Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.     

A complex genomic rearrangement involving the endothelin 3 locus causes dermal hyperpigmentation in the chicken

Dermal hyperpigmentation or Fibromelanosis (FM) is one of the few examples of skin pigmentation phenotypes in the chicken, where most other pigmentation variants influence feather color and patterning. The Silkie chicken is the most widespread and well-studied breed displaying this phenotype. The presence of the dominant FM allele results in extensive pigmentation of the dermal layer of skin and the majority of internal connective tissue. Here we identify the causal mutation of FM as an inverted duplication and junction of two genomic regions separated by more than 400 kb in wild-type individuals. One of these duplicated regions contains endothelin 3 (EDN3), a gene with a known role in promoting melanoblast proliferation. We show that EDN3 expression is increased in the developing Silkie embryo during the time in which melanoblasts are migrating, and elevated levels of expression are maintained in the adult skin tissue. We have examined four different chicken breeds from both Asia and Europe displaying dermal hyperpigmentation and conclude that the same structural variant underlies this phenotype in all chicken breeds. This complex genomic rearrangement causing a specific monogenic trait in the chicken illustrates how novel mutations with major phenotypic effects have been reused during breed formation in domestic animals.     

A retrospective analysis of the haemodynamic and metabolic effects of fluid resuscitation in Vietnamese adults with severe falciparum malaria

Background

Optimising the fluid resuscitation of patients with severe malaria is a simple and potentially cost-effective intervention. Current WHO guidelines recommend central venous pressure (CVP) guided, crystalloid based, resuscitation in adults.

Methods

Prospectively collected haemodynamic data from intervention trials in Vietnamese adults with severe malaria were analysed retrospectively to assess the responses to fluid resuscitation.

Results

43 patients were studied of whom 24 received a fluid load. The fluid load resulted in an increase in cardiac index (mean increase: 0.75 L/min/m² (95% Confidence interval (CI): 0.41 to 1.1)), but no significant change in acid-base status post resuscitation (mean increase base deficit 0.6 mmol/L (95% CI: −0.1 to 1.3). The CVP and PAoP (pulmonary artery occlusion pressure) were highly inter-correlated (r_s = 0.7, p<0.0001), but neither were correlated with acid-base status (arterial pH, serum bicarbonate, base deficit) or respiratory status (PaO₂/FiO₂ ratio). There was no correlation between the oxygen delivery (DO₂) and base deficit at the 63 time-points where they were assessed simultaneously (r_s = −0.09, p = 0.46).

Conclusions

In adults with severe falciparum malaria there was no observed improvement in patient outcomes or acid-base status with fluid loading. Neither CVP nor PAoP correlated with markers of end-organ perfusion or respiratory status, suggesting these measures are poor predictors of their fluid resuscitation needs.     

Adenylate kinase 3 sensitizes cells to cigarette smoke condensate vapor induced cisplatin resistance

Background

The major established etiologic risk factor for bladder cancer is cigarette smoking and one of the major antineoplastic agents used for the treatment of advanced bladder cancer is cisplatin. A number of reports have suggested that cancer patients who smoke while receiving treatment have lower rates of response and decreased efficacy of cancer therapies.

Methodology/Principal Findings

In this study, we investigated the effect of cigarette smoke condensate (CSC) vapor on cisplatin toxicity in urothelial cell lines SV-HUC-1 and SCaBER cells. We showed that chronic exposure to CSC vapor induced cisplatin resistance in both cell lines. In addition, we found that the expression of mitochondrial-resident protein adenylate kinase-3 (AK3) is decreased by CSC vapor. We further observed that chronic CSC vapor-exposed cells displayed decreased cellular sensitivity to cisplatin, decreased mitochondrial membrane potential (ΔΨm) and increased basal cellular ROS levels compared to unexposed cells. Re-expression of AK3 in CSC vapor-exposed cells restored cellular sensitivity to cisplatin. Finally, CSC vapor increased the growth of the tumors and also curtail the response of tumor cells to cisplatin chemotherapy in vivo.

Conclusions/Significance

The current study provides evidence that chronic CSC vapor exposure affects AK3 expression and renders the cells resistant to cisplatin.     

A novel isoform of glucan, water dikinase phosphorylates pre-phosphorylated alpha-glucans and is involved in starch degradation in Arabidopsis

An Arabidopsis thaliana gene encoding a homologue of the potato alpha-glucan, water dikinase GWD, previously known as R1, was identified by screening the Arabidopsis genome and named AtGWD3. The AtGWD3 cDNA was isolated, heterologously expressed and the protein was purified to apparent homogeneity to determine the enzymatic function. In contrast to the potato GWD protein, the AtGWD3 primarily catalysed phosphorylation at the C-3 position of the glucose unit of preferably pre-phosphorylated amylopectin substrate with long side chains. An Arabidopsis mutant, termed Atgwd3, with downregulated expression of the AtGWD3 gene was analysed. In Atgwd3 the amount of leaf starch was constantly higher than wild type during the diurnal cycle. Compared with wild-type leaf starch, the level of C-3 phosphorylation of the glucosyl moiety of starch in this mutant was reduced. Taken together, these data indicate that the C-3 linked phospho-ester in starch plays a so far unnoticed specific role in the degradation of transitory starch.     

Infection of nonhuman primates with recombinant human metapneumovirus lacking the SH, G, or M2-2 protein categorizes each as a nonessential accessory protein and identifies vaccine candidates

Recombinant human metapneumovirus (HMPV) in which the SH, G, or M2 gene or open reading frame was deleted by reverse genetics was evaluated for replication and vaccine efficacy following topical administration to the respiratory tract of African green monkeys, a permissive primate host. Replication of the deltaSH virus was only marginally less efficient than that of wild-type HMPV, whereas the deltaG and deltaM2-2 viruses were reduced sixfold and 160-fold in the upper respiratory tract and 3,200-fold and 4,000-fold in the lower respiratory tract, respectively. Even with the highly attenuated mutants, there was unequivocal HMPV replication at each anatomical site in each animal. Thus, none of these three proteins is essential for HMPV replication in a primate host, although G and M2-2 increased the efficiency of replication. Each gene-deletion virus was highly immunogenic and protective against wild-type HMPV challenge. The deltaG and deltaM2-2 viruses are promising vaccine candidates that are based on independent mechanisms of attenuation and are appropriate for clinical evaluation.