BACKGROUND: We aimed to clarify the incidence and the clinicopathological value of non-muscle myoglobin (Mb) in a large cohort of non-invasive and invasive breast cancer cases. METHODS: Matched pairs of breast tissues from 10 patients plus 17 breast cell lines were screened by quantitative PCR for Mb mRNA. In addition, 917 invasive and 155 non-invasive breast cancer cases were analysed by immunohistochemistry for Mb expression and correlated to clinicopathological parameters and basal molecular characteristics including oestrogen receptor-a (ERa)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1a (HIF-1a), HIF-2a, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERa or FASN was followed up by double immunofluorescence. Finally, the effects of estradiol treatment and FASN inhibition on Mb expression in breast cancer cells were analysed. RESULTS: Myoglobin mRNA was found in a subset of breast cancer cell lines; in microdissected tumours Mb transcript was markedly upregulated. In all, 71% of tumours displayed Mb protein expression in significant correlation with a positive hormone receptor status and better prognosis. In silico data mining confirmed higher Mb levels in luminal-type breast cancer. Myoglobin was also correlated to FASN, HIF-2a and CAIX, but not to HIF-1a or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERa or FASN and Mb. In addition, Mb levels were modulated on estradiol treatment and FASN inhibition in a cell model. CONCLUSION: We conclude that in breast cancer, Mb is co-expressed with ERa and co-regulated by oestrogen signalling and can be considered a hallmark of luminal breast cancer phenotype. This and its possible new role in fatty acid metabolism may have fundamental implications for our understanding of Mb in solid tumours.

Background:The role of MB in tumors cells is yet unclear. Results: MB is induced by hypoxia in breast cancer cell lines, possibly by an alternative transcription start site. Knockdown of MB in breast cancer cells is functionally relevant and significantly alters cellular respiration. Conclusion: MB might impair mitochondria in hypoxic cancer cells. Significance: MB might have tumor-suppressive functions, not described so far.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) has been associated with tumour suppression in various cancers. However, its putative role in bladder cancer is completely unknown. Therefore, we initiated a study analysing ITIH5 expression as well as its prognostic and functional impact on human urothelial cancers (UCs). Expression analysis showed a clear down-regulation of ITIH5 mRNA in 61% (n = 45) of UCs, especially in muscle-invasive tumours (P < 0.001). ITIH5 loss in UCs was further evident on protein level (65.5%, n = 55) as detected by immunohistochemistry. DNA methylation analysis demonstrated tumour-specific ITIH5 promoter methylation in 50% of papillary none-invasive pTa (n = 30) and 68% of invasive (n = 28) UCs. Aberrant ITIH5 promoter methylation in bladder tumours was tightly linked (P < 0.001) with loss of ITIH5 mRNA expression, which was furthermore functionally confirmed by demethylation analysis in cell lines. Pyrosequencing analysis revealed that ITIH5 promoter hypermethylation was closely associated with progressive bladder cancers. Subsequently, a large cohort (n = 120) of clinically challenging pT1 high-grade UC was analysed for ITIH5 expression. Of clinical significance, we found an association between loss of ITIH5 expression and unfavourable prognosis of UC patients without distant metastasis at first diagnosis (recurrence-free survival; hazard ratio: 4.35, P = 0.048). Functionally, ITIH5 re-expression in human RT112 bladder cancer cells led to both suppression of cell migration and inhibition of colony spreading. Hence, we provide evidence that down-regulation of ITIH5 by aberrant DNA hypermethylation may provoke invasive phenotypes in human bladder cancer. Moreover, ITIH5 protein might become a prognostic biomarker for relapse risk stratification in high-grade UC patients.

Although the release of nitric oxide (NO) from biomaterials has been shown to reduce the foreign body response (FBR), the optimal NO release kinetics and doses remain unknown. Herein, polyurethane-coated wire substrates with varying NO release properties were implanted into porcine subcutaneous tissue for 3, 7, 21 and 42 d. Histological analysis revealed that materials with short NO release durations (i.e., 24 h) were insufficient to reduce the collagen capsule thickness at 3 and 6 weeks, whereas implants with longer release durations (i.e., 3 and 14 d) and greater NO payloads significantly reduced the collagen encapsulation at both 3 and 6 weeks. The acute inflammatory response was mitigated most notably by systems with the longest duration and greatest dose of NO release, supporting the notion that these properties are most critical in circumventing the FBR for subcutaneous biomedical applications (e.g., glucose sensors).

Myosin is an actin-based motor protein that generates force by cycling between actin-attached (strong binding: ADP or rigor) and actin-detached (weak binding: ATP or ADP⅐P i ) states during its ATPase cycle. However, it remains unclear what specific conformational changes in the actin binding site take place on binding to actin, and how these structural changes lead to product release and the production of force and motion. We studied the dynamics of the actin binding region of myosin V by using fluorescence resonance energy transfer ( actin ͉ energy transfer ͉ motor proteins ͉ myosin ͉ structural dynamics A current challenge in biophysics is to understand the mechanism of how motor proteins such as myosin convert chemical energy into mechanical work through a cyclic interaction with actin filaments. Numerous structure/function studies have converged on a structural model for force generation, where conformational changes in the active site are coupled to a large rotation of the light-chain binding region, also known as the lever arm hypothesis (1, 2). Myosin alters its affinity for actin in a nucleotide-dependent manner, from strong actin binding states (ADP or rigor state) to weak actin binding states (ATP or ADP⅐P i state), and thus phosphate release is believed to be associated with a large increase in actin affinity. Based on the high-resolution x-ray structure of myosin II (3) it was predicted that the actin binding cleft may open during ATP-induced dissociation of actomyosin and close during the release of the hydrolysis products induced by actin binding. The crystal structure of myosin V in the absence of nucleotide (rigor) demonstrated a closed conformation of the actin binding cleft (4, 5), and this structure fit quite well into the electron microscopy image reconstructions of the actomyosin rigor complex (6). Further studies of myosin II (7) and myosin V (8) by electron microscopy image reconstruction have demonstrated conformational changes in the actin binding cleft in different nucleotide states. Recently, the closed-cleft conformation was also observed in crystallographic studies of molluscan myosin II, wherein a ''counterclockwise'' orientation of the cleft rather than the extent of its closure was proposed to be critical for forming the strong binding rigor conformation (9).By placing fluorescent probes in the actin binding cleft it was directly demonstrated that the cleft opens during ATP-induced dissociation of actomyosin (10, 11). However, there is currently no direct evidence that describes the kinetics of actin binding cleft closure in relationship to actin-activated phosphate release. In addition, there is a lack of information about how conformational changes in the cleft are coupled to structural changes in the nucleotide binding region. Most models of the actomyosin cross-bridge cycle suggest that myosin binds to actin through both ionic and hydrophobic interactions that stabilize a structural change in the actin binding region, such as closure of the actin binding cleft (1, 2). This st...

BackgroundExtracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.Methods ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.Results ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.ConclusionsOur results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0610-2) contains supplementary material, which is available to authorized users.

Blood-based early detection of breast cancer has recently gained novel momentum, as liquid biopsy diagnostics is a fast emerging field. In this study, we aimed to identify secreted proteins which are up-regulated both in tumour tissue and serum samples of breast cancer patients compared to normal tissue and sera. Based on two independent tissue cohorts (n = 75 and n = 229) and one serum cohort (n = 80) of human breast cancer and healthy serum samples, we characterised AGR3 as a novel potential biomarker both for breast cancer prognosis and early breast cancer detection from blood. AGR3 expression in breast tumours is significantly associated with oestrogen receptor α (P<0.001) and lower tumour grade (P<0.01). Interestingly, AGR3 protein expression correlates with unfavourable outcome in low (G1) and intermediate (G2) grade breast tumours (multivariate hazard ratio: 2.186, 95% CI: 1.008-4.740, P<0.05) indicating an independent prognostic impact. In sera analysed by ELISA technique, AGR3 protein concentration was significantly (P<0.001) elevated in samples from breast cancer patients (n = 40, mainly low stage tumours) compared to healthy controls (n = 40). To develop a suitable biomarker panel for early breast cancer detection, we measured AGR2 protein in human serum samples in parallel. The combined AGR3/AGR2 biomarker panel achieved a sensitivity of 64.5% and a specificity of 89.5% as shown by receiver operating characteristic (ROC) curve statistics. Thus our data clearly show the potential usability of AGR3 and AGR2 as biomarkers for blood-based early detection of human breast cancer.

Glenohumeral internal rotation deficit (GIRD) is an adaptive process in which the throwing shoulder experiences a loss of internal rotation (IR). GIRD has most commonly been defined by a loss of >20° of IR compared to the contralateral shoulder. Total rotational motion of the shoulder is the sum of internal and external rotation and may be more important than the absolute value of IR loss. Pathologic GIRD has been defined as a loss of IR combined with a loss of total rotational motion. The leading pathologic process in GIRD is posterior capsular and rotator-cuff tightness, due to the repetitive cocking that occurs with the overhead throwing motion. GIRD has been associated with numerous pathologic conditions, including posterior superior labral tears, partial articular-sided rotator-cuff tears, and superior labral anterior-to-posterior tears. The mainstay of treatment for patients with GIRD is posterior capsular stretching and strengthening to improve scapular mechanics. In patients who fail nonoperative therapy, shoulder arthroscopy can be performed. Arthroscopic surgery in the high-level throwing athlete should be to restore them to their functional baseline with the minimum amount of intervention possible.