Pancreatic Cancer Tumorigenesis Research Articles

PAF1/HIF1α axis rewires the glycolytic metabolism to fuel aggressiveness of pancreatic cancer

BackgroundPAF1/PD2 deregulation contributes to tumorigenesis, drug resistance, and cancer stem cell maintenance in Pancreatic Cancer (PC). Recent studies demonstrate that metabolic reprogramming plays a role in PC progression, but the mechanism is poorly understood. Here, we focused on examining the role of PAF1/PD2 in the metabolic rewiring of PC.MethodsCell lines were transfected with shRNAs to knockdown PAF1/PD2. Metabolic genes regulated by PAF1/PD2 were identified by qPCR/western blot, and metabolic assays were performed. Immunoprecipitations/ChIP were performed to identify PAF1/PD2 protein partners and confirm PAF1/HIF1α sub-complex binding to LDHA.ResultsPAF1 and LDHA showed progressively increased expression in human pancreatic tumor sections. Aerobic glycolysis genes were downregulated in PAF1-depleted PC cells. Metabolic assays indicated a decreased lactate production and glucose uptake in knockdown cells. Furthermore, PAF1/PD2 depletion showed a reduced glycolytic rate and increased oxidative phosphorylation by ECAR and OCR analysis. Interestingly, we identified that HIF1α interacts and co-localizes with PAF1, specifically in PC cells. We also observed that the PAF1/PD2-HIF1α complex binds to the LDHA promoter to regulate its expression, reprogramming the metabolism to utilize the aerobic glycolysis pathway preferentially.ConclusionOverall, the results indicate that PAF1/PD2 rewires PC metabolism by interacting with HIF1α to regulate the expression of LDHA.

Resistance to gemcitabine is mediated by the circ_0036627/miR-145/S100A16 axis in pancreatic cancer.

The development of gemcitabine (GEM) resistance severely limits the treatment efficacy in pancreatic cancer (PC) and increasing evidence highlights the vital roles of circular RNAs (circRNAs) in the tumorigenesis, progression and drug resistance of PC. However, the circRNAs underlying GEM resistance development of PC remains to be clarified. The current research aims to unveil the roles of circ_0036627 in dictating the aggressiveness and GEM sensitivity in PC. We reported the increased expression of circ_0036627 in PC tissues and PC cell lines. Elevated circ_0036627 expression level was correlated with advanced tumour grade and poor overall survival in PC patients. Functional assays and in vivo experiments demonstrated that circ_0036627 overexpression was required for the proliferation, migration invasion and GEM resistance in PC cells. circ_0036627 knockdown suppressed tumour development invivo. The molecular analysis further showed that circ_0036627 increased S100A16 expression by sponging microRNA-145 (miR-145), a tumour-suppressive miRNA that could significantly attenuate PC cell proliferation, migration, invasion and GEM resistance. Furthermore, our findings suggested that S100A16 acted as an oncogenic factor to promote aggressiveness and GEM resistance in PC cells. In conclusion, the current findings provide new mechanistic insights into PC aggressiveness and GEM resistance, suggesting the critical role of circ_0036627/miR-145/S100A16 axis in PC progression and drug resistance development and offering novel therapeutic targets for PC therapy.

OTUB1/NDUFS2 axis promotes pancreatic tumorigenesis through protecting against mitochondrial cell death

Pancreatic cancer is one of the most fatal cancers in the world. A growing number of studies have begun to demonstrate that mitochondria play a key role in tumorigenesis. Our previous study reveals that NDUFS2 (NADH: ubiquinone oxidoreductase core subunit S2), a core subunit of the mitochondrial respiratory chain complex I, is upregulated in Pancreatic adenocarcinoma (PAAD). However, its role in the development of PAAD remains unknown. Here, we showed that NDUFS2 played a critical role in the survival, proliferation and migration of pancreatic cancer cells by inhibiting mitochondrial cell death. Additionally, protein mass spectrometry indicated that the NDUFS2 was interacted with a deubiquitinase, OTUB1. Overexpression of OTUB1 increased NDUFS2 expression at the protein level, while knockdown of OTUB1 restored the effects in vitro. Accordingly, overexpression and knockdown of OTUB1 phenocopied those of NDUFS2 in pancreatic cancer cells, respectively. Mechanically, NDUFS2 was deubiquitinated by OTUB1 via K48-linked polyubiquitin chains, resulted in an elevated protein stability of NDUFS2. Moreover, the growth of OTUB1-overexpressed pancreatic cancer xenograft tumor was promoted in vivo, while the OTUB1-silenced pancreatic cancer xenograft tumor was inhibited in vivo. In conclusion, we revealed that OTUB1 increased the stability of NDUFS2 in PAAD by deubiquitylation and this axis plays a pivotal role in pancreatic cancer tumorigenesis and development.

Abstract 468: Associations of circulating miRNAs with pancreatic cancer risk differ by years between blood collection & and cancer diagnosis

Abstract Pancreatic adenocarcinoma (PDAC) is one of the most fatal cancers and currently used biomarkers for PDAC diagnosis are not adequate for early detection. Therefore, new biomarkers for PDAC early detection are urgently needed. We conducted a large-scale study to prospectively evaluate the association of circulating miRNAs with PDAC risk. Specially, we aimed to examine whether the associations of miRNAs expression levels and changes of those levels from blood collection to cancer diagnosis (BCCD) with PDAC risk. We used pre-diagnostic plasma samples of 1307 PDAC cases from 5 cohort studies (290 from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial, 395 from the Shanghai Women’s and Men’s Health Studies, 154 from the Southern Community Cohort Study, and 468 from the Multiethnic Cohort). Cancer free controls (n=1307) were selected and individually matched to index cases in each cohort. All miRNAs were measured by the NanoString nCounter Analysis System using the Human v3 miRNA Expression panel (798 miRNAs total). Data were pooled and quantile normalized prior to analysis. Associations of circulating miRNAs with PDAC risk were assessed by odds ratio per decile change in miRNA levels using conditional logistic regression. Fold-change of miRNA for cases versus controls by BCCD was analyzed via linear regression. Median time of BCCD was 7.13 years (range: 0.03-19.61 years). We first conducted the association analysis stratified by BCCD: < 5 years, 5-10 years, and >10 years. We found 20 miRNAs were significantly associated with PDAC risk in the <5 years after FDR correction. In the analysis of regressing fold-change of these 20 miRNAs on BCCD, we found the fold change of miR-155-5p was inversely associated with BCCD (β=-0.04, P<0.01), i.e., the case-control difference was larger when blood was drawn closer to PDAC diagnosis, suggesting its potential utility as disease biomarkers for early detection. Conversely, the fold-change of miR-93-5p (β=0.04, P=0.01), miR-223-3p (β=0.03, P=0.02), let-7i-5p (β=0.03, P<0.01), and miR-191-5p (β=0.05, P<0.01) were positively associated with BCCD, i.e., the case-control difference increased with an increasing the time interval of BCCD, indicating they are more likely biomarkers for PDAC risk. Ingenuity pathway analyses of these five significant miRNAs revealed that they were significantly enriched in ribonucleotide reductase signaling pathways at p<0.05. Notably, the silencing of ribonucleotide reductase M2 subunit suppresses tumorigenesis in pancreatic cancer by deactivating the PI3K/AKT/mTOR pathway, further supporting our findings. Findings of this large-scale multiethnic study suggest that changes in circulating levels of let-7i-5p, miR-155-5p, miR-93-5p, miR-191-5p, and miR-223-3p may help identify individuals at an elevated risk of developing PDAC, allowing for screening tests and close surveillance. Citation Format: Hui Cai, Veronica Wendy Setiawan, Xingyi Guo, Jie Wu, Rachael Z Stolzenberg-Solomon, Claire Zhu, Yu-Tang Gao, Jordan Berlin, Fei Ye, Qiuyin Cai, Wei Zheng, Xiao-ou Shu. Associations of circulating miRNAs with pancreatic cancer risk differ by years between blood collection & and cancer diagnosis [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 468.

Abstract 1645: Telomere-related genes associated with clinical-pathological in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) stands as one of the leading causes of cancer deaths worldwide. Studies have suggested that telomeres shortening is linked to genetic instability, higher risk of developing pancreatic cancer and pancreatic tumorigenesis. However, previous research has predominantly focused on either telomere length or specific telomere-related genes in pancreatic cancer. Therefore, it is essential to elucidate the association between telomere-related genes and pancreatic cancer to identify novel therapeutic strategies and establish prognostic biomarkers. Telomere-relevant genes were downloaded from the TelNet database. Expression data from primary tumour of The Cancer Genome Atlas-PAAD (TCGA-PAAD) cohort, the Genome Sequence Archive CRA001160 dataset and the GTEx database were obtained for screen for differentially expressed genes (DEGs). Gene set enrichment analisis was performed using the Enrichr software. The gene essentiality queried from the gene dependency database of the Achilles-DepMap project was also assessed. Finally, data of The Cancer Genome Atlas-PanCancer CaPa cohort and healthy pancreas from the GTEx database, obtained from the UCSC Xena project, were used to correlate gene expression with stemness signatures and clinical-pathological variables. We identified 300 telomere-associated DEGs, Of these, 73 genes were common to studies and they were to be enriched in biological processes related to the cell cycle and telomere homeostasis. The differentially expresed of ASS1, S100P, IGF2BP2, MET, ABCC3 and RAC1 genes were significantly associated with overall survival and recurrence-free survival, conferring them prognostic value. Furthermore, the expression profiles of these genes are significantly associated with the histological grade of the disease, as well as with tumour T-N classification, the presence of residual tumour, the degree of response to treatment and stemness signatures. Among these, three genes of particular interest (KLF3, RAC1 and ABI1) were identified and shown to be highly essential in pancreatic cancer cell lines. In summary, we have identified a telomere gene-related signature for pancreatic cancer, highlighting the prognostic value of KLF3, RAC1, and ABI1 genes. This signature is significantly associated with clinical-pathological variables of the disease, potentially offering new insights for individualized therapy. Citation Format: Erika Curiel Gómez, Vilma Maldonado Lagunas, Jorge Meléndez Zajgla. Telomere-related genes associated with clinical-pathological in 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 1645.

Fundamental insights and molecular interactions in pancreatic cancer: Pathways to therapeutic approaches

The gastrointestinal tract can be affected by a number of diseases that pancreatic cancer (PC) is a malignant manifestation of them. The prognosis of PC patients is unfavorable and because of their diagnosis at advanced stage, the treatment of this tumor is problematic. Owing to low survival rate, there is much interest towards understanding the molecular profile of PC in an attempt in developing more effective therapeutics. The conventional therapeutics for PC include surgery, chemotherapy and radiotherapy as well as emerging immunotherapy. However, PC is still incurable and more effort should be performed. The molecular landscape of PC is an underlying factor involved in increase in progression of tumor cells. In the presence review, the newest advances in understanding the molecular and biological events in PC are discussed. The dysregulation of molecular pathways including AMPK, MAPK, STAT3, Wnt/β-catenin and non-coding RNA transcripts has been suggested as a factor in development of tumorigenesis in PC. Moreover, cell death mechanisms such as apoptosis, autophagy, ferroptosis and necroptosis demonstrate abnormal levels. The EMT and glycolysis in PC cells enhance to ensure their metastasis and proliferation. Furthermore, such abnormal changes have been used to develop corresponding pharmacological and nanotechnological therapeutics for PC.

Abstract PR11: KRAS mutant-specific interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic function

Abstract Mutational activation of the KRAS proto-oncogene is the initiating mutational event in pancreatic ductal adenocarcinoma (PDAC) and ~90% of patients harbor KRAS mutations. PDAC is marred by a 12% five-year overall survival rate, and developing effective therapeutic strategies remains a priority. Accumulating evidence suggests that all KRAS mutations are not created equal. While the KRASG12R mutation is rare in lung and colorectal cancers (<1%), it is the third most common KRAS mutation in PDAC, accounting for approximately 20% of all cases. However, KRASG12R cannot interact with the lipid kinase PI3Ka, a well-characterized RAS effector necessary for KRAS-driven tumorigenesis. Previous studies in mice models of cancer have demonstrated that ablation of the KRAS:PI3Ka interaction limits tumorigenesis. Despite the inability of KRASG12R to activate PI3Ka directly, AKT signaling is robustly activated in KRASG12R-mutant PDAC. The mechanisms that allow the KRASG12R mutant to overcome the inability to activate PI3K and promote PDAC are unclear. We recently developed a Ptf1a-CREERT2;KrasLSL-G12R genetically engineered mouse model to study KRASG12R in an in vivo context. This model does not develop pancreas lesions or tumors. We have uncovered two unique characteristics of human PDAC that we hypothesize allows for the KRASG12R mutation to produce tumors only in human pancreatic tissue. First, we have found that all four PI3K isoforms are overexpressed in human PDAC, and the PI3Kd and PI3Kg isoforms are specifically upregulated in KRASG12R-mutant PDAC. Second, PTEN is oxidized in PDAC. PTEN oxidation results in an intramolecular disulfide bond, which inhibits the phosphatase activity of PTEN and leads to hyperactivated PI3K signaling. Critically, mouse pancreas tissue only expresses the PI3Ka/b isoforms and PTEN is in the reduced state, demonstrating two significant differences between mouse models and human disease. Additionally, we have determined that PTEN becomes fully oxidized in nutrient-restricted medium (low glucose/glutamine), a common strategy employed to mimic the pancreatic tumor microenvironment in cell culture. Using PTEN oxidation-resistant variants, we demonstrate that KRASG12R-mutant PDAC cell lines are reliant on oxidized (inactivated) PTEN for proliferation. To confirm that PTEN inactivation can aid KRASG12R-mediated tumorigenesis in mouse models, we generated at KrasG12R/+;Ptenfl/fl genetically engineered mouse model, and the results of this model will be presented herein. Thus, our data demonstrate that increased PI3K isoform expression, coupled with PTEN oxidation, creates a unique environment that allows KRASG12R to initiate and promote pancreatic tumorigenesis. As PTEN oxidation overcomes the need for KRAS to activate PI3K signaling in PDAC, these results indicate that directly targeting KRAS alone will be insufficient at reducing tumor growth in humans and clinically successful therapeutic strategies will have to develop alternative methods to reduce PI3K signaling in addition to directly targeting KRAS activity. Citation Format: Kamala Sundararaj, Rachel Burge, Samaneh Saberikashani, Lucas Bialousow, Amanda Linke, Merissa Smith, Michael C. Ostrowski, John P. O'Bryan, G. Aaron Hobbs. KRAS mutant-specific interactions reveal mechanisms in pancreatic cancer tumorigenesis and metabolic function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR11.

Abstract C100: Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis

Abstract Biological stresses that are cell-intrinsic (e.g., oncogene-induced imbalances in protein translation, lipid synthesis, mitochondrial dysfunction) or cell-extrinsic (e.g., nutrient deprivation, hypoxia, drug exposure) disrupt cellular function and viability if not mitigated. The Integrated Stress Response (ISR) consists of upstream regulatory kinases (PERK, GCN2, HRI, PKR) that respond to such stresses in part by activating the ATF4 transcription factor. ATF4 then rewires nutrient uptake, cellular metabolism, mitochondrial function, and protein chaperone networks to restore cellular homeostasis. We have shown that ATF4 is upregulated in mouse and human pancreatic ductal adenocarcinoma (PDAC), even compared to normal acinar cells where ATF4 is expressed at relatively high levels. To investigate the role of ATF4 in PDAC, we generated mice with inducible acinar-specific deletion of Atf4 using Ptf1a-CreERT to delete Atf4 following treatment of adult Atf4-floxed mice with tamoxifen. In contrast to previous studies showing germline deletion of Atf4 in mice caused premature mortality at approximately 3 weeks of age due to pancreatic insufficiency, inducible deletion of Atf4 in adult acinar cells does not cause pancreatic deficiency or animal demise. Rather, adult Ptf1a-CreERT;Atf4fl/fl (CA) mice are viable although more sensitive to cerulein-induced pancreatitis. Specifically, compared to control (WT) mice, CA mice exhibit more extensive acinar-ductal metaplasia (ADM), including more extensive expression of cytokeratin-19 (CK19), increased cell death and take longer to recover normal pancreatic architecture following in vivo cerulein challenge. Using ex vivo acinar explant assays derived from WT and CA mice, we further show that induction of ADM in WT acinar cells with TGF-a is associated with down-regulation of Atf4 and Atf4 target genes in WT acinar explants while CA explants undergo accelerated ADM. Furthermore, expression of exogenous Atf4 in acinar cells inhibited ADM. These in vivo and ex vivo findings are consistent with down-regulation of Atf4 being required for ADM. Given the tumor-promoting role of ADM in the early stages of PDAC, we next crossed the CA mice to LSL-KRasG12D mice to generate LSL-KRasG12D;Ptf1a-CreERT;Atf4fl/fl (KCA) mice to examine how loss of Atf4 affected pancreatic intraepithelial neoplasia (PanIN) and PDAC formation. Surprisingly, given the critical need to down-regulate Atf4 in ADM, we show for the first time that genetic deletion of Atf4 in pancreatic acinar cells blocks both PanIN and PDAC formation in KCA mice compared to age-matched KC mice. Rather, KCA mice developed a fatty pancreas devoid of tumor with very few CK19-positive cells forming. These results suggest that Atf4 is critical for PanIN formation and prevents PDAC. This also suggests that Atf4 plays a different role in PanIN formation than in ADM where its down-regulation was critical and indicates a dynamic role for Atf4 depending on oncogenic versus proteotoxic stress, as will be discussed. Citation Format: Leah M. Ziolkowski, Kay F. Macleod. Novel functions of ATF4 in early stages of pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C100.

A novel NR4A2-HuR axis promotes pancreatic cancer growth and tumorigenesis that is inhibited by NR4A2 antagonists

A novel NR4A2-HuR axis promotes pancreatic cancer growth and tumorigenesis that is inhibited by NR4A2 antagonists

Overexpression of ZNF488 supports pancreatic cancer cell proliferation and tumorigenesis through inhibition of ferroptosis via regulating SCD1-mediated unsaturated fatty acid metabolism

BackgroundPancreatic cancer is a malignancy with high mortality. Once diagnosed, effective treatment strategies are limited and the five-year survival is extremely poor. Recent studies have shown that zinc finger proteins play important roles in tumorigenesis, including pancreatic cancer. However, it remains unknown on the clinical significance, function and underlying mechanisms of zinc finger protein 488 (ZNF488) during the development of pancreatic cancer.MethodsThe clinical relevance of ZNF488 and stearoyl-CoA desaturase 1 (SCD1) was examined by analyzing the data from The Cancer Genome Atlas (TCGA) and immunohistochemical staining of the tissue microarray. Gain-of-function and loss-of-function experiments were performed by transfecting the cells with overexpressing lentivirus and siRNAs or shRNA lentivirus, respectively. The function of ZNF488 in pancreatic cancer was assessed by CCK8, colony formation, EdU staining, PI/Annexin V staining and xenografted tumorigenesis. Chip-qPCR assay was conducted to examine the interaction between ZNF488 and the promoter sequence of SCD1. Transcription activity was measured by dual luciferase reporter assay. mRNA and protein expression was detected by qRT-PCR and immunoblotting experiment, respectively. Fatty acid was quantified by gas chromatography mass spectrometry.ResultsZNF488 was overexpressed in pancreatic cancer samples compared with normal tissues. High expression of ZNF488 predicted the poor prognosis of the patients. In vitro, ZNF488 upregulation contributed to the EuU cooperation, proliferation and colony formation of MIAPaCa-2 and PANC-1 cells. Based on PI/Annexin V and trypan blue staining results, we showed that ZNF488 suppressed the ferroptosis and apoptosis of pancreatic cancer cells. Mechanistically, ZNF488 directly interacted with the promoter sequence of SCD1 gene and promoted its transcription activity, which resulted in enhanced palmitoleic and oleic acid production, as well as the peroxidation of fatty acid. In vivo, ZNF488 overexpression promoted the xenograted tumorigenesis of PANC-1, which was reversed by SCD1 knockdown. Importantly, combination of erastin and SCD1 inhibitors A939572 completely blunted the growth of ZNF488 overexpressed MIAPaCa-2 and PANC-1 cells. Usage of A939572 or erastin recovered the sensitivity of pancreatic cancer cells to the treatment of gemcitabine. Lastly, we found a positive correlation between ZNF488 and SCD1 in pancreatic cancer patients based on TCGA and immunohistochemical staining results.ConclusionOverexpression of ZNF488 suppresses the ferroptosis and apoptosis to support the growth and tumorigenesis of pancreatic cancer through augmentation of SCD1-mediated unsaturated fatty acid metabolism. Combination of SCD1 inhibitors, ferroptosis inducers or gemcitabine could be applied for the treatment of pancreatic cancer with overexpression of ZNF488.

Combination therapy of niclosamide with gemcitabine inhibited cell proliferation and apoptosis via Wnt/β-catenin/c-Myc signaling pathway by inducing β-catenin ubiquitination in pancreatic cancer

ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with high morbidity and mortality rates worldwide. Owing to a lack of therapeutic options, the overall survival rate of patients with pancreatic cancer is low. Gemcitabine has been mainly used to treat patients with pancreatic cancer, but its efficacy is limited by chemoresistance. Therefore, a novel therapeutic agent for PDAC therapy is urgently needed. An anthelminthic drug, niclosamide, has already been researched in breast, lung, colon, and pancreatic cancer as an anti-cancer purpose by re-positioning its original purpose. However, combination therapy of gemcitabine and niclosamide was not informed yet. Here, we found that niclosamide co-administered with gemcitabine significantly inhibited tumorigenesis of pancreatic cancer compared to gemcitabine alone. Further, combining niclosamide and gemcitabine inhibited cell proliferation and induced apoptosis. Niclosamide induced cell cycle arrest at the G1 phase, and the levels of CDK4/6 and cyclin D1 were lowered after gemcitabine treatment. In addition, the combination of these chemical compounds more effectively increased the binding level of activated β-catenin destruction complex and β-catenin to enable phosphorylation, compared to gemcitabine alone. After phosphorylation, niclosamide – gemcitabine upregulated the ubiquitin level, which caused phosphorylated β-catenin to undergo proteasomal degradation; the combination was more potent than gemcitabine alone. Finally, the combination more effectively suppressed tumor growth in vivo, compared to gemcitabine alone. Altogether, our results indicate that niclosamide synergistically enhances the antitumor effect of gemcitabine in pancreatic cancer, by inducing the degradation of β-catenin with ubiquitination. Therefore, this drug combination can potentially be used in PDAC therapy.

A novel role of TGFBI in macrophage polarization and macrophage-induced pancreatic cancer growth and therapeutic resistance

Tumor-associated macrophages (TAMs), as a major and essential component of tumor microenvironment (TME), play a critical role in orchestrating pancreatic cancer (PaC) tumorigenesis from initiation to angiogenesis, growth, and systemic dissemination, as well as immunosuppression and resistance to chemotherapy and immunotherapy; however, the critical intrinsic factors responsible for TAMs reprograming and function remain to be identified. By performing single-cell RNA sequencing, transforming growth factor-beta-induced protein (TGFBI) was identified as TAM-producing factor in murine PaC tumors. TAMs express TGFBI in human PaC and TGFBI expression is positively related with human PaC growth. By inducing TGFBI loss-of-function in macrophage (MΦs) in vitro with siRNA and in vivo with Cre-Lox strategy in our developed TGFBI-floxed mice, we demonstrated disruption of TGFBI not only inhibited MΦ polarization to M2 phenotype and MΦ-mediated stimulation on PaC growth, but also significantly improved anti-tumor immunity, sensitizing PaC to chemotherapy in association with regulation of fibronectin 1, Cxcl10, and Ccl5. Our studies suggest that targeting TGFBI in MΦ can develop an effective therapeutic intervention for highly lethal PaC.

PSMC2 is a Novel Prognostic Biomarker and Predicts Immunotherapeutic Responses: From Pancreatic Cancer to Pan-Cancer.

Proteasome 26S subunit ATPase 2 (PSMC2) is a part of the 19S regulatory complex, which catalyzes the unfolding and transport of substrates into the 20S proteasome. Our previous research demonstrated that PSMC2 participates in the tumorigenesis and progression of pancreatic cancer (PC). However, no systematic analysis has been conducted to conclude its expression pattern and correlation with tumor immunity. To investigate the expression level of PSMC2 in PC, its prognostic value and its relationship with tumor immunity. In numerous public and internal cohorts, the expression, prognostic significance, and immunological connections of PSMC2 in PC were investigated. Additionally, using data from The Cancer Genome Atlas (TCGA), a pan-cancer analysis was carried out to examine PSMC2's immunological assocaition, and the predictive power of PSMC2 for immunotherapy was also evaluated in numerous public cohorts. PSMC2 was overexpressed in tumor tissues and linked to unfavorable prognosis in PC. PSMC2 was not only positively correlated with TIICs, also positively correlated with immune checkpoints in PC. In addition to PC, PSMC2 was expected to be an indicator of high immunogenicity in most cancer types. Importantly, PSMC2 could predict the immunotherapeutic responses in various cancer types, including urothelial carcinoma and breast cancer. From PC to pan-cancer analysis, we report that PSMC2 is a novel prognostic biomarker in multiple cancer types. PSMC2 is related to the immuno-hot phenotype and predicts the outcome of immunotherapy. Therefore, the current study emphasizes that cancer patients with high PMSC2 expression should actively receive immunotherapy to improve their prognosis.

LncRNAs associated with vascular mimicry establish a novel molecular subtype and prognostic model for pancreatic cancer.

Vascular mimicry (VM) epitomizes an innovative tumor angiogenesis pathway, potentially serving as an alternate conduit under the assumption of traditional tumor angiogenesis pathway inhibition. The role of VM in pancreatic cancer (PC), however, remains unexplored. Using differential analysis and Spearman correlation, we identified key long non-coding RNAs (lncRNAs) signatures in PC from the collected set of VM-associated genes in the literature. We identified optimal clusters using the non-negative matrix decomposition (NMF) algorithm, and then compared clinicopathological features and prognostic differences between clusters. We also assessed tumor microenvironmental (TME) differences between clusters using multiple algorithms. Using univariate Cox regression analyses as well as lasso regression, we constructed and validated new lncRNA prognostic risk models for PC. We used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to analyze model-enriched functions and pathways. Nomograms were then developed to predict patient survival in association with clinicopathological factors. In addition, single-cell RNA-sequencing (scRNA-seq) analysis was used to analyze the expression patterns of VM-related genes and lncRNAs in the PC of TME. Finally, we used the Connectivity Map (cMap) database to predict local anaesthetics that could modify the VM of PC. In this study, we developed a novel three-cluster molecular subtype using the identified VM-associated lncRNA signatures of PC. The different subtypes have significantly different clinical characteristics and prognostic value, and also show differential treatment response and TME. Following an in-depth analysis, we constructed and validated a novel prognostic risk model for PC based on the VM-associated lncRNA signatures. Enrichment analysis suggested that high riskscores were significantly associated with functions and pathways, including extracellular matrix remodeling, et al. In addition, we predicted eight local anaesthetics that could modulate VM in PC. Finally, we discovered differential expression of VM-related genes and lncRNAs across various cell types within pancreatic cancer. VM has a critical role in PC. This study pioneers the development of a VM-based molecular subtype that demonstrates substantial differentiation in PC populations. Furthermore, we highlighted the significance of VM within the immune microenvironment of PC. Moreover, VM might contribute to PC tumorigenesis through its mediation of mesenchymal remodeling and endothelial transdifferentiation-related pathways, which offers a new perspective on its role in PC.

A bioinformatics analysis, pre-clinical and clinical conception of autophagy in pancreatic cancer: Complexity and simplicity in crosstalk

Pancreatic cancer (PC) is a serious gastrointestinal tract disease for which the 5-year survival rate is less than 10%, even in developed countries such as the USA. The genomic profile alterations and dysregulated biological mechanisms commonly occur in PC. Macroautophagy/autophagy is a cell death process that is maintained at a basal level in physiological conditions, whereas its level often changes during tumorigenesis. The function of autophagy in human cancers is dual and can be oncogenic and onco-suppressor. Autophagy is a potent controller of tumorigenesis in PC. The supportive autophagy in PC escalates the growth rate of PC cells and its suppression can mediate cell death. Autophagy also determines the metastasis of PC cells, and it can control the EMT in affecting migration. Moreover, starvation and hypoxia can stimulate glycolysis, and glycolysis induction can be mediated by autophagy in enhancing tumorigenesis in PC. Furthermore, protective autophagy stimulates drug resistance and gemcitabine resistance in PC cells, and its inhibition can enhance radiosensitivity. Autophagy can degrade MHC-I to mediate immune evasion and also regulates polarization of macrophages in the tumor microenvironment. Modulation of autophagy activity is provided by silibinin, ursolic acid, chrysin and huaier in the treatment of PC. Non-coding RNAs are also controllers of autophagy in PC and its inhibition can improve therapy response in patients. Moreover, mitophagy shows dysregulation in PC, which can enhance the proliferation of PC cells. Therefore, a bioinformatics analysis demonstrates the dysregulation of autophagy-related proteins and genes in PC as biomarkers.

P53 inhibitor iASPP is an unexpected suppressor of KRAS and inflammation-driven pancreatic cancer

Oncogenic KRAS activation, inflammation and p53 mutation are key drivers of pancreatic cancer (PC) development. Here we report iASPP, an inhibitor of p53, as a paradoxical suppressor of inflammation and oncogenic KRASG12D-driven PC tumorigenesis. iASPP suppresses PC onset driven by KRASG12D alone or KRASG12D in combination with mutant p53R172H. iASPP deletion limits acinar-to-ductal metaplasia (ADM) in vitro but accelerates inflammation and KRASG12D-induced ADM, pancreatitis and PC tumorigenesis in vivo. KRASG12D/iASPPΔ8/Δ8 tumours are well-differentiated classical PCs and their derivative cell lines form subcutaneous tumours in syngeneic and nude mice. Transcriptomically, either iASPP deletion or p53 mutation in the KRASG12D background altered the expression of an extensively overlapping gene set, comprised primarily of NF-κB and AP1-regulated inflammatory genes. All these identify iASPP as a suppressor of inflammation and a p53-independent oncosuppressor of PC tumorigenesis.

The role of amino acid metabolism alterations in pancreatic cancer: From mechanism to application.

The incidence of pancreatic cancer is increasing in both developed and developing Nations. In recent years, various research evidence suggested that reprogrammed metabolism may play a key role in pancreatic cancer tumorigenesis and development. Therefore, it has great potential as a diagnostic, prognostic and therapeutic target. Amino acid metabolism is deregulated in pancreatic cancer, and changes in amino acid metabolism can affect cancer cell status, systemic metabolism in malignant tumor patients and mistakenly involved in different biological processes including stemness, proliferation and growth, invasion and migration, redox state maintenance, autophagy, apoptosis and even tumor microenvironment interaction. Generally, the above effects are achieved through two pathways, energy metabolism and signal transduction. This review aims to highlight the current research progress on the abnormal alterations of amino acids metabolism in pancreatic cancer, how they affect tumorigenesis and development of pancreatic cancer and the application prospects of them as diagnostic, prognostic and therapeutic targets.

Clustering analysis and prognostic model based on PI3K/AKT-related genes in pancreatic cancer.

Pancreatic cancer is one of most aggressive malignancies with a dismal prognosis. Activation of PI3K/AKT signaling is instrumental in pancreatic cancer tumorigenesis. The aims of this study were to identify the molecular clustering, prognostic value, relationship with tumor immunity and targeting of PI3K/AKT-related genes (PARGs) in pancreatic cancer using bioinformatics. The GSEA website was searched for PARGs, and pancreatic cancer-related mRNA data and clinical profiles were obtained through TCGA downloads. Prognosis-related genes were identified by univariate Cox regression analysis, and samples were further clustered by unsupervised methods to identify significant differences in survival, clinical information and immune infiltration between categories. Next, a prognostic model was constructed using Lasso regression analysis. The model was well validated by univariate and multivariate Cox regression analyses, Kaplan-Meier survival analysis and ROC curves, and correlations between risk scores and patient pathological characteristics were identified. Finally, GSEA, drug prediction and immune checkpoint protein analyses were performed. Pancreatic cancers were divided into Cluster 1 (C1) and Cluster 2 (C1) according to PARG mRNA expression. C1 exhibited longer overall survival (OS) and higher immune scores and CTLA4 expression, whereas C2 exhibited more abundant PD-L1. A 6-PARG-based prognostic model was constructed to divide pancreatic cancer patients into a high-risk score (HRS) group and a low-risk score (LRS) group, where the HRS group exhibited worse OS. The risk score was defined as an independent predictor of OS. The HRS group was significantly associated with pancreatic cancer metastasis, aggregation and immune score. Furthermore, the HRS group exhibited immunosuppression and was sensitive to radiotherapy and guitarbine chemotherapy. Multidrug sensitivity prediction analysis indicated that the HRS group may be sensitive to PI3K/AKT signaling inhibitors (PIK-93, GSK2126458, CAL-101 and rapamycin) and ATP concentration regulators (Thapsigargin). In addition, we confirmed the oncogenic effect of protein phosphatase 2 regulatory subunit B'' subunit alpha (PPP2R3A) in pancreatic cancer in vitro and in vivo. PARGs predict prognosis, tumor immune profile, radiotherapy and chemotherapy drug sensitivity and are potential predictive markers for pancreatic cancer treatment that can help clinicians make decisions and personalize treatment.

Abstract 1485: Elucidating the role of VGLL1 in pancreatic cancer tumorigenesis

Abstract Vestigial-like 1 (VGLL1) is a co-transcriptional activator that binds to TEA domain-containing transcription factors (TEADs). Its expression is upregulated in a variety of cancers and is correlated with decreased overall patient survival. In normal tissues, VGLL1 is most highly expressed in placental trophoblast cells, which share attributes of rapid cellular proliferation and invasion with cancer cells. Binding of VGLL1 to TEADs shows structural similarity to co-transcriptional activators in the Hippo pathway, yes-associated protein 1 (YAP) and WW Domain containing transcription regulator 1 (WWTR1/TAZ). YAP/TAZ has been implicated in tumorigenesis in many cancer types, and this structural relationship has provided insights into the functional role of VGLL1. The impact of VGLL1 expression in pancreatic cancer has not been fully elucidated and no VGLL1-targeted therapy currently exists. The aim of this study was to evaluate the function and downstream targets of VGLL1 in pancreatic cancer cells. Ectopic expression of VGLL1 in human pancreatic cell lines PANC1 and SU8686 stimulated cancer cell proliferation and invasion, consistent with findings from other cancer types. ChIP-seq and Nanostring assays were also performed to identify VGLL1 downstream target genes and pathways. Interestingly, analysis of ChIP-seq data from VGLL1-transduced PANC1 cells identified four novel transcription factors with a VGLL1 binding motif, in addition to TEAD4 which has been previously reported. Furthermore, gene array analysis showed approximately 150 differentially expressed genes in mouse pancreatic cancer cells transduced with VGLL1, including genes involved in regulation of cell cycle and apoptosis. Ectopic VGLL1 expression resulted in the increased expression of AP-1, MMP9, and VEGF, genes associated with cancer progression. Furthermore, VGLL1 decreased the expression of cyclin-dependent kinase inhibitors such as p15, p16 and p21 that are associated with reduced cancer cell proliferation. To identify potential regulatory features of VGLL1, we performed mass spectrometry to identify phosphorylation sites and potential binding partners. Our preliminary data suggests that 14-3-3 binds to VGLL1, an intriguing finding since 14-3-3 is also known to regulate YAP/TAZ by sequestering it in the cytoplasm upon being phosphorylated. Our results demonstrate that VGLL1 interacts with specific transcription factors such as TEAD4 in pancreatic cancer cells to modulate the expression of genes expected to promote cell proliferation and invasion. In addition, we have identified a potential regulatory molecule for VGLL1 that plays a critical role in inhibiting other co-transcription activators from translocating into the nucleus. It is hoped that this work will set the stage for the development of VGLL1-targeted therapies with which to treat patients with pancreatic cancer. Citation Format: Heather M. Sonnemann, Barbara Pazdrak, Barbara Nassif, Yimo Sun, Lama Elzohary, Amjad Talukder, Arjun Katailiha, Salah-Eddine Bentebibel, Adi Diab, Gregory Lizee. Elucidating the role of VGLL1 in pancreatic cancer tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1485.

Abstract 3701: Targeting wild-type IDH1 enhances chemosensitivity in pancreatic cancer

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer mortality in the United States. The poor prognosis is in part due to development of chemotherapy resistance, despite improvements in multiagent regimens. Our previous work demonstrated that oxidative stress plays an important role in drug resistance. Wild-type isocitrate dehydrogenase 1 (IDH1) is an important enzyme that generates cytosolic NADPH to maintain redox homeostasis and protect cancer cells from oxidative damage. Additionally, we demonstrated that ivosidenib (AG-120), an FDA-approved mutant IDH1 inhibitor, is actually a potent inhibitor of wild-type IDH1, under low magnesium and nutrient levels that are present in the tumor microenvironment. Methods: We evaluated IDH1 expression in PDAC using The Cancer Genome Atlas (TCGA). Cell viability was assessed by Trypan blue and PicoGreen in drug combination assays. Cellular reactive oxygen species (ROS) levels were determined by the DCFDA method. To further assess the therapeutic potential of AG-120 in combination with chemotherapy, tumor volume analyses were performed using patient-derived xenografts (PDX) in athymic nude mice, and survival studies were performed in C57BL/6J mice transplanted with orthotopic murine pancreatic cancer. Results: Analysis of TCGA data indicated that IDH1 is overexpressed in pancreatic cancer tumors. Treatment of MiaPaca2 and Panc1 cancer cells with 5- fluorouracil (5-FU) induced expression of wild-type IDH1 in vitro. Short-term cell viability data demonstrated that targeting IDH1 with AG-120 when combined with DNA-damaging agents (5-FU, oxaliplatin) had a synergistic effect with a positive synergy score and Bliss score greater than 1. Additionally, we assessed long-term cell survival using colony formation assays, which yielded a dramatic reduction in cell survival for both Panc1 and MiaPaCa-2 cells when 5-FU was combined with AG-120, as compared to single-agent controls. Inhibiting IDH1 impairs the ability of pancreatic cancer cells to scavenge ROS levels, enhances chemotherapy-induced apoptosis in pancreatic cancer cells via ROS-mediated damage in vitro. Both PDX tumor volume studies and overall survival analyses revealed that the combination of these AG-120 and chemotherapy synergistically enhanced anti-tumor activity and doubled the survival benefits as compared to single-agent alone. Conclusion: IDH1 plays a critical role in tumorigenesis and chemoresistance in pancreatic cancer. Our data demonstrate that IDH1 inhibition with AG-120 may enhance chemotherapy efficacy and represents an important area for future investigation in the form of clinical trials. Citation Format: Mehrdad Zarei, Omid Hajihassani, Jonathan J. Hue, Hallie J. Graor, Arian Hajihassani, Alexander W. Loftus, Luke D. Rothermel, Jordan M. Winter. Targeting wild-type IDH1 enhances chemosensitivity in pancreatic cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3701.