The Transcription Factor TFAP2A Induces M2 Macrophage Polarization by Upregulating EPS8L3 Expression, Thereby Accelerating the Malignant Progression of Pancreatic Ductal Adenocarcinoma.

Tumor microenvironment (TME) exerts key roles in pancreatic ductal adenocarcinoma (PDAC) development. However, the factors regulating the cross-talk between PDAC cells and TME are largely unknown. In the present study, we identified a long noncoding RNA (lncRNA) KLHDC7B divergent transcript (KLHDC7B-DT), which was up-regulated in PDAC and correlated with poor survival of PDAC patients. Functional assays demonstrated that KLHDC7B-DT enhanced PDAC cell proliferation, migration, and invasion. Mechanistically, KLHDC7B-DT was found to directly bind IL-6 promoter, induce open chromatin structure at IL-6 promoter region, activate IL-6 transcription, and up-regulate IL-6 expression and secretion. The expression of KLHDC7B-DT was positively correlated with IL-6 in PDAC tissues. Via inducing IL-6 secretion, KLHDC7B-DT activated STAT3 signaling in PDAC cells in an autocrine manner. Furthermore, KLHDC7B-DT also activated STAT3 signaling in macrophages in a paracrine manner, which induced macrophage M2 polarization. KLHDC7B-DT overexpressed PDAC cells-primed macrophages promoted PDAC cell proliferation, migration, and invasion. Blocking IL-6/STAT3 signaling reversed the effects of KLHDC7B-DT on macrophage M2 polarization and PDAC cell proliferation, migration, and invasion. In conclusion, KLHDC7B-DT enhanced malignant behaviors of PDAC cells via IL-6-induced macrophage M2 polarization and IL-6-activated STAT3 signaling in PDAC cells. The cross-talk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.

Background: Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to therapy. Stromal cancer-associated fibroblasts (CAFs) play a key role in promoting tumor progression and chemoresistance, by creating a fibrotic microenvironment that impedes drug access and feeds PDAC cells nutrients and pro-tumor signals. CRO-67 (Patent PCT/AU2023/050505) is a novel drug developed with Noxopharm Ltd using a rational medicinal chemistry design to improve bioavailability of chromans which have potent anti-cancer activity. Using our patient-derived PDAC tumor in a dish model (explants; maintain multicellular architecture and fibrosis) we previously showed that CRO-67 has both anti-tumor and CAF reprogramming capacity in 4 patient explants [Cancer Res (2022) 82 (22_Supplement):C073]. Aims: 1) To expand evaluation of CRO-67 in additional patient-derived PDAC tumor explants given PDAC heterogeneity. 2) Assess the effect of CRO-67 on PDAC cell and CAF function. 3) Validate the therapeutic potential of CRO-67 on PDAC growth in vivo. Methods: 1) PDAC tumor samples collected from 7 patients undergoing pancreatic resection. Tumor explants (1–2mm diameter) were cultured on gelatin sponges, treated with CRO-67 (0–50μg/mL) every 3 days and fixed on day 12. Therapeutic response assessed by immunohistochemistry for cytokeratin (PDAC cells), α-smooth muscle actin (CAFs), bromodeoxyuridine (proliferation) and TUNEL (cell death). 2) PDAC cells (MiaPaCa-2) and patient-derived CAFs were treated with CRO-67 (1.5μM) for 48h before proliferation analysis (IncuCYTE S3) and for 24h before apoptosis (Annexin V/DAPI staining; flow cytometry) and cell cycle (DAPI staining; flow cytometry) analysis. 3) Subcutaneous PDAC (BxPC3 human cells) tumors in mice were treated with CRO-67 (2.5mg/kg Intraperitoneally, twice a day) for 21 days and tumor volume measured (calipers). Results: 1) CRO-67 treatment decreased tumor and CAF cell frequency in 6/6 (no quantifiable tumor in patient 7 explants) and 7/7 patient PDAC explants, respectively. It also decreased explant cell proliferation and increased cell death versus controls. 2) CRO-67 completely abolished proliferation of MiaPaCa-2 and reduced the average growth rate of CAFs by 81.3±8.8% (p=0.0025; n=5), increased apoptosis in MiaPaCa-2 by 455.7±21.4% (p=0.003; n=3) and in CAFs by 172.6±13.1% (p=0.0067; n=5) and increased the fraction of cells in G2/M cell cycle phase by 163.8±12.8% (p<0.0001; n=3) in MiaPaCa-2 and by 74.6±8.6% (p=0.0015; n=5) in CAFs versus controls. 3) CRO-67 reduced PDAC tumors in vivo by 56.7±6.6% (p=0.0013; n=9 mice/group) versus controls. Conclusions: CRO-67: 1) reduced PDAC cells and CAFs in human PDAC explants via reduced proliferation and increased death; 2) reduced proliferation of PDAC cells and CAFs in vitro, increased apoptosis and disrupted cell cycle progression through G2/M phase; 3) reduced PDAC tumor growth in vivo. Implication: We predict CRO-67 is a potential ‘dual cell’ therapy with direct anti-tumor effects and CAF reprogramming capacity, warranting further investigation in vivo. Citation Format: Keilah Garcia Netto, Shannon Chiang, John Kokkinos, Koroush S. Haghighi, Aparna Raina, Omali Pitiyarachchi, Janet Youkhana, Quach Truong, Daniel Wenholz, Xiang Li, Olivier Laczka, Naresh Kumar, John Wilkinson, David Goldstein, George Sharbeen, Phoebe A. Phillips. CRO67 has therapeutic potential against pancreatic tumor cells and cancer associated fibroblasts [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 B062.

Increasing evidence indicates that numerous microRNAs (miRNAs) are altered in pancreatic ductal adenocarcinoma (PDAC), and their alterations significantly influence the malignant behaviour of PDAC. Therefore, identifying miRNAs associated with PDAC and their biological roles in the disease may provide promising therapeutic opportunities. Alteration of the expression of miRNA‑766 (miR‑766) has been previously reported in several types of human malignancy. However, to the best of our knowledge, whether miR‑766 exhibits different expression patterns in PDAC and its underlying functions in the progression of PDAC remain to be elucidated. In the present study, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) was used to detect miR‑766 expression levels in PDAC tissues and cell lines. The effects of miR‑766 upregulation on PDAC cell proliferation and invasion were evaluated using MTT and invasion assays, respectively. The mechanisms underlying the role of miR‑766 in PDAC cells were explored using bioinformatics analysis, luciferase reporter assay, RT‑qPCR and western blot analysis. It was found that miR‑766 was significantly downregulated in PDAC tissues and cell lines. The detailed roles of miR‑766 in the progression of PDAC were characterised using Panc‑1 and Aspc‑1 cell lines. The results revealed that the upregulation of miR‑766 restricted the proliferation and invasion of PDAC cells. Through a series of experiments, it was found that E26 transformation specific‑1 (ETS1) was a direct target of miR‑766 in PDAC cells. Furthermore, ETS1 knockdown simulated the inhibitory effects of the overexpression of miR‑766 on PDAC cells, whereas the effects of miR‑766 restoration on the PDAC cells were reversed by overexpressing ETS1. In conclusion, the findings of the present study demonstrate that miR‑766 offers potential as a therapeutic target for patients with PDAC.

Arginine is an amino acid critical for various cellular processes, not only protein synthesis but also metabolism of other essential metabolites, like polyamines, as well as a signaling factor for pathways such as the growth regulator mTOR. Previously, our group measured arginine levels in the interstitial fluid of tumors (TIF) of pancreatic ductal adenocarcinoma (PDAC) murine models and found extremely low arginine levels (2-5 uM) in the tumor microenvironment (TME). Despite near complete absence of this critical nutrient in the TME, pancreatic tumors exhibit aggressive growth. We have sought to understand both how the PDAC TME becomes arginine limited and how PDAC cells adapt to proliferate in the absence of arginine. Using genetically engineered mice, we find that arginase activity in the myeloid compartment of PDAC tumors is responsible for arginine depletion in the TME. Staining of Arg1+ myeloid populations in human PDAC samples suggest a similar mechanism reduces arginine availability in human PDAC tumors as well. We then leveraged our newfound knowledge of PDAC TIF composition to develop a novel ex vivo cell culture media formulation with physiologically relevant nutrient levels and monitored arginine acquisition pathways using isotope tracing and metabolomics assays to determine how PDAC cells cope with arginine deprivation. Under TME nutrient conditions, PDAC cells consume available citrulline and use it to produce arginine by de novo synthesis. Starving cells of citrulline or genetically perturbing arginosuccinate synthase (ASS1), key enzyme in arginine biosynthesis, significantly reduces PDAC cellular arginine and proliferative capacity. Immunohistochemical analysis of both human and mouse PDAC tumors indicates that the de novo arginine synthesis pathway is highly expressed in PDAC but not in untransformed pancreas, suggesting a key role for this pathway in PDAC progression. Altogether, we find that myeloid-derived arginase challenges PDAC cells by limiting arginine availability and suggest that de novo arginine synthesis may be a critical metabolic pathway that enables PDAC tumors to cope with this metabolic challenge. Citation Format: Juan Apiz-Saab, Alex Muir. Myeloid-derived arginase depletes microenvironmental arginine in PDAC tumors and leads to activation of arginine de novo biosynthesis in cancer cells [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 2177.

A novel DDIT3 activator dehydroevodiamine effectively inhibits tumor growth and tumor cell stemness in pancreatic cancer

Background The hsa_circRNA_103809 (circ_0072088) has been an emerging tumour regulator in human cancers, and is identified as one most aberrantly expressed circRNA in patients with pancreatic ductal adenocarcinoma (PDAC). However, the role of circ_0072088 remains unclear in PDAC cells. Methods Expression of circ_0072088, microRNA (miR)-545-3p and solute carrier family 7 member 11 (SLC7A11) was detected by real-time quantitative PCR and western blotting. Cell progression was measured by cell counting kit (CCK)-8 assay, transwell assays and flow cytometry, as well as xenograft tumour models. Glycolysis was evaluated by commercial assay kits. The interaction among circ_0072088, miR-545-3p and SLC7A11 was confirmed by dual-luciferase reporter assay. Results Circ_0072088 was upregulated in PDAC tumours and cells; besides, high circ_0072088 level was associated with high tumour-node-metastasis (TNM) stage. The circ_0072088 siRNA suppressed cell viability, migration, invasion, extracellular acidification rate (ECAR), lactate production, glucose uptake, and ATP generation, but promoted apoptosis rate and oxygen consumption rate (OCR) in SW1990 and PANC-1 cells. In vivo, circ_0072088 knockdown retarded tumour growth of PANC-1 cells. Overexpressing miR-545-3p mimicked circ_0072088 siRNA-induced actions, and inhibited cell progression and glycolysis of SW1990 and PANC-1 cells. Moreover, SLC7A11 downregulation could be mediated by both circ_0072008 siRNA and miR-545-3p mimic, and participating in suppressive role in cell progression and glycolysis of SW1990 and PANC-1 cells. In mechanism, miR-545-3p was targeted by circ_0072008, and SLC7A11 was target of miR-545-3p. Conclusion Circ_0072088 elicited oncogenic role in malignant cell progression and glycolysis of PDAC cells through circ_0072088/miR-545-3p/SLC7A11 pathway. Highlights Circ_0072088 was upregulated in PDAC tumours and was associated with high tumour burden. Blocking circ_0072088 suppressed cell proliferation, migration, invasion, and glycolysis in PDAC cells. Circ_0072088 could directly regulate miR-545-3p, and SLC7A11 was a target of miR-545-3p. Restoring miR-545-3p mimicked the effects of circ_0072088 knockdown in PDAC cell in vitro.

The majority of pancreatic ductal adenocarcinoma (PDAC) patients present with late-stage, metastatic disease that is often resistant to therapeutics, resulting in the lowest survival rate of all major cancers. Metabolic rewiring in response to oncogenic signals plays a critical role in PDAC cell survival, tumor growth, and metastasis. In contrast to normal epithelial cells, these metabolic alterations make PDAC tumors dependent on glutamine for survival, highlighting a unique metabolic vulnerability that can be therapeutically exploited. However, during times of nutrient stress, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids, including glutamine, in a process called, macropinocytosis. Macropinocytosis occurs downstream of oncogenic KRAS, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, underscoring the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process and the contribution of macropinocytic signaling to aggressive cancer phenotypes remains poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that regulates a wide variety of cell signaling pathways, including KRAS, and is commonly deregulated in human PDAC tumors. Recently, PP2A has been implicated as an important regulator of macropinocytosis, but the mechanism by which this occurs is unknown. We demonstrate that the genetic loss of the specific PP2A subunit, B56a, accelerates the formation of KRAS-driven pancreatic lesions in an in vivo mouse model, implicating this subunit as a critical negative regulator of KRAS phenotypes during PDAC progression. Here, we show that acute PP2A-B56a activation prevents the fusion of macropinosomes with lysosomes, robbing PDAC cells of critical metabolic nutrients and driving cell death. Consistent with these findings, PP2A activation also reduces glutamine levels and suppresses oxidative phosphorylation. Finally, we determined that PP2A activating compounds can function synergistically with metabolic inhibitors, supporting a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging. Citation Format: Garima Baral, Claire M. Pfeffer, Indiraa Doraivel, Brittany L. Allen-Petersen. PP2A activation alters macropinosome processing in pancreatic cancer cells leading to metabolic stress and cancer cell death [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 A054.

Pancreatic ductal adenocarcinoma (PDAC) is the seventh most common cause of cancer-related mortality. Despite different methods of treatment, nearly more than 90% of patients with PDAC die shortly after diagnosis. Contrary to promising results in other cancers, immune checkpoint inhibitors (ICIs) showed limited success in PDAC. Recent studies have shown that noncoding RNAs (ncRNAs) are extensively involved in PDAC cell-immune cell interaction and mediate immune evasion in this vicious cancer. PDAC cells recruit numerous ncRNAs to widely affect the phenotype and function of immune cells through various mechanisms. For instance, PDAC cells upregulate miR-301a and downregulate miR-340 to induce M2 polarization of macrophages or overexpress miR-203, miR-146a, and miR-212-3p to downregulate toll-like receptor 4 (TLR4), CD80, CD86, CD1a, major histocompatibility complex (MHC) II, and CD83, thereby evading recognition by dendritic cells. By downregulating miR-4299 and miR-153, PDAC cells can decrease the expression of NK group 2D (NKG2D) and MHC class I chain-related molecules A and B (MICA/B) to blunt the natural killer (NK) cell response. PDAC cells also highly express lncRNA AL137789.1, hsa_circ_0046523, lncRNA LINC00460, and miR-155-5p to upregulate immune checkpoint proteins and escape T cell cytotoxicity. On the other hand, ncRNAs derived from suppressive immune cells promote proliferation, invasion, and drug resistance in PDAC cells. ncRNAs can be applied to overcome resistance to ICIs, monitor the immune microenvironment of PDAC, and predict response to ICIs. This Review article comprehensively discusses recent findings regarding the roles of ncRNAs in the immune evasion of PDAC.

BackgroundThis study investigated the molecular mechanism of long intergenic non-protein coding RNA 1605 (LINC01605) in the process of tumor growth and liver metastasis of pancreatic ductal adenocarcinoma (PDAC).MethodsLINC01605 was filtered out with specificity through TCGA datasets (related to DFS) and our RNA-sequencing data of PDAC tissue samples from Renji Hospital. The expression level and clinical relevance of LINC01605 were then verified in clinical cohorts and samples by immunohistochemical staining assay and survival analysis. Loss- and gain-of-function experiments were performed to estimate the regulatory effects of LINC01605 in vitro. RNA-seq of LINC01605-knockdown PDAC cells and subsequent inhibitor-based cellular function, western blotting, immunofluorescence and rescue experiments were conducted to explore the mechanisms by which LINC01605 regulates the behaviors of PDAC tumor cells. Subcutaneous xenograft models and intrasplenic liver metastasis models were employed to study its role in PDAC tumor growth and liver metastasis in vivo.ResultsLINC01605 expression is upregulated in both PDAC primary tumor and liver metastasis tissues and correlates with poor clinical prognosis. Loss and gain of function experiments in cells demonstrated that LINC01605 promotes the proliferation and migration of PDAC cells in vitro. In subsequent verification experiments, we found that LINC01605 contributes to PDAC progression through cholesterol metabolism regulation in a LIN28B-interacting manner by activating the mTOR signaling pathway. Furthermore, the animal models showed that LINC01605 facilitates the proliferation and metastatic invasion of PDAC cells in vivo.ConclusionsOur results indicate that the upregulated lncRNA LINC01605 promotes PDAC tumor cell proliferation and migration by regulating cholesterol metabolism via activation of the mTOR signaling pathway in a LIN28B-interacting manner. These findings provide new insight into the role of LINC01605 in PDAC tumor growth and liver metastasis as well as its value for clinical approaches as a metabolic therapeutic target in PDAC.

Histone deacetylase 9 (HDAC9) is involved in a variety of malignant tumors, and leads to malignant tumor development and poor prognosis. However, the association between HDAC9 expression, and the prognosis and clinicopathological features of patients with pancreatic ductal adenocarcinoma (PDAC) remains unclear. The present study used reverse transcription-quantitative PCR, western blotting and immunohistochemistry to detect the expression level of HDAC9 in PDAC tumors and cell lines. The Kaplan-Meier method and Pearson's χ2 test were applied to evaluate the prognostic impact of HDAC9. The present study investigated the effect of HDAC9 on the biological function of PDAC cells. The present results indicated that HDAC9 was highly expressed in PDAC tissue and PDAC cell lines (P<0.05). HDAC9 expression level in tumor tissues was negatively associated with tumor size (P=0.026), T stage (P=0.014) and N stage (P=0.004). Kaplan-Meier analysis suggested that patients with high HDAC9 had shorter recurrence-free survival (RFS; P=0.017) and disease-specific survival (DSS; P=0.022). Moreover, the present results suggested that T stage, N stage and HDAC9 expression level were independent predictive factors for RFS and DSS in patients with PDAC. In addition, silencing HDAC9 significantly inhibited the proliferation and migration of PDAC cells. The present results indicated that high expression levels of HDAC9 were associated with tumor progression and poor prognosis; thus, HDAC9 may serve as a prognostic predictor of PDAC.

Obesity is a risk factor and poor prognostic factor for pancreatic ductal adenocarcinoma (PDAC), but the underlying mechanisms remain unclear. PDAC cells and obese visceral adipocytes (O-Ad) derived from mice and humans were used to analyze interactions between the two cell types, and human microvascular endothelial cells were used for angiogenesis assay. A xenograft mouse model with subcutaneously injected PDAC cells was used for animal studies. The relationship between visceral fat and prognosis was analyzed using resected tissues from PDAC patients with and without obesity. Conditioned media (CM) from O-Ad significantly increased PDAC cell growth and migration and angiogenic capacity in both human and mice cells, and blocking osteopontin (OPN) in O-Ad canceled O-Ad-induced effects in both mouse and human cells. In addition, O-Ad directly increased the migratory and tube-forming capacities of endothelial cells, while blocking OPN canceled these effects. O-Ad increased AKT phosphorylation and VEGFA expression in both PDAC and endothelial cells, and OPN inhibition in O-Ad canceled those O-Ad-induced effects. In the xenograft model, PDAC tumor volume was significantly increased in obese mice compared with lean mice, whereas blocking OPN significantly inhibited obesity-accelerated tumor growth. OPN expression in adipose tissues adjacent to human PDAC tumor was significantly higher in obese patients than in non-obese patients. In PDAC patients with obesity, high OPN expression in adipose tissues was significantly associated with poor prognosis. Obese adipocytes trigger aggressive transformation in PDAC cells to induce PDAC progression and accelerate angiogenesis via OPN secretion.

Introduction: Pancreatic ductal adenocarcinoma (PDAC) has the highest mortality rate among major cancers in the United States, and the 5-year survival rate for patients with metastatic PDAC (mPDAC) is only at 3%. Past studies have shown that the paracrine secretion of soluble factors by endothelial cells (ECs) created a unique niche and promoted the survival of cancer cells (cell growth or chemoresistance) in other types of cancer. The liver is the main site of distant metastases in mPDAC, but the influence of the liver EC microenvironment on mPDAC has not been elucidated. In this study, we determined the paracrine effects of liver ECs on the survival of PDAC and identify involved mechanism. Methods: Primary liver ECs were isolated from non-neoplastic liver tissues to mimic the liver EC microenvironment. Conditioned medium (CM) from liver ECs were collected and then applied to PDAC cells, with CM from PDAC as control CM. Effects of CM on PDAC cell proliferation were measured by the MTT assay. Changes in phosphorylation of receptor tyrosine kinases (RTK) between PDAC CM and EC CM treated PADC cells were determined by a Phospho-RTK Array kit and then validated by Western blotting. Involved RKTs were blocked by antibodies for determining their roles in mediating EC effects on PDAC cells. Lastly, A xenograft tumor model was used to establish PDAC tumors and then treated xenograft mice were subcutaneously injected by either PADC CM or EC CM, and tumors growth was monitored and recorded to evaluate the effect of ECs on PADCs. Results: Compared to PDAC CM, EC CM promoted proliferation in 4 different PDAC cells. We found that human epidermal growth factor receptor 3 (HER3 or ERBB3) was only expressed and activated in BxPC-3 cells (HER3+ve), in which the HER3-AKT signaling pathway was activated by EC CM. Furthermore, blocking HER3 activation with a humanized HER3 antibody, seribantumab, completely blocked EC CM-induced AKT activation and cell proliferation. Moreover, depletion of neuregulin (NRG) from EC CM attenuated HER3-AKT activation and indicated that EC-secreted NRG might play a role in promoting PDAC growth. It is interesting that ERK activation was also observed but was not affected by HER3 inhibition. It implied that EGFR signaling pathway might also be involved in EC CM induced PDAC cell growth. Moreover, the combination of cetuximab, trastuzumab and seribantumab yielded the best inhibitory ability on EC CM promoted cell growth as compared to antibody alone or the combination of two of them. Finally, EC CM promoted PDAC tumor growth was also observed in BxPC-3 derived xenograft mouse model. Conclusions: Our results demonstrated that liver EC-secreted factors promoted PDAC growth either in vitro or in vivo, and HER3 was expressed in a subset of PDAC cells and mediated EC-induced proliferation. Moreover, EGFR pathway may also play a role in EC induced cell growth and needs to be addressed in the future study. Our findings suggest a potential of using the combination of HER antibodies/inhibitors for treating patients with HER3+ve mPDACs. Citation Format: Wei Zhang, Michel’le Wright, Moeez Rathore, Ali Vaziri-Gohar, Jordan Winter, Rui Wang. The role of liver endothelium on pancreatic cancer growth [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-131.

Advancing age is a major independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC). However, the ways in which aging alters cells that eventually comprise the PDAC tumor microenvironment (TME) are incompletely understood. Fibroblasts comprise a large part of PDAC tumors, can alter the behavior of cancer cells, and impact treatment responses. Our studies investigate how normal pancreatic fibroblasts acquire tumor-promoting properties during aging and alter the tumorigenic properties of PDAC both in vitro and in vivo. To determine if PDAC tumor growth is altered by an aged microenvironment, we performed orthotopic injection of KPC and Panc02 PDAC cells into the pancreata of aged (&amp;gt;64-week-old) and young (6-8-week-old) C57Bl/6J mice. Tumors in aged mice are significantly larger compared to those in young mice, and there is increased incidence of liver metastases in the aged mouse cohort. We next queried whether aged pancreatic fibroblasts may contribute to this increase in tumor growth. We generated a panel of aged (donors &amp;gt;55 years old) and young (donors &amp;lt;35 years old) normal human pancreatic fibroblasts to determine the effects of healthy aging fibroblasts on PDAC cell behavior. Conditioned media from or co-culture with aged human pancreatic fibroblasts increases the proliferation, migration, and invasion of PDAC cells compared to young fibroblasts. Aged fibroblast conditioned media activated AKT signaling and enhances features of EMT and stemness. Proteomic analysis of conditioned media reveals a significantly altered secretome in aged fibroblasts, with a number of known tumor-promoting proteins released at higher levels from aged compared to young fibroblasts. We examined the contribution of GDF-15, secreted by aged fibroblasts at relatively high levels, as a paracrine modulator of PDAC growth and progression. Addition of recombinant GDF-15 to young fibroblast conditioned media increases proliferation and invasion of PDAC cells to levels similar to aged fibroblast conditioned media. Treatment of young mice with recombinant GDF-15 increases tumor growth and AKT phosphorylation in vivo. Additionally, inhibition of AKT reduces tumor size in aged but not young mice, suggesting that the aged microenvironment at least in part exerts its pro-tumor effects through AKT. In conclusion, we show that aged, non-cancer-associated pancreatic fibroblasts have the potential to promote growth, migration, and invasiveness in multiple models of pancreatic ductal adenocarcinoma. Further studies examining fibroblast-secreted factors and other changes in the aged TME are ongoing. Citation Format: Daniel J. Zabransky, Yash Chhabra, Mitchell Fane, Emma Kartalia, James Leatherman, Jacquelyn Zimmerman, Elizabeth Jaffee, Ashani Weeraratna. Aged pancreatic fibroblasts enhance pancreatic cancer growth and progression [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 1337.

Enhancer of Zeste Homolog 2 Silences MicroRNA-218 in Human Pancreatic Ductal Adenocarcinoma Cells by Inducing Formation of Heterochromatin

Our recent studies established essential and distinct roles for RalA and RalB small GTPase activation in K-Ras mutant pancreatic ductal adenocarcinoma (PDAC) cell line tumorigencity, invasion, and metastasis. However, the mechanism of Ral GTPase activation in PDAC has not been determined. There are four highly related mammalian RalGEFs (RalGDS, Rgl1, Rgl2, and Rgl3) that can serve as Ras effectors. Whether or not they share distinct or overlapping functions in K-Ras-mediated growth transformation has not been explored. We found that plasma membrane targeting to mimic persistent Ras activation enhanced the growth-transforming activities of RalGEFs. Unexpectedly, transforming activity did not correlate directly with total cell steady-state levels of Ral activation. Next, we observed elevated Rgl2 expression in PDAC tumor tissue and cell lines. Expression of dominant negative Ral, which blocks RalGEF function, as well as interfering RNA suppression of Rgl2, reduced PDAC cell line steady-state Ral activity, growth in soft agar, and Matrigel invasion. Surprisingly, the effect of Rgl2 on anchorage-independent growth could not be rescued by constitutively activated RalA, suggesting a novel Ral-independent function for Rgl2 in transformation. Finally, we determined that Rgl2 and RalB both localized to the leading edge, and this localization of RalB was dependent on endogenous Rgl2 expression. In summary, our observations support nonredundant roles for RalGEFs in Ras-mediated oncogenesis and a key role for Rgl2 in Ral activation and Ral-independent PDAC growth.