BRCA1 transcriptionally regulates genes associated with the basal-like phenotype in breast cancer

Expression profiling of BRCA1-deficient tumours has identified a pattern of gene expression similar to basal-like breast tumours. In this study, we examine whether a BRCA1-dependent transcriptional mechanism may underpin the link between BRCA1 and basal-like phenotype. In methods section, the mRNA and protein were harvested from a number of BRCA1 mutant and wild-type breast cancer cell lines and from matched isogenic controls. Microarray-based expression profiling was used to identify potential BRCA1-regulated transcripts. These gene targets were then validated (by in silico analysis of tumour samples) by real-time PCR and Western blot analysis. Chromatin immunoprecipitation (ChIP) assays were used to confirm recruitment of BRCA1 to specific promoters. In results, we demonstrate that functional BRCA1 represses the expression of cytokeratins 5(KRT5) and 17(KRT17) and p-Cadherin (CDH3) in HCC1937 and T47D breast cancer cell lines at both mRNA and protein level. ChIP assays demonstrate that BRCA1 is recruited to the promoters of KRT5, KRT17 and CDH3, and re-ChIP assays confirm that BRCA1 is recruited independently to form c-Myc and Sp1 complexes on the CDH3 promoter. We show that siRNA-mediated inhibition of endogenous c-Myc (and not Sp1) results in a marked increase in CDH3 expression analogous to that observed following the inhibition of endogenous BRCA1. The data provided suggest a model whereby BRCA1 and c-Myc form a repressor complex on the promoters of specific basal genes and represent a potential mechanism to explain the observed overexpression of key basal markers in BRCA1-deficient tumours.

Prediction of BRCA1 status in patients with breast cancer using estrogen receptor and basal phenotype

Purpose: To investigate the proportion of breast cancers arising inpatients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients. Experimental Design: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin. Results: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls. Conclusion: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.

Myoepithelial calls: Pathology, cell separation and markers of myoepithelial differentiation

Until recently the myoepithelial cell has been studied relatively little in terms of its role in breast cancer. A number of malignancies showing myoepithelial differentiation have been reported in the literature, although they are still thought to be relatively rare and only limited studies are published. As a result of recent expression profiling experiments, one type of tumor with myoepithelial features, the so-called 'basal' breast cancer, has received a renewed interest, although it has been known to pathologists for more than two decades. These tumors, which express markers of both luminal and myoepithelial cells, are now being studied using antibodies against some new molecules that have emerged from studies of sorted normal luminal and myoepithelial cells. These immunohistochemical data, combined with genomic studies, may lead to better identification and management of patients with 'basal' tumors.

Cutaneous markers of photo-damage and risk of basal cell carcinoma of the skin : a meta-analysis

BACKGROUND: Epidemiologic research has demonstrated that cutaneous markers of photo-damage are associated with risk of basal cell carcinoma (BCC). However there has been no previous attempt to calculate pooled risk estimates. METHODS: We conducted a systematic review and meta-analysis after extracting relevant studies published up to January 2013 from five electronic databases. Eligible studies were those that permitted quantitative assessment of the association between histologically-confirmed BCC and actinic keratoses, solar elastosis, solar lentigines, or telangiectasia. RESULTS: Seven eligible studies were identified and summary odds ratios (OR) were calculated using both random and quality effects models. Having more than ten actinic keratoses was most strongly associated with BCC, conferring up to a 5-fold increase in risk (OR: 4.97; 95% CI: 3.26, 7.58). Other factors, including solar elastosis, solar lentigines, and telangiectasia had weaker but positive associations with BCC with ORs around 1.5. CONCLUSIONS: Markers of chronic photo-damage are positively associated with BCC. The presence of actinic keratoses was the most strongly associated with BCC of the markers examined. IMPACT: This work highlights the relatively modest association between markers of chronic ultraviolet exposure and BCC.

Corneal nerve structure and function as markers of diabetic neuropathy

Diabetic neuropathy is a significant clinical problem that currently has no effective therapy, and in advanced cases, leads to foot ulceration and lower limb amputation. The accurate detection, characterisation and quantification of this condition are important in order to define at-risk patients, anticipate deterioration, monitor progression and assess new therapies. This thesis evaluates novel corneal methods of assessing diabetic neuropathy. Over the past several years two new non-invasive corneal markers have emerged, and in cross-sectional studies have demonstrated their ability to stratify the severity of this disease. Corneal confocal microscopy (CCM) allows quantification of corneal nerve parameters and non-contact corneal aesthesiometry (NCCA), the presumed functional correlate of corneal structure, assesses the sensitivity of the cornea. Both these techniques are quick to perform, produce little or no discomfort for the patient, and with automatic analysis paradigms developed, are suitable for clinical settings. Each has advantages and disadvantages over established techniques for assessing diabetic neuropathy. New information is presented regarding measurement bias of CCM images, and a unique sampling paradigm and associated accuracy determination method of combinations is described. A novel high-speed corneal nerve mapping procedure has been developed and application of this procedure in individuals with neuropathy has revealed regions of sub-basal nerve plexus that dictate further evaluation, as they appear to show earlier signs of damage than the central region of the cornea that has to date been examined. The discriminative capacity of corneal sensitivity measured by NCCA is revealed to have reasonable potential as a marker of diabetic neuropathy. Application of these new corneal markers for longitudinal evaluation of diabetic neuropathy has the potential to reduce dependence on more invasive, costly, and time-consuming assessments, such as skin biopsy.

Prolonged hyperinsulinemia affects metabolic signal transduction markers in a tissue specific manner

Insulin dysregulation is common in horses although the mechanisms of metabolic dysfunction are poorly understood. We hypothesized that insulin signaling in striated (cardiac and skeletal) muscle and lamellae may be mediated through different receptors as a result of receptor content, and that transcriptional regulation of downstream signal transduction and glucose transport may also differ between tissues sites during hyperinsulinemia. Archived samples from horses treated with a prolonged insulin infusion or a balanced electrolyte solution were used. All treated horses developed marked hyperinsulinemia and clinical laminitis. Protein expression was compared across tissues for the insulin receptor and insulin-like growth factor 1 receptor (IGF-1R) by immunoblotting. Gene expression of metabolic insulin-signaling markers (insulin receptor substrate 1, Akt2, and glycogen synthase kinase 3 beta [GSK-3β]) and glucose transport (basal glucose transporter 1 and insulin-sensitive glucose transporter 4) was evaluated using real-time reverse transcription polymerase chain reaction. Lamellar tissue contained significantly more IGF-1R protein than skeletal muscle, indicating the potential significance of IGF-1R signaling for this tissue. Gene expression of the selected markers of insulin signaling and glucose transport in skeletal muscle and lamellar tissues was unaffected by prolonged hyperinsulinemia. In contrast, the significant upregulation of Akt2, GSK-3β, GLUT1, and GLUT4 gene expression in cardiac tissue suggested that the prolonged hyperinsulinemia induced an increase in insulin sensitivity and a transcriptional activation of glucose transport. Responses to insulin are tissue-specific, and extrapolation of data across tissue sites is inappropriate.

Caractérisation du potentiel métastatique des carcinomes mammaires de phénotype basal

Au Canada, on estime qu'une femme sur neuf développera un cancer du sein et qu'une femme sur vingt-huit en mourra. Les carcinomes mammaires de phénotype basal qui comprennent environ 15 à 25% des cancers envahissants du sein sont des tumeurs malignes ayant un très mauvais pronostic. Dans ce type tumoral, les métastases sont fréquentes et les décès nombreux. La formation des métastases est un processus complexe qui comprend plusieurs étapes; chacune peut être étudiée par des marqueurs spécifiques. Notre hypothèse de recherche est que le carcinome mammaire de phénotype basal possède des caractéristiques spécifiques qui permettent d'expliquer son potentiel métastatique élevé. Six micromatrices tissulaires comprenant un total de 213 tumeurs mammaires ont été confectionnées. L'expression des marqueurs usuels de la métastase et celle de nouveaux marqueurs a été étudiée par des techniques d'immunohistochimie. L'étude comparative de l’expression des marqueurs du potentiel métastatique par les carcinomes mammaires de phénotype basal indique que les protéines Ki-67, EGFR, CD276 et galectine-7 sont étroitement reliées à ce type de cancer. De plus, l'expression du marqueur GATA-3, un marqueur anti-métastatique, fait complètement défaut. Nos résultats confirment que le cancer du sein de phénotype basal possède un plus grand potentiel métastatique que les autres sous-types génétiques et suggèrent que la galectine-7 puisse être impliquée.

Differential effects of apolipoprotein E3 and E4 on markers of oxidative status in macrophages

ApoE is secreted by macrophages at the lesion site of the atherosclerotic plaque, where it is thought to play a protective role against atherosclerosis independently of its effects on lipid metabolism. Of the three common isoforms for apoE, apoE4 is associated with higher risk of cardiovascular disease (CVD). In vitro studies have shown that recombinant apoE may act as an antioxidant in an isoform-dependent manner (E2 > E3 > E4). The oxidative status of the macrophages plays a key role in the process of atherosclerosis. In the present study the possible differential actions of apoE3 and apoE4 on several parameters of oxidative status were determined in stably transfected murine macrophages (RAW 2647-apoE3 and apoE4). No differences between genotypes were observed after peroxide challenge in either protection against cytotoxicity or in cell membrane oxidation, and modest differences were observed in the non-enzymatic antioxidants (glutathione and a-tocopherol) in apoE3 v. apoE4 macrophages. Importantly, cells secreting apoE4 showed increased membrane oxidation under basal conditions, and produced more NO and superoxide anion radicals than the apoE3 macrophages after stimulation. The present data suggest that apoE genotype influences the oxidative status of macrophages, and this could partly contribute to the higher CVD risk observed in apoE4 carriers.

Effect of short-term creatine supplementation on markers of skeletal muscle damage after strenuous contractile activity

The protective effect of short-term creatine supplementation (CrS) upon markers of strenuous contractile activity-induced damage in human and rat skeletal muscles was investigated. Eight Ironman triathletes were randomized into the placebo (Pl; n = 4) and creatine-supplemented (CrS; n = 4) groups. Five days prior to the Ironman competition, the CrS group received creatine monohydrate (20 g day(-1)) plus maltodextrin (50 g) divided in two equal doses. The Pl group received maltodextrin (50 g day(-1)) only. The effect of CrS (5 g day(-1)/kg body weight for 5 days) was also evaluated in a protocol of strenuous contractile activity induced by electrical stimulation in rats. Blood samples were collected before and 36 and 60 h after the competition and were used to determine plasma activities of creatine kinase (CK), lactate dehydrogenase (LDH), aldolase (ALD), glutamic oxaloacetic acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), and C-reactive protein (CRP) level. In rats, plasma activities of CK and LDH, muscle vascular permeability (MVP) using Evans blue dye, muscle force and fatigue were evaluated. Activities of CK, ALD, LDH, GOT, GTP, and levels of CRP were increased in the Pl group after the competition as compared to basal values. CrS decreased plasma activities of CK, LDH, and ALD, and prevented the rise of GOT and GPT plasma activities. In rats, CrS delayed the fatigue, preserved the force, and prevented the rise of LDH and CK plasma activities and MVP in the gastrocnemius muscle. CrS presented a protective effect on muscle injury induced by strenuous contractile activities.

Cell blocks allow reliable evaluation of expression of basal (CK5/6) and luminal (CK8/18) cytokeratins and smooth muscle actin (SMA) in breast carcinoma

Objective:Gene expression studies have revealed several molecular subtypes of breast carcinoma with distinct clinical and biological behaviours. DNA microarray studies correlated with immunohistochemical profiling of breast carcinomas using cytokeratin (CK) markers, Her2/neu, oestrogen receptor (ER), and basal myoepithelial cell markers have identified five breast tumour subtypes: (i) luminal A (ER+; Her2/neu-), (ii) luminal B (ER+; Her2/neu+), (iii) Her2 overexpression (ER-; Her2/neu+), (iv) basal-like (ER-; Her2/neu-, CK5/6 and 14+), and (v) negative for all markers. Luminal carcinomas express cytokeratins in a luminal pattern (CK8/18), and the basal-like type expresses CK5/6 and CK14 or basal epithelial cell markers. CK5/6, CK8/18, and smooth muscle actin (SMA) expression were assessed in cell blocks and compared with expression in surgical specimens.Methods:Sixty-two cases of breast carcinoma diagnosed by fine needle aspiration cytology with cell blocks and available surgical specimens were included. Cell blocks containing at least 10 high-power fields each with at least 10 tumour cells and surgical specimens were immunostained for CK5/6, CK8/18 and SMA.Results:Percentage sensitivity, specificity, positive predictive value, negative predictive value and accuracy were, respectively, 77, 100, 100, 92 and 94 for CK5/6; 98, 66, 96, 80 and 95 for CK8/18; and 92, 96, 85, 98 and 95 for SMA.Conclusion:The identification of CK5/6, CK8/18 and SMA by immunohistochemistry in cell blocks can be a reliable method that yields results close to those obtained in surgical specimens, and can contribute to the classification of breast carcinomas with luminal and basal expression patterns, providing helpful information in the choice of treatment and in the evaluation of prognostic and predictive factors.

Growth rings in cerrado woody species: occurrence and anatomical markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão de citoceratinas de padrão basal (CK5/6), luminal (CK8/18) e actina de músculo liso (1A4) em carcinoma de mama

Pós-graduação em Ginecologia, Obstetrícia e Mastologia - FMB

Proinflammatory and oxidative stress markers in patients submitted to Roux-en-Y gastric bypass after 1 year of follow-up

BACKGROUND/OBJECTIVES: This study examined the effect of weight loss after 3, 6 and 12 months of Roux-en-Y Gastric Bypass (RYGB) on energy intake and on several biomarkers of oxidative stress such as levels of vitamin C, beta-carotene, vitamin E (diet/blood), nitric oxide metabolites (NOx), myeloperoxidase (MPO), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH) and activity of catalase (CAT). SUBJECTS/METHODS: Study with a control group (CG), assessed once, and a bariatric group (BG) assessed at the basal period as well as at 3, 6 and 12 months post-surgery; both groups were composed of 5 men and 31 women (n = 36). Age was 38.7 +/- 9.4 and 39.6 +/- 9.2 years old and body mass index (BMI) was 22.2 +/- 2.1 and 47.6 +/- 9.1 kg/m(2), respectively. The variance measure quoted was SEM. RESULTS: The body weight at 12 months was 35.8 +/- 1.0% (P<0.001) lower than that of the basal period. At the basal period BG showed higher levels of NOx (P=0.007) and TBARS (P<0.001) and lower levels of vitamins C and E (P<0.001) compared with CG. After 3 months the activity of MPO was decreased (P<0.001). Six months after surgery GSH levels were decreased (P=0.037), whereas CAT activity was increased (P=0.029). After 12 months levels of NOx (P=0.004), TBARS (P<0.001), beta-carotene (P<0.001) and vitamin E (P<0.001) were decreased, whereas those of vitamin C (P<0.001) were increased compared with controls. CONCLUSION: RYGB followed by a daily vitamin supplement apparently attenuated pro-inflammatory and oxidative stress markers 1 year after surgery, but additional antioxidant supplementation appears necessary.

Diagnostic utility of immunohistochemistry in distinguishing trichoepithelioma and basal cell carcinoma: evaluation using tissue microarray samples

Trichoepithelioma is a benign neoplasm that shares both clinical and histological features with basal cell carcinoma. It is important to distinguish these neoplasms because they require different clinical behavior and therapeutic planning. Many studies have addressed the use of immunohistochemistry to improve the differential diagnosis of these tumors. These studies present conflicting results when addressing the same markers, probably owing to the small number of basaloid tumors that comprised their studies, which generally did not exceed 50 cases. We built a tissue microarray with 162 trichoepithelioma and 328 basal cell carcinoma biopsies and tested a panel of immune markers composed of CD34, CD10, epithelial membrane antigen, Bcl-2, cytokeratins 15 and 20 and D2-40. The results were analyzed using multiple linear and logistic regression models. This analysis revealed a model that could differentiate trichoepithelioma from basal cell carcinoma in 36% of the cases. The panel of immunohistochemical markers required to differentiate between these tumors was composed of CD10, cytokeratin 15, cytokeratin 20 and D2-40. The results obtained in this work were generated from a large number of biopsies and resulted in the confirmation of overlapping epithelial and stromal immunohistochemical profiles from these basaloid tumors. The results also corroborate the point of view that trichoepithelioma and basal cell carcinoma tumors represent two different points in the differentiation of a single cell type. Despite the use of panels of immune markers, histopathological criteria associated with clinical data certainly remain the best guideline for the differential diagnosis of trichoepithelioma and basal cell carcinoma. Modern Pathology (2012) 25, 1345-1353; doi: 10.1038/modpathol.2012.96; published online 8 June 2012

Stem cell pathways in the basal-like breast carcinoma: the role of beta-catenin and HIF-1alpha

Basal-like tumor is an aggressive breast carcinoma subtype that displays an expression signature similar to that of the basal/myoepithelial cells of the breast tissue. Basal-like carcinoma are characterized by over-expression of the Epidermal Growth Factor receptor (EGFR), high frequency of p53 mutations, cytoplasmic/nuclear localization of beta-catenin, overexpression of the Hypoxia inducible factor (HIF)-1alpha target Carbonic Anhydrase isoenzime 9 (CA9) and a gene expression pattern similar to that of normal and cancer stem cells, including the over-expression of the mammary stem cell markers CD44. In this study we investigated the role of p53, EGFR, beta-catenin and HIF-1alpha in the regulation of stem cell features and genes associated with the basal-like gene expression profile. The findings reported in this investigation indicate that p53 inactivation in ductal breast carcinoma cells leads to increased EGFR mRNA and protein levels. In our experimental model, EGFR overexpression induces beta-catenin cytoplasmatic stabilization and transcriptional activity and, by that, leads to increased aggressive features including mammosphere (MS) forming and growth capacity, invasive potential and overexpression of the mammary stem cell gene CD44. Moreover we found that EGFR/beta-catenin axis promotes hypoxia survival in breast carcinoma cells via increased CA9 expression. Indeed beta-catenin positively regulates CA9 expression upon hypoxia exposure. Interestingly we found that beta-catenin inhibits HIF-1alpha transcriptional activity. Looking for the mechanism, we found that CA9 expression is promoted by HIF-1alpha and cytoplasmatic beta-catenin further increased it post-transcriptionally, via direct mRNA binding and stabilization. These data reveal a functional beta-catenin/HIF-1alpha interplay among hallmarks of basal-like tumors and unveil a new functional role for cytoplasmic beta-catenin in the phenotype of such tumors. Therefore it can be proposed that the interplay here described among EGFR/beta-catenin and HIF-1alpha may play a role in breast cancer stem cell survival and function.