The Ability to Generate Senescent Progeny as a Mechanism Underlying Breast Cancer Cell Heterogeneity

Background Breast cancer is a remarkably heterogeneous disease. Luminal, basal-like, "normal-like", and ERBB2+ subgroups were identified and were shown to have different prognoses. The mechanisms underlying this heterogeneity are poorly understood. In our study, we explored the role of cellular differentiation and senescence as a potential cause of heterogeneity. Methodology/Principal Findings A panel of breast cancer cell lines, isogenic clones, and breast tumors were used. Based on their ability to generate senescent progeny under low-density clonogenic conditions, we classified breast cancer cell lines as senescent cell progenitor (SCP) and immortal cell progenitor (ICP) subtypes. All SCP cell lines expressed estrogen receptor (ER). Loss of ER expression combined with the accumulation of p21Cip1 correlated with senescence in these cell lines. p21Cip1 knockdown, estrogen-mediated ER activation or ectopic ER overexpression protected cells against senescence. In contrast, tamoxifen triggered a robust senescence response. As ER expression has been linked to luminal differentiation, we compared the differentiation status of SCP and ICP cell lines using stem/progenitor, luminal, and myoepithelial markers. The SCP cells produced CD24+ or ER+ luminal-like and ASMA+ myoepithelial-like progeny, in addition to CD44+ stem/progenitor-like cells. In contrast, ICP cell lines acted as differentiation-defective stem/progenitor cells. Some ICP cell lines generated only CD44+/CD24-/ER-/ASMA- progenitor/stem-like cells, and others also produced CD24+/ER- luminal-like, but not ASMA+ myoepithelial-like cells. Furthermore, gene expression profiles clustered SCP cell lines with luminal A and "normal-like" tumors, and ICP cell lines with luminal B and basal-like tumors. The ICP cells displayed higher tumorigenicity in immunodeficient mice. Conclusions/Significance Luminal A and "normal-like" breast cancer cell lines were able to generate luminal-like and myoepithelial-like progeny undergoing senescence arrest. In contrast, luminal B/basal-like cell lines acted as stem/progenitor cells with defective differentiation capacities. Our findings suggest that the malignancy of breast tumors is directly correlated with stem/progenitor phenotypes and poor differentiation potential. © 2010 Mumcuoglu et al

A resampling-based meta-analysis for detection of differential gene expression in breast cancer

<p>Abstract</p> <p>Background</p> <p>Accuracy in the diagnosis of breast cancer and classification of cancer subtypes has improved over the years with the development of well-established immunohistopathological criteria. More recently, diagnostic gene-sets at the mRNA expression level have been tested as better predictors of disease state. However, breast cancer is heterogeneous in nature; thus extraction of differentially expressed gene-sets that stably distinguish normal tissue from various pathologies poses challenges. Meta-analysis of high-throughput expression data using a collection of statistical methodologies leads to the identification of robust tumor gene expression signatures.</p> <p>Methods</p> <p>A resampling-based meta-analysis strategy, which involves the use of resampling and application of distribution statistics in combination to assess the degree of significance in differential expression between sample classes, was developed. Two independent microarray datasets that contain normal breast, invasive ductal carcinoma (IDC), and invasive lobular carcinoma (ILC) samples were used for the meta-analysis. Expression of the genes, selected from the gene list for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes were tested on 10 independent primary IDC samples and matched non-tumor controls by real-time qRT-PCR. Other existing breast cancer microarray datasets were used in support of the resampling-based meta-analysis.</p> <p>Results</p> <p>The two independent microarray studies were found to be comparable, although differing in their experimental methodologies (Pearson correlation coefficient, R = 0.9389 and R = 0.8465 for ductal and lobular samples, respectively). The resampling-based meta-analysis has led to the identification of a highly stable set of genes for classification of normal breast samples and breast tumors encompassing both the ILC and IDC subtypes. The expression results of the selected genes obtained through real-time qRT-PCR supported the meta-analysis results.</p> <p>Conclusion</p> <p>The proposed meta-analysis approach has the ability to detect a set of differentially expressed genes with the least amount of within-group variability, thus providing highly stable gene lists for class prediction. Increased statistical power and stringent filtering criteria used in the present study also make identification of novel candidate genes possible and may provide further insight to improve our understanding of breast cancer development.</p

Involvement of miR-770-5p in trastuzumab response in HER2 positive breast cancer cells.

miRNAs may play effective roles in breast cancer so modulating their expression levels could have therapeutic benefits. Recent studies have found the combination of miRNA-based therapeutics with conventional drugs as promising. This study aimed to find drug-responsive miRNAs, and explore their anticancer activities in HER2+ breast cancer cells and regulatory role in the trastuzumab response. qRT-PCR-array analysis was performed with effective concentrations of tamoxifen and trastuzumab treated BT-474, SK-BR-3 and MCF-7 cells. Motility and invasion analyses were performed with wound healing and xCELLigence impedance-based assays respectively. Viability of cells following mimic transfection and drug treatment was assessed by WST-1 assay. Western blot analysis was used to assess miR-770-5p regulation of proteins and their phosphorylated forms. The clinical relevance of miR-770-5p was examined by TCGA data analysis. The qRT-PCR-array results indicated that miR-770-5p was responsive in a drug and cell line independent manner. Overexpression of miR-770-5p inhibited the motility and cell invasion through regulation of AKT and ERK proteins. Additionally, miR-770-5p potentiated the effectiveness of trastuzumab. Thus, regulating the expression level of miR-770-5p in combination with trastuzumab treatment may simultaneously inhibit the downstream elements of PI3K and MAPK signalling, thereby blocking the proliferation, motility and invasion capacities of HER2+ breast cancer cells

miR-770-5p regulates EMT and invasion in TNBC cells by targeting DNMT3A

© 2021 Elsevier Inc.MicroRNAs (miRNAs) are shown to regulate various processes in cancer like motility and invasion that are key features of the metastatic triple negative breast cancer (TNBCs). Epithelial-mesenchymal transition (EMT) is one of the well-defined cellular transitioning processes characterized with reduced E-cadherin expression and increased mesenchymal molecules such as Vimentin or Snail thereby gives the cells mobility and invasive character. Aberrant DNA methylation by DNA methyltransferases (DNMTs) plays an important role in carcinogenesis. It is well known that DNMTs are required for transcriptional silencing of tumor-associated genes. DNMT3A-induced promoter hypermethylation of E-cadherin has also been known to improve cancer metastasis. Our results indicated that miR-770-5p could downregulate Vimentin and Snail expression levels, while increasing or restoring the expression of E-Cadherin hence, leading to inhibition of EMT phenotypes along with motility and invasion. Specifically, we showed that overexpression of miR-770-5p restored the expression of E-Cadherin in MDA-MB-231 cells via directly targeting DNMT3A. We also observed the change in the spindled shapes showing the loss of mesenchymal characteristics and gain of epithelial phenotype in miR-770-5p overexpressing cells. When considered together, our results show that miR-770-5p could effectively inhibit invasion potential driven by EMT

Overexpression of β-Arrestins inhibits proliferation and motility in triple negative breast cancer cells

Abstract β-Arrestins (βArrs) are intracellular signal regulating proteins. Their expression level varies in some cancers and they have a significant impact on cancer cell function. In general, the significance of βArrs in cancer research comes from studies examining GPCR signalling. Given the diversity of different GPCR signals in cancer cell regulation, contradictory results are inevitable regarding the role of βArrs. Our approach examines the direct influence of βArrs on cellular function and gene expression profiles by changing their expression levels in breast cancer cells, MDA-MB-231 and MDA-MB-468. Reducing expression of βArr1 or βArr2 tended to increase cell proliferation and invasion whereas increasing their expression levels inhibited them. The overexpression of βArrs caused cell cycle S-phase arrest and differential expression of cell cycle genes, CDC45, BUB1, CCNB1, CCNB2, CDKN2C and reduced HER3, IGF-1R, and Snail. Regarding to the clinical relevance of our results, low expression levels of βArr1 were inversely correlated with CDC45, BUB1, CCNB1, and CCNB2 genes compared to normal tissue samples while positively correlated with poorer prognosis in breast tumours. These results indicate that βArr1 and βArr2 are significantly involved in cell cycle and anticancer signalling pathways through their influence on cell cycle genes and HER3, IGF-1R, and Snail in TNBC cells

Construction of miRNA-miRNA networks revealing the complexity of miRNA-mediated mechanisms in trastuzumab treated breast cancer cell lines

<div><p>Trastuzumab is a monoclonal antibody frequently used to prevent the progression of HER2+ breast cancers, which constitute approximately 20% of invasive breast cancers. microRNAs (miRNAs) are small, non-coding RNA molecules that are known to be involved in gene regulation. With their emerging roles in cancer, they are recently promoted as potential candidates to mediate therapeutic actions by targeting genes associated with drug response. In this study we explored miRNA-mediated regulation of trastuzumab mechanisms by identifying the important miRNAs responsible for the drug response via homogenous network analysis. Our network model enabled us to simplify the complexity of miRNA interactions by connecting them through their common pathways. We outlined the functionally relevant miRNAs by constructing pathway-based miRNA-miRNA networks in SKBR3 and BT474 cells, respectively. Identification of the most targeted genes revealed that trastuzumab responsive miRNAs favourably regulate the repression of targets with longer 3’UTR than average considered to be key elements, while the miRNA-miRNA networks highlighted central miRNAs such as hsa-miR-3976 and hsa-miR-3671 that showed strong interactions with the remaining members of the network. Furthermore, the clusters of the miRNA-miRNA networks showed that trastuzumab response was mostly established through cancer related and metabolic pathways. hsa-miR-216b was found to be the part of the most powerful interactions of metabolic pathways, which was defined in the largest clusters in both cell lines. The network based representation of miRNA-miRNA interactions through their shared pathways provided a better understanding of miRNA-mediated drug response and could be suggested for further characterization of miRNA functions.</p></div

20 responsive miRNAs with the greatest difference in expression levels in trastuzumab treated SKBR3 and BT474 cell lines.

<p>20 responsive miRNAs with the greatest difference in expression levels in trastuzumab treated SKBR3 and BT474 cell lines.</p

15 central nodes with the highest degree scores in trastuzumab treated SKBR3 and BT474 miRNA networks.

<p>All the nodes values were statistically significant with P-value <0.05.</p

The second and third largest clusters in the SKBR3 miRNA-miRNA network.

<p>(A) The most powerful interaction is consisted of one thick edge (red) presented between hsa-miR-3942-5p and hsa-miR-298. The edges are made of the cancer related pathways that control majority of the interactions between the other nodes in the cluster. The afermentioned pathways are shown in red boxes in left side. (B) In the last cluster, the interactions are determined by the upregulated miRNAs mostly and they are related to each other through path:hsa04810 (Regulation of actin cytoskeleton) and path:hsa05200 (pathways in cancer) (The edge weight minumum value = 2.9, P<0.05 for each miRNA pair, red nodes:upregulated miRNAs, green nodes: downregulated miRNAs).</p

The miRNA-miRNA network in BT474 cell.

<p>In BT474 cells, 150 trastuzumab responsive miRNAs are defined as functionally relevant with each other. Each node represents a responsive miRNA and the nodes are sized by their degree centrality scores. hsa-miR-3671 is the most central node in the network with the degree centrality score of 19 (P<0.05 for each miRNA pair, red nodes:upregulated miRNAs, green nodes: downregulated miRNAs).</p