Altering bioelectricity on inhibition of human breast cancer cells

Background

Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer.

Methods

In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive).

Results

Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10–20 μM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 μM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil.

Conclusions

Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.

     

Male breast cancer: thirteen years experience of a single center

Background

This retrospective study analysed the epidemiological, clinical, and therapeutic profiles of breast cancer in males.

Methods

We report our experience at the Hospital of the University of Baskent, where 20 cases of male breast cancer were observed and treated between 1995–2008.

Results

Median age at presentation was 66,7 ± 10,9 years. Average follow-up was 63 ± 18,5 months. The main presenting symptom was a mass in 65% of cases (13 patients). Ýnvasive ductal carcinoma was the most frequent pathologic type (70% of cases).

Conclusion

Male breast cancer patients have an incidence of prostate cancer higher than would be predicted in the general population. Cause of men have a higher rate of ER positivity the responses with hormonal agents are good.

     

Untargeted and stable isotope-assisted metabolomic analysis of MDA-MB-231 cells under hypoxia

Introduction

Hypoxia commonly occurs in cancers and is highly related with the occurrence, development and metastasis of cancer. Treatment of triple negative breast cancer remains challenge. Knowledge about the metabolic status of triple negative breast cancer cell lines in hypoxia is valuable for the understanding of molecular mechanisms of this tumor subtype to develop effective therapeutics.

Objectives

Comprehensively characterize the metabolic profiles of triple negative breast cancer cell line MDA-MB-231 in normoxia and hypoxia and the pathways involved in metabolic changes in hypoxia.

Methods

Differences in metabolic profiles affected pathways of MDA-MB-231 cells in normoxia and hypoxia were characterized using GC–MS based untargeted and stable isotope assisted metabolomic techniques.

Results

Thirty-three metabolites were significantly changed in hypoxia and nine pathways were involved. Hypoxia increased glycolysis, inhibited TCA cycle, pentose phosphate pathway and pyruvate carboxylation, while increased glutaminolysis in MDA-MB-231 cells.

Conclusion

The current results provide metabolic differences of MDA-MB-231 cells in normoxia and hypoxia conditions as well as the involved metabolic pathways, demonstrating the power of combined use of untargeted and stable isotope-assisted metabolomic methods in comprehensive metabolomic analysis.

     

Silencing CDK4 radiosensitizes breast cancer cells by promoting apoptosis

Background

The discovery of molecular markers associated with various breast cancer subtypes has greatly improved the treatment and outcome of breast cancer patients. Unfortunately, breast cancer cells acquire resistance to various therapies. Mounting evidence suggests that resistance is rooted in the deregulation of the G1 phase regulatory machinery.

Methods

To address whether deregulation of the G1 phase regulatory machinery contributes to radiotherapy resistance, the MCF10A immortalized human mammary epithelial cell line, ER-PR-Her2+ and ER-PR-Her2- breast cancer cell lines were irradiated. Colony formation assays measured radioresistance, while immunocytochemistry, Western blots, and flow cytometry measured the cell cycle, DNA replication, mitosis, apoptosis, and DNA breaks.

Results

Molecular markers common to all cell lines were overexpressed, including cyclin A1 and cyclin D1, which impinge on CDK2 and CDK4 activities, respectively. We addressed their potential role in radioresistance by generating cell lines stably expressing small hairpin RNAs (shRNA) against CDK2 and CDK4. None of the cell lines knocked down for CDK2 displayed radiosensitization. In contrast, all cell lines knocked down for CDK4 were significantly radiosensitized, and a CDK4/CDK6 inhibitor sensitized MDA-MB-468 to radiation induced apoptosis. Our data showed that silencing CDK4 significantly increases radiation induced cell apoptosis in cell lines without significantly altering cell cycle progression, or DNA repair after irradiation. Our results indicate lower levels of phospho-Bad at ser136 upon CDK4 silencing and ionizing radiation, which has been shown to signal apoptosis.

Conclusion

Based on our data we conclude that knockdown of CDK4 activity sensitizes breast cancer cells to radiation by activating apoptosis pathways.

     

Successful pregnancy after breast cancer therapy: dream or reality?

Background

Nowadays, more breast cancer patients want to have children after the diagnosis of cancer. The purpose of this study is to review the possibility and risks of giving birth among women with breast cancer previously treated by chemotherapy.

Case presentation

Two young women aged 28 and 34 respectively, were treated in our clinic for breast cancer, the first (negative hormonal receptors) by surgery, chemotherapy and radiotherapy and the second (positive hormonal receptors) by surgery, radiotherapy and tamoxifen. They both became pregnant, 1 and 8 years after completion of the therapy respectively.

Results

Laboratory testing during pregnancy was negative in both cases and after an uneventful course each woman gave birth to a perfectly healthy child. The first patient breastfed her baby for three months, while the second one did not breastfeed her baby at all.

Conclusion

Women undergoing chemotherapy for breast cancer can maintain their fertility and get pregnant. Previous chemotherapy for breast cancer does not present any supplementary risks for the child's mental or physical health.

     

Comprehensive quantitative lipidomic approach to investigate serum phospholipid alterations in breast cancer

Introduction

Globally, breast cancer is the most common cancer in women and ranks second most common cause of cancer related mortality. Although efforts are made by researchers in molecular characterization of breast cancer using “-OMIC’S” approaches, limited work has explored to understand the phospholipid alterations in breast cancer.

Objectives

This study aims to explore five classes of serum phospholipid alterations in breast cancer towards discrimination of breast cancer from benign and healthy controls.

Methods

Twenty eight each of breast cancer patients and age-matched benign and healthy control serum samples were used to identify alterations of phospholipids using liquid chromatography-multiple reaction monitoring-mass spectrometry. Both multivariate and univariate statistical analyses were applied to investigate breast cancer associated phospholipid alterations. Differentially expressed phospholipid species were further confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Results

Among the identified and quantified 200 phospholipids, 25 phospholipids were found to be statistically significant (VIP > 1.4 and ANOVA p < 0.05) in the serum of women with breast cancer when compared with benign and healthy controls. Comparison of serum phospholipids of breast cancer patients and healthy controls revealed 12 phospholipids were found to be differentially expressed in which six were up-regulated and six were down-regulated. While comparative analysis of breast cancer serum against benign showed an increased concentration of six phospholipids in breast cancer samples. Further, significantly altered phospholipids were structurally characterized by enhanced product ion scanning.

Conclusion

Our results demonstrate that some of the differentially regulated phospholipids identified in our study such as PE (14:1/16:0), PC (18:0/18:0), LPE 14:0, PE (20:0/22:2) could be a panel of potential signature which can discriminate breast cancer from benign and healthy controls. These findings also provide insight into lipidomic information that can be used for monitoring of breast cancer progression.

     

Breast cancer cell invasion mediated by Gα12 signaling involves expression of interleukins-6 and −8, and matrix metalloproteinase-2

Background

Recent studies on the involvement of the G12 family of heterotrimeric G proteins (Gα12 and Gα13, the products of the GNA12 and GNA13 genes, respectively) in oncogenic pathways have uncovered a link between G12 signaling and cancer progression. However, despite a well characterized role of Rho GTPases, the potential role of secreted factors in the capacity of G12 signaling to promote invasion of cancer cells is just beginning to be addressed.

Methods

MDA-MB-231 and MCF10A breast cancer cell lines were employed as a model system to explore the involvement of secreted factors in G12-stimulated cell invasion. Factors secreted by cells expressing dominant-active Gα12 were identified by protein array, and their involvement in breast cancer cell invasion was assessed through both RNAi-mediated knockdown and antibody neutralization approaches. Bioinformatics analysis of the promoter elements of the identified factors suggested NF-κB elements played a role in their enhanced expression, which was tested by chromatin immunoprecipitation.

Results

We found that signaling through the Gα12 in MDA-MB-231 and MCF10A breast cancer cell lines enhances expression of interleukins (IL)-6 and ?8, and matrix metalloproteinase (MMP)-2, and that these secreted factors play a role in G12-stimulated cell invasion. Furthermore, the enhanced expression of these secreted factors was found to be facilitated by the activation of their corresponding promoters, where NF-κB seems to be one of the major regulators. Inhibition of IL-6 and IL-8, or MMP-2 activity significantly decreased Gα12-mediated cell invasion.

Conclusions

These studies confirm and extend findings that secreted factors contribute to the oncogenic potential of G12 signaling, and suggest potential therapeutic targets to control this process.

     

Three stage axillary lymphatic massage optimizes sentinel lymph node localisation using blue dye

Aims

This paper describes a simple technique of axillary and breast massage which improves the successful identification of blue sentinel nodes using patent blue dye alone.

Methods

Patent blue dye was injected in the subdermal part of the retroaroelar area in 167 patients having surgical treatment for invasive breast cancer. Three stage axillary lymphatic massage was performed prior to making the axillary incision for sentinel lymph node biopsy. All patients had completion axillary sampling or clearance.

Results

A blue lymphatic duct leading to lymph nodes of the first drainage was identified in 163 (97%) of the patients. Results are compared with 168 patients who had sentinel lymph node biopsy using blue dye without axillary massage. Allergic reactions were observed in four patients (1.2%).

Conclusion

Three stage axillary lymphatic massage improves the successful identification of a blue sentinel lymph node in breast cancer patients.

     

Akt1 inhibition promotes breast cancer metastasis through EGFR-mediated β-catenin nuclear accumulation

Background

Knockdown of Akt1 promotes Epithelial-to-Mesenchymal Transition in breast cancer cells. However, the mechanisms are not completely understood.

Methods

Western blotting, immunofluorescence, luciferase assay, real time PCR, ELISA and Matrigel invasion assay were used to investigate how Akt1 inhibition promotes breast cancer cell invasion in vitro. Mouse model of lung metastasis was used to measure in vivo efficacy of Akt inhibitor MK2206 and its combination with Gefitinib.

Results

Knockdown of Akt1 stimulated β-catenin nuclear accumulation, resulting in breast cancer cell invasion. β-catenin nuclear accumulation induced by Akt1 inhibition depended on the prolonged activation of EGFR signaling pathway in breast cancer cells. Mechanistic experiments documented that knockdown of Akt1 inactivates PIKfyve via dephosphorylating of PIKfyve at Ser³¹⁸ site, resulting in a decreased degradation of EGFR signaling pathway. Inhibition of Akt1 using MK2206 could induce an increase in the expression of EGFR and β-catenin in breast cancer cells. In addition, MK2206 at a low dosage enhance breast cancer metastasis in a mouse model of lung metastasis, while an inhibitor of EGFR tyrosine kinase Gefitinib could potentially suppress breast cancer metastasis induced by Akt1 inhibition.

Conclusion

EGFR-mediated β-catenin nuclear accumulation is critical for Akt1 inhibition-induced breast cancer metastasis.

     

Effects of α-zearalenol on the metabolome of two breast cancer cell lines by 1H-NMR approach

Introduction

Zearalenone (ZEN) is one of the most widely distributed toxins that contaminates many crops and foods. Its major metabolites are α-Zearalenol (α-zol) and β-Zearalenol. Previous studies showed that ZEN and α-zol have estrogenic properties and are able to induce growth promoting effect in breast tissues.

Objectivies

Considering that tumorigenesis is dependent on the reprogramming of cellular metabolism and that the evaluation of the cellular metabolome is useful to understand the metabolic changes that can occur during the cancer development and progression or after treatments, aim of our work is to study, for the first time, the effects of α-zol on the metabolomic profile of an estrogen positive breast cancer cell line, MCF-7, and of an estrogen negative breast cancer cell lines MDA-MB231.

Methods

Firstly, we tested the effects of α-zol on the cell viability after 24, 48 and 72 h of treatments with 10^?10, 10^?8 and 10^?6 M concentrations on breast cancer MCF-7 and MDA-MB231 cell lines in comparison to human non-cancerous breast MCF10A cell line. Then, we evaluated cell cycle progression, levels of reactive oxygen species (ROS) and the metabolomic profiling by ¹H-NMR approach on MCF-7 and MDA-MB231 before and after 72 h treatments. Principal component analysis was used to compare the obtained spectra.

Results

α-zol is resulted able to induce: (i) an increase of the cell viability on MCF-7 cells mainly after 72 h treatment, (ii) a slight decrease of the cell viability on MDA-MB231 cells, and (iii) an increase of cells in S phase of the cell cycle and of ROS only in MCF-7 cells. Moreover, the evaluation of metabolomics profile evidenced that after treatment with α-zol the levels of some metabolites increased in MCF-7 cells whereas decreased slightly in MDA-MB231 cells.

Conclusions

Our results showed that α-zol was able to increase the protein biosynthesis as well as the lipid metabolism in MCF-7 cells, and, hence, to induce an estrogen positive breast cancer progression.

     

Comparisons of gene coexpression network modules in breast cancer and ovarian cancer

Background

Breast cancer and ovarian cancer are hormone driven and are known to have some predisposition genes in common such as the two well known cancer genes BRCA1 and BRCA2. The objective of this study is to compare the coexpression network modules of both cancers, so as to infer the potential cancer-related modules.

Methods

We applied the eigen-decomposition to the matrix that integrates the gene coexpression networks of both breast cancer and ovarian cancer. With hierarchical clustering of the related eigenvectors, we obtained the network modules of both cancers simultaneously. Enrichment analysis on Gene Ontology (GO), KEGG pathway, Disease Ontology (DO), and Gene Set Enrichment Analysis (GSEA) in the identified modules was performed.

Results

We identified 43 modules that are enriched by at least one of the four types of enrichments. 31, 25, and 18 modules are enriched by GO terms, KEGG pathways, and DO terms, respectively. The structure of 29 modules in both cancers is significantly different with p-values less than 0.05, of which 25 modules have larger densities in ovarian cancer. One module was found to be significantly enriched by the terms related to breast cancer from GO, KEGG and DO enrichment. One module was found to be significantly enriched by ovarian cancer related terms.

Conclusion

Breast cancer and ovarian cancer share some common properties on the module level. Integration of both cancers helps identifying the potential cancer associated modules.

     

A lost opportunity for science: journals promote data sharing in metabolomics but do not enforce it

Introduction

Data sharing is being increasingly required by journals and has been heralded as a solution to the ‘replication crisis’.

Objectives

(i) Review data sharing policies of journals publishing the most metabolomics papers associated with open data and (ii) compare these journals’ policies to those that publish the most metabolomics papers.

Methods

A PubMed search was used to identify metabolomics papers. Metabolomics data repositories were manually searched for linked publications.

Results

Journals that support data sharing are not necessarily those with the most papers associated to open metabolomics data.

Conclusion

Further efforts are required to improve data sharing in metabolomics.

     

Network-based logistic regression integration method for biomarker identification

Background

Many mathematical and statistical models and algorithms have been proposed to do biomarker identification in recent years. However, the biomarkers inferred from different datasets suffer a lack of reproducibilities due to the heterogeneity of the data generated from different platforms or laboratories. This motivates us to develop robust biomarker identification methods by integrating multiple datasets.

Methods

In this paper, we developed an integrative method for classification based on logistic regression. Different constant terms are set in the logistic regression model to measure the heterogeneity of the samples. By minimizing the differences of the constant terms within the same dataset, both the homogeneity within the same dataset and the heterogeneity in multiple datasets can be kept. The model is formulated as an optimization problem with a network penalty measuring the differences of the constant terms. The L₁ penalty, elastic penalty and network related penalties are added to the objective function for the biomarker discovery purpose. Algorithms based on proximal Newton method are proposed to solve the optimization problem.

Results

We first applied the proposed method to the simulated datasets. Both the AUC of the prediction and the biomarker identification accuracy are improved. We then applied the method to two breast cancer gene expression datasets. By integrating both datasets, the prediction AUC is improved over directly merging the datasets and MetaLasso. And it’s comparable to the best AUC when doing biomarker identification in an individual dataset. The identified biomarkers using network related penalty for variables were further analyzed. Meaningful subnetworks enriched by breast cancer were identified.

Conclusion

A network-based integrative logistic regression model is proposed in the paper. It improves both the prediction and biomarker identification accuracy.

     

Study of the tumor microenvironment during breast cancer progression

Background

Different cells and mediators in the tumor microenvironment play important roles in the progression of breast cancer. The aim of this study was to determine the composition of the microenvironment during tumor progression in order to discover new related biomarkers and potentials for targeted therapy.

Methods

In this study, breast cancer biopsies from four different stages, and control breast biopsies were collected. Then, the mRNA expression of several markers related to different CD4⁺ T cell subsets including regulatory T cells (Treg), T helper (Th) type 1, 2 and 17 were determined. In addition, we investigated the expression of two inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators including FASL, IDO, SOCS1, VEGF, and CCR7.

Results

The results showed that the expression of Th1 and Th17 genes was decreased in tumor tissues compared to control tissues. In addition, we found that the gene expression related to these two cell subsets decreased during cancer progression. Moreover, the expression level of TNF-α increased with tumor progression.

Conclusion

We conclude that the expression of genes related to immune response and inflammation is different between tumor tissues and control tissues. In addition, this difference was perpetuated through the different stages of cancer.

     

IL-1 does not reverse the anti-proliferative effect of aspirin in breast cancer cells

Objectives

Interleukin- 1 (IL-1) is a multifunctional proinflammatory cytokine. There have been studies suggesting a role in affecting growth and invasiveness of malignant breast cells by either blocking or stimulating growth of cultured MCF-7 breast cancer cells. This effect may be mediated by induction of COX-2. Aspirin is an inhibitor of COX-2 and has been implicated, with other non-steroidal anti-inflammatory drugs (NSAIDS) in prevention and treatment of breast cancer. In this study the in vitro effects of IL-1 and aspirin on growth of MCF-7 human breast cancer cells was examined.

Methods

MCF-7 cells were treated with various concentrations of IL-1 and aspirin alone and in combination. Cell growth was assessed by cell number measurement.

Results

Aspirin significantly decreased growth rate in a dose-dependant manner, alone and as a combined treatment with IL-1 with a maximum reduction in growth rate at 300 mg/ml (P < 0.05). Treatment with IL-1 alone showed no significant effect on growth rate of MCF-7 cells (P > 0.05).

Conclusion

This study confirms that aspirin suppresses the proliferation rate of MCF-7 cells both as a single agent and in combination with IL-1. It also suggests that IL-1 alone does not stimulate or inhibit growth of MCF-7 cells.

     

Models of cell signaling uncover molecular mechanisms of high-risk neuroblastoma and predict disease outcome

Background

Despite the progress in neuroblastoma therapies the mortality of high-risk patients is still high (40–50%) and the molecular basis of the disease remains poorly known. Recently, a mathematical model was used to demonstrate that the network regulating stress signaling by the c-Jun N-terminal kinase pathway played a crucial role in survival of patients with neuroblastoma irrespective of their MYCN amplification status. This demonstrates the enormous potential of computational models of biological modules for the discovery of underlying molecular mechanisms of diseases.

Results

Since signaling is known to be highly relevant in cancer, we have used a computational model of the whole cell signaling network to understand the molecular determinants of bad prognostic in neuroblastoma. Our model produced a comprehensive view of the molecular mechanisms of neuroblastoma tumorigenesis and progression.

Conclusion

We have also shown how the activity of signaling circuits can be considered a reliable model-based prognostic biomarker.

Reviewers

This article was reviewed by Tim Beissbarth, Wenzhong Xiao and Joanna Polanska. For the full reviews, please go to the Reviewers’ comments section.

     

Identification of cancer subtypes from single-cell RNA-seq data using a consensus clustering method

Background

Human cancers are complex ecosystems composed of cells with distinct molecular signatures. Such intratumoral heterogeneity poses a major challenge to cancer diagnosis and treatment. Recent advancements of single-cell techniques such as scRNA-seq have brought unprecedented insights into cellular heterogeneity. Subsequently, a challenging computational problem is to cluster high dimensional noisy datasets with substantially fewer cells than the number of genes.

Methods

In this paper, we introduced a consensus clustering framework conCluster, for cancer subtype identification from single-cell RNA-seq data. Using an ensemble strategy, conCluster fuses multiple basic partitions to consensus clusters.

Results

Applied to real cancer scRNA-seq datasets, conCluster can more accurately detect cancer subtypes than the widely used scRNA-seq clustering methods. Further, we conducted co-expression network analysis for the identified melanoma subtypes.

Conclusions

Our analysis demonstrates that these subtypes exhibit distinct gene co-expression networks and significant gene sets with different functional enrichment.

     

The risks of cancer development in systemic lupus erythematosus (SLE) patients: a systematic review and meta-analysis

Background

Although accumulating data have suggested the development of cancer in systemic lupus erythematosus (SLE) patients, these results remain inconsistent. To examine such a putative association, this analysis reports the association between SLE and the risks of 24 cancer types.

Methods

Online databases PubMed, EMBASE, and Web of Science were searched comprehensively for eligible studies, published up to 15 May 2018. Pooled standardized incidence rates (SIRs) with 95% confidence intervals (CIs) were utilized to reveal their associations.

Results

A total of 24 eligible studies were ultimately enrolled. Our results indicated that SLE was associated with increased risk of overall cancers, cancer risk in both genders, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, leukemia, multiple myeloma, cervix, vagina/vulva, renal, bladder, esophagus, gastric, hepatobiliary, lung, oropharynx, larynx, non-melanoma skin, and thyroid cancers. Additionally, SLE could reduce the risk of prostate cancer and cutaneous melanoma; however, it was not significantly associated with breast, uterus, ovarian, pancreatic, colorectal, or brain cancers.

Conclusions

Our results shed light SLE being correlated with increased risk for 16 involved cancers and decreased risk for prostate cancer and cutaneous melanoma. This comprehensive meta-analysis provides epidemiological evidence supporting the associations between SLE and cancer risk. This evidence could be utilized to drive public policies and to help guide personalized medicine to better manage SLE and reduce associated cancer morbidity and mortality.

     

Breast cancer incidence in Greek women in relation to ABO blood groups and Rh factor

Aim

To investigate the correlation between breast cancer in Greek women and ABO blood groups.

Material-methods

In 166 female patients with breast cancer factors such as blood group, histological type, family history, presence or absence of nodal and/or distant metastases were examined. These patients had similar demographic, clinical, surgical, immunohistochemical, laboratory, and follow-up data and this group is representative of general population of women in Greece.

Results

The ductal type of breast cancer was differentially distributed in blood groups Rh (+) (P ≤ 0.001). In patients with A (+) blood group the ductal type of breast cancer was present in 49.6% of cases, in relation to the other blood groups and in AB blood group the same type occurred rarely (3.6%). Rh (+) women with positive family history were more often found in A blood group. The relative risk of metastasis in Rh (-) patients was 4.2 times higher than that in Rh (+) patients. Among Rh (+) patients, the relative risk of metastasis was 1.29 times higher in A blood group than in other blood groups.

Conclusion

Blood group A is often associated with ductal breast cancer (49.6%), in contrast to the other blood groups and particularly to blood group AB (3.6%). Blood group A and, particularly, A (-) has the worst prognosis of all.