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Delineating genetic alterations for tumor progression in the MCF10A series of breast cancer cell lines.

Kadota M, Yang HH, Gomez B, Sato M, Clifford RJ, Meerzaman D, Dunn BK, Wakefield LM, Lee MP - PLoS ONE (2010)

Bottom Line: Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a.We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line.These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Population Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
To gain insight into the role of genomic alterations in breast cancer progression, we conducted a comprehensive genetic characterization of a series of four cell lines derived from MCF10A. MCF10A is an immortalized mammary epithelial cell line (MEC); MCF10AT is a premalignant cell line generated from MCF10A by transformation with an activated HRAS gene; MCF10CA1h and MCF10CA1a, both derived from MCF10AT xenografts, form well-differentiated and poorly-differentiated malignant tumors in the xenograft models, respectively. We analyzed DNA copy number variation using the Affymetrix 500 K SNP arrays with the goal of identifying gene-specific amplification and deletion events. In addition to a previously noted deletion in the CDKN2A locus, our studies identified MYC amplification in all four cell lines. Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a. We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line. Furthermore, we found that RUNX1 expression was reduced in high-grade primary breast tumors compared to low/mid-grade tumors. Mutational analysis identified an activating PIK3CA mutation, H1047R, in MCF10CA1h and MCF10CA1a, which correlates with an increase of AKT1 phosphorylation at Ser473 and Thr308. Furthermore, we showed increased expression levels for genes located in the genomic regions with copy number gain. Thus, our genetic analyses have uncovered sequential molecular events that delineate breast tumor progression. These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors.

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Expression of RUNX1 gene in primary breast tumors.Figure 3A shows qRT-PCR analysis of the RUNX1 mRNA expressions in primary breast tumors and the adjacent normal samples. 25 of 29 our analyzed tumors have histological grade information. High histological grade tumors (n = 16) have significant reduction of RUNX1 expression compared to low/mid grade tumors (n = 9) or to the adjacent normal samples (n = 11). Data are summarized by box plots. The box represents gene expression values between 1st and 3rd quartiles; the line denotes median. Gene expression difference between high-grade (grade 3) and low/mid-grade (grade 1 plus grade 2) is significant (pvalue = 0.044, by t-test). Figure 3B shows RUNX1 mRNA expression in Ivshina breast microarray dataset. Ivshina breast dataset consists of 289 tumors (68 Grade 1, 166 Grade 2, and 55 Grade 3). Affymetrix U133A array expression data showed progressive reduction of RUNX1 gene (reporter: 210365_at) from Grade 1 to Grade 2 to Grade 3. The progressive reduction of RUNX1 expression in higher grade tumors is highly significant (p-value = 3×10−13 by linear regression analysis). Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for this analysis.
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pone-0009201-g003: Expression of RUNX1 gene in primary breast tumors.Figure 3A shows qRT-PCR analysis of the RUNX1 mRNA expressions in primary breast tumors and the adjacent normal samples. 25 of 29 our analyzed tumors have histological grade information. High histological grade tumors (n = 16) have significant reduction of RUNX1 expression compared to low/mid grade tumors (n = 9) or to the adjacent normal samples (n = 11). Data are summarized by box plots. The box represents gene expression values between 1st and 3rd quartiles; the line denotes median. Gene expression difference between high-grade (grade 3) and low/mid-grade (grade 1 plus grade 2) is significant (pvalue = 0.044, by t-test). Figure 3B shows RUNX1 mRNA expression in Ivshina breast microarray dataset. Ivshina breast dataset consists of 289 tumors (68 Grade 1, 166 Grade 2, and 55 Grade 3). Affymetrix U133A array expression data showed progressive reduction of RUNX1 gene (reporter: 210365_at) from Grade 1 to Grade 2 to Grade 3. The progressive reduction of RUNX1 expression in higher grade tumors is highly significant (p-value = 3×10−13 by linear regression analysis). Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for this analysis.

Mentions: We recently reported DNA copy number analysis for 161 primary breast tumors using the Affymetrix 500 K SNP arrays [14]. Using this dataset, we analyzed DNA copy number of RUNX1 in the primary tumors. Our analysis found no evidence of genomic alteration of the RUNX1 gene. However, tumor heterogeneity poses a significant challenge to the detection of intragenic deletions that are present in only a portion of a tumor sample. To investigate gene expression of RUNX1, we performed quantitative RT-PCR (qRT-PCR) analysis for 29 primary tumors from which we were able to isolate high quality RNA. We also did qRT-PCR for 11 RNA samples isolated from normal tissues adjacent to the tumors. We found that RUNX1 expression was down-regulated significantly in high-grade tumors compared to low/mid-grade tumors (Figure 3A, p-value = 0.044 by t-test). Thus, our qRT-PCR analysis of RUNX1 expression in primary tumors corroborated the study in the MCF10A series of cell lines and supported the idea that RUNX1 is a potential tumor suppressor gene involved in carcinogenic progression in this model of breast cancer.


Delineating genetic alterations for tumor progression in the MCF10A series of breast cancer cell lines.

Kadota M, Yang HH, Gomez B, Sato M, Clifford RJ, Meerzaman D, Dunn BK, Wakefield LM, Lee MP - PLoS ONE (2010)

Expression of RUNX1 gene in primary breast tumors.Figure 3A shows qRT-PCR analysis of the RUNX1 mRNA expressions in primary breast tumors and the adjacent normal samples. 25 of 29 our analyzed tumors have histological grade information. High histological grade tumors (n = 16) have significant reduction of RUNX1 expression compared to low/mid grade tumors (n = 9) or to the adjacent normal samples (n = 11). Data are summarized by box plots. The box represents gene expression values between 1st and 3rd quartiles; the line denotes median. Gene expression difference between high-grade (grade 3) and low/mid-grade (grade 1 plus grade 2) is significant (pvalue = 0.044, by t-test). Figure 3B shows RUNX1 mRNA expression in Ivshina breast microarray dataset. Ivshina breast dataset consists of 289 tumors (68 Grade 1, 166 Grade 2, and 55 Grade 3). Affymetrix U133A array expression data showed progressive reduction of RUNX1 gene (reporter: 210365_at) from Grade 1 to Grade 2 to Grade 3. The progressive reduction of RUNX1 expression in higher grade tumors is highly significant (p-value = 3×10−13 by linear regression analysis). Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for this analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2821407&req=5

pone-0009201-g003: Expression of RUNX1 gene in primary breast tumors.Figure 3A shows qRT-PCR analysis of the RUNX1 mRNA expressions in primary breast tumors and the adjacent normal samples. 25 of 29 our analyzed tumors have histological grade information. High histological grade tumors (n = 16) have significant reduction of RUNX1 expression compared to low/mid grade tumors (n = 9) or to the adjacent normal samples (n = 11). Data are summarized by box plots. The box represents gene expression values between 1st and 3rd quartiles; the line denotes median. Gene expression difference between high-grade (grade 3) and low/mid-grade (grade 1 plus grade 2) is significant (pvalue = 0.044, by t-test). Figure 3B shows RUNX1 mRNA expression in Ivshina breast microarray dataset. Ivshina breast dataset consists of 289 tumors (68 Grade 1, 166 Grade 2, and 55 Grade 3). Affymetrix U133A array expression data showed progressive reduction of RUNX1 gene (reporter: 210365_at) from Grade 1 to Grade 2 to Grade 3. The progressive reduction of RUNX1 expression in higher grade tumors is highly significant (p-value = 3×10−13 by linear regression analysis). Oncomine™ (Compendia Bioscience, Ann Arbor, MI) was used for this analysis.
Mentions: We recently reported DNA copy number analysis for 161 primary breast tumors using the Affymetrix 500 K SNP arrays [14]. Using this dataset, we analyzed DNA copy number of RUNX1 in the primary tumors. Our analysis found no evidence of genomic alteration of the RUNX1 gene. However, tumor heterogeneity poses a significant challenge to the detection of intragenic deletions that are present in only a portion of a tumor sample. To investigate gene expression of RUNX1, we performed quantitative RT-PCR (qRT-PCR) analysis for 29 primary tumors from which we were able to isolate high quality RNA. We also did qRT-PCR for 11 RNA samples isolated from normal tissues adjacent to the tumors. We found that RUNX1 expression was down-regulated significantly in high-grade tumors compared to low/mid-grade tumors (Figure 3A, p-value = 0.044 by t-test). Thus, our qRT-PCR analysis of RUNX1 expression in primary tumors corroborated the study in the MCF10A series of cell lines and supported the idea that RUNX1 is a potential tumor suppressor gene involved in carcinogenic progression in this model of breast cancer.

Bottom Line: Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a.We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line.These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Population Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland, United States of America.

ABSTRACT
To gain insight into the role of genomic alterations in breast cancer progression, we conducted a comprehensive genetic characterization of a series of four cell lines derived from MCF10A. MCF10A is an immortalized mammary epithelial cell line (MEC); MCF10AT is a premalignant cell line generated from MCF10A by transformation with an activated HRAS gene; MCF10CA1h and MCF10CA1a, both derived from MCF10AT xenografts, form well-differentiated and poorly-differentiated malignant tumors in the xenograft models, respectively. We analyzed DNA copy number variation using the Affymetrix 500 K SNP arrays with the goal of identifying gene-specific amplification and deletion events. In addition to a previously noted deletion in the CDKN2A locus, our studies identified MYC amplification in all four cell lines. Additionally, we found intragenic deletions in several genes, including LRP1B in MCF10CA1h and MCF10CA1a, FHIT and CDH13 in MCF10CA1h, and RUNX1 in MCF10CA1a. We confirmed the deletion of RUNX1 in MCF10CA1a by DNA and RNA analyses, as well as the absence of the RUNX1 protein in that cell line. Furthermore, we found that RUNX1 expression was reduced in high-grade primary breast tumors compared to low/mid-grade tumors. Mutational analysis identified an activating PIK3CA mutation, H1047R, in MCF10CA1h and MCF10CA1a, which correlates with an increase of AKT1 phosphorylation at Ser473 and Thr308. Furthermore, we showed increased expression levels for genes located in the genomic regions with copy number gain. Thus, our genetic analyses have uncovered sequential molecular events that delineate breast tumor progression. These events include CDKN2A deletion and MYC amplification in immortalization, HRAS activation in transformation, PIK3CA activation in the formation of malignant tumors, and RUNX1 deletion associated with poorly-differentiated malignant tumors.

Show MeSH
Related in: MedlinePlus