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PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes.

Git A, Spiteri I, Blenkiron C, Dunning MJ, Pole JC, Chin SF, Wang Y, Smith J, Livesey FJ, Caldas C - Breast Cancer Res. (2008)

Bottom Line: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire.We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells.Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. Anna.Git@cancer.org.uk

ABSTRACT

Introduction: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation.

Methods: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction.

Results: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components.

Conclusion: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.

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Analysis of microRNA (miRNA) gene expression in breast cancer cell lines and normal tissue. (a) Global miRNA heatmap. Log2 ratios of spots with intensity (A values) greater than 6 were used in CIMminer clustering analysis using correlation distances. (b) Heatmap of differentially expressed miRNAs. miRNA genes for which at least one value differed with log2 greater than 2.5 from the pool (either upregulated or downregulated) were used in CIMminer clustering analysis using correlation distance (upper). The PN miRNA cluster is indicated by a star and is enlarged (lower). The cell type origin (B, basal; L, luminal; N, normal; S?, possible stem cell) and the status of oestrogen receptor/progesterone receptor (ER/PR) are indicated on the left. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.
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Figure 2: Analysis of microRNA (miRNA) gene expression in breast cancer cell lines and normal tissue. (a) Global miRNA heatmap. Log2 ratios of spots with intensity (A values) greater than 6 were used in CIMminer clustering analysis using correlation distances. (b) Heatmap of differentially expressed miRNAs. miRNA genes for which at least one value differed with log2 greater than 2.5 from the pool (either upregulated or downregulated) were used in CIMminer clustering analysis using correlation distance (upper). The PN miRNA cluster is indicated by a star and is enlarged (lower). The cell type origin (B, basal; L, luminal; N, normal; S?, possible stem cell) and the status of oestrogen receptor/progesterone receptor (ER/PR) are indicated on the left. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.

Mentions: Using an in-house array, we examined the expression of 171 miRNAs with a pooled cancer cell line RNA sample as reference. Clustering analysis of the 129 miRNAs with reliable readings was performed using several algorithms. A typical outcome is presented in Figure 2a. The main feature consistent with all algorithms used was the separation of normal breast tissue and PMC42 from all other cell lines. Importantly, the separation of PMC42 from other cell lines held true even when the normal sample was omitted from the analysis (data not shown), suggesting that its profile is inherently different and its separation is not driven by a minor similarity to the vastly different normal sample. On the other branch, the clustering did not partition according to tissue type (colon versus breast), progenitor type (normal versus cancer), ER/PR status, or luminal/basal nature of the cell, regardless of the algorithm employed. This was strikingly emphasised by MT3, an ER/PR-negative colon cancer cell line, sharing a branch with HB4a, an ER/PR-positive immortalised breast luminal epithelium line. It is also noteworthy that, although the mRNA expression of HCC1419 was closest to MDAMB361, its miRNA profile was most similar to that of MCF7, suggesting that the two profiles are not co-regulated.


PMC42, a breast progenitor cancer cell line, has normal-like mRNA and microRNA transcriptomes.

Git A, Spiteri I, Blenkiron C, Dunning MJ, Pole JC, Chin SF, Wang Y, Smith J, Livesey FJ, Caldas C - Breast Cancer Res. (2008)

Analysis of microRNA (miRNA) gene expression in breast cancer cell lines and normal tissue. (a) Global miRNA heatmap. Log2 ratios of spots with intensity (A values) greater than 6 were used in CIMminer clustering analysis using correlation distances. (b) Heatmap of differentially expressed miRNAs. miRNA genes for which at least one value differed with log2 greater than 2.5 from the pool (either upregulated or downregulated) were used in CIMminer clustering analysis using correlation distance (upper). The PN miRNA cluster is indicated by a star and is enlarged (lower). The cell type origin (B, basal; L, luminal; N, normal; S?, possible stem cell) and the status of oestrogen receptor/progesterone receptor (ER/PR) are indicated on the left. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2481505&req=5

Figure 2: Analysis of microRNA (miRNA) gene expression in breast cancer cell lines and normal tissue. (a) Global miRNA heatmap. Log2 ratios of spots with intensity (A values) greater than 6 were used in CIMminer clustering analysis using correlation distances. (b) Heatmap of differentially expressed miRNAs. miRNA genes for which at least one value differed with log2 greater than 2.5 from the pool (either upregulated or downregulated) were used in CIMminer clustering analysis using correlation distance (upper). The PN miRNA cluster is indicated by a star and is enlarged (lower). The cell type origin (B, basal; L, luminal; N, normal; S?, possible stem cell) and the status of oestrogen receptor/progesterone receptor (ER/PR) are indicated on the left. PN miRNA, microRNA specifically expressed in PMC42 and Normal breast tissue.
Mentions: Using an in-house array, we examined the expression of 171 miRNAs with a pooled cancer cell line RNA sample as reference. Clustering analysis of the 129 miRNAs with reliable readings was performed using several algorithms. A typical outcome is presented in Figure 2a. The main feature consistent with all algorithms used was the separation of normal breast tissue and PMC42 from all other cell lines. Importantly, the separation of PMC42 from other cell lines held true even when the normal sample was omitted from the analysis (data not shown), suggesting that its profile is inherently different and its separation is not driven by a minor similarity to the vastly different normal sample. On the other branch, the clustering did not partition according to tissue type (colon versus breast), progenitor type (normal versus cancer), ER/PR status, or luminal/basal nature of the cell, regardless of the algorithm employed. This was strikingly emphasised by MT3, an ER/PR-negative colon cancer cell line, sharing a branch with HB4a, an ER/PR-positive immortalised breast luminal epithelium line. It is also noteworthy that, although the mRNA expression of HCC1419 was closest to MDAMB361, its miRNA profile was most similar to that of MCF7, suggesting that the two profiles are not co-regulated.

Bottom Line: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire.We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells.Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Oncology, Breast Cancer Functional Genomics Laboratory, Cancer Research UK Cambridge Research Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. Anna.Git@cancer.org.uk

ABSTRACT

Introduction: The use of cultured cell lines as model systems for normal tissue is limited by the molecular alterations accompanying the immortalisation process, including changes in the mRNA and microRNA (miRNA) repertoire. Therefore, identification of cell lines with normal-like expression profiles is of paramount importance in studies of normal gene regulation.

Methods: The mRNA and miRNA expression profiles of several breast cell lines of cancerous or normal origin were measured using printed slide arrays, Luminex bead arrays, and real-time reverse transcription-polymerase chain reaction.

Results: We demonstrate that the mRNA expression profiles of two breast cell lines are similar to that of normal breast tissue: HB4a, immortalised normal breast epithelium, and PMC42, a breast cancer cell line that retains progenitor pluripotency allowing in-culture differentiation to both secretory and myoepithelial fates. In contrast, only PMC42 exhibits a normal-like miRNA expression profile. We identified a group of miRNAs that are highly expressed in normal breast tissue and PMC42 but are lost in all other cancerous and normal-origin breast cell lines and observed a similar loss in immortalised lymphoblastoid cell lines compared with healthy uncultured B cells. Moreover, like tumour suppressor genes, these miRNAs are lost in a variety of tumours. We show that the mechanism leading to the loss of these miRNAs in breast cancer cell lines has genomic, transcriptional, and post-transcriptional components.

Conclusion: We propose that, despite its neoplastic origin, PMC42 is an excellent molecular model for normal breast epithelium, providing a unique tool to study breast differentiation and the function of key miRNAs that are typically lost in cancer.

Show MeSH
Related in: MedlinePlus