Signaling In Pancreatic Ductal Adenocarcinoma Research Articles

Dual-Hit Strategy for Therapeutic Targeting of Pancreatic Cancer in Patient-Derived Xenograft Tumors.

Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) "primes" PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT). We aimed to identify the signal derangements responsible for EMT induction and reverse them and devise approaches to stratify SHHi-responsive tumors noninvasively based on clinically-quantifiable parameters. Animals underwent diffusion-weighted magnetic resonance (DW-MR) imaging for measurement of intratumor diffusivity. In parallel, tissue-level deposition of nanoparticle probes was quantified as a marker of vascular permeability/perfusion. Transcriptomic and bioinformatic analysis was employed to investigate SHHi-induced gene reprogramming and identify key "nodes" responsible for EMT induction. Multiple patient tumor isolates responded to short-term SHH inhibitor exposure with increased vascular patency and permeability, with proportionate increases in tumor diffusivity. Nonresponding PDXs did not. SHHi-treated tumors showed elevated FGF drive and distinctly higher nuclear localization of fibroblast growth factor receptor (FGFR1) in EMT-polarized tumor cells. Pan-FGFR inhibitor NVP-BGJ398 (Infigratinib) reversed the SHHi-induced EMT marker expression and nuclear FGFR1 accumulation without compromising the enhanced permeability effect. This dual-hit strategy of SMO and FGFR inhibition provides a clinically-translatable approach to compromise the profound impermeability of PDAC tumors. Furthermore, clinical deployment of DW-MR imaging could fulfill the essential clinical-translational requirement for patient stratification.

Abstract A025: Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression

Abstract Among all cancer types, pancreatic ductal adenocarcinoma (PDAC) has one of the worst outcomes. In recent studies, metabolic and microenvironmental reprogramming in PDAC has been shown to facilitate tumor progression by promoting immune escape mechanisms, however these mechanisms are still poorly understood. Recently, our lab established that autotaxin (ATX) - lysophosphatidic acid (LPA) signaling in PDAC enhances tumorigenicity, but the immunomodulatory function of this signal pathway remains largely unexplored. We now show that ablation of the LPA-producing enzyme autotaxin (ATX) in PDAC cells can reduce tumor burden in mice and reprogram the tumor immune microenvironment by increasing tumor eosinophil infiltration. The functional role of eosinophils in the context of PDAC is a poorly understood area. The presence of eosinophils in pancreatic cancer has been reported in a few case reports: for example, prior studies identified neutrophils and eosinophils together as a group, expressing high levels of CD66b. We confirmed the presence of eosinophils in PDAC in mice as well as patients, and we found that rare patient samples with the highest intratumoral eosinophil abundance had the longest survival rates. Genetic or pharmacologic ATX inhibition increased intratumoral eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. When eosinophils were present, primary tumor growth was modestly reduced, but metastatic spread was greatly reduced. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop wherein ATX-LPA signaling negatively regulates activity of AP-1 transcription factor c-Jun, in turn suppressing expression of potent eosinophil chemoattractant CCL11 (Eotaxin-1). This is the first study that highlights the anti-tumor potential of eosinophils in pancreatic cancer. Citation Format: Sohinee Bhattacharyya, Chet Oon, Holly Sandborg, Terry Morgan, Mara H. Sherman. Autotaxin-lysolipid signaling suppresses a CCL11- eosinophil axis to promote pancreatic cancer progression [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 A025.

An ERK1/2-driven RNA-binding switch in nucleolin drives ribosome biogenesis and pancreatic tumorigenesis downstream of RAS oncogene.

Oncogenic RAS signaling reprograms gene expression through both transcriptional and post-transcriptional mechanisms. While transcriptional regulation downstream of RAS is relatively well characterized, how RAS post-transcriptionally modulates gene expression to promote malignancy remains largely unclear. Using quantitative RNA interactome capture analysis, we here reveal that oncogenic RAS signaling reshapes the RNA-bound proteomic landscape of pancreatic cancer cells, with a network of nuclear proteins centered around nucleolin displaying enhanced RNA-binding activity. We show that nucleolin is phosphorylated downstream of RAS, which increases its binding to pre-ribosomal RNA (rRNA), boosts rRNA production, and promotes ribosome biogenesis. This nucleolin-dependent enhancement of ribosome biogenesis is crucial for RAS-induced pancreatic cancer cell proliferation and can be targeted therapeutically to inhibit tumor growth. Our results reveal that oncogenic RAS signaling drives ribosome biogenesis by regulating the RNA-binding activity of nucleolin and highlight a crucial role for this mechanism in RAS-mediated tumorigenesis.

Abstract 3825: Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the lethal malignancies due to the acquisition of intrinsic or acquired resistance to chemotherapeutic drugs, such as Gemcitabine (Gem). Recent evidence revealed that calcium signaling significantly contributes to Gem-associated chemoresistance (Gem resistance) in PDAC cells. Andrographis, a labdane diterpenoid isolated from the traditional herb Andrographis paniculate, is known to have a potent calcium channel-blocking potential. Accordingly, we hypothesized that Andrographis might facilitate the reversal of Gem resistance in PDAC. Methods: The critical regulatory pathways associated with the Gem resistance in PDAC were identified by analyzing publicly available cell line-based transcriptomic profiling. Gem resistant (Gem-R) PDAC cells (Gem-R MIA PaCa-2 and BxPC-3) were established by continuously culturing cells with Gem. A series of cell culture experiments were performed using Gem-R PDAC cells to evaluate the Andrographis-mediated reversal of Gem resistance in PDAC cells. At last, the cell culture findings were validated in patient-derived PDAC organoids. Results: Transcriptomic profiling identified the calcium signaling pathway as one of the key regulators of Gem resistance (Fold enrichment: 2.8, P =0.002). The combination of Andrographis and Gem revealed a superior anti-cancer potential in Gem-R PDAC cells on cell viability, clonogenicity, and migration compared to individual treatments with Andrographis or Gem. The combined treatment potentiated cellular apoptosis in Gem-R MIA PaCa-2 (Andro, Gem vs. Combination, 20.3%, 10.9% vs. 31.4%) and Gem-R BxPC-3 (Andro, Gem vs. Combination, 11.3%, 6.8% vs. 16.1%). The fluo-4 assay demonstrated that Andrographis decreased intracellular calcium concentration in Gem-R PDAC cells, while the combined treatment with Andrographis and Gem decreased its concentration more in Gem-R MIA PaCa-2 (fold change [FC] =0.79 vs. Andrographis; FC =0.54 vs. Gem) and Gem-R BxPC-3 (FC =0.80 vs. Andrographis; FC =0.73 vs. Gem). Finally, the combined treatment with Andrographis and Gem showed anti-cancer activity in patient-derived PDAC organoids. Conclusions: We have firstly investigated the Andrographis-mediated reversal of chemoresistance against Gem in PDAC cells, possibly through the regulation of cellular apoptosis and calcium signaling, using a systematic series of Gem resistant cell cultures and patient-derived tumor organoid models. Our findings could provide evidence of the combined treatment with Andrographis and Gem as a complementary therapeutic modality against PDAC. Citation Format: Keisuke Okuno, Caiming Xu, Masanori Tokunaga, Cristina Fillat, Haiyong Han, Yusuke Kinugasa, Ajay Goel. Andrographis reverses gemcitabine-associated chemoresistance by regulating calcium signaling in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3825.

RBPJ Deficiency Sensitizes Pancreatic Acinar Cells to KRAS-Mediated Pancreatic Intraepithelial Neoplasia Initiation

Development of pancreatic ductal adenocarcinoma (PDAC) is a multistep process intensively studied; however, precocious diagnosis and effective therapy still remain unsatisfactory. The role for Notch signaling in PDAC has been discussed controversially, as both cancer-promoting and cancer-antagonizing functions have been described. Thus, an improved understanding of the underlying molecular mechanisms is necessary. Here, we focused on RBPJ, the receiving transcription factor in the Notch pathway, examined its expression pattern in PDAC, and characterized its function in mouse models of pancreatic cancer development and in the regeneration process after acute pancreatitis. Conditional transgenic mouse models were usedfor functional analysis of RBPJ in the adult pancreas, initiation of PDAC precursor lesions, and pancreatic regeneration. Pancreata and primary acinar cells were tested for acinar-to-ductal metaplasia together with immunohistology and comprehensive transcriptional profiling by RNA sequencing. We identified reduced RBPJ expression in a subset of human PDAC specimens. Ptf1α-CreERT-driven depletion of RBPJ in transgenic mice revealed that its function is dispensable for the homeostasis and maintenance of adult acinar cells. However, primary RBPJ-deficient acinar cells underwent acinar-to-ductal differentiation in exvivo. Importantly, oncogenic KRAS expression in the context of RBPJ deficiency facilitated the development of pancreatic intraepithelial neoplasia lesions with massive fibrotic stroma formation. Interestingly, RNA-sequencing data revealed a transcriptional profile associated with the cytokine/chemokine and extracellular matrix changes. In addition, lack of RBPJ delays the course of acute pancreatitis and critically impairs it in the context of KRASG12D expression. Our findings imply that downregulation of RBPJ in PDAC patients derepresses Notch targets and promotes KRAS-mediated pancreatic acinar cells transformation and desmoplasia development.

Suppression of Wnt/β-catenin Signaling in PDAC via METTL16-mediated N6-methyladenosine Modification of DVL2.

Pancreatic cancer is a formidable cause of cancer-related deaths worldwide and has witnessed a more than twofold increase in incidence over the last 25 years. The most frequently occurring form of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC), accounting for the majority of pancreatic cancer cases. N6-methyladenosine (m6A), the most abundant transcript modification, has been implicated in the pathogenesis of numerous human cancers, including pancreatic cancer. Despite this, the functional role of methyltransferase-like 16 (METTL16), a critical m6A methyltransferase, in PDAC remains elusive. In this study, we demonstrate that METTL16 expression is significantly diminished in PDAC, rendering it a promising prognostic indicator. Strikingly, both in vitro and in vivo assays revealed accelerated metastasis and invasion of PDAC cells upon METTL16 knockdown, while overexpression of METTL16 exerted an opposite effect. Mechanistically, METTL16 regulates DVL2 expression by suppressing its translation via m6A modification, thereby regulating Wnt/β-catenin signaling., Our results unveil the downregulation of METTL16 as a concomitant increase in DVL2 levels via m6A modification promoting the progression of PDAC. Thus, we propose METTL16 as a novel therapeutic candidate for targeted PDAC treatment.

Abstract C065: Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer

Abstract Background: Unfortunately, the impact of immunotherapy in pancreatic cancer has, to date, been limited. Nonetheless, despite lack of success with checkpoint blockade alone, there is hope that combinations of agents may unlock the potential for immunotherapy success in pancreatic cancer. FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase that is elevated in human Pancreatic Ductal Adenocarcinoma (PDAC) tissue and correlates with high levels of fibrosis and poor CD8 cytotoxic T cell infiltration. We, and others, have already demonstrated a role for FAK in promoting tumor evasion by inducing an immunosuppressive microenvironment, specifically by regulation of cytokines such as CCL5. This has led to trials of the FAK inhibitor (defactinib) in conjunction with immunotherapy. Here we set out to improve our understanding of the role of FAK in regulating chemokine/cytokine mediated signaling in PDAC with a view to identify clinically targetable pathways with potential therapeutic benefit to pancreatic cancer patients. Methods: We combined CRISPR, NanoString, Forward Phase Protein Arrays (FPPA) and ELISA with in vivo experiments using a transplantable KrasG12Dp53R172H mouse model of Pancreatic Cancer and validated findings using human patient-derived PDAC cell lines. Results: We used CRISPR-Cas9 gene editing to deplete FAK gene expression in Panc47 cells, a syngeneic cell line isolated from PDAC arising in fully back-crossed C57BL/6 KPC mice, and reconstituted wild-type FAK (FAK-WT) expression into a clonal Panc47 FAK null (FAK-/-) cell line. Subsequent NanoString and FPPA analyses of secreted cytokines in Panc47 FAK-WT and Panc47 FAK-/- cell lines identified CXCL16 as one of the most significantly increased chemokines upon FAK depletion. Similar results were observed when CRISPR mediated FAK depletion was performed in patient-derived PDAC cell lines. Previous work in our lab has shown that depletion of Signal Transducer and Activator of Transcription-3 (STAT3), a key mediator of immune evasion, in combination with FAK depletion in orthotopically implanted PDAC tumors significantly increases CD8 T-cell infiltration into murine tumors when compared with FAK-/- tumors alone. Here we identify that dual FAK/STAT3 depletion further elevates CXCL16 expression and show that CXCR6, the receptor for CXCL16, is associated with both murine and human CD8 T-cells. Conclusions: These findings identify a FAK-dependent CXCL16:CXCR6 paracrine signaling axis associated with elevated recruitment of CD8 T-cells into PDAC tumors. This requires the dual targeting of FAK and STAT3, identifying a novel therapeutic strategy that warrants further investigation. Citation Format: Jane L. Parry, Marta Canel, Alan Serrels. Dissecting the role of CXCL16 downstream of FAK inhibition in the anti-tumor immune response against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C065.

Tumor and stroma COL8A1 secretion induces autocrine and paracrine progression signaling in pancreatic ductal adenocarcinoma

Several collagen subtypes are involved in pancreatic ductal adenocarcinoma (PDAC) desmoplasia, which constrains therapeutic efficacy. We evaluated collagen type VIII alpha 1 chain (COL8A1), whose function in PDAC is currently unknown. We identified COL8A1 expression in 7 examined PDAC cell lines by microarray analysis, western blotting, and RT‒qPCR. Higher COL8A1 expression occurred in 2 gemcitabine-resistant PDAC cell lines; pancreas tissue (n=15) from LSL-KrasG12D/+; p48-Cre mice with advanced PDAC predisposition; and PDAC parenchyma and stroma of a patient tissue microarray (n=82). Bioinformatic analysis confirmed higher COL8A1 expression in PDAC patient tissue available from TCGA (n=183), GTEx (n=167), and GEO (n=261) databases. siRNA or lentiviral sh-mediated COL8A1 inhibition in PDAC cells reduced migration, invasion and gemcitabine resistance and resulted in lower cytidine deaminase and thymidine kinase 2 expression and was rescued by COL8A1-secreting cancer-associated fibroblasts (CAFs). The activation of COL8A1 expression involved cJun/AP-1, as demonstrated by CHIP assay and siRNA inhibition. Downstream of COL8A1, activation of ITGB1 and DDR1 receptors and PI3K/AKT and NF-κB signaling occurred, as detected by expression, adhesion and EMSA binding studies. Orthotopic transplantation of PDAC cells with downregulated COL8A1 expression resulted in reduced tumor xenograft growth and lower gemcitabine resistance but was prevented by cotransplantation of COL8A1-secreting CAFs. Most importantly, COL8A1 expression in PDAC patient tissues from our clinic (n=84) correlated with clinicopathological data, and we confirmed these findings by the use of patient data (n=177) from the TCGA database. These findings highlight COL8A1 expression in tumor and stromal cells as a new biomarker for PDAC progression.

Berberine Overcomes Gemcitabine-Associated Chemoresistance through Regulation of Rap1/PI3K-Akt Signaling in Pancreatic Ductal Adenocarcinoma.

Gemcitabine (Gem)-based chemotherapy is one of the first-line treatments for pancreatic ductal adenocarcinoma (PDAC). However, its clinical effect is limited due to development of chemoresistance. Various naturally occurring compounds, including Berberine (BBR), provide an anti-cancer efficacy with time-tested safety, individually and in combination with chemotherapeutic drugs. Accordingly, we hypothesized that BBR might enhance the chemosensitivity to Gem in PDAC. In this study, cell culture studies using MIA PaCa-2 and BxPC-3 cells, followed by analysis in patient-derived organoids were performed to evaluate the anti-cancer effects of BBR in PDAC. Considering that cancer is a significant manifestation of increased chronic inflammatory stress, systems biology approaches are prudent for the identification of molecular pathways and networks responsible for phytochemical-induced anti-cancer activity, we used these approaches for BBR-mediated chemosensitization to Gem. Firstly, Gem-resistant (Gem-R) PDAC cells were established, and the combination of BBR and Gem revealed superior anti-cancer efficacy in Gem-R cells. Furthermore, the combination treatment induced cell cycle arrest and apoptosis in Gem-R PDAC cells. Transcriptomic profiling investigated the Rap1 and PI3K-Akt signaling pathway as a key regulator of Gem-resistance and was a key mediator for BBR-mediated chemosensitization in PDAC cells. All cell culture-based findings were successfully validated in patient-derived organoids. In conclusion, we demonstrate that BBR-mediated reversal of chemoresistance to Gem manifests through Rap1/PI3K-Akt signaling in PDAC.

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

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, characterized by a high metastatic burden. RIO Kinase 3 (RIOK3) has been shown to promote invasion and metastasis of PDAC by cytoskeleton remodeling, but the exact mechanism is still unknown. In this study, we analyzed transcriptome sequencing data from RIOK3 stable knockdown PANC-1 cells and TCGA-PDAC data and discovered that RIOK3 was substantially related to focal adhesion signaling in PDAC. Additionally, silencing RIOK3 dramatically decreased Focal Adhesion Kinase (FAK) protein expression and phosphorylation (Tyr397 and Tyr925 sites). Immunoprecipitation assay verified the interaction of RIOK3 and FAK. Furthermore, RIOK3 considerably increased the protein stability of FAK protein but not FAK-Y925F protein. The biological function of RIOK3 in increasing PDAC cell invasion and migration was shown to be dependent on FAK activation. Moreover, we discovered that RIOK3 mutations were mainly characterized by amplification. RIOK3 mRNA was found to be significantly elevated in PDAC tissues and was associated with a poor prognosis. Furthermore, RIOK3 mRNA was significantly upregulated in later T-stage, pre-existing lymph node metastases, and later pathological stage samples. Overall, our study found that RIOK3 promotes PDAC cell invasion and metastasis by stabilizing FAK protein expression and upregulating its phosphorylation. This also provides a new target for therapeutic modalities targeting FAK.

HOXA10 promote pancreatic cancer progression via directly activating canonical NF-κB signaling pathway.

Although transcription factor homeobox A10 (HOXA10) plays an important role in regulating the development of the pancreas, a pathway of HOXA10 participates in pancreatic ductal adenocarcinoma (PDAC) progression has not been revealed. Immunohistochemistry assays were applied to demonstrate the relationship between HOXA10 expression and PDAC progression. Functional assays were used to illustrate the oncogenic role of HOXA10 in PDAC progression. Regulatory mechanisms of HOXA10 induced IKKβ gene transcription and the nuclear transcription factor kappa B (NF-κB) signal pathways activation were also investigated in PDAC cells. In the current study, we show that HOXA10 expression increased in PDAC with higher tumor stage and poor patient survival in public RNA-seq data suggesting HOXA10 is associated with PDAC progression. HOXA10 promotes PDAC cell proliferation, anchorage colony formation, and xenograft growth by activating canonical NF-κB signaling both in vitro and in vivo. Mechanically, HOXA10 up-regulates IKKβ gene transcription directly and subsequently sustain the activation of NF-κB independent of tumor necrosis factor-alpha in PDAC cells. Collectively, up-regulation of HOXA10 gene expression promote cell growth and tumor progression through directly activating canonical NF-κB signaling in PDAC.

RelA Is an Essential Target for Enhancing Cellular Responses to the DNA Repair/Ref-1 Redox Signaling Protein and Restoring Perturbated Cellular Redox Homeostasis in Mouse PDAC Cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a poor response to current treatment regimens. The multifunctional DNA repair-redox signaling protein Ref-1 has a redox signaling function that activates several transcriptional factors (TFs) including NF-κB (RelA), STAT3, AP-1. These have been implicated in signaling in PDAC and associated with cancer progression and therapy resistance. Numerous studies have shown a role for RelA in PDAC inflammatory responses and therapy resistance, little is known as to how these inflammatory responses are modulated through Ref-1 redox signaling pathways during pancreatic pathogenesis. RelA and STAT3 are two major targets of Ref-1 and are important in PDAC pathogenesis. To decipher the mechanistic role of RelA in response to Ref-1 inhibition, we used PDAC cells (KC3590) from a genetically engineered Kras G12D-driven mouse model that also is functionally deficient for RelA (Parent/Vector) or KC3590 cells with fully functional RelA added back (clone 13; C13). We demonstrated that RelA deficient cells are more resistant to Ref-1 redox inhibitors APX3330, APX2009, and APX2014, and their sensitivity is restored in the RelA proficient cells. Knockdown of STAT3 did not change cellular sensitivity to Ref-1 redox inhibitors in either cell type. Gene expression analysis demonstrated that Ref-1 inhibitors significantly decreased IL-8, FOSB, and c-Jun when functional RelA is present. We also demonstrated that PRDX1, a known Ref-1 redox modulator, contributes to Ref-1 inhibitor cellular response. Knockdown of PRDX1 when functional RelA is present resulted in dramatically increased PDAC killing in response to Ref-1 inhibitors. The enhanced cell killing was not due to increased intracellular ROS production. Although Ref-1 inhibition decreased the NADP/NADPH ratio in the cells, the addition of PRDX1 knockdown did not further this redox imbalance. This data suggests that the mechanism of cell killing following Ref-1 inhibition is at least partially mediated through RelA and not STAT3. Further imbalancing of the redox signaling through disruption of the PRDX1-Ref-1 interaction may have therapeutic implications. Our data further support a pivotal role of RelA in mediating Ref-1 redox signaling in PDAC cells with the Kras G12D genotype and provide novel therapeutic strategies to combat PDAC drug resistance.

Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human cancers. Transforming Growth Factor Beta (TGF-β) is a cytokine that switches from a tumor-suppressor at early stages to a tumor promoter in the late stages of tumor development, by yet unknown mechanisms. Tumor associated MUC1 is aberrantly glycosylated and overexpressed in >80% of PDAs and is associated with poor prognosis. MUC1 expression is found in the early stages of PDA development with subsequent increase in later stages. Analysis of human PDA samples from TCGA database showed significant differences in gene expression and survival profiles between low and high MUC1 samples. Further, high MUC1 expression was found to positively correlate to TGF-βRII expression and negatively correlate to TGF-βRI expression in PDA cell lines. We hypothesized that MUC1 overexpression induces TGF-β mediated non-canonical signaling pathways which is known to be associated with poor prognosis. In this study, we report that MUC1 overexpression in PDA cells directly activates the JNK pathway in response to TGF-β, and leads to increased cell viability via up-regulation and stabilization of c-Myc. Conversely, in low MUC1 expressing PDA cells, TGF-β preserves its tumor-suppressive function and inhibits phosphorylation of JNK and stabilization of c-Myc. Knockdown of MUC1 in PDA cells also results in decreased phosphorylation of JNK and c-Myc in response to TGF-β treatment. Taken together, the results indicate that overexpression of MUC1 plays a significant role in switching the TGF-β function from a tumor-suppressor to a tumor promoter by directly activating JNK. Lastly, we report that high-MUC1 PDA tumors respond to TGF-β neutralizing antibody in vivo showing significantly reduced tumor growth while low-MUC1 tumors do not respond to TGF-β neutralizing antibody further confirming our hypothesis.

CXCL12 in Pancreatic Cancer: Its Function and Potential as a Therapeutic Drug Target.

Simple SummaryPancreatic cancer is a challenging disease to treat effectively. Fibroblasts associated with pancreatic cancer contribute to disease progression by secreting factors that enhance tumor cell survival and help tumor cells avoid detection by the immune system. This overview focuses on a chemokine, CXCL12, produced by cancer-associated fibroblasts and how CXCL12 signaling enhances pancreatic cancer progression by contributing to various hallmarks of cancer including, but not limited to, tumor growth and evasion of immune response. These pro-oncogenic functions of CXCL12 make it an attractive target in pancreatic cancer. We discuss the different approaches in development to therapeutically target CXCL12 and finally propose a novel approach, the use of the farnesyl transferase inhibitor tipifarnib to inhibit CXCL12 expression in pancreatic fibroblasts.Pancreatic ductal adenocarcinoma (PDAC) is a disease with limited therapeutic options and dismal long-term survival. The unique tumor environment of PDAC, consisting of desmoplastic stroma, immune suppressive cells, and activated fibroblasts, contributes to its resistance to therapy. Activated fibroblasts (cancer-associated fibroblasts and pancreatic stellate cells) secrete chemokines and growth factors that support PDAC growth, spread, chemoresistance, and immune evasion. In this review, we focus on one such chemokine, CXCL12, secreted by the cancer-associated fibroblasts and discuss its contribution to several of the classical hallmarks of PDAC and other tumors. We review the various therapeutic approaches in development to target CXCL12 signaling in PDAC. Finally, we propose an unconventional use of tipifarnib, a farnesyl transferase inhibitor, to inhibit CXCL12 production in PDAC.

Abstract PO-073: Inactivation of Notch4 attenuated pancreatic tumorigenesis in mice

Abstract Expression of the Notch family of receptors are often upregulated in pancreatic ductal adenocarcinoma (PDAC), however, the functional impacts of the Notch signaling network on pancreatic tumorigenesis remain unresolved. In this study, we focused on Notch4, which had not been investigated in PDAC. Leveraging the conventional Notch4 deficient mouse line and previously established genetically engineered mouse models (GEMM) for PDAC, we generated KC (LSL-KrasG12D;p48-Cre), N4−/−KC (Notch4−/−;LSL-KrasG12D;p48-Cre), PKC (p16flox/flox;LSL-KrasG12D;p48-Cre), and N4−/−PKC GEMMs (Notch4−/−; p16flox/lox;LSL-KrasG12D;p48-Cre). We performed caerulein treatment in both KC and N4−/−KC mice, and compared the development of acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) between them. The ADM/PanIN lesions were significantly smaller in the N4−/−KC than in the KC GEMM (p=0.01), suggesting that Notch4 deficiency attenuated early pancreatic tumorigenesis. This in vivo result was confirmed by in vitro ADM induction of the explant cultures of mouse pancreatic acinar cells. The number of ADM structures in the N4−/−KC acinar cultures was significantly lower than the KC acinar cultures (p<0.001). To evaluate the role of Notch4 in the later stage of pancreatic tumorigenesis, we compared the histological progression and overall survival between the PKC and N4−/−PKC mice. We found that N4−/−PKC mice had better prognosis (p=0.012) and less tumor burden (PanIN: p=0.018 (2 months), PDAC: p=0.039 (5 months)) compared to the PKC GEMM. RNA-Seq analysis of pancreatic tumor cell lines derived from the PKC and N4−/−PKC GEMMs revealed 408 genes were differentially expressed (FDR<0.05) and the genes related to the NGF processing as novel downstream effectors of the Notch4 signaling pathway(p<0.001). Our study is a novel biological investigation that demonstrated that Notch4 signaling possesses tumor promoting function in pancreatic tumorigenesis. Our study revealed a novel association between the NGF processing pathway and Notch4 signaling in PDAC. Citation Format: Kiyoshi Saeki, Wanglong Qiu, Richard Friedman, Carrie Shawber, Jan Kitajewski, Jianhua Hu, Gloria H. Su. Inactivation of Notch4 attenuated pancreatic tumorigenesis in mice [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-073.

Abstract 117: Inhibition of Hedgehog signaling alters fibroblast composition in pancreatic cancer

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog (HH) pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of HH signaling in PDAC have been contradictory, with HH signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how HH pathway inhibition reprograms the PDAC microenvironment. Experimental Design: We used a combination of pharmacologic inhibition, gain- and loss- of-function genetic experiments, CyTOF, and single cell RNA-sequencing to study the roles of HH signaling in PDAC. Results: We find that HH signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAFs) compared to inflammatory CAFs (iCAFs). SHH overexpression promotes tumor growth, while HH pathway inhibition with the Smoothened antagonist LDE225 impairs tumor growth. Further, HH pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in Tregs, consistent with increased immune suppression. Conclusions: HH pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment. Citation Format: Nina G. Steele, Giulia Biffi, Samantha Kemp, Yaqing Zhang, Donovan Drouillard, LiJyun Syu, Yuan Hao, Tobiloba Oni, Erin Brosnan, Ela Elyada, Abhishek Doshi, Christa Hansma, Carlos Espinoza, Ahmed Abbas, Stephanie The, Valerie Irizarry-Negron, Christopher Halbrook, Nicole Franks, Megan Hoffman, Eileen Carpenter, Zeribe Nwosu, Youngkyu Park, Howard Crawford, Costas Lyssiotis, Timothy Frankel, Arvind Rao, Filip Bednar, Andrzej Dlugosz, Jonathan Preall, David Tuveson, Benjamin Allen, Marina Pasca di Magliano. Inhibition of Hedgehog signaling alters fibroblast composition in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 117.

The Dickkopf1 and FOXM1 positive feedback loop promotes tumor growth in pancreatic and esophageal cancers

Dickkopf1 (DKK1) is overexpressed in various cancers and promotes cancer cell proliferation by binding to cytoskeleton-associated protein 4 (CKAP4). However, the mechanisms underlying DKK1 expression are poorly understood. RNA sequence analysis revealed that expression of the transcription factor forkhead box M1 (FOXM1) and its target genes concordantly fluctuated with expression of DKK1 in pancreatic ductal adenocarcinoma (PDAC) cells. DKK1 knockdown decreased FOXM1 expression and vice versa in PDAC and esophageal squamous cell carcinoma (ESCC) cells. Inhibition of either the DKK1-CKAP4-AKT pathway or the ERK pathway suppressed FOXM1 expression, and simultaneous inhibition of both pathways showed synergistic effects. A FOXM1 binding site was identified in the 5ʹ-untranslated region of the DKK1 gene, and its depletion decreased DKK1 expression and cancer cell proliferation. Clinicopathological and database analysis revealed that PDAC and ESCC patients who simultaneously express DKK1 and FOXM1 have a poorer prognosis. Multivariate analysis demonstrated that expression of both DKK1 and FOXM1 is the independent prognostic factor in ESCC patients. Although it has been reported that FOXM1 enhances Wnt signaling, FOXM1 induced DKK1 expression independently of Wnt signaling in PDAC and ESCC cells. These results suggest that DKK1 and FOXM1 create a positive feedback loop to promote cancer cell proliferation.

1190-P: Diabetes Activates Pancreatic Stellate Cells through RAGE Signaling in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive carcinoma with poor prognosis. Its poor prognosis is ascribed to the extensive fibrotic stroma. Pancreatic stellate cells (PSCs) are major contributors to PDAC fibrosis. Type 2 diabetes (T2D) is an established risk factor for PDAC. The common mechanistic link might be the accumulation of advanced glycation end-products (AGEs) and the signaling through receptor for advanced glycation end-products (RAGE), the receptor for AGEs. Nevertheless, the role of RAGE in PSCs activation was not evaluated in PDAC with T2D. First, PSCs were isolated from male obese T2D model, db/db (db) and db/m (C) mice at 12 weeks of age. mRNA expression of the makers for PSCs activation like TGF-β1, αSMA and collagen type1A1 were significantly increased in PSCs isolated from db compared to C (p<0.01, respectively). To evaluate the implication of RAGE, PSCs isolated from C57BL/6 (WT) and RAGE deleted mice (RKO) were stimulated with 12.5-100 μg/ml AGEs. AGEs increased mRNA expression of TGF-β1, αSMA and collagen type1A1 in WT-PSCs with dose dependent manner (p<0.01, respectively) despite no increase in mRNA expressions in RKO-PSCs. Furthermore, PSCs were isolated from high fat diet induced obese WT and RKO for 8 weeks. The mRNA expressions of TGF-β1, αSMA and collagen type1A1 were comparable between two groups. We evaluated surgically resected human PDAC subjects with T2D (PDT2D, 33 cases) and without T2D (PDNT2D, 83cases). αSMA-labeled PSCs significantly surrounded the ducts with dysplasia in PDT2D compared to PDNT2D (p<0.05). The nuclear expression of NF-κB in stromal cells, which was downstream molecules of RAGE, was significantly higher in PDT2D than PDNT2D (p<0.05). Collectively, RAGE signaling was activated in PSCs in T2D, which could be implicated in severe stromal fibrosis in PDAC. Inhibition of RAGE signaling in PSCs may link to a new therapy, in which the stromal fibrosis and cytokine production are reduced in PDAC with T2D. Disclosure C. Uchida: None. H. Mizukami: None. K. Kudo: None. Y. Yamamoto: None. Funding Japan Society for the Promotion of Science (JP17K17579)

Bioinformatics and Functional Analyses Implicate Potential Roles for EOGT and L-fringe in Pancreatic Cancers.

Notch signaling receptors, ligands, and their downstream target genes are dysregulated in pancreatic ductal adenocarcinoma (PDAC), suggesting a role of Notch signaling in pancreatic tumor development and progression. However, dysregulation of Notch signaling by post-translational modification of Notch receptors remains poorly understood. Here, we analyzed the Notch-modifying glycosyltransferase involved in the regulation of the ligand-dependent Notch signaling pathway. Bioinformatic analysis revealed that the expression of epidermal growth factor (EGF) domain-specific O-linked N-acetylglucosamine (EOGT) and Lunatic fringe (LFNG) positively correlates with a subset of Notch signaling genes in PDAC. The lack of EOGT or LFNG expression inhibited the proliferation and migration of Panc-1 cells, as observed by the inhibition of Notch activation. EOGT expression is significantly increased in the basal subtype, and low expression of both EOGT and LFNG predicts better overall survival in PDAC patients. These results imply potential roles for EOGT- and LFNG-dependent Notch signaling in PDAC.

Inhibition of Hedgehog Signaling Alters Fibroblast Composition in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by an extensive fibroinflammatory stroma, which includes abundant cancer-associated fibroblast (CAF) populations. PDAC CAFs are heterogeneous, but the nature of this heterogeneity is incompletely understood. The Hedgehog pathway functions in PDAC in a paracrine manner, with ligands secreted by cancer cells signaling to stromal cells in the microenvironment. Previous reports investigating the role of Hedgehog signaling in PDAC have been contradictory, with Hedgehog signaling alternately proposed to promote or restrict tumor growth. In light of the newly discovered CAF heterogeneity, we investigated how Hedgehog pathway inhibition reprograms the PDAC microenvironment. We used a combination of pharmacologic inhibition, gain- and loss-of-function genetic experiments, cytometry by time-of-flight, and single-cell RNA sequencing to study the roles of Hedgehog signaling in PDAC. We found that Hedgehog signaling is uniquely activated in fibroblasts and differentially elevated in myofibroblastic CAFs (myCAF) compared with inflammatory CAFs (iCAF). Sonic Hedgehog overexpression promotes tumor growth, while Hedgehog pathway inhibition with the smoothened antagonist, LDE225, impairs tumor growth. Furthermore, Hedgehog pathway inhibition reduces myCAF numbers and increases iCAF numbers, which correlates with a decrease in cytotoxic T cells and an expansion in regulatory T cells, consistent with increased immunosuppression. Hedgehog pathway inhibition alters fibroblast composition and immune infiltration in the pancreatic cancer microenvironment.