The Hippo in the room: Targeting the Hippo signalling pathway for osteosarcoma therapies.

Osteosarcoma (OS) is a primary malignant bone tumour which usually occurs in children and adolescents. OS is primarily a result of chromosomal aberrations, a combination of acquired genetic changes and, hereditary, resulting in the dysregulation of cellular functions. The Hippo signalling pathway regulates cell and tissue growth by modulating cell proliferation, differentiation, and migration in developing organs. Mammalian STE20-like 1/2 (MST1/2) protein kinases are activated by neurofibromatosis type 2, Ras association domain family member 2, kidney and brain protein, or other factors. Interactions between MST1/2 and salvador family WW domain-containing protein 1 activate large tumour suppressor kinase 1/2 proteins, which in turn phosphorylate the downstream Yes-associated protein 1/transcriptional coactivator with PDZ-binding motif (YAP/TAZ). Moreover, dysregulation of this pathway can lead to aberrant cell growth, resulting in tumorigenesis. Interestingly, small molecules targeting the Hippo signalling pathways, through affecting YAP/TAZ cellular localisation and their interaction with members of the TEA/ATTS domain family of transcriptional enhancers are being developed and hold promise for the treatment of OS. This review discusses the existing knowledge about the involvement of the Hippo signalling cascade in OS and highlights several small molecule inhibitors as potential novel therapeutics.

Update on genomic and molecular landscapes of well-differentiated liposarcoma and dedifferentiated liposarcoma.

Well-differentiated liposarcoma (WDLPS) is the most frequent subtype of liposarcoma and may transform into dedifferentiated liposarcoma (DDLPS) which is a more aggressive subtype. Retroperitoneal lesions of WDLPS/DDLPS tend to recur repeatedly due to incomplete resections, and adjuvant chemotherapy and radiotherapy have little effect on patient survival. Consequently, identifying therapeutic targets and developing targeted drugs is critical for improving the outcome of WDLPS/DDLPS patients. In this review, we summarised the mutational landscape of WDLPS/DDLPS from recent studies focusing on potential oncogenic drivers and the development of molecular targeted drugs for DDLPS. Due to the limited number of studies on the molecular networks driving WDLPS to DDLPS development, we looked at other dedifferentiation-related tumours to identify potential parallel mechanisms that could be involved in the dedifferentiation process generating DDLPS.

Identification of novel sarcoma risk genes using a two-stage genome wide DNA sequencing strategy in cancer cluster families and population case and control cohorts.

BACKGROUND: Although familial clustering of cancers is relatively common, only a small proportion of familial cancer risk can be explained by known cancer predisposition genes. METHODS: In this study we employed a two-stage approach to identify candidate sarcoma risk genes. First, we conducted whole exome sequencing in three multigenerational cancer families ascertained through a sarcoma proband (n = 19) in order to prioritize candidate genes for validation in an independent case-control cohort of sarcoma patients using family-based association and segregation analysis. The second stage employed a burden analysis of rare variants within prioritized candidate genes identified from stage one in 560 sarcoma cases and 1144 healthy ageing controls, for which whole genome sequence was available. RESULTS: Variants from eight genes were identified in stage one. Following gene-based burden testing and after correction for multiple testing, two of these genes, ABCB5 and C16orf96, were determined to show statistically significant association with cancer. The ABCB5 gene was found to have a higher burden of putative regulatory variants (OR = 4.9, p-value = 0.007, q-value = 0.04) based on allele counts in sarcoma cases compared to controls. C16orf96, was found to have a significantly lower burden (OR = 0.58, p-value = 0.0004, q-value = 0.003) of regulatory variants in controls compared to sarcoma cases. CONCLUSIONS: Based on these genetic association data we propose that ABCB5 and C16orf96 are novel candidate risk genes for sarcoma. Although neither of these two genes have been previously associated with sarcoma, ABCB5 has been shown to share clinical drug resistance associations with melanoma and leukaemia and C16orf96 shares regulatory elements with genes that are involved with TNF-alpha mediated apoptosis in a p53/TP53-dependent manner. Future genetic studies in other family and population cohorts will be required for further validation of these novel findings.

The endoplasmic reticulum-associated protein, OS-9, behaves as a lectin in targeting the immature calcium-sensing receptor.

The mechanisms responsible for the processing and quality control of the calcium-sensing receptor (CaSR) in the endoplasmic reticulum (ER) are largely unknown. In a yeast two-hybrid screen of the CaSR C-terminal tail (residues 865-1078), we identified osteosarcoma-9 (OS-9) protein as a binding partner. OS-9 is an ER-resident lectin that targets misfolded glycoproteins to the ER-associated degradation (ERAD) pathway through recognition of specific N-glycans by its mannose-6-phosphate receptor homology (MRH) domain. We show by confocal microscopy that the CaSR and OS-9 co-localize in the ER in COS-1 cells. In immunoprecipitation studies with co-expressed OS-9 and CaSR, OS-9 specifically bound the immature form of wild-type CaSR in the ER. OS-9 also bound the immature forms of a CaSR C-terminal deletion mutant and a C677A mutant that remains trapped in the ER, although binding to neither mutant was favored over wild-type receptor. OS-9 binding to immature CaSR required the MRH domain of OS-9 indicating that OS-9 acts as a lectin most likely to target misfolded CaSR to ERAD. Our results also identify two distinct binding interactions between OS-9 and the CaSR, one involving both C-terminal domains of the two proteins and the other involving both N-terminal domains. This suggests the possibility of more than one functional interaction between OS-9 and the CaSR. When we investigated the functional consequences of altered OS-9 expression, neither knockdown nor overexpression of OS-9 was found to have a significant effect on CaSR cell surface expression or CaSR-mediated ERK1/2 phosphorylation.

The cardiac L-type calcium channel alpha subunit is a target for direct redox modification during oxidative stress-the role of cysteine residues in the alpha interacting domain.

Cardiovascular disease is the leading cause of death in the Western world. The incidence of cardiovascular disease is predicted to further rise with the increase in obesity and diabetes and with the aging population. Even though the survival rate from ischaemic heart disease has improved over the past 30 years, many patients progress to a chronic pathological condition, known as cardiac hypertrophy that is associated with an increase in morbidity and mortality. Reactive oxygen species (ROS) and calcium play an essential role in mediating cardiac hypertrophy. The L-type calcium channel is the main route for calcium influx into cardiac myocytes. There is now good evidence for a direct role for the L-type calcium channel in the development of cardiac hypertrophy. Cysteines on the channel are targets for redox modification and glutathionylation of the channel can modulate the function of the channel protein leading to the onset of pathology. The cysteine responsible for modification of L-type calcium channel function has now been identified. Detailed understanding of the role of cysteines as possible targets during oxidative stress may assist in designing therapy to prevent the development of hypertrophy and heart failure.

Gain-of-function Lyn induces anemia: appropriate Lyn activity is essential for normal erythropoiesis and Epo receptor signaling.

Lyn is involved in erythropoietin (Epo)-receptor signaling and erythroid homeostasis. Downstream pathways influenced following Lyn activation and their significance to erythropoiesis remain unclear. To address this, we assessed a gain-of-function Lyn mutation (Lyn(up/up)) on erythropoiesis and Epo receptor signaling. Adult Lyn(up/up) mice were anemic, with dysmorphic red cells (spherocyte-like, acanthocytes) in their circulation, indicative of hemolytic anemia and resembling the human disorder chorea acanthocytosis. Heterozygous Lyn(+/up) mice became increasingly anemic with age, indicating that the mutation was dominant. In an attempt to overcome this anemia, extramedullary erythropoiesis was activated. As the mice aged, the levels of different immature erythroid populations changed, indicating compensatory mechanisms to produce more erythrocytes were dynamic. Changes in Epo signaling were observed in Lyn(+/up) erythroid cell lines and primary CD71(+) Lyn(up/up) erythroblasts, including significant alterations to the phosphorylation of Lyn, the Epo receptor, Janus kinase 2, Signal Transducer and Action of Transcription-5, GRB2-associated-binding protein-2, Akt, and Forkhead box O3. As a consequence of altered Lyn signaling, Lyn(+/up) cells remained viable in the absence of Epo but displayed delayed Epo-induced differentiation. These data demonstrate that Lyn gene dosage and activity are critical for normal erythropoiesis; constitutively active Lyn alters Epo signaling, which in turn produces erythroid defects.

Lyn kinase plays important roles in erythroid expansion, maturation and erythropoietin receptor signalling by regulating inhibitory signalling pathways that control survival.

Erythroid homoeostasis is primarily controlled by Epo (erythropoietin) receptor signalling; however, the Lyn tyrosine kinase plays an important subsidiary role in regulating the erythroid compartment. Nonetheless, specific erythroid pathways that require Lyn activity and their biological significance remain unclear. To address this, we asked what consequence loss of Lyn had on the ex vivo expansion and maturation of splenic erythroid progenitors and Epo receptor signalling. Pharmacological inhibition of Lyn with PP2 inhibited the survival of terminally differentiated erythroblasts. Less committed erythroid progenitors expanded well, whereas early splenic Lyn(-/-) erythroblasts had attenuated ex vivo expansion, and late stage Lyn(-/-) erythroblasts were retarded in completing morphological maturation ex vivo. Furthermore, immortalized Lyn(-/-) erythroblasts were slower growing, less viable and inhibited in their differentiation. Signalling studies showed that Lyn was required for both positive GAB2/Akt/FoxO3 (forkhead box O3) survival signals as well as negative feedback of JAK2 (Janus kinase 2)/STAT5 (signal transducer and activator of transcription 5) and ERK1/2 (extracellular-signal-regulated kinase 1/2) signals via SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase 1). During differentiation, Lyn controls survival and cell cycle exit as demonstrated by reduced STAT5 and FoxO3/GSKα/ß (glycogen synthase kinase α/ß) phosphorylation and diminished p27(Kip1) induction in Lyn-deficient erythroblasts. Lyn deficiency alters the balance of pro- and anti-apoptotic molecules (BAD and BclXL), thereby reducing survival and preventing cell cycle exit. Consequently, Lyn facilitates normal erythrocyte production by influencing different stages of erythroid progenitor expansion, and mature cell development and survival signalling.

The adaptor protein 14-3-3 binds to the calcium-sensing receptor and attenuates receptor-mediated Rho kinase signalling.

A yeast two-hybrid screen performed to identify binding partners of the CaR (calcium-sensing receptor) intracellular tail identified the adaptor protein 14-3-3θ as a novel binding partner that bound to the proximal membrane region important for CaR expression and signalling. The 14-3-3θ protein directly interacted with the CaR tail in pull-down studies and FLAG-tagged CaR co-immunoprecipitated with EGFP (enhanced green fluorescent protein)-tagged 14-3-3θ when co-expressed in HEK (human embryonic kidney)-293 or COS-1 cells. The interaction between the CaR and 14-3-3θ did not require a putative binding site in the membrane-proximal region of the CaR tail and was independent of PKC (protein kinase C) phosphorylation. Confocal microscopy demonstrated co-localization of the CaR and EGFP-14-3-3θ in the ER (endoplasmic reticulum) of HEK-293 cells that stably expressed the CaR (HEK-293/CaR cells), but 14-3-3θ overexpression had no effect on membrane expression of the CaR. Overexpression of 14-3-3θ in HEK-293/CaR cells attenuated CaR-mediated Rho signalling, but had no effect on ERK (extracellular-signal-regulated kinase) 1/2 signalling. Another isoform identified from the library, 14-3-3ζ, exhibited similar behaviour to that of 14-3-3θ with respect to CaR tail binding, cellular co-localization and impact on receptor-mediated signalling. However, unlike 14-3-3θ, this isoform, when overexpressed, significantly reduced CaR plasma membrane expression. Results indicate that 14-3-3 proteins mediate CaR-dependent Rho signalling and may modulate the plasma membrane expression of the CaR.

Targeting Lyn tyrosine kinase through protein fusions encompassing motifs of Cbp (Csk-binding protein) and the SOCS box of SOCS1.

The tyrosine kinase Lyn is involved in oncogenic signalling in several leukaemias and solid tumours, and we have previously identified a pathway centred on Cbp [Csk (C-terminal Src kinase)-binding protein] that mediates both enzymatic inactivation, as well as proteasomal degradation of Lyn via phosphorylation-dependent recruitment of Csk (responsible for phosphorylating the inhibitory C-terminal tyrosine of Lyn) and SOCS1 (suppressor of cytokine signalling 1; an E3 ubiquitin ligase). In the present study we show that fusing specific functional motifs of Cbp and domains of SOCS1 together generates a novel molecule capable of directing the proteasomal degradation of Lyn. We have characterized the binding of pY (phospho-tyrosine) motifs of Cbp to SFK (Src-family kinase) SH2 (Src homology 2) domains, identifying those with high affinity and specificity for the SH2 domain of Lyn and that are preferred substrates of active Lyn. We then fused them to the SB (SOCS box) of SOCS1 to facilitate interaction with the ubiquitination-promoting elongin B/C complex. As an eGFP (enhanced green fluorescent protein) fusion, these proteins can direct the polyubiquitination and proteasomal degradation of active Lyn. Expressing this fusion protein in DU145 cancer cells (but not LNCaP or MCF-7 cells), that require Lyn signalling for survival, promotes loss of Lyn, loss of caspase 3, appearance of an apoptotic morphology and failure to survive/expand. These findings show how functional domains of Cbp and SOCS1 can be fused together to generate molecules capable of inhibiting the growth of cancer cells that express high levels of active Lyn.

Injury to the tunica media initiates atherogenesis in the presence of hyperlipidemia.

Background and aims: Fatty streaks initiating the formation of atheromatous plaque appear in the tunica intima. The tunica media is not known to be a nidus for lipid accumulation initiating atherogenesis. We assessed changes to the tunica media in response to a micro-injury produced in the pig aorta. In addition, we assessed human carotid endarterectomy plaques for indication of atheroma initiation in the tunica media. Methods: Three healthy landrace female pigs underwent laparotomy to inject autologous blood and create micro-hematomas at 6 sites within the tunica media of the infrarenal abdominal aorta. These pigs were fed a high-fat diet (HFD) for 4-12 weeks. Post-mortem aortas from all pigs, including a control group of healthy pigs, were serially stained to detect lipid deposits, vasa vasora (VV), immune cell infiltration and inflammatory markers, as well as changes to the vascular smooth muscle cell (vSMC) compartment. Moreover, 25 human carotid endarterectomy (CEA) specimens were evaluated for their lipid composition in the tunica media and intima. Results: High lipid clusters, VV density, and immune cell infiltrates were consistently observed at 5 out of 6 injection sites under prolonged hyperlipidemia. The hyperlipidemic diet also affected the vSMC compartment in the tunica media adjacent to the tunica adventitia, which correlated with VV invasion and immune cell infiltration. Analysis of human carotid specimens post-CEA indicated that 32% of patients had significantly greater atheroma in the tunica media than in the arterial intima. Conclusion: The arterial intima is not the only site for atherosclerosis initiation. We show that injury to the media can trigger atherogenesis.

Integrating novel signaling pathways involved in erythropoiesis.

Many extrinsic and intrinsic factors control the development of red blood cells from committed progenitors, with the Erythropoietin-receptor (Epo-R) signaling network being the primary controlling molecular hub. Although much is understood about erythroid signaling pathways, new and intriguing factors that influence different aspects of erythroid cell development are still being uncovered. New extrinsic effectors include hypoxia and polymeric IgA1 (pIgA1), and new Epo-R signaling pathway components include Lyn/Cbp and Lyn/Liar. Hypoxia directly activates committed erythroid progenitors to expand, whereas pIgA1 activates the Akt and MAP-Kinase (MAPK) pathways through transferrin receptors on more mature erythroid cells. The Lyn/Cbp pathway controls the activity and protein levels of Lyn through recruitment of Csk and SOCS1, as well as feeding into the control of other pathways mediated by recruitment of ras-GAP, PI3-kinase, PLCγ, Fes, and EBP50. Nuclear/cytoplasmic shuttling of Lyn and other signaling molecules is influenced by Liar and results in regulation of their intersecting signaling pathways. The challenge of future research is to flesh out the details of these new signaling regulators/networks and integrate their influences during the different stages of erythropoiesis.

Functions of the Lyn tyrosine kinase in health and disease.

Src family kinases such as Lyn are important signaling intermediaries, relaying and modulating different inputs to regulate various outputs, such as proliferation, differentiation, apoptosis, migration and metabolism. Intriguingly, Lyn can mediate both positive and negative signaling processes within the same or different cellular contexts. This duality is exemplified by the B-cell defect in Lyn-/- mice in which Lyn is essential for negative regulation of the B-cell receptor; conversely, B-cells expressing a dominant active mutant of Lyn (Lynup/up) have elevated activities of positive regulators of the B-cell receptor due to this hyperactive kinase. Lyn has well-established functions in most haematopoietic cells, viz. progenitors via influencing c-kit signaling, through to mature cell receptor/integrin signaling, e.g. erythrocytes, platelets, mast cells and macrophages. Consequently, there is an important role for this kinase in regulating hematopoietic abnormalities. Lyn is an important regulator of autoimmune diseases such as asthma and psoriasis, due to its profound ability to influence immune cell signaling. Lyn has also been found to be important for maintaining the leukemic phenotype of many different liquid cancers including acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and B-cell lymphocytic leukaemia (BCLL). Lyn is also expressed in some solid tumors and here too it is establishing itself as a potential therapeutic target for prostate, glioblastoma, colon and more aggressive subtypes of breast cancer. LAY To relay information, a cell uses enzymes that put molecular markers on specific proteins so they interact with other proteins or move to specific parts of the cell to have particular functions. A protein called Lyn is one of these enzymes that regulate information transfer within cells to modulate cell growth, survival and movement. Depending on which type of cell and the source of the information input, Lyn can positively or negatively regulate the information output. This ability of Lyn to be able to both turn on and turn off the relay of information inside cells makes it difficult to fully understand its precise function in each specific circumstance. Lyn has important functions for cells involved in blood development, including different while blood cells as well as red blood cells, and in particular for the immune cells that produce antibodies (B-cells), as exemplified by the major B-cell abnormalities that mice with mutations in the Lyn gene display. Certain types of leukaemia and lymphoma appear to have too much Lyn activity that in part causes the characteristics of these diseases, suggesting it may be a good target to develop new anti-leukaemia drugs. Furthermore, some specific types, and even specific subtypes, of solid cancers, e.g. prostate, brain and breast cancer can also have abnormal regulation of Lyn. Consequently, targeting this protein in these cancers could also prove to be beneficial.

Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling.

The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

A non-synonymous coding change in the CYP19A1 gene Arg264Cys (rs700519) does not affect circulating estradiol, bone structure or fracture.

BACKGROUND: The biosynthesis of estrogens from androgens is catalyzed by aromatase P450 enzyme, coded by the CYP19A1 gene on chromosome 15q21.2. Genetic variation within the CYP19A1 gene sequence has been shown to alter the function of the enzyme. The aim of this study is to investigate whether a non-synonymous Arg264Cys (rs700519) single nucleotide polymorphism (SNP) is associated with altered levels of circulating estradiol, areal bone mineral density or fracture. METHODS: This population- based study of 1,022 elderly Caucasian women (mean age 74.95 ± 2.60 years) was genotyped for the rs700519 SNP were analyzed to detect any association with endocrine and bone phenotypes. RESULTS: The genotype frequencies were 997 wildtype (97.6%), 24 heterozygous (2.3%) and 1 homozygous (0.1%). When individuals were grouped by genotype, there was no association between the polymorphism and serum estradiol (wildtype 27.5 ± 16.0; variants 31.2 ± 18.4, P = 0.27). There was also no association seen on hip bone mineral density (wildtype 0.81 ± 0.12; 0.84 ± 0.14 for variants, P = 0.48) or femoral neck bone mineral density (0.69 ± 0.10 for wildtype; 0.70 ± 0.12 for variants, P = 0.54) before or after correction of the data with age, height, weight and calcium therapy. There were also no associations with quantitative ultrasound measures of bone structure (broadband ultrasound attenuation, speed of sound and average stiffness). CONCLUSIONS: In a cohort of 1,022 elderly Western Australian women, the presence of Arg264Cys (rs700519) polymorphism was not found to be associated with serum estradiol, bone structure or phenotypes.

Liar, a novel Lyn-binding nuclear/cytoplasmic shuttling protein that influences erythropoietin-induced differentiation.

Erythropoiesis is primarily controlled by erythropoietin (Epo), which stimulates proliferation, differentiation, and survival of erythroid precursors. We have previously shown that the tyrosine kinase Lyn is critical for transducing differentiation signals emanating from the activated Epo receptor. A yeast 2-hybrid screen for downstream effectors of Lyn identified a novel protein, Liar (Lyn-interacting ankyrin repeat), which forms a multiprotein complex with Lyn and HS1 in erythroid cells. Interestingly, 3 of the ankyrin repeats of Liar define a novel SH3 binding region for Lyn and HS1. Liar also contains functional nuclear localization and nuclear export sequences and shuttles rapidly between the nucleus and cytoplasm. Ectopic expression of Liar inhibited the differentiation of normal erythroid progenitors, as well as immortalized erythroid cells. Significantly, Liar affected Epo-activated signaling molecules including Erk2, STAT5, Akt, and Lyn. These results show that Liar is a novel Lyn-interacting molecule that plays an important role in regulating intracellular signaling events associated with erythroid terminal differentiation.

SCIMP is a spatiotemporal transmembrane scaffold for Erk1/2 to direct pro-inflammatory signaling in TLR-activated macrophages.

Immune cells are armed with Toll-like receptors (TLRs) for sensing and responding to pathogens and other danger cues. The role of extracellular-signal-regulated kinases 1/2 (Erk1/2) in TLR signaling remains enigmatic, with both pro- and anti-inflammatory functions described. We reveal here that the immune-specific transmembrane adaptor SCIMP is a direct scaffold for Erk1/2 in TLR pathways, with high-resolution, live-cell imaging revealing that SCIMP guides the spatial and temporal recruitment of Erk2 to membrane ruffles and macropinosomes for pro-inflammatory TLR4 signaling. SCIMP-deficient mice display defects in Erk1/2 recruitment to TLR4, c-Fos activation, and pro-inflammatory cytokine production, with these effects being phenocopied by Erk1/2 signaling inhibition. Our findings thus delineate a selective role for SCIMP as a key scaffold for the membrane recruitment of Erk1/2 kinase to initiate TLR-mediated pro-inflammatory responses in macrophages.

Evidence of altered guinea pig ventricular cardiomyocyte protein expression and growth in response to a 5 min in vitro exposure to H(2)O(2).

Oxidative stress and alterations in cellular calcium homeostasis are associated with the development of cardiac hypertrophy. However, the early cellular mechanisms for the development of hypertrophy are not well understood. Guinea pig ventricular myocytes were exposed to 30 microM H(2)O(2) for 5 min followed by 10 units/mL catalase to degrade the H(2)O(2), and effects on protein expression were examined 48 h later. Transient exposure to H(2)O(2) increased the level of protein synthesis more than 2-fold, assessed as incorporation of [(3)H]leucine (n = 12; p < 0.05). Cell size was increased slightly, but there was no evidence of major cytoskeletal disorganization assessed using fluorescence microscopy. Changes in the expression of individual proteins were assessed using iTRAQ protein labeling followed by mass spectrometry analysis (LC-MALDI-MSMS); 669 proteins were identified, and transient exposure of myocytes to H(2)O(2) altered expression of 35 proteins that were predominantly mitochondrial in origin, including TCA cycle enzymes and oxidative phosphorylation proteins. Consistent with changes in the expression of mitochondrial proteins, transient exposure of myocytes to H(2)O(2) increased the magnitude of the mitochondrial NADH signal 10.5 +/- 2.3% compared to cells exposed to 0 microM H(2)O(2) for 5 min followed by 10 units/mL catalase (n = 8; p < 0.05). In addition, metabolic activity was significantly increased in the myocytes 48 h after transient exposure to H(2)O(2), assessed as formation of formazan from tetrazolium salt. We conclude that a 5 min exposure of ventricular myocytes to 30 microM H(2)O(2) is sufficient to significantly alter protein expression, consistent with the development of hypertrophy in the myocytes. Changes in mitochondrial protein expression and function appear to be early sequelae in the development of hypertrophy.

Src family kinases: regulation of their activities, levels and identification of new pathways.

While the Src family of protein tyrosine kinases (SFK), and the main ancillary molecules involved in their regulation, have been studied for many years, the details of their interplay are not fully understood and thus remain under active investigation. Additionally, new players that coordinate their regulation and direct their signalling cascades are also being uncovered, shedding new light on the complexity of these signalling networks. Through the utilization of novel interaction assays, several new interconnecting mediators that are helping to show the elegance of Src family kinase regulation have been discovered. This review outlines SFK regulation, the discovery of the Csk binding protein (Phosphoprotein Associated with Glycosphingolipid-enriched microdomains, Cbp/PAG), and its role in regulating SFK kinase activity status, as well as protein levels. Further, details of the methods used to identify this dual mode of regulation can be applied to delineate the full gamut of SH2/SH3-directed SFK pathways and, indeed, those of any tyrosine kinase. Using Lyn as a model SFK, we and others have shown that Cbp recruits negative regulators of COOH-terminal Src kinase (Csk)/Csk-like protein-tyrosine kinase (Ctk) after Lyn is activated and bound to Cbp. Lyn phosphorylates Cbp on multiple tyrosine residues, including two that can bind Lyn's SH2 domain with high affinity. Lyn also phosphorylates Y314, which recruits Csk/Ctk to phosphorylate Lyn at its Y508 negative site, allowing an inactive conformation to form. However, the pY508 site has a low affinity for Lyn's SH2 domain, while the Cbp sites have high affinity. Thus, until these Cbp sites are dephosphorylated, Lyn can remain active. Intriguingly, phosphorylated Y314 also binds the suppressor of cytokine signalling 1 (SOCS1), resulting in elevated ubiquitination and degradation of Lyn. Thus, a single phosphotyrosine residue within Cbp co-ordinates a two-phase process involving distinct negative regulatory pathways that allow inactivation, followed by degradation, of SFKs.

The use of whole exome sequencing and murine patient derived xenografts as a method of chemosensitivity testing in sarcoma.

BACKGROUND: Soft tissue and bone sarcoma represent a broad spectrum of different pathology and genetic variance. Current chemotherapy regimens are derived from randomised trials and represent empirical treatment. Chemosensitivity testing and whole exome sequencing (WES) may offer personalized chemotherapy treatment based on genetic mutations. METHODS: A pilot, prospective, non-randomised control experimental study was conducted. Twelve patients with metastatic bone or soft tissue sarcoma that had failed first line chemotherapy treatment were enrolled for this study. Human tissue taken at surgical biopsy under general anaesthetic was divided between two arms of the trial. Subsections of the tumour were used for WES and the remainder was implanted subcutaneously in immunodeficient mice (PDX). Results of WES were analysed using a bioinformatics pipeline to identify mutations conferring susceptibility to kinase inhibitors and common chemotherapeutic agents. PDX models exhibiting successful growth underwent WES of the tumour and subsequent chemosensitivity testing. RESULTS: WES was successful in all 12 patients, with successful establishment PDX tumours models in seven patients. WES identified potential actionable therapeutics in all patients. Significant variation in predicted therapeutics was demonstrated between three PDX samples and their matched tumour samples. CONCLUSION: Analysis of WES of fresh tumour specimens via a bioinformatics pipeline may identify potential actionable chemotherapy agents. Further research into this field may lead to the development of personalized cancer therapy for sarcoma.

Author Correction: Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel.

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