Expression In Pancreatic Cancer Cells Research Articles

TYROBP promotes the spread of pancreatic cancer by causing M2 TAM polarization.

M2-polarized tumor-associated macrophages (M2 TAMs) are known to promote cancer progression, and exosomes are crucial mediators of communication within the tumor microenvironment (TME). However, the specific role of exosomes derived from M2 TAMs in pancreatic cancer (PC) progression remains poorly understood. Tyrosine kinase binding protein (TYROBP, also known as DAP12 for DNAX activating protein-12) is a transmembrane signal transduction polypeptide that interacts with immune cell receptors, influencing cellular functions via signal transduction pathways. TYROBP is prominently found in M2 TAMs exosomes, facilitating its transfer to PC cells and suggesting a potential role in PC pathogenesis. This study initially confirmed the presence of TYROBP in M2 TAMs exosomes and its transfer to PC cells via exosomes. The impact of TYROBP on PC proliferation, apoptosis, migration, and invasion was investigated. Special attention was given to TYROBP's influence on PC metastasis and its underlying mechanisms, focusing particularly on the CD44/AKT/ERK signaling pathway. TYROBP expression in PC cells did not significantly affect tumor cell proliferation or apoptosis but demonstrated a notable inhibitory effect on migration and invasion, which was mediated through the CD44/AKT/ERK pathway. Both in vivo and in vitro experiments consistently showed that TYROBP enhanced PC metastasis. This study elucidates that TYROBP plays a direct role in promoting PC metastasis through its association with M2 TAMs polarization. Therefore, TYROBP represents a potential novel therapeutic target for interventions aimed at combatting PC progression.

Abstract A052: Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy

Abstract Introduction: PDAC is marked by a fibroinflammatory stroma and thrives on interactions between cancer cells and their microenvironment, including CAFs and immune cells, driving disease progression and treatment resistance. Using ChIP-seq on resected human PDAC samples, we identified MICAL2 as a super-enhancer-associated gene and its role in tumor progression. MICAL2 is a flavin monooxygenase that promotes actin depolymerization and SRF transcription. Here, we evaluated how tumor cell-expressed MICAL2 rewires the PDAC microenvironment through the IL1-a/p38 MAP kinase/STAT3 axis. Methods: KPC-Shcontrol (ShCNT) and MICAL2 (M2KD) were orthotopically injected to assess tumor growth. Sc-RNA seq, immunophenotyping, and immunohistochemistry were performed on ShCNT and M2KD tumors. Atomic force microscopy was done to assess tissue stiffness. CD8+ specific antibodies were used for T-cell depletion. Adoptive transfer of CD8+ T cells enriched from M2KD tumors was performed by tail vein injection. Anti-PD1 and anti-IL-1a antibodies were injected IP. RNA-Seq analysis was performed on human PDAC cells (AsPC-1 ShCNT and M2KD) and validation of IL-1α and IL-1R expression was done in human and mouse pancreas control, KD, OE cancer cell lines, and PSCs by q-PCR. We exposed PSCs to conditioned media from cancer cells with and without MICAL2 and checked the expression of genes related to inflammation and fibrosis by qPCR. Results: Orthotopic injections of KPC-M2KD cells led to decreased tumor growth compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Sc-RNA and immunohistochemistry revealed reduced PDPN+ and a-SMA+ CAFs in M2KD tumors. We also observed less collagen and fibronectin deposition in M2KD tumors resulting in reduced tissue stiffness as measured by atomic force microscopy. Flow cytometry and immunofluorescence showed increased intertumoral infiltration of cytotoxic and effector CD8+T cells in M2KD tumors. CD8+T cell depletion reversed growth inhibition in M2KD tumors. Adoptive transfer of CD8+T cells enriched from M2KD tumors impeded control tumor growth. RNA-seq revealed decreased IL1-α expression in M2KD cells (p<0.05). IL1R expression was reduced on PSCs when cocultured with M2KD vs. control cell media. PSCs co-cultured with M2KD cancer cells also showed significant downregulation of genes including IL-6, Cxcl1, and LIF changes that were rescued by adding recombinant IL-1α in M2KD cells. Western blot showed MICAL2 regulates the phosphorylation of p38 MAP kinase which in turn regulates the expression of IL1-α and STAT3 activation confirmed by treating dox-inducible ShCNT and ShM2KD cancer cells with p38 inhibitor. Treating M2KD tumors with immune checkpoint blockade PD-1 alone significantly reduced tumor size and 50% of M2KD tumor-bearing mice had complete tumor regression after treatment with PD-1 and neutralizing IL-1a antibodies. Conclusion: MICAL2 mediates tumor-stromal crosstalk through the IL1-a/p38/STAT3 axis and creates an immunosuppressive environment in PDAC. MICAL2 may be a potential immunomodulatory target for pancreatic cancer therapy. Citation Format: Bharti Garg, Evangeline Mose, Edgar Esparaza, Jay Patel, Rithika Medari, Alexei Martsinkovskiy, Carrie Bishop, Gissele Gonzalez, Adam Engler, Parag Katira, Herve Tiriac, Andrew M Lowy. Silencing MICAL2 Expression in Pancreatic Cancer Cells rewires the tumor microenvironment through the IL1-a/p38 MAP kinase/STAT-3 axis and Sensitizes Tumors to Immune Checkpoint Blockade therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A052.

Curcumin Inhibits the Development of Pancreatic Cancer by Targeting the circ_0079440/miR-522-3p/EIF4A1 Pathway.

Pancreatic cancer (PC) is a common gastrointestinal cancer with high invasiveness and high mortality. Curcumin is a natural polyphenol with anti-tumor activity against different cancers, including PC. Curcumin has been verified to mediate the expression of circular RNAs (circRNAs) to inhibit tumor development. This study aimed to explore the function and regulatory mechanism of curcumin on circ_0079440 in PC. PC cells were treated with different concentrations of curcumin (0, 5, 10 or 15 μM) for 24 h. Gene expression in PC cells and tissues was detected using RT-qPCR. Cell malignant phenotypes were determined by functional assays. The levels of EMT-related proteins were tested using western blot. RNA interaction was determined using RNA pulldown assay, luciferase reporter assay and RIP assay. The results showed that curcumin suppressed cell proliferative, migratory, and invasive capabilities, and weakened epithelial-mesenchymal transition (EMT) in a concentration-dependent way. Circ_0079440 was expressed at a high level in PC and its level was reduced via curcumin administration in PC cells. Rescue assays showed that circ_0079440 overexpression reversed the suppressive effects of curcumin on PC cell malignant phenotypes. Furthermore, in the xenograft mouse models, curcumin treatment inhibited tumor growth and metastasis, and circ_0079440 upregulation reversed the function of curcumin. Additionally, circ_0079440 was revealed to bind to miR-522-3p to upregulate eukaryotic initiation factor 4A1 (EIF4A1) expression in PC cells. EIF4A1 expression was also downregulated by curcumin, and EIF4A1 overexpression abolished the suppressive functions of curcumin. Moreover, EIF4A overexpression or miR-522-3p inhibition counteracted the anti-tumor effects of circ_0079440 depletion on PC development. To sum up, curcumin suppresses PC development by targeting the circ_0079440/miR-522-3p/EIF4A1 pathway, which might provide novel therapeutic targets for treatment of PC.

Tumor-derived exosome PPP3CB induce gemcitabine resistance by regulating miR-298/STAT3 in pancreatic cancer

PurposeDue to resistance to gemcitabine (GEM), patients with pancreatic cancer (PC) usually have poor prognosis and low survival rate. The purpose of our research was to explore the impact of exosome PPP3CB on GEM resistance in PC, and concurrently analyze the regulatory role of the miR-298/STAT3 signaling pathway. MethodsExosomes isolated from PC cells were verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting (WB). The interaction between PPP3CB and miR-298 was verified using dual-luciferase reporter gene assay, followed by evaluation of cell growth and death using CCK8 assay, EdU staining, and flow cytometry. ResultsIncreased PPP3CB expression was observed in GEM-resistant PC cells. Exosomes from PC cells and GEM-resistant PC cells were successfully extracted by ultra-high speed centrifugation. Confocal microscopy showed internalization of fluorescein amide (FAM)-labeled GEM-resistant exosomes by PC cells. PPP3CB enhanced the proliferation of GEM-resistant PC cells and inhibited their apoptosis, whereas down-regulation of PPP3CB promoted the death of PC cells and inhibited the proliferation of GEM-resistant PC cells, and enhance the susceptibility of PC cells to GEM. Additionally, PPP3CB positively regulated STAT3 expression in PC cells by down-regulating miR-298, thus promoting the growth and inhibiting the death of PC cells. ConclusionPC cell-derived exosome PPP3CB enhances STAT3 expression by downregulating miR-298, stimulating cell growth, and suppressing cell death, thereby increasing the resistance of PC cells to GEM.

SIRT7 knockdown promotes gemcitabine sensitivity of pancreatic cancer cell via upregulation of GLUT3 expression

Gemcitabine serves as a first-line chemotherapeutic treatment for pancreatic cancer (PC), but it is prone to rapid drug resistance. Increasing the sensitivity of PC to gemcitabine has long been a focus of research. Fasting interventions may augment the effects of chemotherapy and present new options. SIRT7 is known to link metabolism with various cellular processes through post-translational modifications. We found upregulation of SIRT7 in PC cells is associated with poor prognosis and gemcitabine resistance. Cross-analysis of RNA-seq and ATAC-seq data suggested that GLUT3 might be a downstream target gene of SIRT7. Subsequent investigations demonstrated that SIRT7 directly interacts with the enhancer region of GLUT3 to desuccinylate H3K122. Our group's another study revealed that GLUT3 can transport gemcitabine in breast cancer cells. Here, we found GLUT3 KD reduces the sensitivity of PC cells to gemcitabine, and SIRT7 KD-associated gemcitabine-sensitizing could be reversed by GLUT3 KD. While fasting mimicking induced upregulation of SIRT7 expression in PC cells, knocking down SIRT7 enhanced sensitivity to gemcitabine through upregulating GLUT3 expression. We further confirmed the effect of SIRT7 deficiency on the sensitivity of gemcitabine under fasting conditions using a mouse xenograft model. In summary, our study demonstrates that SIRT7 can regulate GLUT3 expression by binding to its enhancer and altering H3K122 succinylation levels, thus affecting gemcitabine sensitivity in PC cells. Additionally, combining SIRT7 knockdown with fasting may improve the efficacy of gemcitabine. This unveils a novel mechanism by which SIRT7 influences gemcitabine sensitivity in PC and offer innovative strategies for clinical combination therapy with gemcitabine.

Thymoquinone affects hypoxia-inducible factor-1α expression in pancreatic cancer cells via HSP90 and PI3K/AKT/mTOR pathways.

Pancreatic cancer (PC) is associated with some of the worst prognoses of all major cancers. Thymoquinone (TQ) has a long history in traditional medical practice and is known for its anti-cancer, anti-inflammatory, anti-fibrosis and antioxidant pharmacological activities. Recent studies on hypoxia-inducible factor-1α (HIF-1α) and PC have shown that HIF-1α affects the occurrence and development of PC in many aspects. In addition, TQ could inhibit the development of renal cancer by decreasing the expression of HIF-1α. Therefore, we speculate whether TQ affects HIF-1α expression in PC cells and explore the mechanism. To elucidate the effect of TQ in PC cells and the regulatory mechanism of HIF-1α expression. Cell counting kit-8 assay, Transwell assay and flow cytometry were performed to detect the effects of TQ on the proliferative activity, migration and invasion ability and apoptosis of PANC-1 cells and normal pancreatic duct epithelial (hTERT-HPNE) cells. Quantitative real-time polymerase chain reaction and western blot assay were performed to detect the expression of HIF-1α mRNA and protein in PC cells. The effects of TQ on the HIF-1α protein initial expression pathway and ubiquitination degradation in PANC-1 cells were examined by western blot assay and co-immunoprecipitation. TQ significantly inhibited proliferative activity, migration, and invasion ability and promoted apoptosis of PANC-1 cells; however, no significant effects on hTERT-HPNE cells were observed. TQ significantly reduced the mRNA and protein expression levels of HIF-1α in PANC-1, AsPC-1, and BxPC-3 cells. TQ significantly inhibited the expression of the HIF-1α initial expression pathway (PI3K/AKT/mTOR) related proteins, and promoted the ubiquitination degradation of the HIF-1α protein in PANC-1 cells. TQ had no effect on the hydroxylation and von Hippel Lindau protein mediated ubiquitination degradation of the HIF-1α protein but affected the stability of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90, thus promoting its ubiquitination degradation. The regulatory mechanism of TQ on HIF-1α protein expression in PC cells was mainly to promote the ubiquitination degradation of the HIF-1α protein by inhibiting the interaction between HIF-1α and HSP90; Secondly, TQ reduced the initial expression of HIF-1α protein by inhibiting the PI3K/AKT/mTOR pathway.

Blockade of histamine receptor H1 augments immune checkpoint therapy by enhancing MHC-I expression in pancreatic cancer cells

BackgroundAlthough immune checkpoint blockade (ICB) therapy has proven to be extremely effective at managing certain cancers, its efficacy in treating pancreatic ductal adenocarcinoma (PDAC) has been limited. Therefore, enhancing the effect of ICB could improve the prognosis of PDAC. In this study, we focused on the histamine receptor H1 (HRH1) and investigated its impact on ICB therapy for PDAC.MethodsWe assessed HRH1 expression in pancreatic cancer cell (PCC) specimens from PDAC patients through public data analysis and immunohistochemical (IHC) staining. The impact of HRH1 in PCCs was evaluated using HRH1 antagonists and small hairpin RNA (shRNA). Techniques including Western blot, flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-PCR), and microarray analyses were performed to identify the relationships between HRH1 and major histocompatibility complex class I (MHC-I) expression in cancer cells. We combined HRH1 antagonism or knockdown with anti-programmed death receptor 1 (αPD-1) therapy in orthotopic models, employing IHC, immunofluorescence, and hematoxylin and eosin staining for assessment.ResultsHRH1 expression in cancer cells was negatively correlated with HLA-ABC expression, CD8+ T cells, and cytotoxic CD8+ T cells. Our findings indicate that HRH1 blockade upregulates MHC-I expression in PCCs via cholesterol biosynthesis signaling. In the orthotopic model, the combined inhibition of HRH1 and αPD-1 blockade enhanced cytotoxic CD8+ T cell penetration and efficacy, overcoming resistance to ICB therapy.ConclusionsHRH1 plays an immunosuppressive role in cancer cells. Consequently, HRH1 intervention may be a promising method to amplify the responsiveness of PDAC to immunotherapy.

MIF/NR3C2 axis regulates glucose metabolism reprogramming in pancreatic cancer through MAPK-ERK and AP-1 pathways.

Inflammation and aberrant cellular metabolism are widely recognized as hallmarks of cancer. In pancreatic ductal adenocarcinoma (PDAC), inflammatory signaling and metabolic reprogramming are tightly interwoven, playing pivotal roles in the pathogenesis and progression of the disease. However, the regulatory functions of inflammatory mediators in metabolic reprogramming in pancreatic cancer have not been fully explored. Earlier, we demonstrated that pro-inflammatory mediator macrophage migration inhibitory factor (MIF) enhances disease progression by inhibiting its downstream transcriptional factor nuclear receptor subfamily 3 group C member 2 (NR3C2). Here, we provide evidence that MIF and NR3C2 interactively regulate metabolic reprogramming, resulting in MIF-induced cancer growth and progression in PDAC. MIF positively correlates with the HK1 (hexokinase 1), HK2 (hexokinase 2) and LDHA (lactate dehydrogenase) expression and increased pyruvate and lactate production in PDAC patients. Additionally, MIF augments glucose uptake and lactate efflux by upregulating HK1, HK2 and LDHA expression in pancreatic cancer cells in vitro and in mouse models of PDAC. Conversely, a reduction in HK1, HK2 and LDHA expression is observed in tumors with high NR3C2 expression in PDAC patients. NR3C2 suppresses HK1, HK2 and LDHA expression, thereby inhibiting glucose uptake and lactate efflux in pancreatic cancer. Mechanistically, MIF-mediated regulation of glycolytic metabolism involves the activation of the mitogen-activated protein kinase-ERK signaling pathway, whereas NR3C2 interacts with the activator protein 1 to regulate glycolysis. Our findings reveal an interactive role of the MIF/NR3C2 axis in regulating glucose metabolism supporting tumor growth and progression and may be a potential target for designing novel approaches for improving disease outcome.

Abstract 1594: Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response

Abstract Background: While T-cell-based immunotherapy emerged as a promising tool for cancer management, clinical trials and translational studies revealed that cancer cells develop resistance by modulating tumor microenvironment and cell-intrinsic mechanisms. Pancreatic cancer (PC), consisting of dense stroma, immunosuppressive environment, and aberrant mucin expression, has a dismal survival rate and responds poorly to immunotherapies. MUC4, a member of the mucin family, has been reported to block lymphokine-activated killer cells and induce apoptosis of cytotoxic T-cells, suggesting its possible role in immune modulation. However, how the T-cell secretome influences MUC4 expression in PC and its role in modulating the T-cell response is poorly investigated. Method: The scRNA-seq data analysis and primary T-cell conditioned media (CM) were utilized to investigate the MUC4 and T-cell crosstalk. T-cell CM-treated murine PC cell line (KCT-3266) was analyzed by RNA-seq analysis followed by MUC4 silencing studies to investigate its role in evading T-cell response. The subcutaneous murine model was utilized to investigate the influence of MUC4 KO on immune cells and cytotoxic cell infiltration. The RNA isolated from tumor tissues from KrasG12D/+, Trp53R127H/+, Pdx-1-Cre (KPC), and Muc4 knockout (KPMC) murine models were used for PanCancer immune profiling. Results: The scRNA-seq analysis suggests that intratumoral T-cells positively correlate with MUC4 expression in PC patients. The activated T-cell CM consisting of predominantly IL-2, IFN-γ, and TNF-α cytokines, significantly induce the expression of MUC4 transcriptionally and translationally in both human (SW1990 and COLO357) and murine (KCT-3248 and KCT3266) PC cell lines. RNA-seq analysis from T-cell CM-treated cancer cells revealed that T-cell secretome induced the pathways related to cancer cell death. The CRISPR knockout of MUC4 in PC cells showed increased expression of cleaved caspase-3 after T-cell CM treatment, indicating that MUC4 plays a protective role against the T-cell CM-mediated killing. Subcutaneous implantation of MUC4 proficient and deficient PC cells on the flanks of C57BL/6 immunocompetent mice demonstrated a significantly higher infiltration of CD3 positive and cytotoxic T-cell infiltration in the MUC4 deficient tumors, resulting in significantly lower tumor weight. The PanCancer immune profiling also showed that depletion of Muc4 increases the T-cells and cytotoxic T-cell signaling scores in the KPMC tumor tissues compared to KPC. Conclusion: T-cell secretome induces MUC4 expression in pancreatic cancer cells, and the increased MUC4 expression reduces T-cell infiltration and protects PC cells against T-cell-mediated killing. Citation Format: Xiaoqi Li, Imran Khan, Rachel Kehrberg, Zahraa Wajih Alsafwani, Rakesh Bhatia, Sushil Kumar, Surinder K. Batra. Pancreatic cancer and T-cell crosstalk-induced MUC4 expression attenuates T-cell-mediated response [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 1594.

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 3250: LNA gapmer to gastrin inhibits growth and metastases of human pancreatic cancer in mice

Abstract Background: Pancreatic cancer has a poor prognosis and novel strategies are needed to improve the survival. Gastrin stimulates growth of pancreatic cancer by an autocrine mechanism and RNAi techniques that decrease gastrin expression have been shown to inhibit growth of this malignancy in vitro. Locked nucleic acid (LNA) Gapmers are antisense oligonucleotides (ASO) that have long half-life and stability in vivo, hence represent a potential method to deliver RNAi for cancer therapeutics. We developed an LNA Gapmer that selectively downregulates gastrin expression in pancreatic cancer cells in culture. The goal of this project was to test this anti-gastrin Gapmer in vivo as a novel therapy for pancreatic cancer. Methods: The anti-gastrin LNA Gapmer was modified using thiol-click chemistry to conjugate a peptide to the 5’ end and render the Gapmer target-specific with the ability to bind selectively to the cholecystokinin-B receptor that is over-expressed in pancreatic cancer. Forty athymic nude mice with established AsPC-1 human pancreatic orthotopic tumors were divided into four groups of equal tumor size. Mice (N=10 per group) were treated with PBS (controls), an untargeted LNA-Gapmer, the CCK-BR targeted Gapmer (60nM), and the CCK-BR targeted Gapmer (120nM) intraperitoneally twice a week for 4 weeks. Growth was monitored by luciferase IVIS imaging. At necropsy, tumors were excised, and metastases counted. Results: Mean tumor mass was 43% smaller in mice treated with the targeted Gapmer-120nM (P=0.0007). Untargeted and targeted Gapmer 60nM slightly decreased tumor mass but this difference was not significant. Compared to PBS control-treated mice, metastases were significantly less in mice treated with the untargeted Gapmer by -39% (p=0.01); in the targeted Gapmer-60nM by -44% (p=0.003), and with the targeted Gapmer-120nM by -72% (p<0.0001). Gastrin mRNA analysis of the tumors by qRT-PCR confirmed downregulation of gastrin. Conclusion: Novel LNA Gapmers that downregulate gastrin mRNA expression decrease growth and metastases of pancreatic cancer in a dose-related fashion. Citation Format: Godhanjali Chekuri, Wenqiang Chen, Narayan Shivapurkar, Hong Cao, Ruvanthi N. Kularatne, Stephan Stern, Jill P. Smith. LNA gapmer to gastrin inhibits growth and metastases of human pancreatic cancer in mice [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 3250.

HMGA2 alleviates ferroptosis by promoting GPX4 expression in pancreatic cancer cells

Pancreatic cancer is one of the most malignant tumor types and is characterized by high metastasis ability and a low survival rate. As a chromatin-binding protein, HMGA2 is widely overexpressed and considered an oncogene with various undefined regulatory mechanisms. Herein, we demonstrated that HMGA2 is highly expressed in pancreatic cancer tissues, mainly distributed in epithelial cells, and represents a subtype of high epithelial–mesenchymal transition. Deletion of HMGA2 inhibits tumor malignancy through cell proliferation, metastasis, and xenograft tumor growth in vivo. Moreover, HMGA2 enhanced the cellular redox status by inhibiting reactive oxygen species and promoting glutathione production. Importantly, ferroptotic cell death was significantly ameliorated in cells overexpressing HMGA2. Conversely, HMGA2 deletion exacerbated ferroptosis. Mechanistically, HMGA2 activated GPX4 expression through transcriptional and translational regulation. HMGA2 binds and promotes cis-element modification in the promoter region of the GPX4 gene by enhancing enhancer activity through increased H3K4 methylation and H3K27 acetylation. Furthermore, HMGA2 stimulated GPX4 protein synthesis via the mTORC1-4EBP1 and -S6K signaling axes. The overexpression of HMGA2 alleviated the decrease in GPX4 protein levels resulting from the pharmacologic inhibition of mTORC1. Conversely, compared with the control, HMGA2 deletion more strongly reduced the phosphorylation of 4EBP1 and S6K. A strong positive correlation between HMGA2 and GPX4 expression was confirmed using immunohistochemical staining. We also demonstrated that HMGA2 mitigated the sensitivity of cancer cells to combination treatment with a ferroptosis inducer and mTORC1 inhibition or gemcitabine. In summary, our results revealed a regulatory mechanism by which HMGA2 coordinates GPX4 expression and underscores the potential value of targeting HMGA2 in cancer treatment.

Long non-coding RNA TINCR suppresses growth and epithelial-mesenchymal transition by inhibiting Wnt/β-catenin signaling pathway in human pancreatic cancer PANC-1 cells: Insights from in vitro and in vivo studies.

There is increasing evidence that long non-coding RNAs (lncRNAs) play a crucial role in the development and progression of malignant tumors, particularly pancreatic cancer. In this study, the influence of the lncRNA TINCR on the behavior of human pancreatic cancer cells was investigated with the aim of deciphering its role in growth, migration, and invasion. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate TINCR expression in pancreatic cancer cells. Ectopic expression of TINCR in PANC-1 cells was induced to evaluate the effects on cell viability and apoptosis, examining the apoptotic genes Bax and Bcl-2. Migration and invasion assays were used to measure the impact of TINCR on these cellular processes. In vivo studies using a xenograft mouse model examined the effects of TINCR on tumor growth, epithelial-to-mesenchymal transition (EMT) markers, and the Wnt/β-catenin signaling pathway. PANC-1 cells showed strikingly low TINCR expression compared to other pancreatic cancer cell lines. Ectopic TINCR expression reduced the viability of PANC-1 cells primarily by inducing apoptosis, as evidenced by increased Bax and decreased Bcl-2 expression. Overexpression of TINCR significantly increased the percentage of apoptotic cells. It also decreased the migration and invasion ability of PANC-1 cells, as demonstrated in wound healing and transwell assays. In addition, overexpression of TINCR-suppressed proteins is associated with the Wnt/β-catenin signaling pathway in PANC-1 cells. In the xenograft mouse model, overexpression of TINCR inhibited tumor growth, EMT markers, and proteins associated with the Wnt/β-catenin pathway. This study sheds light on the tumour-suppressive role of TINCR in PANC-1 cells and suggests its potential as a therapeutic target. These results shed light on the molecular mechanisms underlying the impact of TINCR on pancreatic cancer and offer promising opportunities for innovative therapeutic strategies to improve outcomes in this serious malignancy.

Abstract A039: Silencing MICAL2 expression in pancreatic cancer cells reduces IL-1A expression and inhibits tumor growth through a CD8+ T cell dependent mechanism

Abstract Introduction: Pancreatic cancer is characterized by desmoplastic, fibroinflammatory stroma. The crosstalk between cancer cells and their surrounding tumor microenvironment (TME) promotes disease progression, metastasis, and chemoresistance. We identified MICAL2 as a super-enhancer-associated gene in pancreatic cancer. MICAL2 (molecule interacting with Cas-L) proteins are flavin monooxygenases that promote actin depolymerization and indirectly regulate SRF transcription. In other work, we have found that MICAL2 promotes pancreatic cancer growth and progression. This study evaluated how MICAL2 pancreatic cancer cell expression modulates the PDAC tumor microenvironment. Methods: RNA-Seq analysis performed on human pancreas cancer cells (AsPC-1) before and after MICAL2 KD revealed differential regulation of IL-1α. We validated the expression of IL-1α in multiple human and mouse pancreas cancer cell lines by q-PCR. We next exposed human (h) and mouse (m) PSCs to conditioned media (CM) from cancer cells before and after MICAL2 KD and checked the expression of a-SMA, fibronectin, and Col1A and IL-6 genes by qPCR. KrasG12D/+; Trp53R172H/+; Pdx1-cre (KPC) cells with and without MICAL2 were orthotopically injected to assess the in vivo tumor growth and metastasis. Sc-RNA seq and Immunophenotyping were performed on Shcontrol (ShCNT) and MICAL2 (M2KD) KD tumors. CD8+ T cell depletion was done using an antibody-mediated approach. The adoptive transfer of CD8+ T cells extracted from MICAL2 KD tumors was done by tail vein injection. Results: MICAL2 KD resulted in the downregulation of the IL-1α gene in both human and mouse pancreas cancer cell lines (50% reduction, p<0.05). Human and mouse pancreatic stellate cells (hPSCs and mPSCs) co-cultured with cancer cells CM showed significant downregulation of a-SMA, fibronectin, Col1A, and IL-6 upon MICAL2 KD as compared to hPSCs and mPSCs co-cultured with ShCNT cells. Orthotopic injections of KPC MICAL2 KD cells led to decreased tumor growth as compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Immunofluorescence (IF) revealed less α-SMA, PDGF-α, and IL-6 expression and reduced deposition of collagen and fibronectin in MICAL2 KD tumors. Single-cell RNA seq and flow cytometry studies of tumors harvested from both groups showed significantly increased infiltration of activated cytotoxic CD8+ T cells in MICAL2 KD tumors which was further confirmed by IF. Antibody-mediated depletion of CD8+ T cells in MICAL2 KD tumors abrogated their reduced tumor growth in both immune-hot and immune-cold KPC cell lines. Adoptive transfer of CD8+ T cells extracted from M2KD tumors significantly reduced ShCNT flank tumor growth. Conclusion: These data reveal that MICAL2 expression mediates tumor-stromal crosstalk through IL1A and creates an immunosuppressive environment in PDAC and suggests MICAL2 may be an attractive immunomodulatory target for pancreatic cancer therapy. Ongoing work is focused on dissecting the role of MICAL2 in priming the metastatic niche through the remodeling of the TME. Citation Format: Bharti Garg, EvanGeline Mose, Edgar Esparza, Jay Patel, Sarah Sass, Alexei Martsinkovskiy, Dawn Jacquish, Herve Tiriac, Andrew M. Lowy. Silencing MICAL2 expression in pancreatic cancer cells reduces IL-1A expression and inhibits tumor growth through a CD8+ T cell dependent mechanism [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 A039.

Vasohibin-2-Targeting Therapies for the Treatment of Pancreatic Ductal Adenocarcinoma.

As pancreatic ductal adenocarcinoma (PDAC) is extremely malignant and refractory, therapeutic options for this cancer are anticipated worldwide. We isolated vasohihibin-2 (VASH2) and observed its overexpression in various types of cancer. We then noticed that upregulated expression of VASH2 in patients with PDAC resulted in a conspicuous reduction in the postoperative survival period and further revealed that the abrogation of Vash2 expression in pancreatic cancer cells inhibited its growth and metastasis and augmented tumor infiltration of CD8+ cells in the mouse model. We developed VASH2-targeting therapies, 2',4'-BNA-based antisense oligonucleotide targeting VASH2 (VASH2-ASO) as a nucleotide-based therapy, and VASH2-peptide vaccine as an antibody-based therapy. We also showed that the VASH2-peptide vaccine inhibited PDAC metastasis in an orthotopic mouse model. Here, we expanded our analysis of the efficacy of VASH2-targeting therapies for PDAC. VASH2-ASO treatment inhibited the growth of primary tumors by reducing tumor angiogenesis, normalizing tumor vessels, preventing ascites accumulation and distant metastasis to the liver and lungs, and augmenting the infiltration of CD8+ cells in metastatic tumors. VASH2-peptide vaccine did not affect the infiltration of CD8+ cells into tumors. The present study revealed that VASH2-targeting therapies are promising options for the treatment of PDAC. VASH2-ASO therapy can be administered at any stage of PDAC. Because of the increase of CD8+ cell infiltration, the combination therapy with immune checkpoint inhibitors may be an attractive option. The VASH2-peptide vaccine therapy may be useful for preventing metastasis and/or recurrence after successful initial treatment.

Curcumin inhibits the invasion and migration of pancreatic cancer cells by upregulating TFPI-2 to regulate ERK- and JNK-mediated epithelial-mesenchymal transition.

Pancreatic cancer (PC) is one of the most deadly human malignancies. Curcumin is a natural polyphenolic compound with wide-ranging pharmacological effects. Growing evidence suggests that curcumin has anticancer activity against PC, but the mechanism remains incompletely elucidated. This study aimed to investigate the effects and mechanisms of curcumin on the invasion and migration of PC cells. Effect of curcumin on tissue factor pathway inhibitor (TFPI)-2 mRNA expression in PC cells was initially identified using qRT-PCR. Cytotoxicity of curcumin was assessed with MTT assays and IC50 was calculated. Involvement of ERK and JNK pathways, as well as protein expression of TFPI-2 and epithelial-mesenchymal transition (EMT)-related markers, were detected using immunoblotting. Invasion and migration of PC cells were examined using Transwell assays. TFPI-2 expression was manipulated by transfection with siRNA and shRNA. Rescue assays were used to validate the effect of curcumin on cell invasion and migration via TFPI-2. Curcumin increased the expression of TFPI-2 mRNA and protein in PC cells and attenuated cell invasion and migration. Curcumin also inhibited ERK and JNK pathways and EMT in PC cells. Knockdown of TFPI-2 partially reversed the inhibition of ERK and JNK pathways and EMT by curcumin. Mechanistically, curcumin upregulated TFPI-2, thereby inhibiting the ERK and JNK pathways, leading to the inhibition of EMT in PC cells. Collectively, curcumin inhibits ERK- and JNK-mediated EMT through upregulating TFPI-2, which in turn suppresses the migration and invasion of PC cells. These findings provide new insights into the antitumor mechanism of curcumin.

IGF2 is upregulated by its antisense RNA to potentiate pancreatic cancer progression.

Pancreatic cancer is a deadly cancer. More and more long noncoding RNAs (lncRNAs) have received confirmation to be dysregulated in tumors and exert the regulatory function. Studies have suggested that lncRNA insulin-like growth factor 2 antisense RNA (IGF2-AS) participates in the development of some cancers. Thus, we attempted to clarify its function in pancreatic cancer. Reverse-transcription quantitative polymerase chain reaction was applied for testing IGF2-AS expression in pancreatic cancer cells. Colony formation and Transwell wound experiments were applied for determining cell proliferative, migratory, and invasive capabilities. The alteration of epithelial-mesenchymal transition (EMT)-related gene level was tested via western blot. The mice model was established for measuring the tumor growth and metastasis. RIP validated the interaction of RNAs. IGF2-AS displays high expression in pancreatic cancer cells. IGF2-AS depletion repressed PC cell proliferative, migratory, invasive capabilities, and EMT process. Furthermore, pancreatic cancer tumor growth and metastasis were also inhibited by IGF2-AS depletion. Additionally, IGF2-AS positively regulated IGF2 level via recruiting HNRNPC. IGF2 overexpression counteracted the functions of IGF2-AS deficiency on pancreatic cancer cell behaviors. Moreover, IGF2R deletion was found to inhibit the positive effect of IGF2 on pancreatic cancer progression. IGF2-AS potentiates pancreatic cancer cell proliferation, tumor growth, and metastasis by recruiting HNRNPC via the IGF2-IGF2R regulatory pathway. These discoveries might offer a novel insight for treatment of PC, which may facilitate targeted therapies of PC in clinical practice.

A novel anti-galectin-9 immunotherapy limits the early progression of pancreatic neoplastic lesions in transgenic mice.

Pancreatic adenocarcinoma (PDAC) is a devastating disease with an urgent need for therapeutic innovation. Immune checkpoint inhibition has shown promise in a variety of solid tumors, but most clinical trials have failed to demonstrate clinical efficacy in PDAC. This low efficacy is partly explained by a highly immunosuppressive microenvironment, which dampens anti-tumor immunity through the recruitment or induction of immunosuppressive cells, particularly regulatory T cells (Tregs). In this context, our laboratory has developed a novel immunotherapeutic strategy aimed at inhibiting the suppressive activity of Tregs, based on a patented (EP3152234B1) monoclonal antibody (mAb) targeting galectin-9 (LGALS9). CD4+ conventional T cells (TCD4 or Tconv), Treg ratio, and LGALS9 expression were analyzed by immunohistochemistry (IHC) and cytometry in blood and pancreas of K-rasLSL.G12D/+;Pdx-1-Cre (KC) and K-rasWildType (WT);Pdx1-Cre (WT) mice aged 4-13 months. Pancreatic intraepithelial neoplasm (PanIN) progression and grade were quantified using FIJI software and validated by pathologists. The anti-galectin-9 mAb was validated for its use in mice on isolated murine C57BL/6 Treg by immunofluorescence staining and cytometry. Its specificity and functionality were validated in proliferation assays on rLGALS9-immunosuppressed murine Tconv and in suppression assays between murine Treg and Tconv. Finally, 2-month-old KC mice were treated with anti-LGALS9 and compared to WT mice for peripheral and infiltrating TCD4, Treg, and PanIN progression. IHC and cytometry revealed a significant increase in LGALS9 expression and Treg levels in the blood and pancreas of KC mice proportional to the stages of precancerous lesions. Although present in WT mice, LGALS9 is expressed at a basal level with low and restricted expression that increases slightly over time, while Treg cells are few in number in their circulation and even absent from the pancreas over time. Using our anti-LGALS9 mAb in mice, it is shown that (i) murine Treg express LGALS9, (ii) the mAb could target and inhibit recombinant murine LGALS9, and (iii) neutralize murine Treg suppressive activity. Finally, the anti-LGALS9 mAb in KC mice reduced (i) LGALS9 expression in pancreatic cancer cells, (ii) the Treg ratio, and (iii) the total surface area and grade of PanIN. We demonstrate for the first time that an anti-LGALS9 antibody, by specifically targeting endogenous LGALS9 tumor and exogenous LGALS9 produced by Treg, was able to limit the progression of pancreatic neoplastic lesions in mice, opening up new prospects for its use as an immunotherapeutic tool in PDAC.

Identification of exosomal miR-484 role in reprogramming mitochondrial metabolism in pancreatic cancer through Wnt/MAPK axis control

The microRNAs (miRNAs) are potent regulators of tumorigenesis in various cancers, especially pancreatic cancer. The abnormal expression of miRNAs can be observed in tumor cells. Noteworthy, miRNAs could be transferred by exosomes as small extracellular vesicles in regulation of carcinogenesis. This research focused on exploring the roles and mechanisms of exosomal miR-484, derived from human bone marrow mesenchymal stem cells (hBMSCs), in the context of molecular interactions and regulation of mitochondrial metabolism. Exosomes were isolated for the examination of miR-484 expression. The impacts of hBMSCs-derived exosomal miR-484 on pancreatic cancer cells were studied using various assays. Evaluation of mitochondrial function and metabolism was performed. Wnt/MAPK pathway-related protein expression was assessed, and an in vivo tumor xenograft model was utilized to examine the functions. Our findings demonstrated a decreased miR-484 expression in pancreatic cancer cells. However, hBMSCs-derived exosomal miR-484 inhibited the proliferation and migration of these cells, while inducing apoptosis. Moreover, miR-484 led to an upsurge in reactive oxygen species production, a decrease in ATP levels, and a disruption in mitochondrial metabolism. In vivo analyses showed that hBMSCs-derived exosomal miR-484 lessened tumor size and weight, while also suppressing the expression of mitochondrial biomarkers. Further, there was a decline in β-catenin and p-p38 protein levels both in vitro and in vivo. The addition of LiCl restored the disrupted mitochondrial metabolism. Conclusively, our results suggest that hBMSCs-derived exosomal miR-484 mitigates the malignant transformation and mitochondrial metabolism of pancreatic cancer by deactivating the Wnt/MAPK pathway.

High expression ITGA2 affects the expression of MET, PD-L1, CD4 and CD8 with the immune microenvironment in pancreatic cancer patients.

Pancreatic cancer is characterized by a grim prognosis and is regarded as one of the most formidable malignancies. Among the genes exhibiting high expression in different tumor tissues, ITGA2 stands out as a promising candidate for cancer therapy. The promotion of cancer in pancreatic cancer is not effective. The objective of this study is to assess the presence of ITGA2, EMT and PD-L1 in pancreatic cancer. We examined the expression of ITGA2, MET, E-cadherin, PD-L1, CD4, and CD8 proteins in 62 pancreatic cancer tissue samples using multi-tissue immunofluorescence and immunohistochemistry techniques. Functional assays, such as the cell migration assay and transwell assay, were used to determine the biological role of ITGA2 in pancreatic cancer. The relationship of ITGA2,EMT and PD-L1 were examined using Western blot analysis and RT-qPCR assay. In our study, we observed the expression of ITGA2, E-cadherin, and PD-L1 in both tumor and stroma tissues of pancreatic cancer. Additionally, a positive correlation between ITGA2, E-cadherin, and PD-L1 in the tumor region (r=0.559, P<0.001 and r=0.511, P<0.001), and PD-L1 in the stroma region (r=0.512, P<0.001).The expression levels of ITGA2, CD4, and CD8 were found to be higher in pancreatic cancer tissues compared to adjacent tissues (P < 0.05). Additionally, ITGA2 was negatively correlated with CD4 and CD8 (r = -0.344, P < 0.005 and r = -0.398, P < 0.005).Furthermore, ITGA2, CD4, and CD8 were found to be correlated with the survival time of patients (P < 0.05). Blocking ITGA2 inhibited the proliferation and invasion ability of pancreatic cancer cells significantly, Additionally, sh-ITGA2 can down-regulate the expression of EMT and PD-L1. We identified a novel mechanism in which ITGA2 plays a crucial role in the regulation of pancreatic cancer growth and invasion. This mechanism involves the upregulation of MET and PD-L1 expression in pancreatic cancer cells. Additionally, we found that increased expression of ITGA2 is associated with a poor prognosis in pancreatic cancer patients. Furthermore, ITGA2 also affects immune regulation in these patients. Therefore, targeting ITGA2 is an effective method to enhance the efficacy of checkpoint immunotherapy and prohibiting tumor growth against pancreatic cancer.