Limits...
Induction of stable drug resistance in human breast cancer cells using a combinatorial zinc finger transcription factor library.

Lee J, Hirsh AS, Wittner BS, Maeder ML, Singavarapu R, Lang M, Janarthanan S, McDermott U, Yajnik V, Ramaswamy S, Joung JK, Sgroi DC - PLoS ONE (2011)

Bottom Line: Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents.Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes.Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.

ABSTRACT
Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust tool for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to fulvestrant, a clinically important anti-endocrine therapeutic agent. From a diverse collection of nearly 400,000 different ZF-TFs, we isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor-positive breast cancer cell lines. Comparative gene expression profile analysis of the six different ZF-TF-transduced breast cancer cell lines revealed five distinct clusters of differentially expressed genes. One cluster was shared among all 6 ZF-TF-transduced cell lines and therefore constituted a common fulvestrant-resistant gene expression signature. Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents. Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes. Finally, we also demonstrated that the common fulvestrant-resistant signature is associated with poor prognosis by interrogating five independent, publicly available human breast cancer gene expression datasets. Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

Show MeSH

Related in: MedlinePlus

Clustering of expression profiles from ZF-TF-induced fulvestrant resistant cells.(A) MCF7 cells transduced with ZF-TF-encoding retrovirus or a control virus. Green box identifies a cluster of 72 genes that are consistently down-regulated by ZF-TF expression- these genes constitute the common fulvestrant-resistant gene expression signature. Black and purple boxes illustrate that gene clusters that are up-regulated by subsets of the six different ZF-TFs. Data represent gene expression profiles from replicate retroviral infections and microarray hybridizations. (B) Pre-existing fulvestrant resistant clones and control MCF-7 cells from Coser et al [19]. Genes are arranged in the same order as in (A). Green box shows that genes down-regulated by all the ZF-TF infections were also down-regulated in the pre-existing fulvestrant resistant sub-population. Black and purple boxes show that some of the gene clusters that were up-regulated by various ZF-TFs were also up-regulated in either two or three (F40-6-V, F40-7-V and F100-16-V) of the four previously described pre-existing fulvestrant resistant subpopulations in the MCF7 cell line [19].
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3139592&req=5

pone-0021112-g003: Clustering of expression profiles from ZF-TF-induced fulvestrant resistant cells.(A) MCF7 cells transduced with ZF-TF-encoding retrovirus or a control virus. Green box identifies a cluster of 72 genes that are consistently down-regulated by ZF-TF expression- these genes constitute the common fulvestrant-resistant gene expression signature. Black and purple boxes illustrate that gene clusters that are up-regulated by subsets of the six different ZF-TFs. Data represent gene expression profiles from replicate retroviral infections and microarray hybridizations. (B) Pre-existing fulvestrant resistant clones and control MCF-7 cells from Coser et al [19]. Genes are arranged in the same order as in (A). Green box shows that genes down-regulated by all the ZF-TF infections were also down-regulated in the pre-existing fulvestrant resistant sub-population. Black and purple boxes show that some of the gene clusters that were up-regulated by various ZF-TFs were also up-regulated in either two or three (F40-6-V, F40-7-V and F100-16-V) of the four previously described pre-existing fulvestrant resistant subpopulations in the MCF7 cell line [19].

Mentions: To determine the transcriptional alterations associated with ZF-TF-induced fulvestrant resistance, we performed comparative gene expression profiling of each of the six ZF-TF-transduced MCF7 cell lines relative to the control MCF7-238 cells. Fulvestrant exposure induces massive cell death in our control MCF7-238 cells. Thus, in order to avoid the confounding effects of cell death-associated gene expression alterations, we performed our expression profiling analysis using MCF7-238 cells and ZF-TF-induced fulvestrant resistant cells grown in the absence of fulvestrant for 3 days. Cluster analysis of the differential expression profiles of the ZF-TF-infected cells and the control cells identified a shared cluster of genes up-regulated by ZF-TF 7, 19 and 70 (Figure 3A, purple rectangle, henceforth gene cluster 1), and distinctive gene expression patterns induced by ZF-TF 64, 83 and 115 (Figure 3A, black rectangles, henceforth gene clusters 2, 3 and 4, respectively). A gene cluster consistently up-regulated in all six fulvestrant resistant cell lines was not identified. However, a 72-gene cluster was consistently down-regulated in all six fulvestrant resistant cell lines (Figure 3A, green rectangle, henceforth gene cluster 5), and this cluster constituted a common fulvestrant-resistant gene expression signature (Table S2).


Induction of stable drug resistance in human breast cancer cells using a combinatorial zinc finger transcription factor library.

Lee J, Hirsh AS, Wittner BS, Maeder ML, Singavarapu R, Lang M, Janarthanan S, McDermott U, Yajnik V, Ramaswamy S, Joung JK, Sgroi DC - PLoS ONE (2011)

Clustering of expression profiles from ZF-TF-induced fulvestrant resistant cells.(A) MCF7 cells transduced with ZF-TF-encoding retrovirus or a control virus. Green box identifies a cluster of 72 genes that are consistently down-regulated by ZF-TF expression- these genes constitute the common fulvestrant-resistant gene expression signature. Black and purple boxes illustrate that gene clusters that are up-regulated by subsets of the six different ZF-TFs. Data represent gene expression profiles from replicate retroviral infections and microarray hybridizations. (B) Pre-existing fulvestrant resistant clones and control MCF-7 cells from Coser et al [19]. Genes are arranged in the same order as in (A). Green box shows that genes down-regulated by all the ZF-TF infections were also down-regulated in the pre-existing fulvestrant resistant sub-population. Black and purple boxes show that some of the gene clusters that were up-regulated by various ZF-TFs were also up-regulated in either two or three (F40-6-V, F40-7-V and F100-16-V) of the four previously described pre-existing fulvestrant resistant subpopulations in the MCF7 cell line [19].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3139592&req=5

pone-0021112-g003: Clustering of expression profiles from ZF-TF-induced fulvestrant resistant cells.(A) MCF7 cells transduced with ZF-TF-encoding retrovirus or a control virus. Green box identifies a cluster of 72 genes that are consistently down-regulated by ZF-TF expression- these genes constitute the common fulvestrant-resistant gene expression signature. Black and purple boxes illustrate that gene clusters that are up-regulated by subsets of the six different ZF-TFs. Data represent gene expression profiles from replicate retroviral infections and microarray hybridizations. (B) Pre-existing fulvestrant resistant clones and control MCF-7 cells from Coser et al [19]. Genes are arranged in the same order as in (A). Green box shows that genes down-regulated by all the ZF-TF infections were also down-regulated in the pre-existing fulvestrant resistant sub-population. Black and purple boxes show that some of the gene clusters that were up-regulated by various ZF-TFs were also up-regulated in either two or three (F40-6-V, F40-7-V and F100-16-V) of the four previously described pre-existing fulvestrant resistant subpopulations in the MCF7 cell line [19].
Mentions: To determine the transcriptional alterations associated with ZF-TF-induced fulvestrant resistance, we performed comparative gene expression profiling of each of the six ZF-TF-transduced MCF7 cell lines relative to the control MCF7-238 cells. Fulvestrant exposure induces massive cell death in our control MCF7-238 cells. Thus, in order to avoid the confounding effects of cell death-associated gene expression alterations, we performed our expression profiling analysis using MCF7-238 cells and ZF-TF-induced fulvestrant resistant cells grown in the absence of fulvestrant for 3 days. Cluster analysis of the differential expression profiles of the ZF-TF-infected cells and the control cells identified a shared cluster of genes up-regulated by ZF-TF 7, 19 and 70 (Figure 3A, purple rectangle, henceforth gene cluster 1), and distinctive gene expression patterns induced by ZF-TF 64, 83 and 115 (Figure 3A, black rectangles, henceforth gene clusters 2, 3 and 4, respectively). A gene cluster consistently up-regulated in all six fulvestrant resistant cell lines was not identified. However, a 72-gene cluster was consistently down-regulated in all six fulvestrant resistant cell lines (Figure 3A, green rectangle, henceforth gene cluster 5), and this cluster constituted a common fulvestrant-resistant gene expression signature (Table S2).

Bottom Line: Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents.Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes.Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, Massachusetts, United States of America.

ABSTRACT
Combinatorial libraries of artificial zinc-finger transcription factors (ZF-TFs) provide a robust tool for inducing and understanding various functional components of the cancer phenotype. Herein, we utilized combinatorial ZF-TF library technology to better understand how breast cancer cells acquire resistance to fulvestrant, a clinically important anti-endocrine therapeutic agent. From a diverse collection of nearly 400,000 different ZF-TFs, we isolated six ZF-TF library members capable of inducing stable, long-term anti-endocrine drug-resistance in two independent estrogen receptor-positive breast cancer cell lines. Comparative gene expression profile analysis of the six different ZF-TF-transduced breast cancer cell lines revealed five distinct clusters of differentially expressed genes. One cluster was shared among all 6 ZF-TF-transduced cell lines and therefore constituted a common fulvestrant-resistant gene expression signature. Pathway enrichment-analysis of this common fulvestrant resistant signature also revealed significant overlap with gene sets associated with an estrogen receptor-negative-like state and with gene sets associated with drug resistance to different classes of breast cancer anti-endocrine therapeutic agents. Enrichment-analysis of the four remaining unique gene clusters revealed overlap with myb-regulated genes. Finally, we also demonstrated that the common fulvestrant-resistant signature is associated with poor prognosis by interrogating five independent, publicly available human breast cancer gene expression datasets. Our results demonstrate that artificial ZF-TF libraries can be used successfully to induce stable drug-resistance in human cancer cell lines and to identify a gene expression signature that is associated with a clinically relevant drug-resistance phenotype.

Show MeSH
Related in: MedlinePlus