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Expression profiles of CD24-/CD44+/ESA+ population in MDA-MB-231 and its highly metastatic variants

ABSTRACT: Breast cancer is a curable disease if it is diagnosed at an early stage. However, only little options are left once the tumor is metastasized to distant organs, and more than 90% of breast cancer death is attributed to metastatic disease. The process of metastasis is highly complex and involves many steps for successful colonization of tumor cells at a target organ. According to the cancer stem cell (CSC) theory, which still remains a hypothesis, these metastatic cells must have stem cell-like capability for their self-renewal in addition to their invasive ability. Therefore, it has been predicted that a “metastatic stem cell”, which is distinct from a cancer stem cell, must exist in the primary tumor mass. To identify genes that are involved in metastasis of CSCs, we isolated CSC populations from a well-established model cell line of breast cancer, MDA-MB231, and that of highly metastatic variants, 231BoM-1833 and 231BrM-2a, using CD24, CD44 and EpCAM (ESA), which have been identified as surface markers for CSCs in breast cancers. Overall yield of CSCs from these cells ranged from 2% to 4%. We then performed global expression profile analysis for these CSCs using the Affymetrix Human Gene 1.0ST array. CSC populations (CD24-/CD44+/ESA+) from MDA-MB231, 231BoM-1833 and 231BrM-2a were isolated by magnetic-activated cell sorting (MACS) using specific antibodies to these surface markers. The total RNA was isolated from the CSC populations using the RNeasy RNA isolation kit (Qiagen). The RNA was then converted to cDNA and they were hybridized to the Human Gene 1.0ST chip (Affymetrix). The data was normalized using the RMA algorithm of the Expression Console software (Affymetrix). A comparison of transcriptional profiles was then performed in CSCs of highly metastatic cell lines (231BoM-1833 and 231BrM-2a) compared to the CSCs of MDA-MB-231.

ORGANISM(S): Homo sapiens

SUBMITTER: Okuda Hiroshi

PROVIDER: S-ECPF-GEOD-25976 | biostudies-other |

REPOSITORIES: biostudies-other

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Project description:Breast cancer is an important worldwide public health concern. The incidence rate of breast cancer increases every year. The primary cause of death is metastasis, a process by which cancer cells spread from a primary site to secondary organs. MicroRNAs (miRs/miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level. Dysregulation of certain miRNAs is involved in carcinogenesis, cancer cell proliferation and metastasis. Therefore, the present study assessed miRNAs associated with breast cancer metastasis using two breast cancer cell lines, the low-metastatic MCF-7 and the highly metastatic MDA-MB-231. miRNA array analysis of both cell lines indicated that 46 miRNAs were differentially expressed when compared between the two cell lines. A total of 16 miRNAs were upregulated in MDA-MB-231 compared with MCF-7 cells, which suggested that their expression levels may be associated with the highly invasive phenotype of MDA-MB-231 cells. Among these miRNAs, miR-222-3p was selected for further study and its expression was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Under both non-adherent and adherent culture conditions, the expression levels of miR-222-3p in the MDA-MB-231 cell line were higher than those noted in the MCF-7 cell line under the same conditions. Suppression of endogenous miR-222-3p expression in MDA-MB-231 cells using a miR-222-3p inhibitor resulted in a 20-40% reduction in proliferation, and a ~30% reduction in migration, which suggested that the aggressive phenotype of MDA-MB-231 cells was partly regulated by miR-222-3p. Bioinformatic analysis of miR-222-3p using TargetScan 8.0, miRDB and PicTar identified 25 common mRNA targets, such as cyclin-dependent kinase inhibitor 1B, ADP-ribosylation factor 4, iroquois homeobox 5 and Bcl2 modifying factor. The results of the present study indicated that miR-222-3p was potentially associated with the proliferation and migratory ability of the MDA-MB-231 cell line.

Project description:BackgroundIncreased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.MethodsThe basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.ResultsLow and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.ConclusionProgressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.

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Expression profiles of CD24-/CD44+/ESA+ population in MDA-MB-231 and its highly metastatic variants

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