Plasma fatty acids composition and estimated delta desaturases activity in women with breast cancer.

INTRODUCTION: Fatty acids (FAs) are the vital constituents of membrane structures. De novo synthesis of FAs includes an enzymatic complex of FA synthase and delta desaturases. These enzymes are overexpressed in tumors, and inhibition of these enzymes is gaining interest. Our aim was to determine if delta desaturase activities are altered in breast cancer (BC) cases and if altered whether delta desaturase activities differ among BC genotypes. MATERIALS AND METHODS: In this observational comparative study, 50 women with BC and 30 control women were recruited for the study. Gas chromatography-flame ionization detector was used to measure the plasma FA levels. Desaturase activities were assessed as product-to-precursor FA ratios. The Wilcoxon signed-rank test was used to compare between two groups, and P ≤ 0.05 was considered as statistically significant. RESULTS: The FA analysis revealed higher levels of monounsaturated FAs (MUFAs) and linolenic acid metabolites (C18:3n-6, C20:4n-6) in BC patients, whereas C20:5n-3 was higher in controls. The Delta 9 desaturase (D9D) and D6D were higher in BC cases suggesting greater conversion saturated FA to MUFA and linoleic acid to its metabolites. D9D-16 activity was statistically significant (P = 0.03) in BC women, particularly in estrogen-receptor-positive patients. CONCLUSION: There is limited evidence to substantiate the link between diet and cancer. The current study showed there is an altered lipid desaturase activity. Nutritional intervention and drugs that target the FA pathway may provide a new approach to prevent and treat BC.

Controlling ERK Activation Dynamics in Mammary Epithelial Cells with Alternating Electric Fields through Microelectrodes.

Amplitude, duration, and frequency of activation of the extracellular-signal-regulated kinase (ERK) pathway code distinct information to instruct cells to migrate, proliferate, or differentiate. Synchronized frequency control of ERK activation would provide a powerful approach to regulate cell behaviors. Here we demonstrated modulation of ERK activities using alternative current (AC) electric fields (EFs) applied through high-k dielectric passivated microelectrodes. Both the amplitude and frequency of ERK activation can be precisely synchronized and modulated. ERK activation in our system is independent of Faradaic currents and electroporation, thus excluding mechanisms of changes in pH, reactive oxygen species, and other electrochemical reaction. Further experiments pinpointed a mechanism of phosphorylation site of epidermal growth factor (EGF) receptor to activate the EGFR-ERK pathway, and independent of EGF. AC EFs thus provide a powerful platform for practical and precise control of EGFR-ERK pathway.

Plasma membrane localization of CYP4Z1 and CYP19A1 and the detection of anti-CYP19A1 autoantibodies in humans.

It is thought that autoantibody (aAb) production can be caused by (aberrant) protein targeting to the plasma surface of cells. We recently demonstrated the presence of the human cytochrome P450 enzyme CYP4Z1 on the plasma membrane of MCF-7 breast cancer cells and the detection of high titers of anti-CYP4Z1 aAbs in breast cancer patients, but not in healthy controls. In the present study we show that cells of the normal breast cell line MCF-10A do not display CYP4Z1 on their surface. By contrast, we detected CYP19A1 (aromatase) on the plasma membrane of both cell lines. Interestingly, the presence of CYPs on the cell surface did not correlate with their relative expression levels in these cell lines. Indirect ELISA experiments demonstrated the presence of anti-CYP19A1 aAbs in female breast cancer patient sera as well as in male and female controls, respectively; aAb titers in all three groups varied considerably and overall, the results obtained for each group were not significantly different from those of either of the other two groups. Based on these data we propose the hypothesis that CYP translocation to the plasma membrane, but not the intracellular expression level, is the crucial precondition for the generation of anti-CYP aAbs.

The KRAS-variant and its impact on normal breast epithelial cell biology.

MicroRNA (miRNA)-binding site variants in 3' untranslated regions (3'UTRs) are a novel class of germ-line, functional mutations, which are now recognized as powerful biomarkers of human cancer risk and biology. The first mutation discovered in this class is the KRAS-variant, a let-7-binding site mutation in the 3'UTR of the KRAS oncogene. The KRAS-variant predicts increased cancer risk for certain populations, is a predictive biomarker of cancer treatment response across cancer types, leads to conserved tumor biology and elevated AKT signaling in KRAS-variant patient tumors, and was recently found to predict elevated TGF-ß and immunosuppression in cancer patients. Based on the functional biology of the KRAS-variant in cancer patients, here we chose to investigate altered normal cellular biology in the presence of the KRAS-variant, through interrogation of an isogenic normal breast epithelial cell line model with and without the KRAS-variant. We find that KRAS-variant normal breast epithelial cells exhibit a mesenchymal phenotype, which appears to be due to numerous molecular changes, including miRNA dysregulation and autocrine pathway alterations, including elevated TGF-ß, resulting in ZEB and SNAIL upregulation. Our findings support the hypothesis that the KRAS-variant has a fundamental biological impact on normal cellular biology, that is conserved in these patients when they develop cancer.

The clinicopathological significance of ubiquitin-conjugating enzyme E2C, leucine-rich repeated-containing G protein-coupled receptor, WW domain-containing oxidoreductase, and vasculogenic mimicry in invasive breast carcinoma.

Ubiquitin-conjugating enzyme E2C (UBE2C), a crucial part of the ubiquitin-conjugating enzyme complex, is reported to promote progression of various cancers. Leucine-rich repeated-containing G protein-coupled receptor (LGR5), a biomarker of cancer stem cells, is reported to be responsible for the initiation and progression of cancers. WW domain-containing oxidoreductase (WWOX), a suppressor of tumor, is reported to inhibit initiation and progression of cancers. Vasculogenic mimicry (VM), a new blood supply pattern, is associated with progression of cancers. However, the clinicopathological significance of UBE2C, LGR5, WWOX, and VM in invasive breast carcinoma (IBC) remains elusive. The aim of this study is to investigate the positive rate of UBE2C, LGR5, WWOX, and VM in IBC and their clinical significance.Positive rates of UBE2C, LGR5, WWOX, and VM in 247 whole IBC samples were detected through immunohistochemistry. Patients data (including clinical, demography, follow-up) were collected.Levels of UBE2C, LGR5, VM, and microvessel density (MVD) were significantly higher, and level of WWOX was significantly lower in IBC specimens when compared with normal mammary gland tissues. Levels of UBE2C, LGR5, VM, and MVD were all positively associated with tumor stages, lymph node metastasis (LNM) stages, tumor grades, and tumor-node-metastasis (TNM) stages, and unfavorably with patients' overall survival (OS) and disease-free survival (DFS). Level of WWOX was negatively associated with tumor stages, LNM stages, grades, and TNM stages, and favorably with patients' OS and DFS. Multivariate analysis indicated that levels of UBE2C, LGR5, VM, MVD, and WWOX, as well as TNM stages were independently prognostic factors for OS and DFS in patients with IBC.UBE2C, LGR5, VM, MVD, and WWOX may be considered as promising indicator of IBC prognosis.

The role of 17ßHSDs in breast tissue and breast cancers.

The family of seventeen beta hydroxysteroid dehydrogenase enzymes has a long and diverse history in breast and breast cancer research. Given the known dependence of the breast on steroid signalling and intracrine steroid metabolism these enzymes are considered to be essential local fine tuners of overall steroid balance in the tissue. This review will cover the current state of knowledge regarding the expression, clinical effect and biological regulation of enzymes in both cancerous and normal states. In addition we will also cover the current state of knowledge regarding 17ßHSD actions in the often neglected adipose and stromal components of tumours.

GCSH antisense regulation determines breast cancer cells' viability.

Since it is known that cancer cells exhibit a preference for increased glycine consumption, the respective glycine metabolizing enzymes are in focus of many research projects. However, no cancer associated studies are available for the Glycine Cleavage System Protein H (GCSH) to date. Our initial analysis revealed a GCSH-overexpression of the protein-coding transcript variant 1 (Tv1) in breast cancer cells and tissue. Furthermore, a shorter (391 bp) transcript variant (Tv*) was amplified with an increased expression in healthy breast cells and a decreased expression in breast cancer samples. The Tv1/Tv* transcript ratio is 1.0 in healthy cells on average, and between 5-10 in breast cancer cells. Thus, a GCSH-equilibrium at the transcript level is likely conceivable for optimal glycine degradation. A possible regulative role of Tv* was proven by Tv1-Tv*-RNA-binding and overexpression studies which consequently led to serious physiological alterations: decreased metabolic activity, release of the lactate dehydrogenase, increased extracellular acidification, and finally necrosis as a result of impaired plasma membranes. In contrast, Tv1-overexpression led to an additional increase in cellular vitality of the tumor cells, primarily due to the acceleration of the mitochondrial glycine decarboxylation activity. Ultimately, we provide the first evidence of a sensitive GCSH-antisense regulation which determines cancerous cell viability.

Chemical Tools for Selective Activity Profiling of Endogenously Expressed MMP-14 in Multicellular Models.

Matrix metalloproteases (MMPs) are a large family of zinc-dependent endopeptidases involved in a diverse set of physiological and pathological processes, most notably in cancer. Current methods for imaging and quantifying MMP activity lack sufficient selectivity and spatiotemporal resolution to allow studies of specific MMP function in vivo. Previously, we reported a strategy for selective targeting of MMPs by engineering a functionally silent cysteine mutation that enables highly specific covalent modification by a designed activity-based probe. Here, we describe the translation of that technology into a mouse model of breast cancer and subsequent demonstration of the utility of the approach for studies of MMP-14 activation in the tumor microenvironment. Using this approach, we find that MMP-14 is active in late stage tumors and is predominantly associated with stromal cell populations that have been activated by specific signaling molecules (e.g., TGFß) produced by tumor cells. Our data demonstrate the applicability of this approach for studies of MMP function in whole organisms and identify important regulatory mechanisms for MMP-14 activity in the tumor microenvironment.

IL-10 suppresses TNF-α-induced expression of human aromatase gene in mammary adipose tissue.

White adipose tissue inflammation is linked with increased aromatase gene expression and estrogen production, a major risk factor for breast cancer in obese postmenopausal women. TNF-α, a proinflammatory cytokine, is a key driver of aromatase promoter I.4-mediated expression in adipose tissue. In this study, we have shown that IL-10, an anti-inflammatory cytokine, suppressed both TNF-α-stimulated human aromatase reporter-luciferase (hARO-Luc) expression in mouse bone marrow mesenchymal stromal cells and aromatase gene expression in human breast adipose stromal cells (ASCs). IL-10 blocked TNF-α-stimulated ERK1/2 activation in ASCs, suggesting an inhibitory effect through the MAPK signaling pathway. The links among obesity, IL-10, and aromatase were confirmed in ovariectomized (OVX) hARO-Luc mice, where increased adiposity was associated with upregulation of aromatase reporter activity and reduced IL-10 level in the mammary fat pad. OVX mice also exhibited changes in gut microbiota, similar to that in obese women, indicating altered immune function. In summary, our results suggest that increased adiposity, induced by the lack of ovarian hormones, results in enhanced expression of aromatase in mammary adipose tissue, mediated by reduction in local IL-10. These findings may bring new insights into the mechanisms involved in the development of postmenopausal breast cancer, as well as novel approaches for prevention.-Martínez-Chacón, G., Brown, K. A., Docanto, M. M., Kumar, H., Salminen, S., Saarinen, N., Mäkelä, S. IL-10 suppresses TNF-α-induced expression of human aromatase gene in mammary adipose tissue.

Advances in targeting HER2-positive breast cancer.

PURPOSE OF REVIEW: Breast cancer is the most common malignancy in women and HER2-positive disease comprises about 15% of all breast cancer cases. Over the last few years, substantial advances have been made in treatment of early breast cancer (EBC) and advanced (ABC) HER2-positive (HER2+) breast cancer. The present review summarizes the major publications on HER2+ breast cancer in the year 2017 and discusses their clinical relevance. RECENT FINDINGS: The clinically most relevant data in 2017 relates to the EBC setting. The St. Gallen Consensus 2017 for the first time recommended neoadjuvant treatment as standard of care in HER2+ EBC. Moreover, the APHINITY trial demonstrated efficacy of adjuvant dual antibody-based HER2 blockade. Data in MBC mostly confirmed established treatment strategies. SUMMARY: Clinical data presented in 2017 for HER2+ breast cancer confirmed current guidelines and extended treatment options for patients. First intriguing steps were made towards de-escalation in the EBC setting.

CBX4 exhibits oncogenic activities in breast cancer via Notch1 signaling.

Polycomb chromobox (CBX) proteins are involved in gene silencing to function as oncogenes or tumor suppressors through the polycomb repressive complex (PRC1). CBX4 has been implicated in the progression of human cancers, but its role and clinical significance in breast cancer remain unclear. Here, we show that CBX4 is up-regulated in breast cancer and exerts oncogenic activities via miR-137-mediated activation of Notch1 signaling pathway. CBX4 expression was increased in breast cancer, compared with the nontumorous tissues. High CBX4 expression was closely correlated with tumor metastasis, advanced clinical stage and poor overall survival in a cohort of 179 patients with breast cancer. In vitro studies demonstrated that CBX4 overexpression enhanced, whereas CBX4 knockdown inhibited cell growth and migration. Mechanistically, in a PRC1-dependent manner, CBX4 inhibited the promoter activity of miR-137 and suppressed its expression. miR-137 decreased the expression of Notch1, Jag1 and Hey2 via targeting their 3'-UTRs. The suppression of Notch1 by siRNA or overexpression of miR-137 markedly attenuated CBX4-promoted phenotypes. Collectively, these findings indicate that CBX4 promotes breast cancer via miR-137-mediated Notch1 signaling. Our data, therefore, suggest that CBX4 serve as a prognostic biomarker and that targeting CBX4/miR-137 axis may provide therapeutic potent in the treatment of breast cancer.

Aldehyde dehydrogenase 1 expression is correlated with poor prognosis in breast cancer.

Breast cancer (BC) is one of the most common cancers worldwide, and is a major cause of death in women. Aldehyde dehydrogenase 1 (ALDH1) is a marker of stem cells and cancer stem cells, and its activity correlates with the outcome of various tumors, including BC. This study aimed to analyze the relationship between ALDH1 expression and clinicopathological characters in BC and the prognostic significance of ALDH1.We used quantitative reverse-transcription PCR (qRT-PCR) to detect ALDHA1 mRNA levels in 25 fresh frozen BC samples and matched noncancerous samples. Immunohistochemistry on tissue microarrays was used to analyze protein expression in 137 paraffin-embedded BC tissues and corresponding noncancerous tissues. STATA 16.0 software was used for statistical analysis.The results suggested that levels of both ALDH1 mRNA and protein in BC were significantly higher than in corresponding adjacent breast samples (3.856 ±â€Š0.3442 vs 1.385 ±â€Š0.1534, P < .001; 52.6% vs 25.5%, P < .001, respectively). ALDH1 protein expression was also significantly associated with histological grade (P  =  .017), tumor size (P  =  .017), and tumor-node-metastasis (TNM) stage (P  =  .038). Multivariate analysis using the Cox regression model demonstrated that ALDH1 expression (P  =  .024), molecular typing (P  =  .046), and TNM classification (P  =  .034) were independent predictive factors for the outcome of BC. Kaplan-Meier analysis and the log-rank test indicated that patients with high ALDH1 expression, triple-negative BC, and advanced TNM stage had a reduced overall survival time.These data suggest that ALDH1 could be used as a prognostic factor for BC and may provide a useful therapeutic target in the treatment of BC.

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis.

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5' end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development.

Estrogen receptor agonists/antagonists in breast cancer therapy: A critical review.

Estrogens display intriguing tissue selective action that is of great biomedical importance in the development of optimal therapeutics for the prevention and treatment of breast cancer. There are also strong evidences to show that both endogenous and exogenous estrogens are involved in the pathogenesis of breast cancer. Tamoxifen has been the only drug of choice for more than 30years to treat patients with estrogen related (ER) positive breast tumors. There is a need therefore, for identifying newer, potential and novel candidates for breast cancer. Keeping this in view, the present review focuses on selective estrogen receptor modulators and estrogen antagonists such as sulfatase and aromatase inhibitors involved in breast cancer therapy. A succinct and critical overview of the structure of estrogen receptors, their signaling and involvement in breast carcinogenesis are herein described.

The Hippo kinases LATS1 and 2 control human breast cell fate via crosstalk with ERα.

Cell fate perturbations underlie many human diseases, including breast cancer. Unfortunately, the mechanisms by which breast cell fate are regulated are largely unknown. The mammary gland epithelium consists of differentiated luminal epithelial and basal myoepithelial cells, as well as undifferentiated stem cells and more restricted progenitors. Breast cancer originates from this epithelium, but the molecular mechanisms that underlie breast epithelial hierarchy remain ill-defined. Here, we use a high-content confocal image-based short hairpin RNA screen to identify tumour suppressors that regulate breast cell fate in primary human breast epithelial cells. We show that ablation of the large tumour suppressor kinases (LATS) 1 and 2 (refs 5, 6), which are part of the Hippo pathway, promotes the luminal phenotype and increases the number of bipotent and luminal progenitors, the proposed cells-of-origin of most human breast cancers. Mechanistically, we have identified a direct interaction between Hippo and oestrogen receptor-α (ERα) signalling. In the presence of LATS, ERα was targeted for ubiquitination and Ddb1-cullin4-associated-factor 1 (DCAF1)-dependent proteasomal degradation. Absence of LATS stabilized ERα and the Hippo effectors YAP and TAZ (hereafter YAP/TAZ), which together control breast cell fate through intrinsic and paracrine mechanisms. Our findings reveal a non-canonical (that is, YAP/TAZ-independent) effect of LATS in the regulation of human breast cell fate.

Overexpression and enhanced specific activity of aldoketo reductases (AKR1B1 & AKR1B10) in human breast cancers.

The incidence of breast cancer in India is on the rise and is rapidly becoming the primary cancer in Indian women. The aldoketo reductase (AKR) family has more than 190 proteins including aldose reductase (AKR1B1) and aldose reductase like protein (AKR1B10). Apart from liver cancer, the status of AKR1B1 and AKR1B10 with respect to their expression and activity has not been reported in other human cancers. We studied the specific activity and expression of AKR1B1 and AKR1B10 in breast non tumor and tumor tissues and in the blood. Fresh post-surgical breast cancer and non-cancer tissues and blood were collected from the subjects who were admitted for surgical therapy. Malignant, benign and pre-surgical chemotherapy samples were evaluated by histopathology scoring. Expression of AKR1B1 and AKR1B10 was carried out by immunoblotting and immunohistochemistry (IHC) while specific activity was determined spectrophotometrically. The specific activity of AKR1B1 was significantly higher in red blood cells (RBC) in all three grades of primary surgical and post-chemotherapy samples. Specific activity of both AKR1B1 and AKR1B10 increased in tumor samples compared to their corresponding non tumor samples (primary surgical and post-chemotherapy). Immunoblotting and IHC data also indicated overexpression of AKR1B1 in all grades of tumors compared to their corresponding non tumor samples. There was no change in the specific activity of AKR1B1 in benign samples compared to all grades of tumor and non-tumors.

[Clinical significance of expression of PI3Kp110α in breast cancer].

Objective: To study the clinical significance of PI3Kp110α expression in breast cancer. Methods: The expressions of PI3Kp110α, HER2, PTEN, p-Akt and Ki-67 in invasive ductal carcinoma (IDC), adjacent ductal carcinoma in situ (DCIS) and normal breast tissue were detected by immunohistochemistry in 102 cases of breast cancer. The expression of PI3Kp110α in IDC was compared with those in DCIS and normal breast tissues. Correlations between expression of PI3Kp110α and expression of HER2, PTEN, p-Akt and Ki-67 index in IDC were analyzed. Correlation between expression of PI3Kp110α and stage of IDC was studied as well. Results: In the normal breast tissues, there were 97 cases (95.1%) with low level and 5 cases (4.9%) with high level expression of PI3Kp110α. In the DCIS tissues, there were 67 cases (65.7%) with low level and 35 cases (34.3%) with high level expression of PI3Kp110α. In the IDC tissues, there were 14 cases (13.7%) with low level and 88 cases (86.3%) with high level expression of PI3Kp110α. The difference between expression of PI3Kp110α in normal breast tissue, DCIS and IDC was significant (P<0.001). In the IDC tissues, expression of PI3Kp110α was negatively correlated with expression of HER2 (rs=-0.213, P=0.032) and PTEN (rs=-0.197, P=0.047), while was not significantly correlated with expression of p-Akt (P=0.119) and Ki-67 index (P=0.636). In contrast, expressions of HER-2 and p-Akt were positively correlated with Ki-67 index (P=0.001, P=0.035), while expression of HER2 was not correlated with p-Akt (P=0.177). Expression of PI3Kp110α was negatively correlated with T stage and TNM stage (P=0.003, P=0.016), while not correlated with N stage and M stage(both P>0.05). Expression of HER-2 was positively correlated with T stage (P=0.037), while not correlated with N stage, M stage and TNM stage (P>0.05 for all). Neither Ki-67 index nor expression of PTEN and p-Akt (P=0.194) were correlated with T stage, N stage, M stage and TNM stage. Conclusions: Expression of PI3Kp110α plays an important role in oncogenesis and development of breast cancer. Based on the fact that expression of PI3Kp110α is negatively correlated with expression of HER2 and PTEN and with T stage and TNM stage, we may conclude that increased expression of PI3Kp110α is another independent pathway in breast cancer development, and breast cancer patients with high expression of PI3Kp110α may have a better prognosis.

Early signaling dynamics of the epidermal growth factor receptor.

Despite extensive study of the EGF receptor (EGFR) signaling network, the immediate posttranslational changes that occur in response to growth factor stimulation remain poorly characterized; as a result, the biological mechanisms underlying signaling initiation remain obscured. To address this deficiency, we have used a mass spectrometry-based approach to measure system-wide phosphorylation changes throughout the network with 10-s resolution in the 80 s after stimulation in response to a range of eight growth factor concentrations. Significant changes were observed on proteins far downstream in the network as early as 10 s after stimulation, indicating a system capable of transmitting information quickly. Meanwhile, canonical members of the EGFR signaling network fall into clusters with distinct activation patterns. Src homology 2 domain containing transforming protein (Shc) and phosphoinositol 3-kinase (PI3K) phosphorylation levels increase rapidly, but equilibrate within 20 s, whereas proteins such as Grb2-associated binder-1 (Gab1) and SH2-containing tyrosine phosphatase (SHP2) show slower, sustained increases. Proximity ligation assays reveal that Shc and Gab1 phosphorylation patterns are representative of separate timescales for physical association with the receptor. Inhibition of phosphatases with vanadate reveals site-specific regulatory mechanisms and also uncovers primed activating components in the network, including Src family kinases, whose inhibition affects only a subset of proteins within the network. The results presented highlight the complexity of signaling initiation and provide a window into exploring mechanistic hypotheses about receptor tyrosine kinase (RTK) biology.

Improved synthesis of MC4-PPEA and the biological evaluation of its hydroxymethyl derivative.

Nicotinamide phosphoribosyltransferase (Nampt) is an intriguing target for the treatment of many diseases, including cancer. Previously, our group demonstrated that carborane clusters may be used to increase the potency of small molecule inhibitors of Nampt over other, similarly sized organic moieties. Herein, we report a greatly improved, gram-scale synthesis of our most potent agent: 1-(4'-(trans-3″-(3‴-pyridyl) acrylamide)butyl)-1,7-dicarba-closo-dodecaborane (MC4-PPEA). Additionally, the carborane moiety of the molecule has been modified with a hydroxymethyl functional group to allow for its covalent attachment to targeted prodrugs, the synthesis of which are underway. Using cell viability assays, we demonstrate that this new derivative exhibits low, to mid-nanomolar potencies against human breast cancer cell lines in vitro.