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Altered regulation of PDK4 expression promotes antiestrogen resistance in human breast cancer cells.

Walter W, Thomalla J, Bruhn J, Fagan DH, Zehowski C, Yee D, Skildum A - Springerplus (2015)

Bottom Line: Using siRNA to knock down PDK4 expression increased TamR-MCF-7 sensitivity to ICI; in contrast adapting cells to growth in glucose depleted media did not affect ICI sensitivity.Wild type MCF-7 cells are reported to be heterozygous for a G to A mutation that results in a substitution of threonine for alanine near PDK4's catalytic site.We found loss of heterozygosity in TamR-MCF-7 cells; TamR-MCF-7 are homozygous for the wild type allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Minnesota Medical School, Duluth Campus, Duluth, MN USA.

ABSTRACT
Acquired or de novo resistance to the selective estrogen receptor modulators tamoxifen and fulvestrant (ICI) is a major barrier to successful treatment of breast cancer. Gene expression patterns in tamoxifen resistant (TamR-MCF-7) cells were compared to their parental cells (MCF-7L) to identify an aberrantly regulated metabolic pathway. TamR-MCF-7 cells are cross resistant to ICI and doxorubicin, and have increased mitochondrial DNA. A small subset of genes had altered expression in TamR-MCF-7 relative to MCF-7L cells. One of the genes, pyruvate dehydrogenase kinase-4 (PDK4), phosphorylates pyruvate dehydrogenase (PDH). PDK4 expression was elevated in TamR-MCF-7 cells; this result was also observed in a second model of acquired antiestrogen resistance. PDK4 expression is controlled in part by glucocorticoid response elements in the PDK4 gene promoter. In MCF-7L cells, PDK4 mRNA expression was insensitive to glucocorticoid receptor agonists, while dexamethasone dramatically increased PDK4 expression in TamR-MCF-7 cells. Using siRNA to knock down PDK4 expression increased TamR-MCF-7 sensitivity to ICI; in contrast adapting cells to growth in glucose depleted media did not affect ICI sensitivity. Despite TamR-MCF-7 cells high levels of PDK4 mRNA relative to MCF-7L, TamR-MCF-7 cells have increased PDH activity. Wild type MCF-7 cells are reported to be heterozygous for a G to A mutation that results in a substitution of threonine for alanine near PDK4's catalytic site. We found loss of heterozygosity in TamR-MCF-7 cells; TamR-MCF-7 are homozygous for the wild type allele. These data support a role for altered regulation of PDH by PDK4 and altered substrate utilization in the development of drug resistance in human breast cancer cells.

No MeSH data available.


Related in: MedlinePlus

Tamoxifen resistant breast cancer cells have elevated mitochondrial DNA and cross resistance to ICI and doxorubicin. a MCF-7L and TamR-MCF-7 cells were treated for 2 days in 1 nM ICI or vehicle control (DMSO), then harvested and fixed in ethanol. Cell cycle phase distribution was determined by flow cytometric measurement of propidium iodide staining per cell. The average percentage of G1, S and G2/M cells are shown; error bars indicate one standard deviation (n = 3). b MCF-7L and TamR-MCF-7 cells were treated ±0.02 mm doxorubicin. After 4 days, total cellular mass was measured by sulforhodamine B staining. The average SRB staining is shown; error bars indicate one standard deviation (n = 6). c MCF-7L and TamR-MCF-7 cells were treated for 2 days with 10 nm 4-hydroxytamoxifen or control (EtOH). Genomic DNA was extracted and used as template for detection of cytochrome B DNA on the mitochondrial genome and pyruvate kinase DNA on the nuclear genome. The average ratio of cytochrome B to pyruvate kinase is shown; error bars indicate one standard deviation (n = 3)
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Fig2: Tamoxifen resistant breast cancer cells have elevated mitochondrial DNA and cross resistance to ICI and doxorubicin. a MCF-7L and TamR-MCF-7 cells were treated for 2 days in 1 nM ICI or vehicle control (DMSO), then harvested and fixed in ethanol. Cell cycle phase distribution was determined by flow cytometric measurement of propidium iodide staining per cell. The average percentage of G1, S and G2/M cells are shown; error bars indicate one standard deviation (n = 3). b MCF-7L and TamR-MCF-7 cells were treated ±0.02 mm doxorubicin. After 4 days, total cellular mass was measured by sulforhodamine B staining. The average SRB staining is shown; error bars indicate one standard deviation (n = 6). c MCF-7L and TamR-MCF-7 cells were treated for 2 days with 10 nm 4-hydroxytamoxifen or control (EtOH). Genomic DNA was extracted and used as template for detection of cytochrome B DNA on the mitochondrial genome and pyruvate kinase DNA on the nuclear genome. The average ratio of cytochrome B to pyruvate kinase is shown; error bars indicate one standard deviation (n = 3)

Mentions: To determine whether cells selected for resistance to the SERM 4-hydroxytamoxifen (TamR-MCF-7) had cross resistance to the pure antiestrogen fulvestrant (ICI), cell cycle phase distribution was compared in TamR-MCF-7 and parental cells (MCF-7L) after treatment with vehicle or 1 nM (ICI) in the presence of 5 % fetal bovine serum. Vehicle treated TamR-MCF-7 cells had an increased G1 phase population and decreased S phase population compared with vehicle treated MCF-7L cells, reflecting a slower rate of proliferation. While MCF-7L cells had an increased fraction of cells in the G1 phase and a decreased fraction in S phase, TamR-MCF-7 cell cycle phase distribution was unaffected by treatment with ICI (Fig. 2a), indicating cross resistance to ICI developed with selection for 4-hyroxytamoxifen resistance. Because resistance to antiestrogen has been associated with resistance to chemotherapy agents (Skildum et al. 2011), we then compared sensitivity to doxorubicin by measuring cell mass after treatment. While 0.02 μM doxorubicin resulted in near complete cytotoxicity of MCF-7L cells, TamR-MCF-7 cell growth was not affected (Fig. 2b).Fig. 2


Altered regulation of PDK4 expression promotes antiestrogen resistance in human breast cancer cells.

Walter W, Thomalla J, Bruhn J, Fagan DH, Zehowski C, Yee D, Skildum A - Springerplus (2015)

Tamoxifen resistant breast cancer cells have elevated mitochondrial DNA and cross resistance to ICI and doxorubicin. a MCF-7L and TamR-MCF-7 cells were treated for 2 days in 1 nM ICI or vehicle control (DMSO), then harvested and fixed in ethanol. Cell cycle phase distribution was determined by flow cytometric measurement of propidium iodide staining per cell. The average percentage of G1, S and G2/M cells are shown; error bars indicate one standard deviation (n = 3). b MCF-7L and TamR-MCF-7 cells were treated ±0.02 mm doxorubicin. After 4 days, total cellular mass was measured by sulforhodamine B staining. The average SRB staining is shown; error bars indicate one standard deviation (n = 6). c MCF-7L and TamR-MCF-7 cells were treated for 2 days with 10 nm 4-hydroxytamoxifen or control (EtOH). Genomic DNA was extracted and used as template for detection of cytochrome B DNA on the mitochondrial genome and pyruvate kinase DNA on the nuclear genome. The average ratio of cytochrome B to pyruvate kinase is shown; error bars indicate one standard deviation (n = 3)
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Fig2: Tamoxifen resistant breast cancer cells have elevated mitochondrial DNA and cross resistance to ICI and doxorubicin. a MCF-7L and TamR-MCF-7 cells were treated for 2 days in 1 nM ICI or vehicle control (DMSO), then harvested and fixed in ethanol. Cell cycle phase distribution was determined by flow cytometric measurement of propidium iodide staining per cell. The average percentage of G1, S and G2/M cells are shown; error bars indicate one standard deviation (n = 3). b MCF-7L and TamR-MCF-7 cells were treated ±0.02 mm doxorubicin. After 4 days, total cellular mass was measured by sulforhodamine B staining. The average SRB staining is shown; error bars indicate one standard deviation (n = 6). c MCF-7L and TamR-MCF-7 cells were treated for 2 days with 10 nm 4-hydroxytamoxifen or control (EtOH). Genomic DNA was extracted and used as template for detection of cytochrome B DNA on the mitochondrial genome and pyruvate kinase DNA on the nuclear genome. The average ratio of cytochrome B to pyruvate kinase is shown; error bars indicate one standard deviation (n = 3)
Mentions: To determine whether cells selected for resistance to the SERM 4-hydroxytamoxifen (TamR-MCF-7) had cross resistance to the pure antiestrogen fulvestrant (ICI), cell cycle phase distribution was compared in TamR-MCF-7 and parental cells (MCF-7L) after treatment with vehicle or 1 nM (ICI) in the presence of 5 % fetal bovine serum. Vehicle treated TamR-MCF-7 cells had an increased G1 phase population and decreased S phase population compared with vehicle treated MCF-7L cells, reflecting a slower rate of proliferation. While MCF-7L cells had an increased fraction of cells in the G1 phase and a decreased fraction in S phase, TamR-MCF-7 cell cycle phase distribution was unaffected by treatment with ICI (Fig. 2a), indicating cross resistance to ICI developed with selection for 4-hyroxytamoxifen resistance. Because resistance to antiestrogen has been associated with resistance to chemotherapy agents (Skildum et al. 2011), we then compared sensitivity to doxorubicin by measuring cell mass after treatment. While 0.02 μM doxorubicin resulted in near complete cytotoxicity of MCF-7L cells, TamR-MCF-7 cell growth was not affected (Fig. 2b).Fig. 2

Bottom Line: Using siRNA to knock down PDK4 expression increased TamR-MCF-7 sensitivity to ICI; in contrast adapting cells to growth in glucose depleted media did not affect ICI sensitivity.Wild type MCF-7 cells are reported to be heterozygous for a G to A mutation that results in a substitution of threonine for alanine near PDK4's catalytic site.We found loss of heterozygosity in TamR-MCF-7 cells; TamR-MCF-7 are homozygous for the wild type allele.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, University of Minnesota Medical School, Duluth Campus, Duluth, MN USA.

ABSTRACT
Acquired or de novo resistance to the selective estrogen receptor modulators tamoxifen and fulvestrant (ICI) is a major barrier to successful treatment of breast cancer. Gene expression patterns in tamoxifen resistant (TamR-MCF-7) cells were compared to their parental cells (MCF-7L) to identify an aberrantly regulated metabolic pathway. TamR-MCF-7 cells are cross resistant to ICI and doxorubicin, and have increased mitochondrial DNA. A small subset of genes had altered expression in TamR-MCF-7 relative to MCF-7L cells. One of the genes, pyruvate dehydrogenase kinase-4 (PDK4), phosphorylates pyruvate dehydrogenase (PDH). PDK4 expression was elevated in TamR-MCF-7 cells; this result was also observed in a second model of acquired antiestrogen resistance. PDK4 expression is controlled in part by glucocorticoid response elements in the PDK4 gene promoter. In MCF-7L cells, PDK4 mRNA expression was insensitive to glucocorticoid receptor agonists, while dexamethasone dramatically increased PDK4 expression in TamR-MCF-7 cells. Using siRNA to knock down PDK4 expression increased TamR-MCF-7 sensitivity to ICI; in contrast adapting cells to growth in glucose depleted media did not affect ICI sensitivity. Despite TamR-MCF-7 cells high levels of PDK4 mRNA relative to MCF-7L, TamR-MCF-7 cells have increased PDH activity. Wild type MCF-7 cells are reported to be heterozygous for a G to A mutation that results in a substitution of threonine for alanine near PDK4's catalytic site. We found loss of heterozygosity in TamR-MCF-7 cells; TamR-MCF-7 are homozygous for the wild type allele. These data support a role for altered regulation of PDH by PDK4 and altered substrate utilization in the development of drug resistance in human breast cancer cells.

No MeSH data available.


Related in: MedlinePlus