Tumor Progression In Pancreatic Ductal Adenocarcinoma Research Articles

Pancreatic Cancer and Venous Thromboembolism.

Pancreatic ductal adenocarcinoma (PDAC) accounts for more than 90% of all pancreatic cancers and is the most fatal of all cancers. The treatment response from combination chemotherapies is far from satisfactory and surgery remains the mainstay of curative strategies. These challenges warrant identifying effective treatments for combating this deadly cancer. PDAC tumor progression is associated with the robust activation of the coagulation system. Notably, cancer-associated thrombosis (CAT) is a significant risk factor in PDAC. CAT is a concept whereby cancer cells promote thromboembolism, primarily venous thromboembolism (VTE). Of all cancer types, PDAC is associated with the highest risk of developing VTE. Hypoxia in a PDAC tumor microenvironment also elevates thrombotic risk. Direct oral anticoagulants (DOACs) or low-molecular-weight heparin (LMWH) are used only as thromboprophylaxis in PDAC. However, a precision medicine approach is recommended to determine the precise dose and duration of thromboprophylaxis in clinical setting.

Glycolysis and tumor progression promoted by the m6A writer VIRMA via m6A-dependent upregulation of STRA6 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3’ UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.

Abstract 4277: CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic cancer progression

Abstract Background: Pancreatic Ductal Adenocarcinoma (PDAC) is characterized by a heightened oncogenic mutational burden, an immunosuppressive tumor microenvironment, and a dense desmoplastic stroma. During the tumor progression from in situ carcinoma to intraductal neoplasia, cancer cells manage to evade the stromal compartment and infiltrate into the ducts. This invasive tumor progression is often associated with the overexpression of extracellular matrix (ECM) modulating genes. Despite this correlation, there are currently no effective and specific inhibitors of ECM-altering pathways. By understanding the signaling mechanism, the ECM remodeling pathways may be intercepted to prevent invasive tumor progression. This study aims to elucidate the role of cAMP-response element binding protein 1 (CREB1) and its downstream effectors in regulating PDAC ECM remodeling. Methods: The Cancer Genome Atlas (TCGA) was quired for CREB1 expression and other ECM modulators in PDAC patients. In vitro, CRISPR/CAS9-based genomic editing was utilized to knockout CREB1 in the murine KPC cell line, on which RNA-seq analysis was performed. To investigate the effects of CREB in PDAC progression, a novel conditional CREB1 knockout (CREBfl/fl) was created in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice (KPCC-/-). Tumors were extracted and histologically evaluated. Immunoblotting and staining were conducted on both in vitro and in vivo samples. Chromatin immunoprecipitation sequencing (ChIP-seq) and qPCR were performed on the KPC CREBKO. Eukaryotic Promoter Database (EPD) and ENCODE databases were explored to evaluate the transcriptional role of CREB in regulating ECM genes. Results: Patient PDAC samples data from TCGA revealed elevated levels of CREB1, ROCK1, and ROCK2 compared to normal pancreatic tissues. When comparing high CREB expression to low CREB expression, upregulation of several ECM genes was noted, including ROCK1 and ROCK2 in patients with high CREB expression. In CREBKO, RNA-seq analysis revealed the downregulation of key ECM genes, including Mmp3, Mmp10, Lama3, and Fn1 compared to CREB wildtype. In our novel murine conditional knockout model, a substantial reduction in fibrosis was observed. In both in vitro and in vivo, immunoblot and staining demonstrated a marked reduction in the expression of ECM remodeling proteins upon CREB deletion. Through ChIP-seq, the direct involvement of CREB1 in the transcriptional regulation of Rock1 and Rock2 was established. EPD and ENCODE were utilized to confirm and validate our CREB regulation experimental findings. Conclusion: These findings demonstrate the role of CREB in PDAC progression, which may provide a potential target to intercept ECM remodeling via inhibition of the CREB-ROCK axis to suppress PDAC tumor progression and invasion. Citation Format: Sudhakar Jinka, Siddharth Mehra, Varunkumar Krishnamoorthy, Anna Bianchi, Karthik Rajkumar, Andrew Adams, Haleh Amirian, Samara Singh, Edmond W. Box, Erin Dickey, Nivelo L. Alberto, Ban Yuguang, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. CREB-ROCK driven extracellular matrix remodeling exasperates pancreatic 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 4277.

Abstract 6674: Fungi utilizes exocytosis pathway to facilitate the release of DAMPs from pancreatic cancer cells

Abstract Recent studies overwhelmingly show that microbiome is pervasive to tumor and play key role in immunotherapy response and reprograming tumor immune microenvironment (TIME). However, it remains unknown how microbiome facilitates the host-immune cell crosstalk and promote tumorigenesis. Our recent study in pancreatic ductal adenocarcinoma (PDAC) show that intratumor fungal microbiome facilitate the release of a damage associated molecular pattern (DAMP) molecule IL-33 from cancer cells. The secreted IL33 from cancer cells in turns recruits ILC2 and TH2 cells which promotes PDAC tumorigenesis. However, the mechanism of fungal mediated IL-33 release from PDAC cells remains unknown. Our follow up studies using high throughput integrated proteomic profiling and immunoprecipitation revealed a group of exocyst complex proteins interacting with IL-33 in the presence of fungus. The exocyst complex consists of eight sub-unit proteins that tethers the secretory vesicles to the plasma membrane and implicated in cellular exocytosis. We found EXOC6B a component of exocyst complex, modulates IL-33 release via exocytosis pathway from the PDAC cells. Genetic depletion of EXOC6B resulted in shunted IL-33 release and significantly abrogated PDAC tumor progression. Mechanistically, we found that mycobiome activates a non-canonical TLR2-CARD9 pathway, thereby driving IL-33 nucleocytoplasmic transport and exocytosis. This study identifies a unique mechanism of EXOC6B mediated release of IL-33 that expands our knowledge on how host-mycobiome interaction shapes PDAC tumor microenvironment by intricate molecular mechanism. Our study identifies EXOC6B as a new therapeutics target involved in the mycobiome driven IL-33 secretion in pancreatic cancer. Citation Format: Aftab Alam, Shyamananda Singh Mayengbam, Sharon Senchanthisai, Bektas Irmak, Prasenjit Dey. Fungi utilizes exocytosis pathway to facilitate the release of DAMPs from pancreatic cancer cells [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 6674.

Abstract A085: First-in-class humanized antibody targeting alternatively spliced tissue factor augments anti-metastatic efficacy of chemotherapy in a preclinical model of pancreatic ductal adenocarcinoma

Abstract Introduction. Alternatively spliced tissue factor (asTF) promotes progression of pancreatic ductal adenocarcinoma (PDAC) via activating b1-integrins on PDAC cell surfaces. hRabMab1, a humanized inhibitory anti-asTF antibody we recently developed, can suppress PDAC primary tumor growth as a single agent. Whether hRabMab1 has the potential to suppress metastases in PDAC is unknown. Aim. To test hRabMab1’s ability to suppress metastatic spread in combination with chemotherapy. Methods. asTF-proficient, KRAS G12V-mutant human PDAC cell line PaCa44 yields aggressive primary orthotopic tumors that spread spontaneously to the relevant anatomical sites, accompanied by severe leukocytosis. 5 × 105 PaCa44 cells were implanted into the pancreata of NOD-SCID mice (n=40). 10 days post-implantation, mice were randomized into 4 cohorts: vehicle; hRabMab1 administered IV at 18 mg/kg; gemcitabine/paclitaxel (gem/paclitaxel, 50 mg/kg/3 mg/kg); and hRabMab1+gem/paclitaxel. Results. Post-mortem analysis of tumor tissue and luciferase imaging of the abdominal cavity showed that gem/paclitaxel alone reduced primary tumor volume, but not metastatic spread; only the combination of hRabMab1 and gem/paclitaxel significantly reduced metastatic spread (p=0.0114 vs vehicle, 1-way ANOVA). RNAseq analysis of primary tumor tissue followed by gene set enrichment of differentially expressed genes showed that the addition of hRabMab1 to gem/paclitaxel downregulated tubulin binding and microtubule motor activity. Genes involved in extracellular matrix/collagen production and neovascularization were upregulated in response to gem/paclitaxel; addition of hRabMab1 to gem/paclitaxel tentatively reduced this compensatory effect. In the liver, hRabMab1 reduced liver metastasis as a single agent (p=0.031 vs vehicle) and in combination with gem/paclitaxel (p=0.0054 vs vehicle, 1-way ANOVA). In the surgical incision site, metastatic penetrance was as follows: vehicle, 100%; hRabMab1, 100%; gem/paclitaxel, 44% (4/9); hRabMab1+gem/paclitaxel, 0% (p=0.037715, gem/paclitaxel vs hRabMab1+gem/paclitaxel, Chi square test). Only the combination of hRabMab1 and gem/paclitaxel eliminated leukocytosis; levels of neutrophils, monocytes, eosinophils, and basophils were significantly lower in the hRabMab1+gem/paclitaxel cohort vs gem/paclitaxel cohort (p=0.035, p=0.051, p=0.032, and p=0.026, respectively). Leukocytes recruited from the circulation fuel tumor progression in PDAC. To address the potential significance of white blood cell (WBC) count normalization in our model, we performed correlation analysis between WBC counts and tumor volumes across all cohorts; there was a highly significant positive correlation between these two parameters (R=0.682, p=0.00002). Conclusions. This is the first study demonstrating that hRabMab1 can suppress metastasis in PDAC. hRabMab1’s ability to improve anti-metastatic efficacy of chemotherapy is potentially significant and warrants further investigation. Citation Format: Clayton S. Lewis, Charles Backman, Sabahat Ahsan, Ashley Cliff, Arthi Hariharan, Jen Jen Yeh, Davendra P. S. Sohal, Vladimir Y. Bogdanov. First-in-class humanized antibody targeting alternatively spliced tissue factor augments anti-metastatic efficacy of chemotherapy in a preclinical model of pancreatic ductal adenocarcinoma [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 A085.

The Role of Noncoding RNA Antisense Transcript of the B-Cell Translocation Gene 3 Regulation of BTG3 in Pancreatic Ductal Adenocarcinoma Tumor Progression

Antisense transcript of the B-cell translocation gene 3 (ASBEL) is a highly conserved antisense non-coding RNA (ncRNA) and participates in a variety of biological processes. However, the ASBEL expression status in pancreatic ductal adenocarcinoma (PDAC) and its correlation with BTG3 expression and tumor cell progression were not completely clear. We conducted cell experiments and animal experiments to confirm that ASBEL plays a crucial role in the tumorigenesis of PDAC by targeting BTG3. ASBEL regulation in PDAC tumorigenesis was evaluated using Western blotting, quantitative polymerase chain reaction, Cell Counting Kit-8 assay, flow cytometry, and cell transfection. We also evaluated the expression of ASBEL and BTG3 in tumor tissues and cells using Western blotting and quantitative real-time polymerase chain reaction. Finally, we explored the role of ASBEL in tumor development by silencing or overexpressing ASBEL gene in AsPC-1 or CFPAC-1 cells, respectively, and evaluated the antitumor activity in vivo using an ASBEL antagonist. Our study revealed the expression of ASBEL in all pancreatic cell lines. The expression level of ASBEL in tumor tissues was found to be higher than that of paracarcinomatous tissues. ASBEL suppresses expression of BTG3, enhances proliferation and suppresses apoptosis, and promotes migration and invasion in pancreatic cancer cell. Antagonist regulates the expression of ASBEL in AsPC-1, and suppresses tumor growth in xenograft mouse model. Our results indicate that ASBEL may play a tumor-promoting factor in PDAC by targeting BTG3 and could be as an important biomarker for PDAC treatment. (Curr Ther Res Clin Exp. 2023; 84:XXX-XXX).

A LETM2-Regulated PI3K-Akt Signaling Axis Reveals a Prognostic and Therapeutic Target in Pancreatic Cancer.

Simple SummaryLEMT2 was a newly discovered protein-encoding gene with little cancer research and an unclear mechanism. This study aimed to illustrate LETM2 as the crucial oncogene for tumor progression in pancreatic ductal adenocarcinoma (PDAC). We analyzed the expression level and prognostic value of LETM2 in multiple cancers using pan-cancer analysis and found that the LETM2 expression was the most significantly related to the dismal prognosis of PDAC. Immunohistochemical analyses showed that high LETM2 expression was correlated with poor outcomes of PDAC. In in vitro and in vivo experiments, LETM2 knockdown significantly inhibited tumor proliferation and metastasis, while LETM2 overexpression exerted the opposite effects. Then, we suggested that LETM2 may facilitate tumor progression by activating downstream PI3K-Akt signaling pathway in PDAC. In conclusion, the study enhanced our understanding of the LETM2-regulated PI3K-Akt signaling axis served as a prognostic and therapeutic target of pancreatic cancer.Pancreatic ductal adenocarcinoma (PDAC) is one of the highest mortalities malignant tumors, which is characterized by difficult diagnosis, rapid progression and high recurrence rate. Nevertheless, PDAC responds poorly to conventional therapies, which highlights the urgency to identify novel prognostic and therapeutic targets. LEMT2 was a newly discovered protein-encoding gene with little cancer research and an unclear mechanism. Thus, this study aimed to illustrate LETM2 as the crucial oncogene for tumor progression in PDAC. In this study, we analyzed the expression level and prognostic value of LETM2 in multiple cancers using pan-cancer analysis. The analyses based on the TCGA-GTEx dataset indicated that the LETM2 expression was obviously elevated in several cancers, and it was the most significantly related to the dismal prognosis of PDAC. Subsequently, we demonstrated the functional role and mechanism of LETM2 by clinical sample evaluation, and in in vitro and in vivo experiments. Immunohistochemical analyses showed that high expression of LETM2 was correlated with poor outcomes of PDAC. Moreover, we demonstrated that LETM2 knockdown significantly inhibited tumor proliferation and metastasis, and promoted cell apoptosis, while LETM2 overexpression exerted the opposite effects. Finally, the impairment caused by LETM2-knockdown could be recovered via excitation of the PI3k-Akt pathway in vitro and in vivo animal models, which suggested that LETM2 could activate the downstream PI3K-Akt pathway to participate in PDAC progression. In conclusion, the study enhanced our understanding of LETM2 as an oncogene hallmark of PDAC. LETM2 may facilitate tumor progression by activating the PI3K-Akt signaling pathway, which provides potential targets for the diagnosis, treatment, and prognosis of pancreatic cancer.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr SY24-03.

Abstract 5607: Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model

Abstract Introduction: An immunosuppressed, apoptosis-resistant phenotype characterizes pancreatic ductal adenocarcinoma (PDAC). Both inflammation and tumorigenesis are influenced by the Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) pathway. TLY012 is pegylated recombinant TRAIL protein and is an orphan drug for systemic sclerosis and chronic pancreatitis. The potential synergy between TLY012 and anti-mouse PD-1 antibody was investigated in a syngeneic immunocompetent heterotopic mouse model. Methods: KPC-luc PDAC cells were subcutaneously implanted into C57Bl/6 mice. Treatment combined 10 mg/kg TLY012 three times per week with 10 mg/kg anti-mouse PD-1 antibody twice weekly and were compared to individual treatment and control groups. Cytokine profiling via 32-plex Luminex immunoassay was completed after 7 days and after three months of treatment. Flow cytometry at three months evaluated NK and T cell profiles in the spleen and tumors. Results: PDAC tumor-bearing mice treated with the combination of anti-mouse PD-1 and TLY012 had slower tumor growth and reduced tumor volume at 70 days compared to either drug alone (all p<0.005). B-cell activating factor, which can be a marker of PDAC tumor progression, was decreased in single and dual treatment groups at three months of treatment. This factor showed the earliest decrease in the dual treatment group, with a cytokine level 44% of that of control mice at 7 days. Long-term dual treatment mice also had the highest levels of proinflammatory cytokines interferon gamma (average 5.6 times control level, p=0.046), CCL5 (average 14.1 times control level, p=0.048), and interleukin-3 (IL-3, average 71.1 times control level, p=0.0053). Flow cytometry showed trends toward decreased circulating regulatory T cells, increased NK cells, and a higher proportion of CD8+ T cells within tumors in the dual treatment group. Conclusions: The combination of anti-mouse PD-1 and TLY012 prevents growth of PDAC in an immunocompetent mouse model. The combination also increases tumor-infiltrating CD8+ T cells, decreases circulating Treg cells and alters cytokine expression. CCL5 is a T-cell chemokine, and both interferon gamma and IL-3 are produced by activated T cells and macrophages, and have proinflammatory, antitumor effects. Combining TLY012 and anti-mouse PD-1 creates these changes in immune cell and cytokine levels, suggesting induction of a more proinflammatory immune environment. The proinflammatory state may contribute to decreased tumor growth. Citation Format: Anna D. Louie, Kelsey E. Huntington, Young Lee, Jared Mompoint, Jinxuan Wu, Seulki Lee, Wafik S. El-Deiry. Combining PD-1 inhibition with the TRAIL receptor agonist TLY012 promotes pancreatic ductal adenocarcinoma tumor regression in an immunocompetent mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5607.

Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

The transcriptional regulator nuclear factor of activated T-cells, cytoplasmic 3 (NFATc3) is constitutively activated in several cancer types and plays important roles in cancer development and progression. Heavily phosphorylated NFATc3 resides in the cytoplasm of resting cells, and dephosphorylated NFATc3 translocates to the nucleus to activate expression of target genes in cells exposed to stimuli, for instance, hypoxia. Apart from phosphorylation, various post-translational modifications have been reported to regulate NFAT transcriptional activity. However, the mechanisms remain elusive. Here, we have demonstrated that NFATc3 is activated in human pancreatic ductal adenocarcinoma (PDAC) cells and that excessive activation of NFATc3 is correlated to advanced stages of PDAC and short survival time of PDAC patients. NFATc3 is deSUMOylated at K384 by SENP3 under hypoxia, which impairs the interaction between NFATc3 and phosphokinase GSK-3β, subsequently decreases NFATc3 phosphorylation and increases its nuclear occupancy. Knockdown of SENP3 greatly decreased hypoxia-induced NFATc3 nuclear occupancy. Our results highlight that SENP3-mediated deSUMOylation acts as an essential modulator of NFATc3, which is instrumental in PDAC tumor progression under hypoxia.

Stromal HIF2 Regulates Immune Suppression in the Pancreatic Cancer Microenvironment

BACKGROUND & AIMS:Pancreatic ductal adenocarcinoma (PDAC) has a hypoxic, immunosuppressive stroma that contributes to its resistance to immune checkpoint blockade therapies. The hypoxia-inducible factors (HIFs) mediate the cellular response to hypoxia, but their role within the PDAC tumor microenvironment remains unknown.METHODS:We used a dual recombinase mouse model to delete Hif1α or Hif2α in α-smooth muscle actin–expressing cancer-associated fibroblasts (CAFs) arising within spontaneous pancreatic tumors. The effects of CAF HIF2α expression on tumor progression and composition of the tumor microenvironment were evaluated by Kaplan-Meier analysis, reverse transcription quantitative real-time polymerase chain reaction, histology, immunostaining, and by both bulk and single-cell RNA sequencing. CAF-macrophage crosstalk was modeled ex vivo using conditioned media from CAFs after treatment with hypoxia and PT2399, an HIF2 inhibitor currently in clinical trials. Syngeneic flank and orthotopic PDAC models were used to assess whether HIF2 inhibition improves response to immune checkpoint blockade.RESULTS:CAF-specific deletion of Hif2α, but not Hif1α, suppressed PDAC tumor progression and growth, and improved survival of mice by 50% (n ¼ 21–23 mice/group, Log-rank P ¼ .0009). Deletion of CAF-HIF2 modestly reduced tumor fibrosis and significantly decreased the intratumoral recruitment of immunosuppressive M2 macrophages and regulatory T cells. Treatment with the clinical HIF2 inhibitor PT2399 significantly reduced in vitro macrophage chemotaxis and M2 polarization, and improved tumor responses to immunotherapy in both syngeneic PDAC mouse models.CONCLUSIONS:Together, these data suggest that stromal HIF2 is an essential component of PDAC pathobiology and is a druggable therapeutic target that could relieve tumor microenvironment immunosuppression and enhance immune responses in this disease.

Transforming growth factor-β challenge alters the N-, O-, and glycosphingolipid glycomes in PaTu-S pancreatic adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis and high mortality. Transforming growth factor-β (TGF-β) plays a key role in PDAC tumor progression, which is often associated with aberrant glycosylation. However, how PDAC cells respond to TGF-β and the role of glycosylation therein is not well known. Here, we investigated the TGF-β-mediated response and glycosylation changes in the PaTu-8955S (PaTu-S) cell line deficient in SMA-related and MAD-related protein 4 (SMAD4), a signal transducer of the TGF-β signaling. PaTu-S cells responded to TGF-β by upregulating SMAD2 phosphorylation and target gene expression. We found that TGF-β induced expression of the mesenchymal marker N-cadherin but did not significantly affect epithelial marker E-cadherin expression. We also examined differences in N-glycans, O-glycans, and glycosphingolipid-linked glycans in PaTu-S cells upon TGF-β stimulation. TGF-β treatment primarily induced N-glycome aberrations involving elevated levels of branching, core fucosylation, and sialylation in PaTu-S cells, in agreement with TGF-β-induced changes in the expression of glycosylation-associated genes. In addition, we observed differences in O glycosylation and glycosphingolipid glycosylation profiles after TGF-β treatment, including lower levels of sialylated Tn antigen and neoexpression of globosides. Furthermore, the expression of transcription factor sex-determining region Y-related high-mobility group box 4 was upregulated upon TGF-β stimulation, and its depletion blocked TGF-β-induced N-glycomic changes. Thus, TGF-β-induced N-glycosylation changes can occur in a sex-determining region Y-related high-mobility group box 4–dependent and SMAD4-independent manner in the pancreatic PaTu-S cancer cell line. Our results open up avenues to study the relevance of glycosylation in TGF-β signaling in SMAD4-inactivated PDAC.

Fungal mycobiome drives IL-33 secretion and type 2 immunity in pancreatic cancer.

TH2 cells and innate lymphoid cells 2 (ILC2) can stimulate tumor growth by secreting pro-tumorigenic cytokines such as interleukin-4 (IL-4), IL-5, and IL-13. However, the mechanisms by which type 2 immune cells traffic to the tumor microenvironment are unknown. Here, we show that oncogenic KrasG12D increases IL-33 expression in pancreatic ductal adenocarcinoma (PDAC) cells, which recruits and activates TH2 and ILC2 cells. Correspondingly, cancer-cell-specific deletion of IL-33 reduces TH2 and ILC2 recruitment and promotes tumor regression. Unexpectedly, IL-33 secretion is dependent on the intratumoral fungal mycobiome. Genetic deletion of IL-33 or anti-fungal treatment decreases TH2 and ILC2 infiltration and increases survival. Consistently, high IL-33 expression is observed in approximately 20% of human PDAC, and expression is mainly restricted to cancer cells. These data expand our knowledge of the mechanisms driving PDAC tumor progression and identify therapeutically targetable pathways involving intratumoral mycobiome-driven secretion of IL-33.

Oligoadenylate synthetases-like is a prognostic biomarker and therapeutic target in pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is fatal cancer that causes death. Early metastasis, resistance to chemotherapy, and lack of treatment contribute to a poor prognosis. Therefore, finding new therapeutic targets and biomarkers is a particularly urgent need to improve the survival of PDAC patients. Oligoadenylate synthetases-like (OASL), an antiviral enzyme produced by interferon (IFN), has been found to be associated with the occurrence and development of multiple cancers. However, its role in PDAC has been less well-studied. The value of OASL in PDAC was evaluated by bioinformatics and in vitro experiments.MethodsThe expression of OASL was evaluated using the Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA) online websites. The survival time was also calculated by GEPIA. The correlation between OASL messenger RNA (mRNA) expression and immune infiltration was analyzed by the Tumor Immune Estimation Resource (TIMER) database. Clinical characteristics were revealed by The Cancer Genome Atlas (TCGA) data. A nomogram and forest plot were constructed based on univariate and multivariate Cox regression. Cell experiments [western blot assays, 3-(4,5-dimethylathiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, transwell assays, flow cytometry assays] were used to verify the value of OASL in PDAC cells (Panc-1, Mia paca-2, and Aspc-1).ResultsA higher expression of OASL was observed in PDAC (P<0.05). Patients with increased expression of OASL had worse overall survival (OS; P<0.05) and disease-specific survival (DSS; P<0.05). The expression of OASL was correlated with T stage (P=0.030) and N stage (P=0.004), radiation therapy (P=0.013), primary therapy outcome (P<0.001), residual tumor (P=0.028), and tumor location (P=0.004) by univariate analysis, which also confirmed that OASL was an independent prognostic factor. Moreover, OASL expression was positively associated with neutrophils. In vitro experiments indicated that knockdown of OASL inhibited cell viability and invasion while increasing apoptosis rate.ConclusionsHigh expression of OASL is associated with poor prognosis. Targeting OASL delays PDAC tumor progression in vitro. We highlight that OASL is a novel prognostic biomarker and therapeutic target of PDAC.

A Distinct Gut Microbial Ecology is Associated with Enhanced Chemosensitivity Following Neutrophil Attenuation in Pancreatic Cancer

Background: Infiltration of innate immune cells, such as neutrophils, in the tumor microenvironment (TME) are associated with worse oncologic outcomes in pancreatic ductal adenocarcinoma (PDAC). Given recent evidence highlighting the complex intersection between chemosensitivity and the gut microbiome in solid tumors, we sought to investigate if the enhanced chemosensitivity observed with neutrophil attenuation in PDAC is associated with a distinct gut microbial ecology. Methods: C57BL/6 mice were orthotopically injected with syngeneic KrasG12D/+;Trp53fl/+;Pdx-Cre(KPC) PDAC cells, and randomized into four treatment groups after 10d tumor growth (n=8-10/arm): vehicle-control, anti-Ly6G antibody treatment alone with neutrophil-attenuating—but not depleting—dosing (25 μg/dose) q3d, gemcitabine(100 mg/kg) and paclitaxel(10 mg/kg) qwk(G/P), and G/P+anti-Ly6G. Fecal samples from mice (n=3/group) were collected 10d after treatment and submitted for shotgun metagenomic sequencing. Results: Bray-Curtis principal coordinate analysis of microbial β-diversity revealed significant differences in microbial composition with neutrophil attenuation and chemotherapy. Abundance of 37 species were differentially significant between G/P+anti-Ly6G and G/P-treated cohorts (p-adj<0.05). Seven Alistipes spp. (class Bacteroidea)—previously associated with chemoresistance in solid cancers—were significantly reduced in the gut microbiome of G/P+anti-Ly6G-treated mice. Bifidobacterium pseudolongum, previously associated with PDAC tumor progression, was the most significantly reduced bacterial species in the gut microbiome of G/P+anti-Ly6G-treated mice when compared with G/P alone-group. Conclusion: Our data unearth potential crosstalk between a distinct but narrow gut microbial ecology and anti-tumor adaptive immunity in response to neutrophil attenuation that is associated with improved chemosensitivity in PDAC. Therapeutic manipulation of this microbial program may mitigate chemoresistance and improve outcomes in PDAC patients.

BZW1 Facilitates Glycolysis and Promotes Tumor Growth in Pancreatic Ductal Adenocarcinoma Through Potentiating eIF2α Phosphorylation

Pancreatic ductal adenocarcinoma (PDAC) is characterized by severe metabolic stress due to fibrosis and poor vascularization. BZW1 is an eIF5-mimic protein involved in tumorigenesis and progression. The aim of this study was to investigate the role of BZW1 in metabolic stress resistance in PDAC. BZW1 expression was evaluated in human PDAC tissue microarray and PDAC cells. Glycolysis regulation of BZW1 and its correlation with glycolysis-related genes was analyzed. Tumor growth, cell proliferation, and apoptosis were evaluated in mice xenograft tumors and patient-derived organoids. The results of bioinformatic screening identified that BZW1 was 1 of the top 3 genes favorable for tumor progression in PDAC. The analysis of our cohort confirmed that BZW1 was overexpressed in human PDAC tissues compared with nontumor tissues, and its abnormal expression was correlated with large tumor size and poor prognosis. BZW1 promoted cell proliferation and inhibited apoptosis in both mouse xenograft models and PDAC-derived organoids via facilitating glycolysis in the oxygen-glucose-deprivation condition. Mechanically, BZW1 served as an adaptor for PKR-like endoplasmic reticulum (ER) kinase (PERK), facilitated the phosphorylation of eIF2α, promoted internal ribosome entry site-dependent translation of HIF1α and c-Myc, and thereby boosted the Warburg effect. In organoid-based xenografts with high BZW1 levels, both the PERK/eIF2α phosphorylation inhibitor GSK2606414 and ISRIB significantly suppressed tumor growth and prolonged animal survival. BZW1 is a key molecule in the internal ribosome entry site-dependent translation of HIF1α/c-Myc and plays crucial roles in the glycolysis of PDAC. BZW1 might serve as a therapeutic target for patients with pancreatic cancer.

Abstract PO-114: STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment

Abstract One of the defining characteristics of pancreatic ductal adenocarcinoma (PDAC) is the formation of a dense stroma comprised of cancer associated fibroblasts (CAFs) and immune cell populations. This stroma is immunosuppressive and can act as a physical barrier against common therapeutic treatments. Attempts to therapeutically target the PDAC stroma have yielded contradictory results, suggesting both tumor promoting and tumor limiting roles for CAFs. These studies emphasize the need to understand important transsignaling pathways between CAFs, tumor cells, and the immune microenvironment. IL-6 is a pleiotropic cytokine involved in several physiological functions and its increased expression is strongly associated with poor survival rates in PDAC patients. STAT3 is a major downstream target of IL-6, and its aberrant activation has been implicated in PDAC tumor progression and immune evasion. IL-6 expression and the IL-6/STAT3 signaling axis in PDAC has been characterized in epithelial tumor cells, however its stromal-specific function on PDAC has yet to be elucidated. We hypothesized that the STAT3 signaling axis in pancreatic CAFs contributes to the immunosuppressive and fibrotic phenotype seen with disease progression. Employing CreLoxP technology, the fibroblast specific protein-1 (Fsp-Cre) transgene was used to conditionally delete STAT3 in fibroblasts in the PdxFlp; KrasG12D; p53frt/frt (KPF) PDAC mouse model developed by our lab. Deletion of STAT3 in fibroblasts significantly increased the survival in a cohort of KPF mice compared to those with intact STAT3. In preliminary investigations, we found an increase in CD8+ T cell infiltration but a decrease in regulatory T cells in the STAT3-deleted cohort. We also observed a decrease in immunosuppressive M2 macrophage populations and an increase in M1 macrophages in the STAT3-deleted cohort. These preliminary results demonstrate a previously unexplored role of IL-6/STAT3 signaling in fibroblasts during PDAC progression. Citation Format: Julia E. Lefler, Michael Ostrowski, Catherine MarElia-Bennett. STAT3 in cancer-associated fibroblasts promotes an immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-114.

CD109 expression in tumor cells and stroma correlates with progression and prognosis in pancreatic cancer.

CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, whose expression is upregulated in some types of malignant tumors. High levels of CD109 in tumor cells have been reported to correlate with poor prognosis; however, significance of CD109 stromal expression in human malignancy has not been elucidated. In this study, we investigated the tumorigenic properties of CD109 in pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analysis of 92 PDAC surgical specimens revealed that positive CD109 expression in tumor cells was significantly associated with poor prognosis (disease-free survival, p = 0.003; overall survival, p = 0.002), and was an independent prognostic factor (disease-free survival, p = 0.0173; overall survival, p = 0.0104) in PDAC. Furthermore, CD109 expression was detected in the stroma surrounding tumor cells, similar to that of α-smooth muscle actin, a histological marker of cancer-associated fibroblasts. The stromal CD109 expression significantly correlated with tumor progression in PDAC (TNM stage, p = 0.033; N factor, p = 0.024; lymphatic invasion, p = 0.028). In addition, combined assessment of CD109 in tumor cells and stroma could identify the better prognosis group of patients from the entire patient population. In MIA PaCa-2 PDAC cell line, we demonstrated the involvement of CD109 in tumor cell motility, but not in PANC-1. Taken together, CD109 not only in the tumor cells but also in the stroma is involved in the progression and prognosis of PDAC, and may serve as a useful prognostic marker in PDAC.

ROBO2 hampers malignant biological behavior and predicts a better prognosis in pancreatic adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDAC) is a fatalmalignant cancer with extremely poor prognosis and high mortality. Genome wide studies show that Slit/Robo signaling pathway takes a major effect in the oncogenesis and progression of pancreatic cancer. However, the function and mechanism of ROBO2 in the development of PDAC remains unclear. Methods In present study, we use Western blot and real-time polymerase chain reaction (RT-PCR) to detect the expression of ROBO2 in pancreatic cell lines. Cell proliferation,Transwellmigration and invasion were conducted inAsPC-1, MIA PaCa-2 and PANC-1cell lines. RNA sequencing, bioinformatics analysisand Western blot were used to explore its mechanism and potential target molecules. The expression of ROBO2 in 95 tumor tissues was detected by immunohistochemistry. Results ROBO2 expression was downregulated in PDAC cell lines and tissue samples. A high expression of ROBO2 was associated with better prognosis. Upregulation of ROBO2 inhibited PDAC cell proliferation, migration, and invasion. However, we found theoppositeresults in the ROBO2 downregulation group. In addition, the function of ROBO2 on cell proliferation was further affirmed by the animal model. Finally, the results of RNA sequencing indicated that ROBO2 partly promoted the antitumor activity by inhibiting ECM1 in PDAC. Conclusions Our work suggests that ROBO2 inhibits tumor progression in PDAC and may serve as a predictive biomarker and therapeutic target in PDAC.

Coagulation Signaling through PAR1 as a Therapeutic Target in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal disease with a 5-year survival rate of less than 10% following diagnosis. The aggressive and invasive properties of pancreatic cancer tumors coupled with poor diagnostic options contribute to the high mortality rate since most patients present with late-stage disease. Accordingly, PDAC is linked to the highest rate of cancer-associated venous thromboembolic disease of all solid tumor malignancies. However, in addition to promoting clot formation, recent studies suggest that the coagulation system in PDAC mediates a reciprocal relationship, whereby coagulation proteases and receptors promote PDAC tumor progression and dissemination. Here, upregulation of tissue factor (TF) by tumor cells can drive local generation of the central coagulation protease thrombin that promotes cell signaling activity through protease-activated receptors (PARs) expressed by both tumor cells and multiple stromal cell subsets. Moreover, the TF-thrombin-PAR1 signaling axis appears to be a major mechanism of cancer progression in general and PDAC in particular. Here, we summarize the current literature regarding the role of PAR1 in PDAC and review possibilities for pharmacologically targeting PAR1 as a PDAC therapeutic approach.