Paige C. Arneson-Wissink

Cancer cachexia is a metabolic syndrome commonly observed in patients with pancreatic ductal adenocarcinoma (PDAC), characterized by wasting of skeletal and cardiac muscle. This condition is associated with the neurohormonal stress response and activation of the sympathetic nervous system. However the impact of cachexia on cardiac adrenergic signalling remains poorly understood. Here we used a preclinical model of PDAC cachexia to investigate how sympathetic input to the heart is altered. We found desensitization of β1‐adrenergic receptor (β1‐AR) in the heart, along with evidence of increased adrenergic tone. Pharmacological blockade with the β1‐selective antagonist metoprolol partially restored adrenergic responsiveness in PDAC mice, suggesting that elevated adrenergic drive contributes to β1‐AR desensitization. The impaired β1‐AR sensitivity blunted the chronotropic response to dobutamine and reduced inotropic reserve under adrenergic stress. Together these findings uncover disruption of cardiac adrenergic signalling in PDAC cachexia. image Key points: Pancreatic cancer‐associated cachexia promotes β1‐adrenergic receptor desensitization in the heart. Pancreatic cancer‐induced β1‐adrenergic receptor desensitization is partly mediated by increased adrenergic transmission to the heart. β1‐adrenergic receptor desensitization in mice with pancreatic cancer reduces contractile reserve under adrenergic stress.

Background: Cachexia is defined by chronic loss of fat and muscle, is a frequent complication of pancreatic ductal adenocarcinoma (PDAC) and negatively impacts patient outcomes. Nutritional supplementation cannot fully reverse tissue wasting, and the mechanisms underlying this phenotype are unclear. This work aims to define the relative contributions of catabolism and anabolism to adipose wasting in PDAC‐bearing mice. Human antigen R (HuR) is an RNA‐binding protein recently shown to suppress adipogenesis. We hypothesize that fat wasting results from a loss of adipose anabolism driven by increased HuR activity in adipocytes of PDAC‐bearing mice. Methods: Adult C57BL/6J mice received orthotopic PDAC cell ( Kras ^G12D ; p53 ^R172H/+ ; Pdx1‐cre ) (PDAC) or PBS (sham) injections. Mice exhibiting moderate cachexia (9 days after injection) were fasted for 24 h, or fasted 24 h and refed 24 h before euthanasia. A separate cohort of PDAC mice were treated with an established HuR inhibitor (KH‐3, 100 mg/kg) and subjected to the fast/refeed paradigm. We analysed body mass, gross fat pad mass and adipose tissue mRNA expression. We quantified lipolytic rate as the normalized quantity of glycerol released from 3T3‐L1 adipocytes in vitro and gonadal fat pads (gWAT) ex vivo. Results: 3T3‐L1 adipocytes treated with PDAC cell conditioned media (CM) had lower expression of lipolysis and lipogenesis genes than control cells and did not display elevated lipolysis as measured by liberated glycerol. PDAC gWAT cultured ex vivo displayed decreased lipolysis compared to sham gWAT (−54.7%). PDAC and sham mice lost equivalent fat mass after a 24 h fast; however, PDAC mice could not restore inguinal fat pads (iWAT) (−40.5%) or gWAT (−31.8%) mass after refeeding. RNAseq revealed 572 differentially expressed genes in gWAT from PDAC compared to sham mice. Downregulated genes ( n = 126) were associated with adipogenesis (adj p = 0.05), and expression of adipogenesis master regulators Pparg and Cebpa were reduced in gWAT from PDAC mice. Immunohistochemistry revealed increased HuR staining in gWAT (+74.9%) and iWAT (+41.2%) from PDAC mice. Inhibiting HuR binding restored lipogenesis in refed animals with a concomitant increase in iWAT mass (+131.7%). Conclusions: Our work highlights deficient adipose anabolism as a driver of reduced lipid content in 3T3‐L1 adipocytes treated with PDAC conditioned media and PDAC mice. The small molecule KH‐3, which disrupts HuR binding, restored adipose anabolism in PDAC mice. This highlights HuR as a potentially targetable regulatory node for adipose anabolism in cancer cachexia.

Background: Cachexia is defined by chronic loss of fat and muscle, is a frequent complication of pancreatic ductal adenocarcinoma (PDAC), and negatively impacts patient outcomes. Nutritional supplementation cannot fully reverse tissue wasting, and the mechanisms underlying this phenotype are unclear. This work aims to define the relative contributions of catabolism and anabolism to adipose wasting in PDAC-bearing mice. Human antigen R (HuR) is an RNA-binding protein recently shown to suppress adipogenesis. We hypothesize that fat wasting results from a loss of adipose anabolism driven by increased HuR activity in adipocytes of PDAC-bearing mice. Methods: Adult C57BL/6J mice received orthotopic PDAC cell ( Kras ^G12D ; p53 ^R172H/+ ; Pdx1-cre ) (PDAC) or PBS (sham) injections. Mice exhibiting moderate cachexia (9 days after injection) were fasted for 24h, or fasted 24h and refed 24h before euthanasia. A separate cohort of PDAC mice were treated with an established HuR inhibitor (KH-3, 100 mg/kg) and subjected to the fast/refeed paradigm. We analyzed body mass, gross fat pad mass, and adipose tissue mRNA expression. We quantified lipolytic rate as the normalized quantity of glycerol released from 3T3-L1 adipocytes in vitro , and gonadal fat pads (gWAT) ex vivo . Results: 3T3-L1 adipocytes treated with PDAC cell conditioned media (CM) had lower expression of lipolysis and lipogenesis genes than control cells, and did not display elevated lipolysis as measured by liberated glycerol. PDAC gWAT cultured ex vivo displayed decreased lipolysis compared to sham gWAT (-54.7%). PDAC and sham mice lost equivalent fat mass after a 24h fast, however, PDAC mice could not restore inguinal fat pads (iWAT) (-40.5%) or gWAT (-31.8%) mass after refeeding. RNAseq revealed 572 differentially expressed genes in gWAT from PDAC compared to sham mice. Downregulated genes (n=126) were associated with adipogenesis (adj p=0.05), and expression of adipogenesis master regulators Pparg and Cebpa were reduced in gWAT from PDAC mice. Immunohistochemistry revealed increased HuR staining in gWAT (+74.9%) and iWAT (+41.2%) from PDAC mice. Inhibiting HuR binding restored lipogenesis in refed animals with a concomitant increase in iWAT mass (+131.7%). Conclusions: Our work highlights deficient adipose anabolism as a driver of reduced lipid content in 3T3-L1 adipocytes treated with PDAC conditioned media and PDAC mice. The small molecule KH-3, which disrupts HuR binding, restored adipose anabolism in PDAC mice. This highlights HuR as a potentially targetable regulatory node for adipose anabolism in cancer cachexia.

Background & Aims: Tumor immune resistance is recognized as a contributor to low survivorship in pancreatic ductal adenocarcinoma (PDAC). The inflammatory cytokine interleukin-6 (IL-6) promotes polarization of CD4 T cell populations away from immune tolerance, and induces differentiation of cytotoxic CD8 T cells. This work aims to test whether IL-6 could stimulate an anti-tumor response in PDAC Methods: We overexpressed IL-6 in multiple Kras ^G12D/+ , Tp53 ^R172H/+, Pdx1-Cre (KPC) cell lines, which were orthotopically implanted in mice (OT-PDAC ^IL6 ). We followed mouse survival and measured tumor growth, tumor histology, and plasma IL-6 at 5 and 10 days after tumor implantation. We measured tumor immune cell infiltration via flow cytometry and histology. We used antibody-based T cell depletion and secondary tumor implantation rechallenge to test the dependency of the durable immune reaction on T cells. We use lipid nanoparticle (LNP)-based delivery of IL-6 mRNA to the pancreas as an orthogonal approach for testing the effect of elevated IL-6 in the tumor microenvironment on anti-tumor T cell invasion. Results: Improved survival occurred in all instances of OT-PDAC ^IL6 , with one cell line (KxPxCx) reproducibly resulting in long-term recurrence-free survival. With KxPxCx cells, circulating IL-6 was 100-fold higher in OT-KxPxCx ^IL6 than in OT-KxPxCx ^parental mice. Flow cytometry revealed increased T cells and NK cells, and decreased T regulatory cells, and we observed significantly increased lymphoid aggregates in OT-KXPXCX ^IL6 as compared to OT--KxPxCx ^parental tumors. Antibody-based CD4 ⁺ and CD8 ⁺ T cell depletion prevented tumor clearance and completely abolished the survival advantage in OT-KxPxCx ^IL6 mice. The anti-tumor immune response to OT-KxPxCx ^IL6 rendered mice immune to re-challenge with OT-KxPxCx ^parental tumors. LNP delivery of IL-6 to the pancreas elevated systemic IL-6 levels ∼50 fold, lowered tumor burden, and increased anti-tumor T cell phenotypes. Conclusions: Locally high IL-6 concentrations potently enhance the T cell-mediated anti-tumor response to PDAC. SYNOPSIS: Interleukin-6 induces rapid and durable T cell-driven immune clearance of pancreatic ductal adenocarcinoma. The anti-tumor immune microenvironment is hallmarked by increased lymphoid aggregate formation, increased CD4 T cell abundance, and decreased Treg abundance.

Background: Patients with pancreatic ductal adenocarcinoma (PDAC) often suffer from cachexia, a wasting syndrome that significantly reduces both quality of life and survival. Although advanced cachexia is associated with inflammatory signalling and elevated muscle catabolism, the early events driving wasting are poorly defined. During periods of nutritional scarcity, the body relies on hepatic ketogenesis to generate ketone bodies, and lipid metabolism via ketogenesis is thought to protect muscle from catabolizing during nutritional scarcity.Methods: We developed an orthotopic mouse model of early PDAC cachexia in 12‐week‐old C57BL/6J mice. Murine pancreatic cancer cells (KPC) were orthotopically implanted into the pancreas of wild‐type, IL‐6^−/−, and hepatocyte STAT3^−/− male and female mice. Mice were subject to fasting, 50% food restriction, ad libitum feeding or ketogenic diet interventions. We measured longitudinal body composition by EchoMRI, body mass and food intake. At the endpoint, we measured tissue mass, tissue gene expression by quantitative real‐time polymerase chain reaction, whole‐body calorimetry, circulating hormone levels, faecal protein and lipid content, hepatic lipid content and ketogenic response to medium‐chain fatty acid bolus. We assessed muscle atrophy in vivo and C2C12 myotube atrophy in vitro.Results: Pre‐cachectic PDAC mice did not preserve gastrocnemius muscle mass during 3‐day food restriction (−13.1 ± 7.7% relative to food‐restricted sham, P = 0.0117) and displayed impaired fatty acid oxidation during fasting, resulting in a hypoketotic state (ketogenic response to octanoate bolus, −83.0 ± 17.3%, P = 0.0328; Hmgcs2 expression, −28.3 ± 7.6%, P = 0.0004). PDAC human patients display impaired fasting ketones (−46.9 ± 7.1%, P < 0.0001) and elevated circulating interleukin‐6 (IL‐6) (12.4 ± 16.5‐fold increase, P = 0.0001). IL‐6^−/− PDAC mice had improved muscle mass (+35.0 ± 3.9%, P = 0.0031) and ketogenic response (+129.4 ± 44.4%, P = 0.0033) relative to wild‐type PDAC mice. Hepatocyte‐specific signal transducer and activator of transcription 3 (STAT3) deletion prevented muscle loss (+9.3 ± 4.0%, P = 0.009) and improved fasting ketone levels (+52.0 ± 43.3%, P = 0.018) in PDAC mice. Without affecting tumour growth, a carbohydrate‐free diet improved tibialis anterior myofibre diameter (+16.5 ± 3.5%, P = 0.0089), circulating ketone bodies (+333.0 ± 117.6%, P < 0.0001) and Hmgcs2 expression (+106.5 ± 36.1%, P < 0.0001) in PDAC mice. Ketone supplementation protected muscle against PDAC‐induced atrophy in vitro (+111.0 ± 17.6%, P < 0.0001 myofibre diameter).Conclusions: In early PDAC cachexia, muscle vulnerability to wasting is dependent on inflammation‐driven metabolic reprogramming in the liver. PDAC suppresses lipid β‐oxidation and impairs ketogenesis in the liver, which is reversed in genetically modified mouse models deficient in IL‐6/STAT3 signalling or through ketogenic diet supplementation. This work establishes a direct link between skeletal muscle homeostasis and hepatic metabolism. Dietary and anti‐inflammatory interventions that restore ketogenesis may be a viable preventative approach for pre‐cachectic patients with pancreatic cancer.

Cancer cachexia, and its associated complications, represent a large and currently untreatable roadblock to effective cancer management. Many potential therapies have been proposed and tested—including appetite stimulants, targeted cytokine blockers, and nutritional supplementation—yet highly effective therapies are lacking. Innovative approaches to treating cancer cachexia are needed. Members of the Kruppel-like factor (KLF) family play wide-ranging and important roles in the development, maintenance, and metabolism of skeletal muscle. Within the KLF family, we identified KLF10 upregulation in a multitude of wasting contexts—including in pancreatic, lung, and colon cancer mouse models as well as in human patients. We subsequently interrogated loss-of-function of KLF10 as a potential strategy to mitigate cancer associated muscle wasting. In vivo studies leveraging orthotopic implantation of pancreas cancer cells into wild-type and KLF10 KO mice revealed significant preservation of lean mass and robust suppression of pro-atrophy muscle-specific ubiquitin ligases Trim63 and Fbxo32, as well as other factors implicated in atrophy, calcium signaling, and autophagy. Bioinformatics analyses identified Transforming growth factor beta (TGF-β), a known inducer of KLF10 and cachexia promoting factor, as a key upstream regulator of KLF10. We provide direct in vivo evidence that KLF10 KO mice are resistant to the atrophic effects of TGF-β. ChIP-based binding studies demonstrated direct binding to Trim63 , a known wasting-associated atrogene. Taken together, we report a critical role for the TGF-β/KLF10 axis in the etiology of pancreatic cancer–associated muscle wasting and highlight the utility of targeting KLF10 as a strategy to prevent muscle wasting and limit cancer-associated cachexia.

SUMMARY: Cancer cachexia is highly prevalent in patients with pancreatic ductal adenocarcinoma (PDAC). Although advanced cachexia is associated with inflammatory signaling, the early events driving wasting are poorly defined. Using an orthotopic mouse model of PDAC, we find that early cachexia is defined by a pronounced vulnerability to undernutrition, characterized by increased skeletal muscle wasting. PDAC suppresses lipid beta oxidation and impairs ketogenesis in the liver, which coordinates the adaptive response to nutritional scarcity. When PDAC mice are fed ketogenic diet, this effect is reversed, and muscle mass is preserved. Furthermore, physiologic levels of ketones are sufficient to protect myotubes against PDAC-associated wasting. Interleukin-6 (IL-6) drives liver metabolic reprogramming, and hepatocyte-specific loss of Signal Transducer and Activator of Transcription 3 (STAT3) is sufficient to prevent PDAC-associated muscle loss. Together, these studies define a key role for the liver in cachexia development and directly link skeletal muscle homeostasis to hepatic lipid oxidation.

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA–treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp , and (c) found that 3-MA–associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.