Preclinical Development of U3-1784, a Novel FGFR4 Antibody Against Cancer, and Avoidance of Its On-target Toxicity.

The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects.U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.

Fibroblast growth factor 23 directly targets hepatocytes to promote inflammation in chronic kidney disease.

Patients with chronic kidney disease (CKD) develop increased levels of the phosphate-regulating hormone, fibroblast growth factor (FGF) 23, that are associated with a higher risk of mortality. Increases in inflammatory markers are another common feature that predicts poor clinical outcomes. Elevated FGF23 is associated with higher circulating levels of inflammatory cytokines in CKD, which can stimulate osteocyte production of FGF23. Here, we studied whether FGF23 can directly stimulate hepatic production of inflammatory cytokines in the absence of α-klotho, an FGF23 coreceptor in the kidney that is not expressed by hepatocytes. By activating FGF receptor isoform 4 (FGFR4), FGF23 stimulated calcineurin signaling in cultured hepatocytes, which increased the expression and secretion of inflammatory cytokines, including C-reactive protein. Elevating serum FGF23 levels increased hepatic and circulating levels of C-reactive protein in wild-type mice, but not in FGFR4 knockout mice. Administration of an isoform-specific FGFR4 blocking antibody reduced hepatic and circulating levels of C-reactive protein in the 5/6 nephrectomy rat model of CKD. Thus, FGF23 can directly stimulate hepatic secretion of inflammatory cytokines. Our findings indicate a novel mechanism of chronic inflammation in patients with CKD and suggest that FGFR4 blockade might have therapeutic anti-inflammatory effects in CKD.

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle.

OBJECTIVE: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle. METHODS: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression. RESULTS: We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes. CONCLUSIONS: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca(2+)-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.

Activation of Cardiac Fibroblast Growth Factor Receptor 4 Causes Left Ventricular Hypertrophy.

Chronic kidney disease (CKD) is a worldwide public health threat that increases risk of death due to cardiovascular complications, including left ventricular hypertrophy (LVH). Novel therapeutic targets are needed to design treatments to alleviate the cardiovascular burden of CKD. Previously, we demonstrated that circulating concentrations of fibroblast growth factor (FGF) 23 rise progressively in CKD and induce LVH through an unknown FGF receptor (FGFR)-dependent mechanism. Here, we report that FGF23 exclusively activates FGFR4 on cardiac myocytes to stimulate phospholipase Cγ/calcineurin/nuclear factor of activated T cell signaling. A specific FGFR4-blocking antibody inhibits FGF23-induced hypertrophy of isolated cardiac myocytes and attenuates LVH in rats with CKD. Mice lacking FGFR4 do not develop LVH in response to elevated FGF23, whereas knockin mice carrying an FGFR4 gain-of-function mutation spontaneously develop LVH. Thus, FGF23 promotes LVH by activating FGFR4, thereby establishing FGFR4 as a pharmacological target for reducing cardiovascular risk in CKD.

Regulation of autophagy and the ubiquitin-proteasome system by the FoxO transcriptional network during muscle atrophy.

Stresses like low nutrients, systemic inflammation, cancer or infections provoke a catabolic state characterized by enhanced muscle proteolysis and amino acid release to sustain liver gluconeogenesis and tissue protein synthesis. These conditions activate the family of Forkhead Box (Fox) O transcription factors. Here we report that muscle-specific deletion of FoxO members protects from muscle loss as a result of the role of FoxOs in the induction of autophagy-lysosome and ubiquitin-proteasome systems. Notably, in the setting of low nutrient signalling, we demonstrate that FoxOs are required for Akt activity but not for mTOR signalling. FoxOs control several stress-response pathways such as the unfolded protein response, ROS detoxification, DNA repair and translation. Finally, we identify FoxO-dependent ubiquitin ligases including MUSA1 and a previously uncharacterised ligase termed SMART (Specific of Muscle Atrophy and Regulated by Transcription). Our findings underscore the central function of FoxOs in coordinating a variety of stress-response genes during catabolic conditions.

Inducible activation of Akt increases skeletal muscle mass and force without satellite cell activation.

A better understanding of the signaling pathways that control muscle growth is required to identify appropriate countermeasures to prevent or reverse the loss of muscle mass and force induced by aging, disuse, or neuromuscular diseases. However, two major issues in this field have not yet been fully addressed. The first concerns the pathways involved in leading to physiological changes in muscle size. Muscle hypertrophy based on perturbations of specific signaling pathways is either characterized by impaired force generation, e.g., myostatin knockout, or incompletely studied from the physiological point of view, e.g., IGF-1 overexpression. A second issue is whether satellite cell proliferation and incorporation into growing muscle fibers is required for a functional hypertrophy. To address these issues, we used an inducible transgenic model of muscle hypertrophy by short-term Akt activation in adult skeletal muscle. In this model, Akt activation for 3 wk was followed by marked hypertrophy ( approximately 50% of muscle mass) and by increased force generation, as determined in vivo by ankle plantar flexor stimulation, ex vivo in intact isolated diaphragm strips, and in single-skinned muscle fibers. No changes in fiber-type distribution and resistance to fatigue were detectable. Bromodeoxyuridine incorporation experiments showed that Akt-dependent muscle hypertrophy was accompanied by proliferation of interstitial cells but not by satellite cell activation and new myonuclei incorporation, pointing to an increase in myonuclear domain size. We can conclude that during a fast hypertrophic growth myonuclear domain can increase without compromising muscle performance.

Smad2 and 3 transcription factors control muscle mass in adulthood.

Loss of muscle mass occurs in a variety of diseases, including cancer, chronic heart failure, aquired immunodeficiency syndrome, diabetes, and renal failure, often aggravating pathological progression. Preventing muscle wasting by promoting muscle growth has been proposed as a possible therapeutic approach. Myostatin is an important negative modulator of muscle growth during myogenesis, and myostatin inhibitors are attractive drug targets. However, the role of the myostatin pathway in adulthood and the transcription factors involved in the signaling are unclear. Moreover, recent results confirm that other transforming growth factor-beta (TGF-beta) members control muscle mass. Using genetic tools, we perturbed this pathway in adult myofibers, in vivo, to characterize the downstream targets and their ability to control muscle mass. Smad2 and Smad3 are the transcription factors downstream of myostatin/TGF-beta and induce an atrophy program that is muscle RING-finger protein 1 (MuRF1) independent. Furthermore, Smad2/3 inhibition promotes muscle hypertrophy independent of satellite cells but partially dependent of mammalian target of rapamycin (mTOR) signaling. Thus myostatin and Akt pathways cross-talk at different levels. These findings point to myostatin inhibitors as good drugs to promote muscle growth during rehabilitation, especially when they are combined with IGF-1-Akt activators.

Akt activation prevents the force drop induced by eccentric contractions in dystrophin-deficient skeletal muscle.

Skeletal muscles of the mdx mouse, a model of Duchenne Muscular Dystrophy, show an excessive reduction in the maximal tetanic force following eccentric contractions. This specific sign of the susceptibility of dystrophin-deficient muscles to mechanical stress can be used as a quantitative test to measure the efficacy of therapeutic interventions. Using inducible transgenesis in mice, we show that when Akt activity is increased the force drop induced by eccentric contractions in mdx mice becomes similar to that of wild-type mice. This effect is not correlated with muscle hypertrophy and is not blocked by rapamycin treatment. The force drop induced by eccentric contractions is similar in skinned muscle fibers from mdx and Akt-mdx mice when stretch is applied directly to skinned fibers. However, skinned fibers isolated from mdx muscles exposed to eccentric contractions in vivo develop less isometric force than wild-type fibers and this force depression is completely prevented by Akt activation. These experiments indicate that the myofibrillar-cytoskeletal system of dystrophin-deficient muscle is highly susceptible to a damage caused by eccentric contraction when elongation is applied in vivo, and this damage can be prevented by Akt activation. Microarray and PCR analyses indicate that Akt activation induces up-regulation of genes coding for proteins associated with Z-disks and costameres, and for proteins with anti-oxidant or chaperone function. The protein levels of utrophin and dysferlin are also increased by Akt activation.

Chromosomal deletions in bladder cancer: shutting down pathways.

Bladder cancer is one of the most common cancers in the world, leading to approximately 145,000 deaths annually. Bladder cancer is typically managed by surgical removal of the tumor; however, the recurrence rate is disappointingly very high, often requiring systemic chemotherapy. Improvement in the diagnosis and prognosis of bladder cancer will only come from a comprehensive understanding of the genetic factors that lead to its development. In this review, we focus on the chromosomal deletions that contribute to the downregulation of tumor suppressor pathways in bladder cancer. Chromosomal deletions are not a random event, since bladder cancer progression has been associated with specific chromosomal deletions and this progression correlates with specific stages of tumor development. The most commonly found chromosomal deletion in all stages of bladder cancer involves deletions in chromosome 9, resulting in the loss of three genes encoding proteins that activate the Rb and p53 tumor suppressors. Additionally, chromosome 9 harbors the TSC1 tumor suppressor which downregulates the well-known anti-apoptotic Akt/mTOR pathway. Hence, deletions on one chromosome may have a crucial influence on the initial steps in tumor development. Other deletions targeting the tumor suppressors Rb, p53, FHIT and LZTS1 occur at later stages of tumor development. Considering the central importance of these tumor suppressor pathways in the formation and evolution of tumors, the time has come to evaluate available drugs in bladder cancer that target the positive regulators of these pathways.

Identification of MMP-15 as an anti-apoptotic factor in cancer cells.

We have performed an in vitro selection for an anti-apoptotic phenotype that resembles the selection process that pre-malignant cells undergo in the initial phase of carcinogenesis in vivo. Using the cervical carcinoma cell line HeLa S3 as a model system, the selection procedure yielded cell clones that displayed increased resistance to apoptosis induced by Fas, tumor necrosis factor-related apoptosis-inducing ligand, and serum starvation. Gene expression profiling using gene family focused cDNA arrays revealed numerous genes that are differentially expressed in HeLa S3 and the resistant subclones and therefore are potentially involved in the definition of sensitivity to apoptotic stimuli. From the genes identified in this functional genomics approach we validated the anti-apoptotic activity of the membrane-anchored matrix metalloproteinase 15 (MMP-15) by means of small interfering RNA-mediated knock-down and ectopic expression in parental HeLa S3 cells and, to confirm a more general significance of our findings, in other cancer cell lines. The in vivo relevance of these findings is supported by the overexpression of MMP-15 in human lung adenocarcinoma compared with normal lung. Because MMP-15 is known to promote invasion, our results suggest that this protease connects metastasis and apoptosis resistance by an unknown regulatory mechanism. Our findings therefore strongly suggest that cancer characteristics such as metastatic potential, which are thought to evolve late in cancer progression, could be manifested early on by selection for an anti-apoptotic phenotype.