Endogenous myoglobin in human breast cancer is a hallmark of luminal cancer phenotype.

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-alpha (ERalpha)/progesteron receptor (PR)/HER2, fatty acid synthase (FASN), hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, glucose transporter 1 (GLUT1) and carbonic anhydrase IX (CAIX). The spatial relationship of Mb and ERalpha 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-2alpha and CAIX, but not to HIF-1alpha or GLUT1, suggesting hypoxia to participate in its regulation. Double immunofluorescence showed a cellular co-expression of ERalpha 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 ERalpha 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.

Hypometabolism as the ultimate defence in stress response: how the comparative approach helps understanding of medically relevant questions.

First conceptualized from breath-hold diving mammals, later recognized as the ultimate cell autonomous survival strategy in anoxia-tolerant vertebrates and burrowing or hibernating rodents, hypometabolism is typically recruited by resilient organisms to withstand and recover from otherwise life-threatening hazards. Through the coordinated down-regulation of biosynthetic, proliferative and electrogenic expenditures at times when little ATP can be generated, a metabolism turned 'down to the pilot light' allows the re-balancing of energy demand with supply at a greatly suppressed level in response to noxious exogenous stimuli or seasonal endogenous cues. A unifying hallmark of stress-tolerant organisms, the adaptation effectively prevents lethal depletion of ATP, thus delineating a marked contrast with susceptible species. Along with disengaged macromolecular syntheses, attenuated transmembrane ion shuttling and PO2 -conforming respiration rates, the metabolic slowdown in tolerant species usually culminates in a non-cycling, quiescent phenotype. However, such a reprogramming also occurs in leading human pathophysiologies. Ranging from microbial infections through ischaemia-driven infarcts to solid malignancies, cells involved in these disorders may again invoke hypometabolism to endure conditions non-permissive for growth. At the same time, their reduced activities underlie the frequent development of a general resistance to therapeutic interventions. On the other hand, a controlled induction of hypometabolic and/or hypothermic states by pharmacological means has recently stimulated intense research aimed at improved organ preservation and patient survival in situations requiring acutely administered critical care. The current review article therefore presents an up-to-date survey of concepts and applications of a coordinated and reversibly down-regulated metabolic rate as the ultimate defence in stress responses.

Endogenous α-calcitonin-gene-related peptide promotes exercise-induced, physiological heart hypertrophy in mice.

AIM: It is unknown how the heart distinguishes various overloads, such as exercise or hypertension, causing either physiological or pathological hypertrophy. We hypothesize that alpha-calcitonin-gene-related peptide (αCGRP), known to be released from contracting skeletal muscles, is key at this remodelling. METHODS: The hypertrophic effect of αCGRP was measured in vitro (cultured cardiac myocytes) and in vivo (magnetic resonance imaging) in mice. Exercise performance was assessed by determination of maximum oxygen consumption and time to exhaustion. Cardiac phenotype was defined by transcriptional analysis, cardiac histology and morphometry. Finally, we measured spontaneous activity, body fat content, blood volume, haemoglobin mass and skeletal muscle capillarization and fibre composition. RESULTS: While αCGRP exposure yielded larger cultured cardiac myocytes, exercise-induced heart hypertrophy was completely abrogated by treatment with the peptide antagonist CGRP(8-37). Exercise performance was attenuated in αCGRP(-/-) mice or CGRP(8-37) treated wild-type mice but improved in animals with higher density of cardiac CGRP receptors (CLR-tg). Spontaneous activity, body fat content, blood volume, haemoglobin mass, muscle capillarization and fibre composition were unaffected, whereas heart index and ventricular myocyte volume were reduced in αCGRP(-/-) mice and elevated in CLR-tg. Transcriptional changes seen in αCGRP(-/-) (but not CLR-tg) hearts resembled maladaptive cardiac phenotype. CONCLUSIONS: Alpha-calcitonin-gene-related peptide released by skeletal muscles during exercise is a hitherto unrecognized effector directing the strained heart into physiological instead of pathological adaptation. Thus, αCGRP agonists might be beneficial in heart failure patients.

Old proteins - new locations: myoglobin, haemoglobin, neuroglobin and cytoglobin in solid tumours and cancer cells.

AIM: The unexpected identification of myoglobin (MB) in breast cancer prompted us to evaluate the clinico-pathological value of MB, haemoglobin (HB) and cytoglobin (CYGB) in human breast carcinoma cases. We further screened for the presence of neuroglobin (NGB) and CYGB in tumours of diverse origin, and assessed the O(2) -response of HB, MB and CYGB mRNAs in cancer cell lines, to better elicit the links between this ectopic globin expression and tumour hypoxia. METHODS: Breast tumours were analysed by immunohistochemistry for HB, MB and CYGB and correlated with clinico-pathological parameters. Screening for CYGB and NGB mRNA expression in tumour entities was performed by hybridization, quantitative PCR (qPCR) and bioinformatics. Hypoxic or anoxic responses of HB, MB and CYGB mRNAs was analysed by qPCR in human Hep3B, MCF7, HeLa and RCC4 cancer cell lines. RESULTS: 78.8% of breast cancer cases were positive for MB, 77.9% were positive for HB and 55.4% expressed CYGB. The closest correlation with markers of hypoxia was observed for CYGB. Compared to the weakly positive status of MB in healthy breast tissues, invasive tumours either lost or up-regulated MB. Breast carcinomas showed the tendency to silence CYGB. HB was not seen in normal tissues and up-regulated in tumours. Beyond breast malignancies, expression levels of NGB and CYGB mRNAs were extremely low in brain tumours (glioblastoma, astrocytoma). NGB was not observed in non-brain tumours. CYGB mRNA, readily detectable in breast cancer and other tumours, is down-regulated in lung adenocarcinomas. Alpha1 globin (α1 globin) and Mb were co-expressed in MCF7 and HeLa cells; CYGB transcription was anoxia-inducible in Hep3B and RCC4 cells. CONCLUSIONS: This is the first time that HB and CYGB are reported in breast cancer. Neither NGB nor CYGB are systematically up-regulated in tumours. The down-regulated CYGB expression in breast and lung tumours is in line with a tumour-suppressor role. Each of the screened cancer cells expresses at least one globin (i.e. main globin species: CYGB in Hep3B; α1 globin + MB in MCF7 and HeLa). Thus, globins exist in a wide variety of solid tumours. However, the generally weak expression of the endogenous proteins in the cancer argues against a significant contribution to tumour oxygenation. Future studies should consider that cancer-expressed globins might function in ways not directly linked to the binding and transport of oxygen.

Hypoxia tolerance in animals: biology and application.

Many invertebrates and ectothermic vertebrates successfully cope with a fluctuating supply of ambient oxygen-and consequently, a highly variable tissue oxygenation-through increasing their antioxidant barriers. During chronic deprivation of oxygen, however, the hypometabolic defense mode of the fruit fly Drosophila, the hypoxia-induced behavioral hypothermia of the crayfish Pacifastacus leniusculus, and the production of ethanol during anoxia by the crucian carp Carassius carassius all indicate that these animals are also capable of utilizing a suite of genetic and physiological defenses to survive otherwise lethal reductions in tissue oxygenation. Normally, much of an organism's gene response to hypoxia is orchestrated via the hypoxia-inducible transcription factor HIF. Recent developments expand our view of HIF function even further by highlighting regulatory roles for HIF in the hypometabolism of insects, in the molting and the normoxic immune response of crustaceans, and in the control-via the downstream effector gene erythropoietin-of the hypoxic ventilatory response and pulmonary hypertension in mammals. These and related topics were collectively presented by the authors in a symposium of the 2008 ICA-CBP conference at Mara National Reserve, Kenya, Africa. This synthesis article communicates the essence of the symposium presentations to the wider community.

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Close tetrapod relationships of the coelacanth Latimeria indicated by haemoglobin sequences.

The origin of tetrapods has been debated for many years. In traditional systematics, the extinct lobe-finned bony fish (Rhipidistia) are regarded as the closest relatives of tetrapods. Among living fish, the coelacanth Latimeria chalumnae (Actinistia), which is the only recent representative of the Crossopterygii (Actinistia and Rhipidistia), the lungfish (Dipnoi) and ray-finned fish (Actinopterygii), have each been considered as sister-groups of the tetrapods. We have now determined the sequence of the alpha- and beta-globin chains of coelacanth haemoglobin and compared them with all known haemoglobins of bony and cartilaginous fish as well as those of tadpoles and adult amphibians. Haemoglobins of bony fish match more closely those of larval than adult amphibians. The beta chains of Latimeria match those of tadpoles more closely (54%) than do those of any other fish, whereas the alpha chains of Latimeria (45.4%), and especially of teleosts (49.2%), are closer to those of larval amphibians than are those of lungfish (39.8%). If only synapomorphous sequence matches (those at derived positions shared by one bony fish and tadpoles but not by any other bony fish) are considered, both Latimeria globin chains have distinctly more identities with phase of tadpoles than do those of any bony fish. Thus the primary structure of Latimeria haemoglobin indicates that the coelacanth is the closest living relative of tetrapods.

Phylogenetic analysis of reptilian hemoglobins: trees, rates, and divergences.

Phylogenetic relationships among reptiles were examined using previously published and newly determined hemoglobin sequences. Trees reconstructed from these sequences using maximum-parsimony, neighbor-joining, and maximum-likelihood algorithms were compared with a phylogenetic tree of Amniota, which was assembled on the basis of published morphological data. All analyses differentiated alpha chains into alphaA and alphaD types, which are present in all reptiles except crocodiles, where only alphaA chains are expressed. The occurrence of the alphaD chain in squamates (lizards and snakes only in this study) appears to be a general characteristic of these species. Lizards and snakes also express two types of beta chains (betaI and betaII), while only one type of beta chain is present in birds and crocodiles. Reconstructed hemoglobin trees for both alpha and beta sequences did not yield the monophyletic Archosauria (i.e., crocodilians + birds) and Lepidosauria (i.e., Sphenodon + squamates) groups defined by the morphology tree. This discrepancy, as well as some other poorly resolved nodes, might be due to substantial heterogeneity in evolutionary rates among single hemoglobin lineages. Estimation of branch lengths based on uncorrected amino acid substitutions and on distances corrected for multiple substitutions (PAM distances) revealed that relative rates for squamate alphaA and alphaD chains and crocodilian beta chains are at least twice as high as those of the rest of the chains considered. In contrast to these rate inequalities between reptilian orders, little variation was found within squamates, which allowed determination of absolute evolutionary rates for this subset of hemoglobins. Rate estimates for hemoglobins of lizards and snakes yielded 1.7 (alphaA) and 3.3 (beta) million years/PAM when calibrated with published divergence time vs. PAM distance correlates for several speciation events within snakes and for the squamate left and right arrow sphenodontid split. This suggests that hemoglobin chains of squamate reptiles evolved approximately 3.5 (alphaA) or approximately 1.7 times (beta) faster than their mammalian equivalents. These data also were used to obtain a first estimate of some intrasquamate divergence times.

Molecular analysis of ependymins from the cerebrospinal fluid of the orders Clupeiformes and Salmoniformes: no indication for the existence of an euteleost infradivision.

Ependymins represent the predominant protein constituents in the cerebrospinal fluid of many teleost fish and they are synthesized in meningeal fibroblasts. Here, we present the ependymin sequences from the herring (Clupea harengus) and the pike (Esox lucius). A comparison of ependymin homologous sequences from three different orders of teleost fish (Salmoniformes, Cypriniformes, and Clupeiformes) revealed the highest similarity between Clupeiformes and Cypriniformes. This result is unexpected because it does not reflect current systematics, in which Clupeiformes belong to a separate infradivision (Clupeomorpha) than Salmoniformes and Cypriniformes (Euteleostei). Furthermore, in Salmoniformes the evolutionary rate of ependymins seems to be accelerated mainly on the protein level. However, considering these inconstant rates, neither neighbor-joining trees nor DNA parsimony methods gave any indication that a separate euteleost infradivision exists.

A "living fossil" sequence: primary structure of the coelacanth (Latimeria chalumnae) hemoglobin--evolutionary and functional aspects.

The coelacanth (Latimeria chalumnae, Actinistia) has a single hemoglobin component. The primary structures of the alpha- and beta-chains are presented. They could be separated by reversed-phase HPLC. Peptides obtained by tryptic digestion of the native and oxidized chains were isolated by reversed-phase HPLC and sequenced in liquid and gas-phase sequenators. The alignment was achieved by employing the N-terminal sequences of the native chains and those of a beta-chain cyanogen bromide peptide as well as fragments obtained by acid hydrolysis. The Latimeria alpha-chains consist of 142 amino-acid residues, due to a fish-specific insertion between positions 46 and 47, whereas the beta-chains are of normal length (146 residues). Latimeria alpha- and beta-chains share 72 (51.1%) and 70 (47.9%) identical residues with human hemoglobin, respectively. Numerous heme contacts and positions involved in subunit interface contacts are replaced. The most interesting of them were studied by molecular modeling. The loss of an alpha 1/beta 2-contact by the exchanges alpha 92(FG4)Arg----Leu and beta 43(CD2)Glu----Lys might be responsible for the easy dissociation of the tetrameric hemoglobin molecule. A comparison of the residues replaced in contact positions with fishes and amphibians revealed the highest number of matches between Latimeria and tadpoles. The same result was obtained by the evaluation of other regions relevant for structure and function of the molecule, like exon-intron boundary regions, phosphate binding sites and salt bridges responsible for the Bohr effect.

Primary structure and oxygen-binding properties of the hemoglobin from the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta). Evidence for phylogenetic isolation.

The primary structures of the alpha- and beta-hemoglobin chains of the lesser hedgehog tenrec (Echinops telfairi, Zalambdodonta) are presented. Chain separation was performed by carboxymethyl-cellulose chromatography. The peptides, obtained by tryptic digestion of the oxidized chains, were prefractionated by gel chromatography and isolated by reversed-phase HPLC. For sequence analysis gas and liquid phase sequencers were employed. The tenrec hemoglobin consists of one alpha- and two beta-chains the latter occurring in a 1:1 ratio and differing in beta 16 Gly/Cys and beta 118 Phe/Leu. Two external cysteine residues at beta 16 and beta 52 cause reversible polymerization to octamers and most likely irreversible formation of higher polymers. A comparison of the whole chains and certain positions of tenrec hemoglobin with those of Insectivora sensu strictu, Scandentia and Proto- and Metatheria corroborates a long and independent evolution of tenrec and its phylogenetic isolation from the Insectivora s.str. (hedgehog, musk shrew and mole). Replacements at positions involved in heme and subunit interface contacts are discussed. Compared to human hemoglobin the tenrec pigment shows a low intrinsic oxygen affinity as well as lower chloride and temperature sensitivities, a reduced Bohr effect and a strong response to 2,3-DPG. The possible adaptive significance of these properties is discussed in relation to the large diurnal body temperature variations seen in tenrecs.

The mini-hemoglobins in neural and body wall tissue of the nemertean worm, Cerebratulus lacteus.

Hemoglobin (Hb) occurs in circulating red blood cells, neural tissue, and body wall muscle tissue of the nemertean worm, Cerebratulus lacteus. The neural and body wall tissue each express single major Hb components for which the amino acid sequences have been deduced from cDNA and genomic DNA. These 109-residue globins form the smallest stable Hbs known. The globin genes have three exons and two introns with splice sites in the highly conserved positions of most globin genes. Alignment of the sequences with those of other globins indicates that the A, B, and H helices are about one-half the typical length. Phylogenetic analysis indicates that shortening results in a small tendency of globins to group together regardless of their actual relationships. The neural and body wall Hbs in situ are half-saturated with O2 at 2.9 and 4.1 torr, respectively. The Hill coefficient for the neural Hb in situ, approximately 2.9, suggests that the neural Hb self-associates in the deoxy state at least to tetramers at the 2-3 mM (heme) concentration estimated in the cells. The Hb must dissociate upon oxygenation and dilution because the weight-average molecular mass of the HbO2 in vitro is only about 18 kDa at 2-3 microM heme concentration. Calculations suggest that the Hb can function as an O2 store capable of extending neuronal activity in an anoxic environment for 5-30 min.