Tara J. Loux

The expression of the receptor for advanced glycation endproducts (RAGE) is permissive for early pancreatic neoplasia

Pancreatic cancer is an almost uniformly lethal disease, characterized by late diagnosis, early metastasis, resistance to chemotherapy, and early mutation of the Kras oncogene. Here we show that the receptor for advanced glycation endproducts (RAGE) is required for the activation of interleukin 6 (IL-6)–mediated mitochondrial signal transducers and activators of transcription 3 (STAT3) signaling in pancreatic carcinogenesis. RAGE expression correlates with elevated levels of autophagy in pancreatic cancer in vivo and in vitro, and this heightened state of autophagy is required for IL-6–induced STAT3 activation. To further explore the intersection of RAGE, autophagy, and pancreatic carcinogenesis, we created a transgenic murine model, backcrossing RAGE-null mice to a spontaneous mouse model of pancreatic cancer, Pdx1-Cre:KrasG12D/+ (KC). Targeted ablation of Rage in KC mice delayed neoplasia development, decreased levels of autophagy, and inhibited mitochondrial STAT3 activity and subsequent ATP production. Our results suggest a critical role for RAGE expression in the earliest stages of pancreatic carcinogenesis, potentially acting as the “autophagic switch,” regulating mitochondrial STAT3 signaling.

Weight‐specific Health‐related Quality of Life in Adolescents With Extreme Obesity

The objectives of this multisite study were to: (i) examine differences by gender and race on generic and weight‐ specific health‐related quality of life (HRQOL) in adolescents with extreme obesity (BMI ≥ 40 kg/m2) and (ii) explore HRQOL differences based on treatment pursued (behavioral vs. bariatric surgery). Study participants included 145 obese adolescents (mean age = 15.3 years; 68% female; 46% black; mean BMI = 50.6) referred to pediatric weight management programs. Participants completed generic (PedsQL) and weight‐specific (Impact of Weight on Quality of Life‐Kids (IWQOL‐Kids)) HRQOL measures. Generic and weight‐specific measures indicated global (e.g., all domains) HRQOL impairment and significant differences by race. Physical, emotional, and social scores of the PedsQL (Ps < 0.01) and the physical comfort and body esteem scores of the IWQOL‐Kids (Ps < 0.001) were significantly higher for black compared to white adolescents with extreme obesity. Extremely obese adolescents pursuing bariatric surgery reported similar HRQOL to adolescents pursuing behavioral treatment (n = 30 matched pairs). HRQOL did not differ for extremely obese adolescents based on type of treatment sought, but race/ethnicity should be considered when characterizing these youth. Although racial differences in adolescent body image/esteem have been reported, it is unknown why black adolescents with extreme obesity would report less impact of weight on their physical functioning. Overall, these data suggest that HRQOL is not homogenous in adolescents with extreme obesity.

The Receptor for Advanced Glycation End Products Promotes Pancreatic Carcinogenesis and Accumulation of Myeloid-Derived Suppressor Cells

Pancreatic ductal adenocarcinoma (PDA) has an aggressive natural history and is resistant to therapy. The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor for many damage-associated molecular pattern molecules. RAGE is overexpressed in both human and murine models of PDA as well as most advanced epithelial neoplasms. The immunosuppressive nature of the PDA microenvironment is facilitated, in part, by the accumulation of regulatory immune cell infiltrates such as myeloid-derived suppressor cells (MDSCs). To study the role of RAGE expression in the setting of mutant Ras-promoted pancreatic carcinogenesis (KC), a triple-transgenic model of spontaneous murine PDA in a RAGE-null background (KCR) was generated. KCR mice had markedly delayed pancreatic carcinogenesis and a significant diminution of MDSCs compared with KC mice at comparable time points postweaning. Although RAGE was not required for the development or suppressor activity of MDSCs, its absence was associated with temporally limited pancreatic neoplasia and altered phenotype and function of the myeloid cells. In lieu of MDSCs, KCR animals at comparable time points exhibited mature CD11b+Gr1−F4/80+ cells that were not immunosuppressive in vitro. KCR mice also maintained a significantly less suppressive milieu evidenced by marked decreases in CCL22 in relation to CXCL10 and diminished serum levels of IL-6.

Regulation of FAT/CD36 mRNA gene expression by long chain fatty acids in the differentiated 3T3-L1 cells

Defects in fatty acid translocase (FAT/CD36) have been identified as a major factor in insulin resistance and defective fatty acid and glucose metabolism. Therefore, understanding of the regulation of FAT/CD36 expression and function is important for a potential therapeutic target for type II diabetes. We differentiated 3T3-L1 preadipocytes into matured adipocytes and examined the roles of insulin and long chain fatty acids on FAT/CD36 expression and function. Our results indicate that FAT/CD36 mRNA expression was not detected at preadipocyte but was significantly increased at matured adipocyte. In fully differentiated 3T3-L1 adipocytes, insulin significantly increased FAT/CD36 mRNA and protein expression in a dose dependent manner. The free fatty acid stearic acid reduced FAT/CD36 mRNA expression while the non-metabolizable free fatty acid alpha-bromopalmitate (2-BP) significantly increased FAT/CD36 mRNA and protein expression. Isoproterenol, in contrast, dose-dependently reduced FAT/CD36 mRNA expression and increased free fatty acid release. Mechanism analysis indicated that the effect of insulin and 2-BP on the FAT/CD36 mRNA gene expression may be mediated through activation of PPAR-γ, suggesting that FAT/CD36 may have important implications in the pathophysiology of defective fatty acid metabolism.

Molecular Mechanism of the Intracellular Segments of the Melanocortin-4 Receptor for NDP−MSH Signaling

Mutations of the human melanocortin-4 receptor (hMC4R) have been previously identified to be the most common cause of monogenic human obesity. Specifically, mutations of the intracellular C terminus and the third intracellular loop of hMC4R have been reported to play an important role in human obesity. However, the molecular basis of these hMC4R intracellular segments in receptor function remains unclear. In this study, we utilized deletions and mutations of specific portions of the hMC4R to determine the molecular mechanism of both the C terminus and the third intracellular loop in receptor signaling. Our results indicate that deletions of the distal 25 (the entire C terminus), 22, 18, 17, 16, and 15 amino acids of the C terminus result in the complete loss of both [Nle(4)-d-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) binding and NDP-MSH-mediated cAMP production. Deletion of the distal 14 amino acids of the C terminus significantly decreases both NDP-MSH binding affinity and potency, but deletion of the distal 13 amino acids of the C terminus does not affect NDP-MSH activity. Further analysis revealed that the proximal 12 amino acids of the C terminus are not only important for receptor signaling but also important for ligand binding. Our results also indicate that the third intracellular loop of the hMC4R is important for receptor signaling but not ligand binding. In summary, our findings suggest that the proximal region of the melanocortin-4 receptor (MC4R) C terminus is crucial not only for receptor signaling but also for ligand binding, while the third intracellular loop is important mainly for receptor signaling.

Extracellular DNA promotes colorectal tumor cell survival after cytotoxic chemotherapy

BACKGROUND Inflammation promotes the growth and survival of malignant cells. Inflammation within the tumor microenvironment is a result of damage-associated molecular patterns released by dead or dying cells that provide survival signals to the surrounding cells. It has been proposed that extracellular DNA can act as a damage-associated molecular pattern given the association between circulating DNA and autoimmune diseases. Herein, we demonstrate a novel role for genomic extracellular DNA binding to the Toll-like receptor (TLR)-9 on tumor cells in response to cytotoxic insult. MATERIALS AND METHODS The colorectal tumor cell line HCCT116 was used to study the role of DNA in tumor cell response to chemotherapy. Cell viability was assessed using CCK-8 assay. Cell death mechanisms were assessed by YOYO-1 and lactate dehydrogenase staining for necrosis and TUNEL staining for apoptosis. Autophagy was measured by LC3 punctate formation. TLR9-short hairpin RNA was used to knockdown TLR-9 and determine its role in tumor cell response to DNA. RESULTS DNA is released from necrotic tumor cells after chemotherapy. Survival after cytotoxic insult is enhanced by the presence of extracellular DNA as a result of inhibition of apoptosis and enhanced autophagy. Knockdown of TLR-9 enhanced apoptosis, diminished autophagy, and decreased survival after cytotoxic insult in the presence or absence of extracellular DNA. CONCLUSIONS DNA in the tumor microenvironment promotes survival through induction of autophagy via TLR-9 signaling. This work has important implications for targeting extracellular DNA, TLR-9, and autophagy during treatment with chemotherapy and enhances our understanding of the role of extracellular DNA in the tumor microenvironment.

NK cells as recipients of cytokine signals

Publisher Summary Cytokines are polypeptide mediators that play pivotal roles in communication between cells and can be broadly distinguished as leadered cytokines, including hematopoietins, colony stimulating factors, chemokines, tumor necrosis factor (TNF) family members, and leaderless interleukin (IL)-1 extended family, fibroblast growth factor family, high mobility group box 1 protein (HMGB1), and others. They are pleiotropic, synergistic, and redundant, conferring substantial evolutionary advantages. Cytokine expression is disturbed in many infectious, inflammatory and autoimmune disease states. Autocrine activities mediated by cytokines in natural killer (NK) cells include the roles of self-secreted interferon (IFN)-γ and IL-10; paracrine mediators include the cross-talk of HMGB1, IL-12 and IL-15 between NK cells and dendritic cells (DCs) at the immunologic synapse, while endocrine activity received by NKs from release of chemokines into the vascular circulation by distant cells is vital for migration of the innate immune response from the bone marrow into the peripheral blood to an area of disease. NK cells recognize stressed cells, entertaining a constitutive ability to mediate cytotoxicity in target cells and secrete cytokines rather rapidly in response. They participate in the innate resistance to intracellular pathogens and malignancies. Their role is critical to expansion of the Th1 biased adaptive immune response and development of secondary lymphatic sites. They also influence hematopoiesis, increasing myelopoiesis and decreasing megacytopoiesis and erythropoiesis.