RNAi mediated silencing of STAT3/PD-L1 in tumor-associated immune cells induces robust anti-tumor effects in immunotherapy resistant tumors.

Malignant tumors are often associated with an immunosuppressive tumor microenvironment (TME), rendering most of them resistant to standard-of-care immune checkpoint inhibitors (CPIs). Signal transducer and activator of transcription 3 (STAT3), a ubiquitously expressed transcription factor, has well-defined immunosuppressive functions in several leukocyte populations within the TME. Since the STAT3 protein has been challenging to target using conventional pharmaceutical modalities, we investigated the feasibility of applying systemically delivered RNA interference (RNAi) agents to silence its mRNA directly in tumor-associated immune cells. In preclinical rodent tumor models, chemically stabilized acylated small interfering RNAs (siRNAs) selectively silenced Stat3 mRNA in multiple relevant cell types, reduced STAT3 protein levels, and increased cytotoxic T cell infiltration. In a murine model of CPI-resistant pancreatic cancer, RNAi-mediated Stat3 silencing resulted in tumor growth inhibition, which was further enhanced in combination with CPIs. To further exemplify the utility of RNAi for cancer immunotherapy, this technology was used to silence Cd274, the gene encoding the immune checkpoint protein programmed death-ligand 1 (PD-L1). Interestingly, silencing of Cd274 was effective in tumor models that are resistant to PD-L1 antibody therapy. These data represent the first demonstration of systemic delivery of RNAi agents to the TME and suggest applying this technology for immuno-oncology applications.

FGF21 protects against ischaemia reperfusion injury in normal and fatty livers.

BACKGROUND: Liver ischaemia/reperfusion (I/R) injury, which is an inevitable clinical problem of liver resection, liver transplantation and haemorrhagic shock. Fibroblast growth factor 21 (FGF21) was intimately coupled with multiple metabolic processes and proved to protect against apoptosis and inflammatory response in hepatocytes during hepatic I/R injury. However, the regulatory mechanisms of FGF21 in hepatic I/R injury remains unknown. Therefore, we hypothesize that FGF21 protects hepatic tissues from I/R injury. METHODS: Blood samples were available from haemangiomas patients undergoing hepatectomy and murine liver I/R model and used to further evaluate the serum levels of FGF21 both in humans and mice. We further explored the regulatory mechanisms of FGF21 in murine liver I/R model by using FGF21-knockout mice (FGF21-KO mice) and FGF21-overexpression transgenic mice (FGF21-OE mice) fed a high-fat or ketogenic diet. RESULTS: Our results show that the circulating levels of FGF21 were robustly decreased after liver I/R in both humans and mice. Silencing FGF21 expression with FGF21-KO mice aggravates liver injury at 6 h after 75 min of partial liver ischaemia, while FGF21-OE mice display alleviated hepatic I/R injury and inflammatory response. Compared with chow diet mice, exogenous FGF21 decreases the levels of aminotransferase, histological changes, apoptosis and inflammatory response in hepatic I/R injury treatment mice with a high-fat diet. Meanwhile, ketogenic diet mice are not sensitive to hepatic I/R injury. CONCLUSIONS: The circulating contents of FGF21 are decreased during liver warm I/R injury and exogenous FGF21 exerts hepatoprotective effects on hepatic I/R injury. Thus, FGF21 regulates hepatic I/R injury and may be a key therapeutic target.

Global deletion of NTPDase3 protects against diet-induced obesity by increasing basal energy metabolism.

BACKGROUND: Ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), also known as CD39L3, is the dominant ectonucleotidase expressed by beta cells in the islet of Langerhans and on nerves. NTPDase3 catalyzes the conversion of extracellular ATP and ADP to AMP and modulates purinergic signaling. Previous studies have shown that NTPDase3 decreases insulin release from beta-cells in vitro. This study aims to determine the impact of NTPDase3 in diet-induced obesity (DIO) and metabolism in vivo. METHODS: We developed global NTPDase3 deficient (Entpd3-/-) and islet beta-cell-specific NTPDase-3 deficient mice (Entpd3flox/flox,InsCre) using Ins1-Cre targeted gene editing to compare metabolic phenotypes with wildtype (WT) mice on a high-fat diet (HFD). RESULTS: Entpd3-/- mice exhibited similar growth rates compared to WT on chow diet. When fed HFD, Entpd3-/- mice demonstrated significant resistance to DIO. Entpd3-/- mice consumed more calories daily and exhibited less fecal calorie loss. Although Entpd3-/- mice had no increases in locomotor activity, the mice exhibited a significant increase in basal metabolic rate when on the HFD. This beneficial phenotype was associated with improved glucose tolerance, but not higher insulin secretion. In fact, Entpd3flox/flox,InsCre mice demonstrated similar metabolic phenotypes and insulin secretion compared to matched controls, suggesting that the expression of NTPDase3 in beta-cells was not the primary protective factor. Instead, we observed a higher expression of uncoupling protein 1 (UCP-1) in brown adipose tissue and an augmented browning in inguinal white adipose tissue with upregulation of UCP-1 and related genes involved in thermogenesis in Entpd3-/- mice. CONCLUSIONS: Global NTPDase3 deletion in mice is associated with resistance to DIO and obesity-associated glucose intolerance. This outcome is not driven by the expression of NTPDase3 in pancreatic beta-cells, but rather likely mediated through metabolic changes in adipocytes.

Liver-derived FGF21 is essential for full adaptation to ketogenic diet but does not regulate glucose homeostasis.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is expressed in several metabolically active tissues, including liver, fat, and acinar pancreas, and has pleiotropic effects on metabolic homeostasis. The dominant source of FGF21 in the circulation is the liver. OBJECTIVE AND METHODS: To analyze the physiological functions of hepatic FGF21, we generated a hepatocyte-specific knockout model (LKO) by mating albumin-Cre mice with FGF21 flox/flox (fl/fl) mice and challenged it with different nutritional models. RESULTS: Mice fed a ketogenic diet typically show increased energy expenditure; this effect was attenuated in LKO mice. LKO on KD also developed hepatic pathology and altered hepatic lipid homeostasis. When evaluated using hyperinsulinemic-euglycemic clamps, glucose infusion rates, hepatic glucose production, and glucose uptake were similar between fl/fl and LKO DIO mice. CONCLUSIONS: We conclude that liver-derived FGF21 is important for complete adaptation to ketosis but has a more limited role in the regulation of glycemic homeostasis.

SGLT2 inhibition reprograms systemic metabolism via FGF21-dependent and -independent mechanisms.

Pharmacologic inhibition of the renal sodium/glucose cotransporter-2 induces glycosuria and reduces glycemia. Given that SGLT2 inhibitors (SGLT2i) reduce mortality and cardiovascular risk in type 2 diabetes, improved understanding of molecular mechanisms mediating these metabolic effects is required. Treatment of obese but nondiabetic mice with the SGLT2i canagliflozin (CANA) reduces adiposity, improves glucose tolerance despite reduced plasma insulin, increases plasma ketones, and improves plasma lipid profiles. Utilizing an integrated transcriptomic-metabolomics approach, we demonstrate that CANA modulates key nutrient-sensing pathways, with activation of 5' AMP-activated protein kinase (AMPK) and inhibition of mechanistic target of rapamycin (mTOR), independent of insulin or glucagon sensitivity or signaling. Moreover, CANA induces transcriptional reprogramming to activate catabolic pathways, increase fatty acid oxidation, reduce hepatic steatosis and diacylglycerol content, and increase hepatic and plasma levels of FGF21. Given that these phenotypes mirror the effects of FGF21 to promote lipid oxidation, ketogenesis, and reduction in adiposity, we hypothesized that FGF21 is required for CANA action. Using FGF21-null mice, we demonstrate that FGF21 is not required for SGLT2i-mediated induction of lipid oxidation and ketogenesis but is required for reduction in fat mass and activation of lipolysis. Taken together, these data demonstrate that SGLT2 inhibition triggers a fasting-like transcriptional and metabolic paradigm but requires FGF21 for reduction in adiposity.

Deficiency of fibroblast growth factor 21 (FGF21) promotes hepatocellular carcinoma (HCC) in mice on a long term obesogenic diet.

OBJECTIVE: Non-alcoholic fatty liver (NAFL) associated with obesity is a major cause of liver diseases which can progress to non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Fibroblast growth factor 21 (FGF21) plays an important role in liver metabolism and is also a potential marker for NAFL. Here we aimed to test the effect of FGF21 deficiency on liver pathology in mice consuming a conventional high fat, high sucrose (HFHS) obesogenic diet for up to 52 weeks. METHODS: C57BL6 WT and FGF21 KO mice were fed a conventional obesogenic diet and were evaluated at 16 and 52 weeks. Evaluation included metabolic assessment, liver pathology, and transcriptomic analysis. RESULTS: With consumption of HFHS diet, FGF21 deficient mice (FGF21 KO) develop excess fatty liver within 16 weeks. Hepatic pathology progresses and at 52 weeks FGF21 KO mice show significantly worse fibrosis and 78% of mice develop HCC; in contrast only 6% of WT mice develop HCC. Well differentiated hepatocellular carcinomas in FGF21 KO mice were characterized by expanded hepatic plates, loss of reticulin network, cytologic atypia, and positive immunostaining for glutamine synthetase. Microarray analysis reveals enrichment of several fibroblast growth factor signaling pathways in the tumors. CONCLUSIONS: In addition to attenuating inflammation and fibrosis in mice under a number of dietary challenges, we show here that FGF21 is required to limit the progression from NAFL to HCC in response to prolonged exposure to an obesogenic diet. The induction of hepatic FGF21 in response to the high fat, high sucrose obesogenic diet may play an important role in limiting progression of liver pathology from NAFL to HCC.

Fibroblast growth factor 21 (FGF21) is robustly induced by ethanol and has a protective role in ethanol associated liver injury.

OBJECTIVE: Excess ethanol consumption has serious pathologic consequences. In humans, repeated episodes of binge drinking can lead to liver damage and have adverse effects on other organs such as pancreas and brain. Long term chronic consumption of ethanol can also result in progressive alcoholic liver disease and cirrhosis. Fibroblast growth factor 21 (FGF21) is a metabolic regulator with multiple physiologic functions. FGF21 is a novel biomarker for non-alcoholic fatty liver disease (NAFLD) in humans and limits hepatotoxicity in mice. Therefore, we explored the possibility that FGF21 plays a role in response to ethanol consumption in both humans and mice. METHODS: We used a binge drinking paradigm in humans to examine the effect of acute ethanol consumption on circulating FGF21. We adapted this paradigm to evaluate the acute response to ethanol in mice. We then examined the role of FGF21 on liver pathology in two models of chronic ethanol consumption in both wild type (WT) mice and mice lacking FGF21 (FGF21-KO). RESULTS: Acute ethanol consumption resulted in a robust induction of serum FGF21 after 6 h in both humans and mice. Serum ethanol peaked at 1 h in both species and was cleared by 6 h. Ethanol clearance was the same in WT and FGF21-KO mice, indicating that FGF21 does not play a major role in ethanol metabolism in a binge paradigm. When FGF21-KO mice were fed the Lieber-DeCarli diet, a high fat diet supplemented with ethanol, a higher mortality was observed compared to WT mice after 16 days on the diet. When FGF21-KO mice consumed 30% ethanol in drinking water, along with a normal chow diet, there was no mortality observed even after 16 weeks, but the FGF21-KO mice had significant liver pathology compared to WT mice. CONCLUSIONS: Acute or binge ethanol consumption significantly increases circulating FGF21 levels in both humans and mice. However, FGF21 does not play a role in acute ethanol clearance. In contrast, chronic ethanol consumption in the absence of FGF21 is associated with significant liver pathology alone or in combination with excess mortality, depending on the type of diet consumed with ethanol. This suggests that FGF21 protects against long term ethanol induced hepatic damage and may attenuate progression of alcoholic liver disease. Further study is required to assess the therapeutic potential of FGF21 in the treatment of alcoholic liver disease.

Elevated Serum Fibroblast Growth Factor 21 in Humans with Acute Pancreatitis.

BACKGROUND: The metabolic regulator Fibroblast Growth Factor 21 (FGF21) is highly expressed in the acinar pancreas, but its role in pancreatic function is obscure. It appears to play a protective role in acute experimental pancreatitis in mice. The aim of this study was to define an association between FGF21 and the course and resolution of acute pancreatitis in humans. METHODS AND PRINCIPAL FINDINGS: Twenty five subjects with acute pancreatitis admitted from May to September 2012 to the Beth Israel Deaconess Medical Center (BIDMC) were analyzed. Serial serum samples were collected throughout hospitalization and analyzed for FGF21 levels by ELISA. Twenty healthy subjects sampled three times over a four week period were used as controls. We found that, in patients with pancreatitis, serum FGF21 rises significantly and peaks four to six days after the maximum lipase level, before slowly declining. Maximum FGF21 levels were significantly greater than baseline levels for acute pancreatitis subjects (1733 vs. 638 pg/mL, P = 0.003). This maximum value was significantly greater than the highest value observed for our control subjects (1733 vs. 322 pg/mL, P = 0.0002). The ratio of active to total FGF21 did not change during the course of the disease (42.5% vs. 44.4%, P = 0.58). Fold increases in FGF21 were significantly greater in acute pancreatitis subjects than the fold difference seen in healthy subjects (4.7 vs. 2.0, P = 0.01). Higher fold changes were also seen in severe compared to mild pancreatitis (18.2 vs. 4.4, P = 0.01). The timing of maximum FGF21 levels correlated with day of successful return to oral intake (R2 = 0.21, P = 0.04). CONCLUSIONS: Our results demonstrate that serum FGF21 rises significantly in humans with acute pancreatitis. The pancreas may be contributing to increased FGF21 levels following injury and FGF21 may play a role in the recovery process.

Fibroblast growth factor 21 has no direct role in regulating fertility in female mice.

OBJECTIVE: Reproduction is an energetically expensive process. Insufficient calorie reserves, signaled to the brain through peripheral signals such as leptin, suppress fertility. Recently, fibroblast growth factor 21 (FGF21) was implicated as a signal from the liver to the hypothalamus that directly inhibits the hypothalamic-gonadotropin axis during fasting and starvation. However, FGF21 itself increases metabolic rate and can induce weight loss, which suggests that the effects of FGF21 on fertility may not be direct and may reflect changes in energy balance. METHODS: To address this important question, we evaluated fertility in several mouse models with elevated FGF21 levels including ketogenic diet fed mice, fasted mice, mice treated with exogenous FGF21 and transgenic mice over-expressing FGF21. RESULTS: We find that ketogenic diet fed mice remain fertile despite significant elevation in serum FGF21 levels. Absence of FGF21 does not alter transient infertility induced by fasting. Centrally infused FGF21 does not suppress fertility despite its efficacy in inducing browning of inguinal white adipose tissue. Furthermore, a high fat diet (HFD) can restore fertility of female FGF21-overexpressing mice, a model of growth restriction, even in the presence of supraphysiological serum FGF21 levels. CONCLUSIONS: We conclude that FGF21 is not a direct physiological regulator of fertility in mice. The infertility observed in FGF21 overexpressing mice is likely driven by the increased energy expenditure and consequent excess calorie requirements resulting from high FGF21 levels.

Fibroblast Growth Factor 21 (FGF21) Protects against High Fat Diet Induced Inflammation and Islet Hyperplasia in Pancreas.

Fibroblast growth factor 21 (FGF21) is an important endocrine metabolic regulator expressed in multiple tissues including liver and adipose tissue. Although highest levels of expression are in pancreas, little is known about the function of FGF21 in this tissue. In order to understand the physiology of FGF21 in the pancreas, we analyzed its expression and regulation in both acinar and islet tissues. We found that acinar tissue express 20-fold higher levels than that observed in islets. We also observed that pancreatic FGF21 is nutritionally regulated; a marked reduction in FGF21 expression was noted with fasting while obesity is associated with 3-4 fold higher expression. Acinar and islet cells are targets of FGF21, which when systemically administered, leads to phosphorylation of the downstream target ERK 1/2 in about half of acinar cells and a small subset of islet cells. Chronic, systemic FGF21 infusion down-regulates its own expression in the pancreas. Mice lacking FGF21 develop significant islet hyperplasia and periductal lymphocytic inflammation when fed with a high fat obesogenic diet. Inflammatory infiltrates consist of TCRb+ Thy1+ T lymphocytes with increased levels of Foxp3+ regulatory T cells. Increased levels of inflammatory cells were coupled with elevated expression of cytokines such as TNFα, IFNγ and IL1ß. We conclude that FGF21 acts to limit islet hyperplasia and may also prevent pancreatic inflammation.

Phosphoproteomic screen identifies potential therapeutic targets in melanoma.

Therapies directed against receptor tyrosine kinases are effective in many cancer subtypes, including lung and breast cancer. We used a phosphoproteomic platform to identify active receptor tyrosine kinases that might represent therapeutic targets in a panel of 25 melanoma cell strains. We detected activated receptors including TYRO3, AXL, MERTK, EPHB2, MET, IGF1R, EGFR, KIT, HER3, and HER4. Statistical analysis of receptor tyrosine kinase activation as well as ligand and receptor expression indicates that some receptors, such as FGFR3, may be activated via autocrine circuits. Short hairpin RNA knockdown targeting three of the active kinases identified in the screen, AXL, HER3, and IGF1R, inhibited the proliferation of melanoma cells and knockdown of active AXL also reduced melanoma cell migration. The changes in cellular phenotype observed on AXL knockdown seem to be modulated via the STAT3 signaling pathway, whereas the IGF1R-dependent alterations seem to be regulated by the AKT signaling pathway. Ultimately, this study identifies several novel targets for therapeutic intervention in melanoma.

Molecular aspects of the interaction of Hoechst-33258 with GC-rich promoter region of c-met.

The c-Met signaling pathway is one of the main regulators of cell proliferation, differentiation, apoptosis, and other cellular processes. As aberrant expression of c-Met has been implicated in tumor growth, invasion, and metastasis, c-Met is recognized as a promising target for cancer therapeutics. In continuation of our search for small molecules with anticancer properties, we investigated the binding of bis-benzimidazole derivative Hoechst-33258 to the promoter region 24RY, -142 to -119 (5'-GGGGCAGAGGCGGGAGGAAACGCG-3', 24R and its complement 5'-CGCGTTTCCTCCCGCCTCTGCCCC-3', 24Y), upstream to the transcriptional start site of the c-met gene. Strong complexation of duplex 24RY-Hoechst is revealed by a high binding constant K, 3.25 x 10(5) M(-1), with significant changes of Delta DeltaG (-10 kcal/mol), Delta DeltaH (-83 kcal/mol), Delta DeltaS (-241 cal/[mol K]), and melting temperature (DeltaT(m) = +8 degrees C). This is accompanied by bathochromic shift of 10 nm, 50% hypochromism at 340 nm, isobestic points at 302 and 369 nm in absorption spectra, and induced emission band at 465 nm in the fluorescence spectra of Hoechst. Molecular modeling data demonstrated that Hoechst binds to consecutive GGs in the minor groove of 24RY containing residues 12-17. Therefore, Hoechst-33258 can be a potential anticancer agent against malignancies associated with c-Met upregulation.

Interaction of actinomycin D with promoter element of c-met and its inhibitory effect on the expression of c-Met.

c-Met, the tyrosine kinase receptor for hepatocyte growth factor plays a pivotal role in normal cellular signaling and overexpression of c-Met protein is reported in several human cancers. Thus, transcriptional regulation of c-met appears to be an attractive target for chemotherapy. Therefore, we selected a 24mer GC rich sequence (24R) from the c-met promoter located at -142 to -119 from transcription start site and studied its interaction with anticancer drug, Actinomycin D. Spectroscopic analysis demonstrated a strong complexation between ActD and 24RY as shown by: (i) a high binding constant, K of 4-5 x 10(5) M(-1) with DeltaDeltaG of -47 +/- 1.5 Kcalmol(-1); (ii) marked increase by +10 degrees C in melting temperature of 24RY; and (iii) significant changes in circular dichroic spectra of both ActD and 24RY. Molecular modeling revealed the preference of ActD to the Sp1 binding site, GGCGGG, in 24RY. Expression of the c-Met was checked in HepG2 cells, a human hepatocellular carcinoma cell line by using western blotting and immunocytochemistry. Downregulation of c-Met expression by as much as 50% was observed in the presence of 20ng/ml (IC(50)) of ActD. Taking into account of the binding studies also, we feel that the down regulation of c-Met perhaps involves binding of ActD to the promoter site of c-met. Therefore, c-met could be a challenging and promising target for therapeutic strategies in combating cancer.