Hepatic signal transducer and activator of transcription-3 signalling drives early-stage pancreatic cancer cachexia via suppressed ketogenesis.

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 ß-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.

Optimization of Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Three-dimensional (3D) cell culture models are widely used in tumor studies to more accurately reflect cell-cell interactions and tumor growth conditions in vivo. 3D anchorage-independent spheroids derived by culturing cells in ultra-low attachment (ULA) conditions is particularly relevant to ovarian cancer, as such cell clusters are often observed in malignant ascites of late-stage ovarian cancer patients. We and others have found that cells derived from anchorage-independent spheroids vary widely in gene expression profiles, proliferative state, and metabolism compared to cells maintained under attached culture conditions. This includes changes in mitochondrial function, which is most commonly assessed in cultured live cells by measuring oxygen consumption in extracellular flux assays. To measure mitochondrial function in anchorage-independent multicellular aggregates, we have adapted the Agilent Seahorse extracellular flux assay to optimize measurements of oxygen consumption and extracellular acidification of ovarian cancer cell spheroids generated by culture in ULA plates. This protocol includes: (i) Methods for culturing tumor cells as uniform anchorage-independent spheroids; (ii) Optimization for the transfer of spheroids to the Agilent Seahorse cell culture plates; (iii) Adaptations of the mitochondrial and glycolysis stress tests for spheroid extracellular flux analysis; and (iv) Suggestions for optimization of cell numbers, spheroid size, and normalization of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values. Using this method, we have found that ovarian cancer cells cultured as anchorage-independent spheroids display altered mitochondrial function compared to monolayer cultures attached to plastic dishes. This method allows for the assessment of mitochondrial function in a more relevant patho/physiological culture condition and can be adapted to evaluate mitochondrial function of various cell types that are able to aggregate into multicellular clusters in anchorage-independence. Graphic abstract: Workflow of the Extracellular Flux Assay to Measure Respiration of Anchorage-independent Tumor Cell Spheroids.

Antisense oligonucleotide-mediated knockdown of Mpzl3 attenuates the negative metabolic effects of diet-induced obesity in mice.

Previously, we demonstrated that global knockout (KO) of the gene encoding myelin protein zero-like 3 (Mpzl3) results in reduced body weight and adiposity, increased energy expenditure, and reduced hepatic lipid synthesis in mice. These mice also exhibit cyclic and progressive alopecia which may contribute to the observed hypermetabolic phenotype. The goal of the current study was to determine if acute and peripherally restricted knockdown of Mpzl3 could ameliorate the negative metabolic effects of exposure to a high-fat and sucrose, energy-dense (HED) diet similar to what was observed in global Mpzl3 KO mice in the absence of a skin phenotype. Mpzl3 antisense oligonucleotide (ASO) administration dose-dependently decreased fat mass and circulating lipids in HED-fed C57BL/6N mice. These changes were accompanied by a decrease in respiratory exchange ratio, a reduction in energy expenditure and food intake, a decrease in expression of genes regulating de novo lipogenesis in white adipose tissue, and an upregulation of genes associated with steroid hormone biosynthesis in liver, thermogenesis in brown adipose tissue and fatty acid transport in skeletal muscle. These data demonstrate that resistance to the negative metabolic effects of HED is a direct effect of Mpzl3 knockdown, rather than compensatory changes that could be associated with deletion of Mpzl3 during development in global KO mice. Inhibiting MPZL3 could be a potential therapeutic approach for the treatment of obesity and associated dyslipidemia.

Extracellular Glutathione Peroxidase GPx3 and Its Role in Cancer.

Mammalian cells possess a multifaceted antioxidant enzyme system, which includes superoxide dismutases, catalase, the peroxiredoxin/thioredoxin and the glutathione peroxidase systems. The dichotomous role of reactive oxygen species and antioxidant enzymes in tumorigenesis and cancer progression complicates the use of small molecule antioxidants, pro-oxidants, and targeting of antioxidant enzymes as therapeutic approaches for cancer treatment. It also highlights the need for additional studies to investigate the role and regulation of these antioxidant enzymes in cancer. The focus of this review is on glutathione peroxidase 3 (GPx3), a selenoprotein, and the only extracellular GPx of a family of oxidoreductases that catalyze the detoxification of hydro- and soluble lipid hydroperoxides by reduced glutathione. In addition to summarizing the biochemical function, regulation, and disease associations of GPx3, we specifically discuss the role and regulation of systemic and tumor cell expressed GPx3 in cancer. From this it is evident that GPx3 has a dichotomous role in different tumor types, acting as both a tumor suppressor and pro-survival protein. Further studies are needed to examine how loss or gain of GPx3 specifically affects oxidant scavenging and redox signaling in the extracellular tumor microenvironment, and how GPx3 might be targeted for therapeutic intervention.

Context-dependent activation of SIRT3 is necessary for anchorage-independent survival and metastasis of ovarian cancer cells.

Tumor cells must alter their antioxidant capacity for maximal metastatic potential. Yet the antioxidant adaptations required for ovarian cancer transcoelomic metastasis, which is the passive dissemination of cells in the peritoneal cavity, remain largely unexplored. Somewhat contradicting the need for oxidant scavenging are previous observations that expression of SIRT3, a nutrient stress sensor and regulator of mitochondrial antioxidant defenses, is often suppressed in many primary tumors. We have discovered that this mitochondrial deacetylase is specifically upregulated in a context-dependent manner in cancer cells. SIRT3 activity and expression transiently increased following ovarian cancer cell detachment and in tumor cells derived from malignant ascites of high-grade serous adenocarcinoma patients. Mechanistically, SIRT3 prevents mitochondrial superoxide surges in detached cells by regulating the manganese superoxide dismutase (SOD2). This mitochondrial stress response is under dual regulation by SIRT3. SIRT3 rapidly increases SOD2 activity as an early adaptation to cellular detachment, which is followed by SIRT3-dependent increases in SOD2 mRNA during sustained anchorage-independence. In addition, SIRT3 inhibits glycolytic capacity in anchorage-independent cells thereby contributing to metabolic changes in response to detachment. While manipulation of SIRT3 expression has few deleterious effects on cancer cells in attached conditions, SIRT3 upregulation and SIRT3-mediated oxidant scavenging are required for anoikis resistance in vitro following matrix detachment, and both SIRT3 and SOD2 are necessary for colonization of the peritoneal cavity in vivo. Our results highlight the novel context-specific, pro-metastatic role of SIRT3 in ovarian cancer.

GPx3 supports ovarian cancer progression by manipulating the extracellular redox environment.

Ovarian cancer remains the most lethal gynecologic malignancy, and is primarily diagnosed at late stage when considerable metastasis has occurred in the peritoneal cavity. At late stage abdominal cavity ascites accumulation provides a tumor-supporting medium in which cancer cells gain access to growth factors and cytokines that promote survival and metastasis. However, little is known about the redox status of ascites, or whether antioxidant enzymes are required to support ovarian cancer survival during transcoelomic metastasis in this medium. Gene expression cluster analysis of antioxidant enzymes identified two distinct populations of high-grade serous adenocarcinomas (HGSA), the most common ovarian cancer subtype, which specifically separated into clusters based on glutathione peroxidase 3 (GPx3) expression. High GPx3 expression was associated with poorer overall patient survival and increased tumor stage. GPx3 is an extracellular glutathione peroxidase with reported dichotomous roles in cancer. To further examine a potential pro-tumorigenic role of GPx3 in HGSA, stable OVCAR3 GPx3 knock-down cell lines were generated using lentiviral shRNA constructs. Decreased GPx3 expression inhibited clonogenicity and anchorage-independent cell survival. Moreover, GPx3 was necessary for protecting cells from exogenous oxidant insult, as demonstrated by treatment with high dose ascorbate. This cytoprotective effect was shown to be due to GPx3-dependent removal of extracellular H2O2. Importantly, GPx3 was necessary for clonogenic survival when cells were cultured in patient-derived ascites fluid. While oxidation reduction potential (ORP) of malignant ascites was heterogeneous in our patient cohort, and correlated positively with ascites iron content, GPx3 was required for optimal survival regardless of ORP or iron content. Collectively, our data suggest that HGSA ovarian cancers cluster into distinct groups of high and low GPx3 expression. GPx3 is necessary for HGSA ovarian cancer cellular survival in the ascites tumor environment and protects against extracellular sources of oxidative stress, implicating GPx3 as an important adaptation for transcoelomic metastasis.

Alterations in nociception and morphine antinociception in mice fed a high-fat diet.

Currently, more than 78.6 million adults in the United States are obese. A majority of the patient population receiving treatment for pain symptoms is derived from this subpopulation. Environmental factors, including the increased availability of food high in fat and sugar, contribute to the continued rise in the rates of obesity. The focus of this study was to investigate whether long-term exposure to a high-fat, energy-dense diet enhances baseline thermal and inflammatory nociception while reducing sensitivity to morphine-induced antinociception. Antinociceptive and hypothermic responses to morphine were determined in male and female C57BL/6N mice fed either a "western-style" diet high in fat and sucrose (HED) or a standard low-fat chow diet for 15 weeks. Antinociception was assessed using both the hot plate and tail flick tests of acute thermal pain and the formalin test of inflammatory pain. Acute administration of morphine dose-dependently increased antinociception in the hot plate and tail flick assays for mice of both sexes fed chow and HED. However, female mice displayed lower antinociceptive response to morphine compared to males in the tail-flick test. Hypothermic responses to acute morphine were also assessed in mice fed chow or HED. Male and female mice fed chow, and female mice fed HED displayed similar hypothermic responses to morphine. However, males fed HED did not exhibit morphine-induced hypothermia. Tolerance to the antinociceptive and hypothermic effects of morphine was assessed after ten days of repeated daily administration (10mg/kg morphine). Male mice fed chow or HED developed tolerance to morphine in the hot plate test. However, females fed HED did not. In the tail flick assay, only mice fed HED developed tolerance to morphine. All groups showed tolerance to morphine-induced hypothermia. In the formalin test, we found that both male and female mice fed HED had reduced sensitivity to the antinociceptive effects of morphine (6mg/kg). Collectively, these data suggest that sensitivity and tolerance to the antinociceptive effects of morphine may be dependent on diet and sex in the hot plate and tail flick thermal pain models, and that the acute antinociceptive effects of morphine in the formalin inflammatory pain model may also be dependent on these two factors. In addition, diet and sex can influence morphine-induced hypothermia. Exposure to an HED may lead to changes in neuronal signaling pathways that alter nociceptive responses to noxious stimuli in a sex-specific manner. Thus, dietary modifications might be a useful way to impact pain therapy.