Sidse Ehmsen

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans.

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.

S100A14 is a novel independent prognostic biomarker in the triple-negative breast cancer subtype.

Triple‐negative breast cancer (TNBC) represents a heterogeneous subgroup with generally poor outcome and lack of an effective targeted therapy. Prognostic or predictive biomarkers to guide treatment decisions for this group of patients are needed. To evaluate the potential of S100A14 protein as a novel biomarker in TNBC, the protein expression of S100A14 was correlated with clinical outcomes in a Pilot Sample set and a Danish cohort of predominantly TNBC patients. Kaplan‐Meier analysis identified a prognostic impact of S100A14 on disease‐free survival and overall survival, showing that tumors with high S100A14 protein expression levels were significantly correlated with poor outcome in TNBC patients (p = 0.017; p = 0.038), particularly those in the basal‐like subgroup (p = 0.006; p = 0.037). Importantly, TNBC patients with high S100A14 expression, but tumor‐negative axillary lymph nodes (N−), had equally poor outcomes as those with tumor‐positive axillary lymph nodes (N+), while TNBC/N− patients with low S100A14 expression had a significantly better disease free survival (p = 0.013). Multivariate analysis revealed that S100A14 is an independent prognostic factor for TNBC patients (p = 0.024; p = 0.05). At the cellular level, S100A14 was found to be expressed in epithelial‐like, but not in mesenchymal‐like, TNBC cells in vitro. S100A14 is an independent prognostic factor in TNBC and a novel potential therapeutic target in TNBC.

Elucidation of altered pathways in tumor-initiating cells of triple-negative breast cancer: A useful cell model system for drug screening

A limited number of cancer cells within a tumor are thought to have self‐renewing and tumor‐initiating capabilities that produce the remaining cancer cells in a heterogeneous tumor mass. Elucidation of central pathways preferentially used by tumor‐initiating cells/cancer stem cells (CSCs) may allow their exploitation as potential cancer therapy targets. We used single cell cloning to isolate and characterize four isogenic cell clones from a triple‐negative breast cancer cell line; two exhibited mesenchymal‐like and two epithelial‐like characteristics. Within these pairs, one, but not the other, resulted in tumors in immunodeficient NOD/Shi‐scid/IL‐2 Rγ null mice and efficiently formed mammospheres. Quantitative proteomics and phosphoproteomics were used to map signaling pathways associated with the tumor‐initiating ability. Signaling associated with apoptosis was suppressed in tumor‐initiating versus nontumorigenic counterparts with pro‐apoptotic proteins, such as Bcl2‐associated agonist of cell death (BAD), FAS‐associated death domain protein (FADD), and myeloid differentiation primary response protein (MYD88), downregulated in tumor‐initiating epithelial‐like cells. Functional studies confirmed significantly lower apoptosis in tumor‐initiating versus nontumorigenic cells. Moreover, central pathways, including β‐catenin and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB)‐related signaling, exhibited increased activation in the tumor‐initiating cells. To evaluate the CSC model as a tool for drug screening, we assessed the effect of separately blocking NF‐κB and Wnt/β‐catenin signaling and found markedly reduced mammosphere formation, particularly for tumor‐initiating cells. Similar reduction was also observed using patient‐derived primary cancer cells. Furthermore, blocking NF‐κB signaling in mice transplanted with tumor‐initiating cells significantly reduced tumor outgrowth. Our study demonstrates that suppressed apoptosis, activation of pathways associated with cell viability, and CSCs are the major differences between tumor‐initiating and nontumorigenic cells independent of their epithelial‐like/mesenchymal‐like phenotype. These altered pathways may provide targets for future drug development to eliminate CSCs, and the cell model may be a useful tool in such drug screenings. Stem Cells 2017;35:1898–1912

Correlation between circulating cell‐free PIK3CA tumor DNA levels and treatment response in patients with PIK3CA‐mutated metastatic breast cancer

Liquid biopsies focusing on the analysis of cell‐free circulating tumor DNA (ctDNA) may have important clinical implications for personalized medicine, including early detection of cancer, therapeutic guidance, and monitoring of recurrence. Mutations in the oncogene, PIK3CA, are frequently observed in breast cancer and have been suggested as a predictive biomarker for PI3K‐selective inhibitor treatment. In this study, we analyzed the presence of PIK3CA mutations in formalin‐fixed, paraffin‐embedded, metastatic tissue and corresponding ctDNA from serum of patients with advanced breast cancer using a highly sensitive, optimized droplet digital PCR (ddPCR) assay. We found 83% of patients with PIK3CA mutation in the metastatic tumor tissue also had detectable PIK3CA mutations in serum ctDNA. Patients lacking the PIK3CA mutation in corresponding serum ctDNA all had nonvisceral metastatic disease. Four patients with detectable PIK3CA‐mutated ctDNA were followed with an additional serum sample during oncological treatment. In all cases, changes in PIK3CA ctDNA level correlated with treatment response. Our results showed high concordance between detection of PIK3CA mutations in tumor tissue and in corresponding serum ctDNA and suggest that serum samples from patients with advanced breast cancer and ddPCR may be used for PIK3CA mutation status assessment to complement imaging techniques as an early marker of treatment response.

CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients: CYPOR is a novel and independent prognostic biomarker of recurrence-free survival in triple-negative breast cancer patients

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple‐negative breast cancer (TNBC). In a mass spectrometry‐based global proteomic study of 44 formalin‐fixed, paraffin‐embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal‐like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence‐free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1–2.85), and this correlation was recapitulated in a cohort of 580 basal‐like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06–1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20–6.19), particularly those who were lymph node tumor‐negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07–4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance.