Lack of Genomic Heterogeneity at High-Resolution aCGH between Primary Breast Cancers and Their Paired Lymph Node Metastases

Lymph-node metastasis (LNM) predict high recurrence rates in breast cancer patients. Systemic treatment aims to eliminate (micro)metastatic cells. However decisions regarding systemic treatment depend largely on clinical and molecular characteristics of primary tumours. It remains, however, unclear to what extent metastases resemble the cognate primary breast tumours, especially on a genomic level, and as such will be eradicated by the systemic therapy chosen. In this study we used high-resolution aCGH to investigate DNA copy number differences between primary breast cancers and their paired LNMs. To date, no recurrent LNM-specific genomic aberrations have been identified using array comparative genomic hybridization (aCGH) analysis. In our study we employ a high-resolution platform and we stratify on different breast cancer subtypes, both aspects that might have underpowered previously performed studies.To test the possibility that genomic instability in triple-negative breast cancers (TNBCs) might cause increased random and potentially also recurrent copy number aberrations (CNAs) in their LNMs, we studied 10 primary TNBC–LNM pairs and 10 ER-positive (ER+) pairs and verified our findings adding additionally 5 TNBC-LNM and 22 ER+-LNM pairs. We found that all LNMs clustered nearest to their matched tumour except for two cases, of which one was due to the presence of two distinct histological components in one tumour. We found no significantly altered CNAs between tumour and their LNMs in the entire group or in the subgroups. Within the TNBC subgroup, no absolute increase in CNAs was found in the LNMs compared to their primary tumours, suggesting that increased genomic instability does not lead to more CNAs in LNMs. Our findings suggest a high clonal relationship between primary breast tumours and its LNMs, at least prior to treatment, and support the use of primary tumour characteristics to guide adjuvant systemic chemotherapy in breast cancer patients.     

Rethinking glycolysis: on the biochemical logic of metabolic pathways

Metabolic pathways may seem arbitrary and unnecessarily complex. In many cases, a chemist might devise a simpler route for the biochemical transformation, so why has nature chosen such complex solutions? In this review, we distill lessons from a century of metabolic research and introduce new observations suggesting that the intricate structure of metabolic pathways can be explained by a small set of biochemical principles. Using glycolysis as an example, we demonstrate how three key biochemical constraints--thermodynamic favorability, availability of enzymatic mechanisms and the physicochemical properties of pathway intermediates--eliminate otherwise plausible metabolic strategies. Considering these constraints, glycolysis contains no unnecessary steps and represents one of the very few pathway structures that meet cellular demands. The analysis presented here can be applied to metabolic engineering efforts for the rational design of pathways that produce a desired product while satisfying biochemical constraints.     

Is One Sputum Specimen as Good as Two during Follow-Up Cultures for Monitoring Multi Drug Resistant Tuberculosis Patients in India?

Background

In India, the Revised National Tuberculosis Control Programme (RNTCP) has adopted the strategy of examining two specimens during follow-up culture examinations to monitor the treatment response of multi-drug resistant tuberculosis (MDR-TB) patients.

Objectives

To determine the incremental yield of the second sputum specimen during follow-up culture examinations among patients with MDR-TB and the effect on case management on changing from two to one specimen follow-up strategy.

Methods

A cross sectional record review of MDR-TB patients registered during 2008–09 under RNTCP was undertaken in three MDR-TB treatment sites of India.

Results

Of 1721 pairs of follow-up sputum culture examinations done among 220 MDR-TB patients, 451(26%) were positive with either of the two specimens; 29(1.7%) were culture positive only on the second specimen indicating the incremental yield. To detect one additional culture positive result on the second specimen, 59 specimens needed to be processed. If we had examined only one specimen, we would have missed 29 culture-positive results. By current RNTCP guidelines, however, a single specimen policy would have altered case management in only 3(0.2%) instances, where patients would have missed a one month extension of the intensive phase of MDR-TB treatment. There is no meaningful advantage in using two specimens for the monitoring of MDR-TB patients. A single specimen policy could be safely implemented with negligible clinical effect on MDR-TB patients and favourable resource implications for RNTCP.     

Short Stature, Onychodysplasia, Facial Dysmorphism, and Hypotrichosis Syndrome Is Caused by a POC1A Mutation

Disproportionate short stature refers to a heterogeneous group of hereditary disorders that are classified according to their mode of inheritance, clinical skeletal and nonskeletal manifestations, and radiological characteristics. In the present study, we report on an autosomal-recessive osteocutaneous disorder that we termed SOFT (short stature, onychodysplasia, facial dysmorphism, and hypotrichosis) syndrome. We employed homozygosity mapping to locate the disease-causing mutation to region 3p21.1-3p21.31. Using whole-exome-sequencing analysis complemented with Sanger direct sequencing of poorly covered regions, we identified a homozygous point mutation (c.512T>C [p.Leu171Pro]) in POC1A (centriolar protein homolog A). This mutation was found to cosegregate with the disease phenotype in two families. The p.Leu171Pro substitution affects a highly conserved amino acid residue and is predicted to interfere with protein function. Poc1, a POC1A ortholog, was previously found to have a role in centrosome stability in unicellular organisms. Accordingly, although centrosome structure was preserved, the number of centrosomes and their distribution were abnormal in affected cells. In addition, the Golgi apparatus presented a dispersed morphology, cholera-toxin trafficking from the plasma membrane to the Golgi was aberrant, and large vesicles accumulated in the cytosol. Collectively, our data underscore the importance of POC1A for proper bone, hair, and nail formation and highlight the importance of normal centrosomes in Golgi assembly and trafficking from the plasma membrane to the Golgi apparatus.     

Thermodynamic constraints shape the structure of carbon fixation pathways

Thermodynamics impose a major constraint on the structure of metabolic pathways. Here, we use carbon fixation pathways to demonstrate how thermodynamics shape the structure of pathways and determine the cellular resources they consume. We analyze the energetic profile of prototypical reactions and show that each reaction type displays a characteristic change in Gibbs energy. Specifically, although carbon fixation pathways display a considerable structural variability, they are all energetically constrained by two types of reactions: carboxylation and carboxyl reduction. In fact, all adenosine triphosphate (ATP) molecules consumed by carbon fixation pathways - with a single exception - are used, directly or indirectly, to power one of these unfavorable reactions. When an indirect coupling is employed, the energy released by ATP hydrolysis is used to establish another chemical bond with high energy of hydrolysis, e.g. a thioester. This bond is cleaved by a downstream enzyme to energize an unfavorable reaction. Notably, many pathways exhibit reduced ATP requirement as they couple unfavorable carboxylation or carboxyl reduction reactions to exergonic reactions other than ATP hydrolysis. In the most extreme example, the reductive acetyl coenzyme A (acetyl-CoA) pathway bypasses almost all ATP-consuming reactions. On the other hand, the reductive pentose phosphate pathway appears to be the least ATP-efficient because it is the only carbon fixation pathway that invests ATP in metabolic aims other than carboxylation and carboxyl reduction. Altogether, our analysis indicates that basic thermodynamic considerations accurately predict the resource investment required to support a metabolic pathway and further identifies biochemical mechanisms that can decrease this requirement.     

HSP60 is transported through the secretory pathway of 3-MCA-induced fibrosarcoma tumour cells and undergoes N-glycosylation

There is increasing evidence localizes the mitochondrial chaperone heat shock protein (HSP)60, outside the cell, where it mediates interactions between immune cells and other body tissues. However, the mechanisms by which HSP60 is secreted into the extracellular environment are not fully understood. Recent studies have shown that HSP60 is actively released by a nonconventional secretion mechanism, the lipid raft-exosome pathway. In the present study, we show for the first time that HSP60, produced by 3-methylcholantrene-induced fibrosarcoma tumour cells, is secreted through the conventional endoplasmic reticulum-Golgi secretory pathway. Confocal microscopy using anti-TGN38 and anti-HSP60 antibodies together with monensin, a Golgi transport inhibitor, demonstrated the relocation of HSP60 to the Golgi of malignant cells but not primary fibroblast cells subjected to heat shock or fibroblast cell lines. Transmission electron microscopy, flow cytometry and cell fractionation of cell treated with brefeldin A, an inhibitor of endoplasmic reticulum to Golgi protein transport, further indicated that HSP60 is present both in the endoplasmic reticulum and the Golgi complex of malignant cells. We found a single mRNA with a mitochondrial targeting sequence encoding for HSP60 in the malignant cells but two HSP60 translation products, namely the native unmodified protein and a protein post-translationally modified by N-glycosylation. The N-glycans observed were composed of high-mannose structures and bi-, tri- and tetra-antennary complex type structures occupying sites of the three potential glycosylation sites present on HSP60. Accordingly, we propose that HSP60 in malignant cells is transported through the endoplasmic reticulum-Golgi secretion pathway, where it acquires N-glycans, and thus can affect the immunological properties of the proteins in the tumour microenvironment.     

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB₁R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB₁R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB₁R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB₁R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB₁R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB₁R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB₁R desensitization. Additionally, presence of DOR facilitates signaling via a new CB₁R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB₁R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.     

Dissecting the autocrine and paracrine roles of the CCR2-CCL2 axis in tumor survival and angiogenesis

The CCL2 CCR2 axis is likely to contributes to the development and progression of cancer diseases by two major mechanisms; autocrine effect of CCL2 as a survival/growth factor for CCR2+ cancer cells and, the attraction of CCR2+ CX₃CR1+tumor associated macrophages that in the absence of CCR2 hardly migrate. Thus far no in vivo system has been set up to differentiate the selective contribution of each of these features to cancer development. Here we employed a chimera animal model in which all non-malignant cells are CCR2−/−, but all cancer cells are CCR2+, combined with an adoptive transfer system of bone marrow (BM) CX₃CR1+ cells from CCR2+ mice harboring a targeted replacement of the CX₃CR1gene by an enhanced green fluorescent protein (EGFP) reporter gene (cx₃cr1 ^gfp), together with the CD45.1 congene. Using this system we dissected the selective contribution of CX₃CR1+CCR2+ cells, which comprise only about 7% of CD11b+ BM cells, to tumor development and angiogenesis. Showing that aside for their direct pro-angiogenic effect they are essential for the recruitment of other CD11b+ cells to the tumor site. We further show that the administration of CCR2-Ig, that selectively and specifically neutralize CCL2, to mice in which CCR2 is expressed only on tumor cells, further suppressed tumor development, implicating for the key role of this chemokine supporting tumor survival in an autocrine manner. This further emphasizes the important role of CCL2 as a target for therapy of cancer diseases.