Øystein Garred

Role of the Disulfide Bond in Shiga Toxin A-chain for Toxin Entry into Cells

Shiga toxin consists of an enzymatically active A-chain and a pentameric binding subunit. The A-chain has a trypsin-sensitive region, and upon cleavage two disulfide bonded fragments, A1 and A2, are generated. To study the role of the disulfide bond, it was eliminated by mutating cysteine 242 to serine. In T47D cells this mutated toxin was more toxic than wild type toxin after a short incubation, whereas after longer incubation times wild type toxin was most toxic. Cells cleaved not only wild type but also mutated A-chain into A1 and A2 fragments. The mutated A-chain was more sensitive than wild type toxin to Pronase, and it was degraded at a higher rate in T47D cells. Subcellular fractionation demonstrated transport of both wild type and mutated toxin to the Golgi apparatus. Brefeldin A, which disrupts the Golgi apparatus, protected not only against Shiga toxin but also against the mutated toxin, indicating involvement of the Golgi apparatus. After prebinding of Shiga(C242S) toxin to wells coated with the Shiga toxin receptor, Gb3, trypsin treatment induced dissociation of A1 from the toxin-receptor complex demonstrating that in addition to stabilizing the A-chain, the disulfide bond prevents dissociation of the A1 fragment from the toxin-receptor complex.

Reconstitution of Clathrin‐Independent Endocytosis at the Apical Domain of Permeabilized MDCK II cells: Requirement for a Rho‐Family GTPase

This paper studies the endocytosis of ricin at the apical pole of polarized MDCK II cells after permeabilization of the cells basolaterally with streptolysin O. Ricin endocytosis after the addition of cytosol with an ATP‐regenerating system was 2–3‐fold higher than after the addition of a transport medium. A similar increase in ricin endocytosis was obtained by reconstitution of dialyzed cytosol with the nonhydrolyzable GTP analog, GTPγS, in the presence of an ATP‐regenerating system. The nonhydrolyzable GDP analog, GDPβS, did not increase ricin uptake. In contrast to the data obtained with ricin, GTPγS was found to inhibit apical transferrin uptake in MDCK II cells transfected with the human transferrin receptor, and the data thus imply that GTPγS supports clathrin‐independent endocytosis. Electron microscopy (EM) demonstrated that free endocytic vesicles were formed from the apical pole of permeabilized MDCK II cells in the presence of GTPγS and that both a ricin–HRP conjugate, HRP, and cationized gold were endocytosed. Ricin endocytosis in the presence of intact cytosol, as well as GTPγS‐stimulated ricin uptake, was inhibited by Clostridium botulinum C3 transferase, an enzyme found to inactivate Rho proteins. The data demonstrate that apical clathrin‐independent endocytosis functions in the presence of GTPγS, and suggest that one or more of the small GTP binding proteins of the Rho family is involved in regulation of the apical clathrin‐independent endocytosis in MDCK II cells.

Direct Transcriptional Consequences of Somatic Mutation in Breast Cancer

Summary Disordered transcriptomes of cancer encompass direct effects of somatic mutation on transcription, coordinated secondary pathway alterations, and increased transcriptional noise. To catalog the rules governing how somatic mutation exerts direct transcriptional effects, we developed an exhaustive pipeline for analyzing RNA sequencing data, which we integrated with whole genomes from 23 breast cancers. Using X-inactivation analyses, we found that cancer cells are more transcriptionally active than intermixed stromal cells. This is especially true in estrogen receptor (ER)-negative tumors. Overall, 59% of substitutions were expressed. Nonsense mutations showed lower expression levels than expected, with patterns characteristic of nonsense-mediated decay. 14% of 4,234 rearrangements caused transcriptional abnormalities, including exon skips, exon reusage, fusions, and premature polyadenylation. We found productive, stable transcription from sense-to-antisense gene fusions and gene-to-intergenic rearrangements, suggesting that these mutation classes drive more transcriptional disruption than previously suspected. Systematic integration of transcriptome with genome data reveals the rules by which transcriptional machinery interprets somatic mutation.

Assessing Invasion Criteria in Fine Needle Aspirates from Breast Carcinoma Diagnosed as DCIS or Invasive Carcinoma

OBJECTIVE To evaluate invasion criteria in fine needle aspiration cytology (FNAC) of histologically diagnosed breast ductal carcinoma in situ (DCIS) and invasive carcinoma and to evaluate their usefulness in identifying an invasive component in addition to DCIS. STUDY DESIGN The material consisted of 331 smears diagnosed as suspicious for or consistent with DCIS and in which histology had shown either DCIS or invasive ductal carcinoma. All smears were reevaluated for the following invasion criteria: invasion of fat or fibrous tissue fragments, fibroblast proliferation, cell-poor elastoid tissue fragments, tubular structures and intracytoplasmic vacuoles. RESULTS All invasion criteria except cytoplasmic vacuoles correlated with invasiveness, but none of them were found exclusively in invasive lesions. Pseudoinvasion in fibrous or fatty tissue fragments were found in 8 cases of histologic pure DCIS. One DCIS (0.4%) revealed > or = 2 invasion features as well as 22 invasive carcinomas (20.7%), representing 7.4% of all cases. CONCLUSION Using established invasion criteria, practically no pure DCIS lesion will be diagnosed as invasive on FNAC, but one will identify only a subset of cases harboring an invasive component.

Metabolic clusters of breast cancer in relation to gene- and protein expression subtypes

BackgroundThe heterogeneous biology of breast cancer leads to high diversity in prognosis and response to treatment, even for patients with similar clinical diagnosis, histology, and stage of disease. Identifying mechanisms contributing to this heterogeneity may reveal new cancer targets or clinically relevant subgroups for treatment stratification. In this study, we have merged metabolite, protein, and gene expression data from breast cancer patients to examine the heterogeneity at a molecular level.MethodsThe study included primary tumor samples from 228 non-treated breast cancer patients. High-resolution magic-angle spinning magnetic resonance spectroscopy (HR MAS MRS) was performed to extract the tumors metabolic profiles further used for hierarchical cluster analysis resulting in three significantly different metabolic clusters (Mc1, Mc2, and Mc3). The clusters were further combined with gene and protein expression data.ResultsOur result revealed distinct differences in the metabolic profile of the three metabolic clusters. Among the most interesting differences, Mc1 had the highest levels of glycerophosphocholine (GPC) and phosphocholine (PCho), Mc2 had the highest levels of glucose, and Mc3 had the highest levels of lactate and alanine. Integrated pathway analysis of metabolite and gene expression data uncovered differences in glycolysis/gluconeogenesis and glycerophospholipid metabolism between the clusters. All three clusters had significant differences in the distribution of protein subtypes classified by the expression of breast cancer-related proteins. Genes related to collagens and extracellular matrix were downregulated in Mc1 and consequently upregulated in Mc2 and Mc3, underpinning the differences in protein subtypes within the metabolic clusters. Genetic subtypes were evenly distributed among the three metabolic clusters and could therefore contribute to additional explanation of breast cancer heterogeneity.ConclusionsThree naturally occurring metabolic clusters of breast cancer were detected among primary tumors from non-treated breast cancer patients. The clusters expressed differences in breast cancer-related protein as well as genes related to extracellular matrix and metabolic pathways known to be aberrant in cancer. Analyses of metabolic activity combined with gene and protein expression provide new information about the heterogeneity of breast tumors and, importantly, the metabolic differences infer that the clusters may be susceptible to different metabolically targeted drugs.

Increased coagulation activity and genetic polymorphisms in the F5, F10 and EPCR genes are associated with breast cancer: a case-control study.

BackgroundThe procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer.Methods366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test.ResultsFour SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status.ConclusionsA procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer.

Apical macropinocytosis in polarized MDCK cells: Regulation by N-ethylmaleimide-sensitive proteins

In cells tested so far endocytosis seems to be dependent on N-ethylmaleimide (NEM)-sensitive proteins, and treatment with NEM results in a complete block of endocytosis. We here demonstrate that treatment of polarized MDCK I cells with NEM strongly increased endocytosis of ricin and horseradish peroxidase at the apical side, and electron microscopy revealed NEM-induced formation of large macropinosomes at the apical pole. The NEM-stimulated apical endocytosis seemed to involve phosphatidylinositol-3 kinase, protein kinase C and phospholipase D and it was dependent on ATP. Moreover, in contrast to endocytosis in nonpolarized cells ricin endocytosis at the basolateral side continued in the presence of NEM whereas endocytosis of transferrin was blocked. Furthermore, recycling of ricin endocytosed in the absence of NEM was not inhibited on either side upon addition of NEM demonstrating the existence of a NEM-resistant fusion machinery. The results suggest that the fusogenic property of both the apical and the basolateral plasma membrane of MDCK cells differs from that typically observed in cells unable to polarize.

Entry of Shiga Toxin into Cells

The effect of Shiga toxin with mutations in the A fragment has been tested on cells in order to get more information about the processing of the A fragment during entry into the cytosol. A mutant with a deletion between the A1 and A2 domain in the A fragment is resistant to cleavage by trypsin and is less toxic than wild type toxin on both Vero and A431 cells. The results support the view that processing of the A fragment is important for the high toxicity of the wild type toxin. A number of cell lines are resistant to Shiga toxin although they bind the toxin. However, A431 cells can be sensitized by butyric acid treatment, and transport of Shiga toxin to the Golgi apparatus seems to be required for the intoxication in the sensitized cells. The role of retrograde transport through the Golgi apparatus to the endoplasmic reticulum (ER) will be discussed.

Erratum: Inflammation of mammary adipose tissue occurs in overweight and obese patients exhibiting early-stage breast cancer

Growing evidence indicates that adiposity is associated with breast cancer risk and negatively affects breast cancer recurrence and survival, a paracrine role of mammary adipose tissue being very likely in this process. In contrast to other adipose depots, occurrence of a sub-inflammatory state of mammary adipose tissue defined by dying adipocytes surrounded by macrophages forming crown-like structures in overweight and obese subjects, remains only partially described. In a general population of breast cancer patients (107 patients) mostly undergoing breast-conserving surgery, we found a positive association between patient’s body composition, breast adipocytes size, and presence of crown-like structures in mammary adipose tissue close to the tumor. Overweight (BMI: 25.0–29.9 kg/m2) and obese (BMI ≥ 30.0 kg/m2) patients have 3.2 and 6.9 times higher odds ratio of crown-like structures respectively, compared with normal weight patients. The relatively small increase in adipocyte size in crown-like structures positive vs. negative patients suggests that mammary adipose tissue inflammation might occur early during hypertrophy. Our results further highlight that body mass index is an adequate predictor of the presence of crown-like structures in mammary adipose tissue among postmenopausal women, whereas in premenopausal women truncal fat percentage might be more predictive, suggesting that mammary adipose tissue inflammation is more likely to occur in patients exhibiting visceral obesity. Finally, the presence of crown-like structures was positively associated with systemic markers such as the Triglyceride/High-density lipoprotein-cholesterol ratio serum C-reactive protein and glucose/(HbA1c) glycated Haemoglobin. These compelling results demonstrate that excess adiposity, even in overweight patients, is associated with mammary adipose tissue inflammation, an event that could contribute to breast cancer development and progression.Immunology: Weight tied to inflammation in fat surrounding tumorOverweight and obese women with breast cancer show more inflammation in their mammary fat tissue, creating an environment favorable to tumor growth. In a study performed at the Oslo University Hospital, Norway, Charlotte Vaysse and colleagues characterized the fat cells found close to the breast tumors of 107 patients with early-stage disease. The researchers showed that overweight and obese women were more likely to have clusters of pro-inflammatory macrophage cells within the fat tissue close to the tumors than normal weight women. They further divided the patients according to whether they’d gone through menopause or not, and found that body mass index was a good predictor of fat cell inflammatory status in postmenopausal women, whereas belly fat percentage was a more accurate measure for premenopausal women. The inflammation brought on by excess weight may contribute to breast cancer development and progression.

Breast cancer stromal elastosis is associated with mammography screening detection, low Ki67 expression and favourable prognosis in a population-based study

BackgroundMammography screen-detected breast cancers have a better prognosis than predicted from established prognostic markers. A search for additional features that are characteristic for these tumours and their prognosis is needed to reduce overtreatment, a recognized challenge in breast cancer patient management today. Here, we have investigated the occurrence and importance of tumour elastosis.MethodsWe performed a population based retrospective study of breast cancers detected in the Norwegian Breast Cancer Screening Programme in Vestfold County during 2004–2009. In total, 197 invasive screen-detected cancers and 75 interval cancers in patients aged 50–69 years were compared with regard to standard clinico-pathological parameters and tumour shape, as well as ER, PR, HER2 and Ki67 expression. In particular, the presence of elastotic material in tumours was graded on a 4-tiered scale (score 0–3).ResultsScreen-detected cancers had a significantly higher content of stromal elastosis than interval cancers (p < 0.001). High content of elastosis (score 3) correlated strongly with stellate tumour shape, low histological grade, and ER+/HER2- status. Further, high elastosis score was significantly associated with lower Ki67 expression. In survival analyses, cases with high elastosis demonstrated increased recurrence free (p = 0.03) and disease-specific survival (p = 0.11) compared to cases with low elastosis.ConclusionThere is a strong correlation between the presence of tumour elastosis, stellate tumour shape and mammography detection of breast cancers. To our knowledge, this is the first time elastosis has been studied in relation to breast cancer detection method. Presence of elastosis is associated with low tumour cell proliferation (Ki67) and a good prognosis.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/13000_2014_230