ß-catenin S45F mutation results in apoptotic resistance.

Wnt/ß-catenin signaling is one of the key cascades regulating embryogenesis and tissue homeostasis; it has also been intimately associated with carcinogenesis. This pathway is deregulated in several tumors, including colorectal cancer, breast cancer, and desmoid tumors. It has been shown that CTNNB1 exon 3 mutations are associated with an aggressive phenotype in several of these tumor types and may be associated with therapeutic tolerance. Desmoid tumors typically have a stable genome with ß-catenin mutations as a main feature, making these tumors an ideal model to study the changes associated with different types of ß-catenin mutations. Here, we show that the apoptosis mechanism is deregulated in ß-catenin S45F mutants, resulting in decreased induction of apoptosis in these cells. Our findings also demonstrate that RUNX3 plays a pivotal role in the inhibition of apoptosis found in the ß-catenin S45F mutants. Restoration of RUNX3 overcomes this inhibition in the S45F mutants, highlighting it as a potential therapeutic target for malignancies harboring this specific CTNNB1 mutation. While the regulatory effect of RUNX3 in ß-catenin is already known, our results suggest the possibility of a feedback loop involving these two genes, with the CTNNB1 S45F mutation downregulating expression of RUNX3, thus providing additional possible novel therapeutic targets for tumors having deregulated Wnt/ß-catenin signaling induced by this mutation.

Management of Cancer Cachexia: Attempting to Develop New Pharmacological Agents for New Effective Therapeutic Options.

Cancer cachexia (CC) is a multifactorial syndrome characterized by systemic inflammation, uncontrolled weight loss and dramatic metabolic alterations. This includes myofibrillar protein breakdown, increased lipolysis, insulin resistance, elevated energy expediture, and reduced food intake, hence impairing the patient's response to anti-cancer therapies and quality of life. While a decade ago the syndrome was considered incurable, over the most recent years much efforts have been put into the study of such disease, leading to the development of potential therapeutic strategies. Several important improvements have been reached in the management of CC from both the diagnostic-prognostic and the pharmacological viewpoint. However, given the heterogeneity of the disease, it is impossible to rely only on single variables to properly treat patients presenting this metabolic syndrome. Moreover, the cachexia symptoms are strictly dependent on the type of tumor, stage and the specific patient's response to cancer therapy. Thus, the attempt to translate experimentally effective therapies into the clinical practice results in a great challenge. For this reason, it is of crucial importance to further improve our understanding on the interplay of molecular mechanisms implicated in the onset and progression of CC, giving the opportunity to develop new effective, safe pharmacological treatments. In this review we outline the recent knowledge regarding cachexia mediators and pathways involved in skeletal muscle (SM) and adipose tissue (AT) loss, mainly from the experimental cachexia standpoint, then retracing the unimodal treatment options that have been developed to the present day.

Targeted Therapies in Veterinary Oncology.

Advances in molecular biology have permitted a much more detailed understanding of cellular dysfunction at the molecular and genetic levels in cancer cells. This has resulted in the identification of novel targets for therapeutic intervention, including proteins that regulate signal transduction, gene expression, and protein turnover. In many instances, small molecules are used to disrupt the function of these targets, often through competitive inhibition of ATP binding or the prevention of necessary protein-protein interactions. More than 40 small molecule inhibitors are now approved to treat a variety of human cancers, substantially impacting patient outcomes.

The TLR7/8/9 Antagonist IMO-8503 Inhibits Cancer-Induced Cachexia.

: Muscle wasting is a feature of the cachexia syndrome, which contributes significantly to the mortality of patients with cancer. We have previously demonstrated that miR-21 is secreted through extracellular vesicles (EV) by lung and pancreatic cancer cells and promotes JNK-dependent cell death through its binding to the TLR7 receptor in murine myoblasts. Here, we evaluate the ability of IMO-8503, a TLR7, 8, and 9 antagonist, to inhibit cancer-induced cachexia. Using EVs isolated from lung and pancreatic cancer cells and from patient plasma samples, we demonstrate that IMO-8503 inhibits cell death induced by circulating miRNAs with no significant toxicity. Intraperitoneal administration of the antagonist in a murine model for Lewis lung carcinoma (LLC-induced cachexia) strongly impaired several cachexia-related features, such as the expression of Pax7 as well as caspase-3 and PARP cleavage in skeletal muscles, and significantly prevented the loss of lean mass in tumor-bearing mice. IMO-8503 also impaired circulating miRNA-induced cell death in human primary myoblasts. Taken together, our findings strongly indicate that IMO-8503 serves as a potential therapy for the treatment of cancer cachexia. SIGNIFICANCE: Cancer-associated cachexia is a significant problem for patients with cancer that remain poorly understood, understudied, and inadequately treated; these findings report a potential new therapeutic for the treatment of TLR7-mediated cancer cachexia.

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.

Classical NF-κB Metabolically Reprograms Sarcoma Cells Through Regulation of Hexokinase 2.

BACKGROUND: Metabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized. METHODS: Classical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XFe24 was used to analyze oxygen consumption rate as a measure of aerobic respiration. RESULTS: Inhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells. CONCLUSION: These findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.

Inflammation induced loss of skeletal muscle.

Inflammation is an important contributor to the pathology of diseases implicated in skeletal muscle dysfunction. A number of diseases and disorders including inflammatory myopathies and Chronic Obstructive Pulmonary Disorder (COPD) are characterized by chronic inflammation or elevation of the inflammatory mediators. While these disease states exhibit different pathologies, all have in common the loss of skeletal muscle mass and a deregulated skeletal muscle physiology. Pro-inflammatory cytokines are key contributors to chronic inflammation found in many of these diseases. This section of the review focuses on some of the known inflammatory disorders like COPD, Rheumatoid Arthritis (RA) and inflammatory myopathies that display skeletal muscle atrophy and also provides the reader an overview of the mediators of inflammation, their signaling pathways, and mechanisms of action. This article is part of a Special Issue entitled "Muscle Bone Interactions".

Microvesicles containing miRNAs promote muscle cell death in cancer cachexia via TLR7.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression and, in cancers, are often packaged within secreted microvesicles. The cachexia syndrome is a debilitating state of cancer that predominantly results from the loss of skeletal muscle mass, which is in part associated with apoptosis. How tumors promote apoptosis in distally located skeletal muscles has not been explored. Using both tumor cell lines and patient samples, we show that tumor-derived microvesicles induce apoptosis of skeletal muscle cells. This proapoptotic activity is mediated by a microRNA cargo, miR-21, which signals through the Toll-like 7 receptor (TLR7) on murine myoblasts to promote cell death. Furthermore, tumor microvesicles and miR-21 require c-Jun N-terminal kinase activity to regulate this apoptotic response. Together, these results describe a unique pathway by which tumor cells promote muscle loss, which might provide a great insight into elucidating the causes and treatment options of cancer cachexia.

Interferon-γ resets muscle cell fate by stimulating the sequential recruitment of JARID2 and PRC2 to promoters to repress myogenesis.

The inflammatory cytokine interferon-γ (IFN-γ) orchestrates a diverse array of fundamental physiological processes. IFN-γ and the class II transactivator (CIITA) play essential roles in inhibiting muscle development during the inflammatory response. We describe the mechanism through which IFN-γ and CIITA inhibit myogenesis by repressing gene expression in muscle cells subjected to inflammation. In mice, the presence of increased amounts of circulating IFN-γ resulted in the increased abundance of Polycomb repressive complex 2 (PRC2) in muscle fibers, a tissue in which PRC2 is not normally present in the adult. We showed that CIITA first interacted with the Jumonji family protein JARID2, a noncatalytic subunit of PRC2, which caused an RNA polymerase II (RNAPII), phosphorylated at serine-5, to pause at target promoters. Additional subunits of the PRC2 complex, including the catalytic subunit EZH2, were then recruited in a JARID2-dependent manner that was concurrent with the loss of RNAPII and the methylation of Lys(27) of histone H3 (H3K27), which is associated with gene repression. IFN-γ and CIITA act to both promote the abundance of PRC2 subunits, which are not normally present during muscle differentation, and recruit the PRC2 complex to block myogenesis. Together, these data indicate that increased amounts of IFN-γ reset myogenic cell fate through a multistep mechanism that culminates in the recruitment of PRC2 to silence muscle-specific genes.

Myogenin recruits the histone chaperone facilitates chromatin transcription (FACT) to promote nucleosome disassembly at muscle-specific genes.

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell.

CIITA is silenced by epigenetic mechanisms that prevent the recruitment of transactivating factors in rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMS) are highly malignant pediatric sarcomas. We have discovered that the gene encoding the major histocompatibilty complex class II transactivator, CIITA, is silenced in cells representing both major subtypes of RMS. Silencing of CIITA prevents the IFN-γ inducible expression of MHC class II genes in these cells. Overexpression of CIITA in these cells can restore MHC expression. We have found that IFN-γ signaling is intact in these cells, but pSTAT1 and IRF1 do not bind to the CIITA PIV promoter. The CIITA promoter is not hypermethylated in RD (ERMS) cells but does show a modestly enhanced methylation status in SJRH30 (ARMS) cells. We have found that histone acetylation, which normally increases on the CIITA PIV promoter following IFN-γ treatment, is blocked in both types of RMS cells. In RD cells, treatment with a histone deacetylase inhibitor (TSA) reverses the silencing of CIITA. In SJRH30 cells, treatment with DNA methyltransferase inhibitors and TSA cooperatively restores CIITA expression. Surprisingly, we have also shown that the expression of two components of the immunoproteasome, which are embedded in the class II locus, is stimulated by IFN-γ in certain RMS cells in the absence of stimulation by CIITA. CIITA overexpression can also activate the expression of these genes, indicating that the immunoproteasome genes LMP2 and LMP7 can be activated by both CIITA dependent and CIITA independent pathways.

Sequential association of myogenic regulatory factors and E proteins at muscle-specific genes.

BACKGROUND: Gene expression in skeletal muscle is controlled by a family of basic helix-loop-helix transcription factors known as the myogenic regulatory factors (MRFs). The MRFs work in conjunction with E proteins to regulate gene expression during myogenesis. However, the precise mechanism by which the MRFs activate gene expression is unclear. In this work, we sought to define the binding profiles of MRFs and E proteins on muscle-specific genes throughout a time course of differentiation. RESULTS: We performed chromatin immunoprecipitation (ChIP) assays for myogenin, MyoD, Myf5 and E proteins over a time course of C2C12 differentiation, resulting in several surprising findings. The pattern of recruitment is specific to each promoter tested. The recruitment of E proteins often coincides with the arrival of the MRFs, but the binding profile does not entirely overlap with the MRF binding profiles. We found that E12/E47 is bound to certain promoters during proliferation, but every gene tested is preferentially bound by HEB during differentiation. We also show that MyoD, myogenin and Myf5 have transient roles on each of these promoters during muscle differentiation. We also found that RNA polymerase II occupancy correlates with the transcription profile of these promoters. ChIP sequencing assays confirmed that MyoD, myogenin and Myf5 co-occupy promoters. CONCLUSIONS: Our data reveal the sequential association of MyoD, myogenin, Myf5 and HEB on muscle-specific promoters. These data suggest that each of the MRFs, including Myf5, contribute to gene expression at each of the geness analyzed here.. The dynamic binding profiles observed suggest that MRFs and E proteins are recruited independently to promoters.

Gamma interferon modulates myogenesis through the major histocompatibility complex class II transactivator, CIITA.

Gamma interferon (IFN-γ) is an inflammatory cytokine that has complex effects on myogenesis. Here, we show that the IFN-γ-induced inhibition of myogenesis is mediated by the major histocompatibility complex (MHC) class II transactivator, CIITA, which binds to myogenin and inhibits its activity. In IFN-γ-treated myoblasts, the inhibition of muscle-specific genes includes the expression of myogenin itself, while in myotubes, myogenin expression is unaffected. Thus, CIITA appears to act by both repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. Stimulation by IFN-γ in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. The IFN-γ-mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-γ. Through overexpression studies, we confirm that the expression of CIITA, independent of IFN-γ, is sufficient to inhibit myogenesis. Through knockdown studies, we also demonstrate that CIITA is necessary for the IFN-γ-mediated inhibition of myogenesis. Finally, we show that CIITA, which lacks DNA binding activity, is recruited to muscle-specific promoters coincident with reductions in RNA polymerase II recruitment. Thus, this work reveals how IFN-γ modulates myogenesis and demonstrates a key role for CIITA in this process.

Transcriptional analysis of the titin cap gene.

Mutations in titin cap (Tcap), also known as telethonin, cause limb-girdle muscular dystrophy type 2G (LGMD2G). Tcap is one of the titin interacting Z-disc proteins involved in the regulation and development of normal sarcomeric structure. Given the essential role of Tcap in establishing and maintaining normal skeletal muscle architecture, we were interested in determining the regulatory elements required for expression of this gene in myoblasts. We have defined a highly conserved 421 bp promoter proximal promoter fragment that contains two E boxes and multiple putative Mef2 binding sequences. This promoter can be activated by MyoD and myogenin in NIH3T3 fibroblast cells, and maintains the differentiated cell-specific expression pattern of the endogenous Tcap in C2C12 cells. We find that while both E boxes are required for full activation by MyoD or myogenin in NIH3T3 cells, the promoter proximal E box has a greater contribution to activation of this promoter in C2C12 cells and to activation by MyoD in NIH3T3 cells. Together, the data suggest an important role for MyoD in activating Tcap expression through the promoter proximal E box. We also show that myogenin is required for normal expression in vivo and physically binds to the Tcap promoter during embryogenesis.