Cancer-associated fibroblast-derived acetate promotes pancreatic cancer development by altering polyamine metabolism via the ACSS2-SP1-SAT1 axis.

The ability of tumour cells to thrive in harsh microenvironments depends on the utilization of nutrients available in the milieu. Here we show that pancreatic cancer-associated fibroblasts (CAFs) regulate tumour cell metabolism through the secretion of acetate, which can be blocked by silencing ATP citrate lyase (ACLY) in CAFs. We further show that acetyl-CoA synthetase short-chain family member 2 (ACSS2) channels the exogenous acetate to regulate the dynamic cancer epigenome and transcriptome, thereby facilitating cancer cell survival in an acidic microenvironment. Comparative H3K27ac ChIP-seq and RNA-seq analyses revealed alterations in polyamine homeostasis through regulation of SAT1 gene expression and enrichment of the SP1-responsive signature. We identified acetate/ACSS2-mediated acetylation of SP1 at the lysine 19 residue that increased SP1 protein stability and transcriptional activity. Genetic or pharmacologic inhibition of the ACSS2-SP1-SAT1 axis diminished the tumour burden in mouse models. These results reveal that the metabolic flexibility imparted by the stroma-derived acetate enabled cancer cell survival under acidosis via the ACSS2-SP1-SAT1 axis.

Structural Basis for Multivalent MUC16 Recognition and Robust Anti-Pancreatic Cancer Activity of Humanized Antibody AR9.6.

Mucin-16 (MUC16) is a target for antibody-mediated immunotherapy in pancreatic ductal adenocarcinoma (PDAC) amongst other malignancies. The MUC16 specific monoclonal antibody AR9.6 has shown promise for PDAC immunotherapy and imaging. Here, we report the structural and biological characterization of the humanized AR9.6 antibody (huAR9.6). The structure of huAR9.6 was determined in complex with a MUC16 SEA (Sea urchin sperm, Enterokinase, Agrin) domain. Binding of huAR9.6 to recombinant, shed, and cell-surface MUC16 was characterized, and anti-PDAC activity was evaluated in vitro and in vivo. huAR9.6 bound a discontinuous, SEA domain epitope with an affinity of ~90 nM. Binding affinity depended on the specific SEA domain(s) present, and glycosylation enhanced affinity by 3-7-fold driven by favorable entropy and enthalpy and via distinct transition state thermodynamic pathways. Treatment with huAR9.6 reduced the in vitro growth, migration, invasion, and clonogenicity of MUC16-positive PDAC cells and patient-derived organoids (PDOs). HuAR9.6 blocked MUC16-mediated ErbB and AKT activation in PDAC cells, PDOs and patient-derived xenografts and induced antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. More importantly, huAR9.6 treatment caused substantial PDAC regression in subcutaneous and orthotopic tumor models. The mechanism of action of huAR9.6 may depend on dense avid binding to homologous SEA domains on MUC16. The results of this study validate the translational therapeutic potential of huAR9.6 against MUC16-positive PDACs.

Engrailed-1 Promotes Pancreatic Cancer Metastasis.

Engrailed-1 (EN1) is a critical homeodomain transcription factor (TF) required for neuronal survival, and EN1 expression has been shown to promote aggressive forms of triple negative breast cancer. Here, it is reported that EN1 is aberrantly expressed in a subset of pancreatic ductal adenocarcinoma (PDA) patients with poor outcomes. EN1 predominantly repressed its target genes through direct binding to gene enhancers and promoters, implicating roles in the activation of MAPK pathways and the acquisition of mesenchymal cell properties. Gain- and loss-of-function experiments demonstrated that EN1 promoted PDA transformation and metastasis in vitro and in vivo. The findings nominate the targeting of EN1 and downstream pathways in aggressive PDA.

radioGWAS: link radiome to genome to discover driver genes with somatic mutations for heterogeneous tumor image phenotype in pancreatic cancer.

Addressing the significant level of variability exhibited by pancreatic cancer necessitates the adoption of a systems biology approach that integrates molecular data, biological properties of the tumors, and clinical features of the patients. In this study, a comprehensive multi-omics methodology was employed to examine a distinctive collection patient dataset containing rapid autopsy tumor and normal tissue samples as well as longitudinal imaging with a focus on pancreatic cancer. By performing a whole exome sequencing analysis on tumor and normal tissues to identify somatic gene variants and a radiomics feature analysis to tumor CT images, the genome-wide association approach established a connection between pancreatic cancer driver genes and relevant radiomics features, enabling a thorough and quantitative assessment of the heterogeneity of pancreatic tumors. The significant association between sets of genes and radiomics features revealed the involvement of genes in shaping tumor morphological heterogeneity. Some results of the association established a connection between the molecular level mechanism and their outcomes at the level of tumor structural heterogeneity. Because tumor structure and tumor structural heterogeneity are related to the patients' overall survival, patients who had pancreatic cancer driver gene mutations with an association to a certain radiomics feature have been observed to experience worse survival rates than cases without these somatic mutations. Furthermore, the outcome of the association analysis has revealed potential gene mutations and radiomics feature candidates that warrant further investigation in future research endeavors.

Tumour extracellular vesicles and particles induce liver metabolic dysfunction.

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs-particularly palmitic acid-induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis.

Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.

MUC16 Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma by Upregulating NRP2-Associated Cell Adhesion.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer, as it commonly metastasizes to the liver resulting in an overall poor prognosis. However, the molecular mechanism involved in liver metastasis remains poorly understood. Here, we aimed to identify the MUC16-mediated molecular mechanism of PDAC-liver metastasis. Previous studies demonstrated that MUC16 and its C-terminal (Cter) domain are involved in the aggressiveness of PDAC. In this study, we observed MUC16 and its Cter expression significantly high in human PDAC tissues, PDAC organoids, and metastatic liver tissues, while no expression was observed in normal pancreatic tissues using IHC and immunofluorescence (IFC) analyses. MUC16 knockdown in SW1990 and CD18/HPAF PDAC cells significantly decreased the colony formation, migration, and endothelial/p-selectin binding. In contrast, MUC16-Cter ectopic overexpression showed significantly increased colony formation and motility in MiaPaCa2 pancreatic cancer cells. Interestingly, MUC16 promoted cell survival and colonization in the liver, mimicking an ex vivo environment. Furthermore, MUC16 enhanced liver metastasis in the in vivo mouse model. Our integrated analyses of RNA-sequencing suggested that MUC16 alters Neuropilin-2 (NRP2) and cell adhesion molecules in pancreatic cancer cells. Furthermore, we identified that MUC16 regulated NRP2 via JAK2/STAT1 signaling in PDAC. NRP2 knockdown in MUC16-overexpressed PDAC cells showed significantly decreased cell adhesion and migration. Overall, the findings indicate that MUC16 regulates NRP2 and induces metastasis in PDAC. IMPLICATIONS: This study shows that MUC16 plays a critical role in PDAC liver metastasis by mediating NRP2 regulation by JAK2/STAT1 axis, thereby paving the way for future therapy efforts for metastatic PDAC.

Small-molecule IKKß activation modulator (IKAM) targets MAP3K1 and inhibits pancreatic tumor growth.

Activation of inhibitor of nuclear factor NF-κB kinase subunit-ß (IKKß), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKß knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKß and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKß is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKß in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKß inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKß but inhibited the IKKß kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKß phosphorylation was inhibited by 39-100, thus we termed it IKKß activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKß levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKß activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

Machine learning analyses of highly-multiplexed immunofluorescence identifies distinct tumor and stromal cell populations in primary pancreatic tumors.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a formidable challenge for patients and clinicians. OBJECTIVE: To analyze the distribution of 31 different markers in tumor and stromal portions of the tumor microenvironment (TME) and identify immune cell populations to better understand how neoplastic, non-malignant structural, and immune cells, diversify the TME and influence PDAC progression. METHODS: Whole slide imaging (WSI) and cyclic multiplexed-immunofluorescence (MxIF) was used to collect 31 different markers over the course of nine distinctive imaging series of human PDAC samples. Image registration and machine learning algorithms were developed to largely automate an imaging analysis pipeline identifying distinct cell types in the TME. RESULTS: A random forest algorithm accurately predicted tumor and stromal-rich areas with 87% accuracy using 31 markers and 77% accuracy using only five markers. Top tumor-predictive markers guided downstream analyses to identify immune populations effectively invading into the tumor, including dendritic cells, CD4+ T cells, and multiple immunoregulatory subtypes. CONCLUSIONS: Immunoprofiling of PDAC to identify differential distribution of immune cells in the TME is critical for understanding disease progression, response and/or resistance to treatment, and the development of new treatment strategies.

Visceral adipose tissue remodeling in pancreatic ductal adenocarcinoma cachexia: the role of activin A signaling.

Pancreatic ductal adenocarcinoma (PDAC) patients display distinct phenotypes of cachexia development, with either adipose tissue loss preceding skeletal muscle wasting or loss of only adipose tissue. Activin A levels were measured in serum and analyzed in tumor specimens of both a cohort of Stage IV PDAC patients and the genetically engineered KPC mouse model. Our data revealed that serum activin A levels were significantly elevated in Stage IV PDAC patients in comparison to age-matched non-cancer patients. Little is known about the role of activin A in adipose tissue wasting in the setting of PDAC cancer cachexia. We established a correlation between elevated activin A and remodeling of visceral adipose tissue. Atrophy and fibrosis of visceral adipose tissue was examined in omental adipose tissue of Stage IV PDAC patients and gonadal adipose tissue of an orthotopic mouse model of PDAC. Remarkably, white visceral adipose tissue from both PDAC patients and mice exhibited decreased adipocyte diameter and increased fibrotic deposition. Strikingly, expression of thermogenic marker UCP1 in visceral adipose tissues of PDAC patients and mice remained unchanged. Thus, we propose that activin A signaling could be relevant to the acceleration of visceral adipose tissue wasting in PDAC-associated cachexia.

Differential expression profile of CXC-receptor-2 ligands as potential biomarkers in pancreatic ductal adenocarcinoma.

The discovery of early detection markers of pancreatic cancer (PC) disease is highly warranted. We analyzed the expression profile of different CXC-receptor-2 (CXCR2) ligands in PC cases for the potential of biomarker candidates. Analysis of different PDAC microarray datasets with matched normal and pancreatic tumor samples and next-generation sequenced transcriptomics data using an online portal showed significantly high expression of CXCL-1, 3, 5, 6, 8 in the tumors of PC patients. High CXCL5 expression was correlated to poor PC patient survival. Interestingly, mRNA and protein expression analysis of human PC cell lines showed higher CXCL2, 3, and 5 expressions in cell lines derived from metastatic sites than primary tumors. Furthermore, we utilized immunohistochemistry (IHC) to evaluate the expression of CXCR2 ligands in the human PC tumors and observed positive staining for CXCL1, 3, and 8 with a higher average IHC composite score of CXCL3 in the PC tissue specimens than the normal pancreas. We also observed an increase in the expression of mouse CXCL1, 3, and 5 in the pre-cancerous lesions of tumors and metastasis tissues derived from the PDX-cre-LSL-KrasG12D mouse model. Together, our data suggest that different CXCR2 ligands show the potential of being utilized as a diagnostic biomarker in PC patients.

Mucin 5AC-Mediated CD44/ITGB1 Clustering Mobilizes Adipose-Derived Mesenchymal Stem Cells to Modulate Pancreatic Cancer Stromal Heterogeneity.

BACKGROUND & AIMS: Secreted mucin 5AC (MUC5AC) promotes pancreatic cancer (PC) progression and chemoresistance, suggesting its clinical association with poor prognosis. RNA sequencing analysis from the autochthonous pancreatic tumors showed a significant stromal alteration on genetic ablation of Muc5ac. Previously, depletion or targeting the stromal fibroblasts showed an ambiguous effect on PC pathogenesis. Hence, identifying the molecular players and mechanisms driving fibroblast heterogeneity is critical for improved clinical outcomes. METHODS: Autochthonous murine models of PC (KrasG12D, Pdx1-Cre [KC] and KrasG12D, Pdx1-Cre, Muc5ac-/- [KCM]) and co-implanted allografts of murine PC cell lines (Muc5ac wild-type and CRISPR/Cas knockout) with adipose-derived mesenchymal stem cells (AD-MSCs) were used to assess the role of Muc5ac in stromal heterogeneity. Proliferation, migration, and surface expression of cell-adhesion markers on AD-MSCs were measured using live-cell imaging and flow cytometry. MUC5AC-interactome was investigated using mass-spectrometry and enzyme-linked immunosorbent assay. RESULTS: The KCM tumors showed a significant decrease in the expression of α-smooth muscle actin and fibronectin compared with histology-matched KC tumors. Our study showed that MUC5AC, carrying tumor secretome, gets enriched in the adipose tissues of tumor-bearing mice and patients with PC, promoting CD44/CD29 (integrin-ß1) clustering that leads to Rac1 activation and migration of AD-MSCs. Furthermore, treatment with KC-derived serum enhanced proliferation and migration of AD-MSCs, which was abolished on Muc5ac-depletion or pharmacologic inhibition of CXCR2 and Rac1, respectively. The AD-MSCs significantly contribute toward α-smooth muscle actin-positive cancer-associated fibroblasts population in Muc5ac-dependent manner, as suggested by autochthonous tumors, co-implantation xenografts, and patient tumors. CONCLUSION: MUC5AC, secreted during PC progression, enriches in adipose and enhances the mobilization of AD-MSCs. On recruitment to pancreatic tumors, AD-MSCs proliferate and contribute towards stromal heterogeneity.

Ubiquitous Aberration in Cholesterol Metabolism across Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer (PC) is characterized by metabolic deregulations that often manifest as deviations in metabolite levels and aberrations in their corresponding metabolic genes across the clinical specimens and preclinical PC models. Cholesterol is one of the critical metabolites supporting PC, synthesized or acquired by PC cells. Nevertheless, the significance of the de novo cholesterol synthesis pathway has been controversial in PC, indicating the need to reassess this pathway in PC. We utilized preclinical models and clinical specimens of PC patients and cell lines and utilized mass spectrometry-based sterol analysis. Further, we also performed in silico analysis to corroborate the significance of de novo cholesterol synthesis pathway in PC. Our results demonstrated alteration in free sterol levels, including free cholesterol, across in vitro, in vivo, and clinical specimens of PC. Especially, our sterol analyses established consistent alterations in free cholesterol across the different PC models. Overall, this study demonstrates the significance and consistency in deviation of cholesterol synthesis pathway in PC while showing the aberrations in sterol metabolite intermediates and the related genes using preclinical models, in silico platforms, and the clinical specimens.

CD73 induces GM-CSF/MDSC-mediated suppression of T cells to accelerate pancreatic cancer pathogenesis.

Metabolic alterations regulate cancer aggressiveness and immune responses. Given the poor response of pancreatic ductal adenocarcinoma (PDAC) to conventional immunotherapies, we investigated the link between metabolic alterations and immunosuppression. Our metabolic enzyme screen indicated that elevated expression of CD73, an ecto-5'-nucleotidase that generates adenosine, correlates with increased aggressiveness. Correspondingly, we observed increased interstitial adenosine levels in tumors from spontaneous PDAC mouse models. Diminishing CD73 by genetic manipulations ablated in vivo tumor growth, and decreased myeloid-derived suppressor cells (MDSC) in orthotopic mouse models of PDAC. A high-throughput cytokine profiling demonstrated decreased GM-CSF in mice implanted with CD73 knockdowns. Furthermore, we noted increased IFN-γ expression by intratumoral CD4+ and CD8+ T cells in pancreatic tumors with CD73 knockdowns. Depletion of CD4+ T cells, but not CD8+ T cells abrogated the beneficial effects of decreased CD73. We also observed that splenic MDSCs from Nt5e knockdown tumor-bearing mice were incompetent in suppressing T cell activation in the ex vivo assays. Replenishing GM-CSF restored tumor growth in Nt5e knockout tumors, which was reverted by MDSC depletion. Finally, anti-CD73 antibody treatment significantly improved gemcitabine efficacy in orthotopic models. Thus, targeting the adenosine axis presents a novel therapeutic opportunity for improving the anti-tumoral immune response against PDAC.

IgE-Based Therapeutic Combination Enhances Antitumor Response in Preclinical Models of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) represents 3% of all cancer cases and 7% of all cancer deaths in the United States. Late diagnosis and inadequate response to standard chemotherapies contribute to an unfavorable prognosis and an overall 5-year survival rate of less than 10% in PDAC. Despite recent advances in tumor immunology, tumor-induced immunosuppression attenuates the immunotherapy response in PDAC. To date, studies have focused on IgG-based therapeutic strategies in PDAC. With the recent interest in IgE-based therapies in multiple solid tumors, we explored the MUC1-targeted IgE potential against pancreatic cancer. Our study demonstrates the notable expression of FceRI (receptor for IgE antibody) in tumors from PDAC patients. Our study showed that administration of MUC1 targeted-IgE (mouse/human chimeric anti-MUC1.IgE) antibody at intermittent levels in combination with checkpoint inhibitor (anti-PD-L1) and TLR3 agonist (PolyICLC) induces a robust antitumor response that is dependent on NK and CD8 T cells in pancreatic tumor-bearing mice. Subsequently, our study showed that the antigen specificity of the IgE antibody plays a vital role in executing the antitumor response as nonspecific IgE, induced by ovalbumin (OVA), failed to restrict tumor growth in pancreatic tumor-bearing mice. Utilizing the OVA-induced allergic asthma-PDAC model, we demonstrate that allergic phenotype induced by OVA cannot restrain pancreatic tumor growth in orthotopic tumor-bearing mice. Together, our data demonstrate the novel tumor protective benefits of tumor antigen-specific IgE-based therapeutics in a preclinical model of pancreatic cancer, which can open new avenues for future clinical interventions.

Plexin-B3 Regulates Cellular Motility, Invasiveness, and Metastasis in Pancreatic Cancer.

The Plexins family of proteins are well-characterized transmembrane receptors of semaphorins, axon guidance cue molecules, that mediate the cell attraction or repelling effects for such cues. Plexins and their ligands are involved in numerous cellular activities, such as motility, invasion, and adhesion to the basement membrane. The detachment of cells and the gain in motility and invasion are hallmarks of the cancer metastasis cascade, thus generating interest in exploring the role of plexins in cancer metastasis. Semaphorin-plexin complexes can act as tumor promoters or suppressors, depending upon the cancer type, and are under investigation for therapeutic purposes. Our group has identified Semaphorin-5A (SEMA5A)/Plexin-B3 as an attractive targetable complex for pancreatic cancer (PC) metastasis. However, our understanding of the Plexin-B3 function and pathological expression in PC is limited, and our present study delineates the role of Plexin-B3 in PC malignancy. We examined the pathological expression of Plexin-B3 in PC tumors and metastasis using a human tissue microarray, disease progression model of PDX-Cre-Kras(G12D) (KC) mice, and different metastatic sites obtained from the KrasG12D; Trp53R172H; Pdx1-Cre (KPC) mice model. We observed a higher Plexin-B3 expression in PC tumor cores than the normal pancreas, and different metastatic sites were positive for Plexin-B3 expression. However, in the KC mice model, the Plexin-B3 expression increased initially and then decreased with the disease progression. Next, to evaluate the functional role of Plexin-B3, we utilized T3M-4- and CD18/HPAF-Control and -Plexin B3 knockdown cells for different in vivo and in vitro studies. The knockdown of Plexin-B3 enhanced the in vitro cellular migration, invasiveness, and impaired colony formation in three-dimensional culture, along with an increase in cellular spread and remodeling of the actin filaments. We also observed a higher metastasis in nude mice injected with T3M-4- and CD18/HPAF-shPlexin-B3 cells compared to their respective control cells. Furthermore, we observed a lower number of proliferating Ki-67-positive cells and higher ALDH1-A1-positive cells in the tumors formed by Plexin-B3 knockdown cells compared to tumors formed by the control cells. Together, our data suggest that the loss of Plexin-B3 is associated with the interference of cell division machinery and the induction of stem cell-like characteristics in PC cells.