flow cytometry. A panel of inflammatory cytokines was finally evaluated through Ella Automated Immunoassay System. Results: In CONTAINER study 113 patients were enrolled and 30 of them underwent surgery. 21 surgical specimens were received from which 18 PDOs were successfully established. EVs with diameter ≥ 70nm were isolated from plasma, EV mean size was similar before and after surgery whereas individual variations in particle concentration and in surface antigen exposure were observed. Correlation with clinical and pathological characteristics of patients will be performed once reached an adequate sample size for statistical analyses. A panel of inflammatory cytokines (IL1β-, IFNγ, TNFα, IL6, IL8, TGFβ1, CRP and Leptin) was assessed in plasma samples obtained before and after surgery. Except for IL1β, a significant individual variation of circulating cytokines before and after surgery was observed. Conclusions: This study will contribute to unveil mechanisms of disease progression and drug resistance in PC. Possible predictive factors and mechanisms of drug resistance will be investigated taking advantage of the information derived from both in vitro models and circulating tumor-related entities. Citation Format: Michela Cortesi, Tania Rossi, Michele Zanoni, Sara Bandini, Carlo Alberto Pacilio, Alessandro Cucchetti, Martina Valgiusti, Chiara Gallio, Alessandro Passardi, Giorgia Gurioli, Sara Bravaccini, Alessandra Dubini, Giovanni Luca Frassineti, Paola Ulivi, Giovanni Martinelli, Giorgio Ercolani, Ilario Giovanni Rapposelli. Integrating patient’s derived organoids and liquid biopsy: An exploratory study of early stage 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 5161.

Abstract 1840: MYBBP1A AMPylated by SELO to influence its function

Abstract Introduction: MYBBP1A, named Myb-binding protein 1A, is a protein that mainly locates in nucleolar and shuttles between the nucleus and cytoplasm, nuclear import may be mediated by KPNA2, however export depend partially on XPO1/CRM1. It activates or represses transcription via interactions with sequence specific DNA-binding proteins. Repression may be mediated at least in part by histone deacetylase activity (HDAC activity). Acts as a corepressor and in concert with CRY1, represses the transcription of the core circadian clock component PER2. Preferentially binds to di-methylated histone H3 'Lys-9' (H3K9me2) on the PER2 promoter. MYBBP1A overexpressed in tumor than normal and have a significantly negative correlation with patient overall survival. We aim to explore the role of MYBBP1A in PDAC progression and underlying mechanism. In our research, we find and prove the novel AMPylating enzyme SELO-AMPylate MYBBP1A inhibits PDAC progression and modified site in S1353 and further influence downstream relative RNA, such as 18s rRNA, 28S rRNA. Methods and Materials: 1. The function of MYBBP1A in tumorigenesis at the organismal and cellular levels. MYBBP1A is widely expressed in different tissues. Firstly, we explored the correlation between MYBBP1A expression level and PDAC through TCGA database and Kaplan-Meier plotter. We then established MYBBP1A gene knockout mice mating with PDX/P53K172H/KRASG12C to induced a primary PDAC model to analyze the progression of pancreatic cancer. The effect of MYBBP1A on tumor cell growth and proliferation was explored through cell proliferation analysis and a nude mouse tumor xenograft model. 2. The MYBBP1A AMPylated by SELO influence MYBBP1A function in cell proliferation was investigated at the molecular and cellular levels. AMPylation-specific antibodies were used to confirmed MYBBP1A AMPylated by SELO in both cell lysates and purified proteins through experiments such as CO-IP and WB. Stably expressed MYBBP1A WT/S1353A were constructed to verify the function in cell proliferation or migration by SELO. The signaling molecules were further explored through CO-IP and WB to identify the changed molecular by SELO-AMPylate-MYBBP1A, revealing the entire signaling pathways of AMPylation-mediated regulation of PDAC proliferation. Conclusion: We have demonstrated the function of MYBBP1A in the formation of pancreatic cancer, and further revealed SELO-AMPylate-MYBBP1A importance in the process of reversing MYBBP1A’s role, which has played a certain significance for the treatment of pancreatic cancer and the exploration of related therapeutic targets. Citation Format: Wu Li. MYBBP1A AMPylated by SELO to influence its function [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 1840.