Selective activation of NF-κB subunits in human breast cancer: Potential roles for NF-κB2/p52 and for Bcl-3

Members of the NF-kappa B/Rel transcription factor family have been shown recently to be required for cellular transformation by oncogenic Ras and by other oncoproteins and to suppress transformation-associated apoptosis. Furthermore, NF-kappa B has been shown to be activated by several oncoproteins including HER2/Neu, a receptor tyrosine kinase often expressed in human breast cancer. Human breast cancer cell lines, human breast tumors and normal adjacent tissue were analysed by gel mobility shift assay, immunoblotting of nuclear extracts and immunohistochemistry for activation of NF-kappa B. Furthermore, RNA levels for NF-kappa B-activated genes were analysed in order to determine if NF-kappa B is functionally active in human breast cancer. Our data indicate that the p65/RelA subunit of NF-kappa B is activated (i.e., nuclear) in breast cancer cell lines. However, breast tumors exhibit an absence or low level of nuclear p65/RelA but show activated c-Rel, p50 and p52 as compared to nontumorigenic adjacent tissue. Additionally, the I kappa B homolog Bcl-3, which functions to stimulate transcription with p50 or p52, was also activated in breast tumors. There was no apparent correlation between estrogen receptor status and levels of nuclear NF-kappa B complexes. Transcripts of NF-kappa B-regulated genes were found elevated in breast tumors, as compared to adjacent normal tissue, indicating functional NF-kappa B activity. These data suggest a potential role for a subset of NF-kappa B and I kappa B family proteins, particularly NF-kappa B/p52 and Bcl-3, in human breast cancer. Additionally, the activation of functional NF-kappa B in these tumors likely involves a signal transduction pathway distinct from that utilized by cytokines

Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy

<p>Abstract</p> <p>Background</p> <p>Duchenne muscular dystrophy (DMD) is an inherited and progressive disease causing striated muscle deterioration. Patients in their twenties generally die from either respiratory or cardiac failure. In order to improve the lifespan and quality of life of DMD patients, it is important to prevent or reverse the progressive loss of contractile function of the heart. Recent studies by our labs have shown that the peptide NBD (Nemo Binding Domain), targeted at blunting Nuclear Factor κB (NF-κB) signaling, reduces inflammation, enhances myofiber regeneration, and improves contractile deficits in the diaphragm in dystrophin-deficient <it>mdx </it>mice.</p> <p>Methods</p> <p>To assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko) mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice.</p> <p>Results</p> <p>At baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice.</p> <p>Conclusions</p> <p>We conclude that NBD can significantly improve cardiac contractile dysfunction in the dko mouse model of DMD and may thus provide a novel therapeutic treatment for heart failure.</p

WNT-1 Signaling Inhibits Apoptosis by Activating β-Catenin/T Cell Factor–Mediated Transcription

Wnt signaling plays a critical role in development and oncogenesis. Although significant progress has been made in understanding the downstream signaling cascade of Wnt signaling, little is known regarding Wnt signaling modification of the cell death machinery. Given that numerous oncogenes transform cells by providing cell survival function, we hypothesized that Wnt signaling may inhibit apoptosis. Here, we report that cells expressing Wnt-1 were resistant to cancer therapy–mediated apoptosis. Wnt-1 signaling inhibited the cytochrome c release and the subsequent caspase-9 activation induced by chemotherapeutic drugs, including both vincristine and vinblastine. Furthermore, we found that Wnt-1–mediated cell survival was dependent on the activation of β-catenin/T cell factor (Tcf) transcription. Inhibition of β-catenin/Tcf transcription by expression of the dominant-negative mutant of Tcf-4 blocked Wnt-1–mediated cell survival and rendered cells sensitive to apoptotic stimuli. These results provide the first demonstration that Wnt-1 inhibits cancer therapy–mediated apoptosis and suggests that Wnt-1 may exhibit its oncogenic potential through a mechanism of anti-apoptosis

Evaluating potential biomarkers of cachexia and survival in skeletal muscle of upper gastrointestinal cancer patients

Background&nbsp; In order to grow the potential therapeutic armamentarium in the cachexia domain of supportive oncology, there is a pressing need to develop suitable biomarkers and potential drug targets. This pilot study evaluated several potential candidate biomarkers in skeletal muscle biopsies from a cohort of upper gastrointestinal cancer (UGIC) patients.&nbsp; Methods&nbsp; One hundred seven patients (15 weight-stable healthy controls (HC) and 92 UGIC patients) were recruited. Mean (standard deviation) weight-loss of UGIC patients was 8.1 (9.3\%). Cachexia was defined as weight-loss &ge;5\%. Rectus&thinsp;abdominis muscle was obtained at surgery and was analysed by western blotting or quantitative real-time&ndash;polymerase chain reaction. Candidate markers were selected according to previous literature and included Akt and phosphorylated Akt (pAkt, n&thinsp;=&thinsp;52), forkhead box O transcription factors (n&thinsp;=&thinsp;59), ubiquitin E3 ligases (n&thinsp;=&thinsp;59, control of muscle anabolism/catabolism), BNIP3 and GABARAPL1 (n&thinsp;=&thinsp;59, as markers of autophagy), myosin heavy-chain (MyHC, n&thinsp;=&thinsp;54), dystrophin (n&thinsp;=&thinsp;39), &beta;-dystroglycan (n&thinsp;=&thinsp;52), and &beta;-sarcoglycan (n&thinsp;=&thinsp;52, as markers of structural alteration in a muscle). Patients were followed up for an average of 1255&thinsp;days (range 581&ndash;1955&thinsp;days) or until death. Patients were grouped accordingly and analysed by (i) all cancer patients vs. HC; (ii) cachectic vs. non-cachectic cancer patients; and (iii) cancer patients surviving &le;1 vs. {\textgreater}1&thinsp;year post operatively.&nbsp; Results&nbsp; Cancer compared with HC patients had reduced mean (standard deviation) total Akt protein [0.49 (0.31) vs. 0.89 (0.17), P&thinsp;=&thinsp;0.001], increased ratio of phosphorylated to total Akt [1.33 (1.04) vs. 0.32 (0.21), P&thinsp;=&thinsp;0.002] and increased expression of GABARAPL1 [1.60 (0.76) vs. 1.10 (0.57), P&thinsp;=&thinsp;0.024]. &beta;-Dystroglycan levels were higher in cachectic compared with non-cachectic cancer patients [1.01 (0.16) vs. 0.87 (0.20), P&thinsp;=&thinsp;0.007]. Survival was shortened in patients with low compared with high MyHC levels (median 316 vs. 1326&thinsp;days, P&thinsp;=&thinsp;0.023) and dystrophin levels (median 341 vs. 660&thinsp;days, P&thinsp;=&thinsp;0.008).&nbsp; Conclusions&nbsp; The present study has identified intramuscular protein level of &beta;-dystroglycan as a potential biomarker of cancer cachexia. Changes in the structural elements of muscle (MyHC or dystrophin) appear to be survival biomarkers

Dystrophin glycoprotein complex dysfunction:a regulatory link between muscular dystrophy and cancer cachexia

SummaryCachexia contributes to nearly a third of all cancer deaths, yet the mechanisms underlying skeletal muscle wasting in this syndrome remain poorly defined. We report that tumor-induced alterations in the muscular dystrophy-associated dystrophin glycoprotein complex (DGC) represent a key early event in cachexia. Muscles from tumor-bearing mice exhibited membrane abnormalities accompanied by reduced levels of dystrophin and increased glycosylation on DGC proteins. Wasting was accentuated in tumor mdx mice lacking a DGC but spared in dystrophin transgenic mice that blocked induction of muscle E3 ubiquitin ligases. Furthermore, DGC deregulation correlated positively with cachexia in patients with gastrointestinal cancers. Based on these results, we propose that, similar to muscular dystrophy, DGC dysfunction plays a critical role in cancer-induced wasting

NF-κB-induced loss of MyoD messenger RNA: Possible role in muscle decay and cachexia

MyoD regulates skeletal muscle differentiation (SMD) and is essential for repair of damaged tissue. The transcription factor nuclear factor kappa B (NF-κB) is activated by the cytokine tumor necrosis factor (TNF), a mediator of skeletal muscle wasting in cachexia. Here, the role of NF-κB in cytokine-induced muscle degeneration was explored. In differentiating C2C12 myocytes, TNF-induced activation of NF-κB inhibited SMD by suppressing MyoD mRNA at the posttranscriptional level. In contrast, in differentiated myotubes, TNF plus interferon-γ (IFN-γ) signaling was required for NF-κB–dependent down-regulation of MyoD and dysfunction of skeletal myofibers. MyoD mRNA was also down-regulated by TNF and IFN-γ expression in mouse muscle in vivo. These data elucidate a possible mechanism that may underlie the skeletal muscle decay in cachexia

IKK/NF-κB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis

Nuclear factor κB (NF-κB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-κB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of κB kinase β (IKKβ), or IKKγ. Similar increases occur in myoblasts lacking RelA/p65 or IKKβ, and muscles from RelA/p65 or IKKβ mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-κB signaling decreases, whereas the induction of alternative members IKKα, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKα regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-κB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis

An NF-κB - EphrinA5-Dependent Communication between NG2+ Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates

SummarySkeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2+ interstitial cells and myoblasts. NF-κB in NG2+ cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2+ cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth

Wnt signaling promotes oncogenic transformation by inhibiting c-Myc–induced apoptosis

Aberrant activation of the Wnt/β-catenin signaling pathway is associated with numerous human cancers and often correlates with the overexpression or amplification of the c-myc oncogene. Paradoxical to the cellular transformation potential of c-Myc is its ability to also induce apoptosis. Using an inducible c-MycER expression system, we found that Wnt/β-catenin signaling suppressed apoptosis by inhibiting c-Myc–induced release of cytochrome c and caspase activation. Both cyclooxygenase 2 and WISP-1 were identified as effectors of the Wnt-mediated antiapoptotic signal. Soft agar assays showed that neither c-Myc nor Wnt-1 alone was sufficient to induce cellular transformation, but that Wnt and c-Myc coordinated in inducing transformation. Furthermore, coexpression of Wnt-1 and c-Myc induced high-frequency and rapid tumor growth in nude mice. Extensive apoptotic bodies were characteristic of c-Myc–induced tumors, but not tumors induced by coactivation of c-Myc and Wnt-1, indicating that the antiapoptotic function of Wnt-1 plays a critical role in the synergetic action between c-Myc and Wnt-1. These results elucidate the molecular mechanisms by which Wnt/β-catenin inhibits apoptosis and provide new insight into Wnt signaling-mediated oncogenesis