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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.

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Fulvestrant resistance induced by 6 different ZF-TFs.(A) Drug sensitivity of fulvestrant-selected MCF7 ZF-TF-transduced cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses selected first in puromycin (the transduction selection marker) and then in fulvestrant for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. Data are representative of triplicate experiments. (B and C) Growth curves of MCF7 and T47D cells in the presence and absence of fulvestrant. Comparison of cell growth rates (cell number, mean +/− SEM, n = 8; time in days as indicated) of MCF7 and T47D cells stably transduced with control retrovirus or one of six different ZF-TF-expressing retroviruses (7, 19, 64, 70, 83 and 115) in the presence (blue line) or absence (pink line) of fulvestrant. (D) Drug sensitivity of fulvestrant non-selected MCF7 ZF-TF cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses and selected in puromycin for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. (E) Drug sensitivity of MCF cells transduced with ZF-TFs lacking the NF-KB p65 activation domain. MCF7 cells infected with retroviruses encoding ZF-TFs (7,19, 64, 70, 83 and 115) lacking the NF-KB p65 activation domain were selected in puromycin for 1 month and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining for visualization.
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pone-0021112-g002: Fulvestrant resistance induced by 6 different ZF-TFs.(A) Drug sensitivity of fulvestrant-selected MCF7 ZF-TF-transduced cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses selected first in puromycin (the transduction selection marker) and then in fulvestrant for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. Data are representative of triplicate experiments. (B and C) Growth curves of MCF7 and T47D cells in the presence and absence of fulvestrant. Comparison of cell growth rates (cell number, mean +/− SEM, n = 8; time in days as indicated) of MCF7 and T47D cells stably transduced with control retrovirus or one of six different ZF-TF-expressing retroviruses (7, 19, 64, 70, 83 and 115) in the presence (blue line) or absence (pink line) of fulvestrant. (D) Drug sensitivity of fulvestrant non-selected MCF7 ZF-TF cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses and selected in puromycin for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. (E) Drug sensitivity of MCF cells transduced with ZF-TFs lacking the NF-KB p65 activation domain. MCF7 cells infected with retroviruses encoding ZF-TFs (7,19, 64, 70, 83 and 115) lacking the NF-KB p65 activation domain were selected in puromycin for 1 month and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining for visualization.

Mentions: DNA encoding ZF-TFs was rescued by PCR from the genomic DNA of pooled fulvestrant-resistant cells. The sequences of the individual ZF-TFs were determined and 46 unique ZF-TF clones identified. These 46 unique ZF-TFs were re-cloned into the retroviral vector and converted into clonal virus stocks that were used to transduce MCF7-R73 cells. These 46 retrovirally transduced cell populations were then challenged with fulvestrant (Figure 1C). Compared with the control MCF7-R73 cells transduced with the NF-KB p65-only retrovirus (hereafter referred to as MCF-238 cells), the MCF7-R73 cells transduced with six of the 46 unique ZF-TFs demonstrated survival and growth in the presence of 100 nM fulvestrant (Figures 2A and B). The sequences of the six ZF-TF arrays conferring fulvestrant resistance are presented in Table 2. To test whether these six ZF-TFs could induce fulvestrant-resistant in cells other than MCF7-R73, T47D breast cancer cells, a second fulvestrant-sensitive ER+ human breast cancer line, were individually transduced with each of the six different ZF-TFs and challenged with fulvestrant. Similar to what was observed with MCF7-R73 cells, the six ZF-TFs conferred resistance to fulvestrant-induced growth inhibition in T47D cells (Figure 2C). Consistent with its reported mechanism of action, fulvestrant suppressed ER−alpha expression in all ZF-TF-induced resistant sublines and the sensitive MCF7-238 control subline (Figure S1). Given that fulvestrant suppressed ER− alpha expression to a level equal to or greater than that observed in the control cells, it is unlikely that drug resistance was caused by enhanced drug metabolism or active drug exclusion. In order to assess the latter possibility, we performed drug sensitivity testing as previously described [20] to 24 chemotherapeutic agents and investigational compounds (Table S1) in three different ZF-TF-transduced fulvestrant-resistant cell lines. Comparison of the control MCF7-R73 cells to the three ZF-TF fulvestrant resistant cell lines revealed no significant difference in the pattern of drug resistance and sensitivity suggesting that the ZF-TFs are not inducing a multi-drug resistance phenotype (Figure 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)

Fulvestrant resistance induced by 6 different ZF-TFs.(A) Drug sensitivity of fulvestrant-selected MCF7 ZF-TF-transduced cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses selected first in puromycin (the transduction selection marker) and then in fulvestrant for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. Data are representative of triplicate experiments. (B and C) Growth curves of MCF7 and T47D cells in the presence and absence of fulvestrant. Comparison of cell growth rates (cell number, mean +/− SEM, n = 8; time in days as indicated) of MCF7 and T47D cells stably transduced with control retrovirus or one of six different ZF-TF-expressing retroviruses (7, 19, 64, 70, 83 and 115) in the presence (blue line) or absence (pink line) of fulvestrant. (D) Drug sensitivity of fulvestrant non-selected MCF7 ZF-TF cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses and selected in puromycin for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. (E) Drug sensitivity of MCF cells transduced with ZF-TFs lacking the NF-KB p65 activation domain. MCF7 cells infected with retroviruses encoding ZF-TFs (7,19, 64, 70, 83 and 115) lacking the NF-KB p65 activation domain were selected in puromycin for 1 month and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining for visualization.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139592&req=5

pone-0021112-g002: Fulvestrant resistance induced by 6 different ZF-TFs.(A) Drug sensitivity of fulvestrant-selected MCF7 ZF-TF-transduced cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses selected first in puromycin (the transduction selection marker) and then in fulvestrant for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. Data are representative of triplicate experiments. (B and C) Growth curves of MCF7 and T47D cells in the presence and absence of fulvestrant. Comparison of cell growth rates (cell number, mean +/− SEM, n = 8; time in days as indicated) of MCF7 and T47D cells stably transduced with control retrovirus or one of six different ZF-TF-expressing retroviruses (7, 19, 64, 70, 83 and 115) in the presence (blue line) or absence (pink line) of fulvestrant. (D) Drug sensitivity of fulvestrant non-selected MCF7 ZF-TF cells. MCF7 cells transduced with one of six different ZF-TF-expressing retroviruses and selected in puromycin for 1 month were grown in the absence of fulvestrant for 7 days and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining and visualization. (E) Drug sensitivity of MCF cells transduced with ZF-TFs lacking the NF-KB p65 activation domain. MCF7 cells infected with retroviruses encoding ZF-TFs (7,19, 64, 70, 83 and 115) lacking the NF-KB p65 activation domain were selected in puromycin for 1 month and then challenged with 100 nM fulvestrant or vehicle (0.1% ethanol) for 21 days followed by crystal violet staining for visualization.
Mentions: DNA encoding ZF-TFs was rescued by PCR from the genomic DNA of pooled fulvestrant-resistant cells. The sequences of the individual ZF-TFs were determined and 46 unique ZF-TF clones identified. These 46 unique ZF-TFs were re-cloned into the retroviral vector and converted into clonal virus stocks that were used to transduce MCF7-R73 cells. These 46 retrovirally transduced cell populations were then challenged with fulvestrant (Figure 1C). Compared with the control MCF7-R73 cells transduced with the NF-KB p65-only retrovirus (hereafter referred to as MCF-238 cells), the MCF7-R73 cells transduced with six of the 46 unique ZF-TFs demonstrated survival and growth in the presence of 100 nM fulvestrant (Figures 2A and B). The sequences of the six ZF-TF arrays conferring fulvestrant resistance are presented in Table 2. To test whether these six ZF-TFs could induce fulvestrant-resistant in cells other than MCF7-R73, T47D breast cancer cells, a second fulvestrant-sensitive ER+ human breast cancer line, were individually transduced with each of the six different ZF-TFs and challenged with fulvestrant. Similar to what was observed with MCF7-R73 cells, the six ZF-TFs conferred resistance to fulvestrant-induced growth inhibition in T47D cells (Figure 2C). Consistent with its reported mechanism of action, fulvestrant suppressed ER−alpha expression in all ZF-TF-induced resistant sublines and the sensitive MCF7-238 control subline (Figure S1). Given that fulvestrant suppressed ER− alpha expression to a level equal to or greater than that observed in the control cells, it is unlikely that drug resistance was caused by enhanced drug metabolism or active drug exclusion. In order to assess the latter possibility, we performed drug sensitivity testing as previously described [20] to 24 chemotherapeutic agents and investigational compounds (Table S1) in three different ZF-TF-transduced fulvestrant-resistant cell lines. Comparison of the control MCF7-R73 cells to the three ZF-TF fulvestrant resistant cell lines revealed no significant difference in the pattern of drug resistance and sensitivity suggesting that the ZF-TFs are not inducing a multi-drug resistance phenotype (Figure 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