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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

Glucose deprivation results in increased PDK4 protein without a change in mRNA. MCF-7L and TamR-MCF-7 cells were treated for 2 days in media containing 4.5 mM glucose or 4.5 mM galactose. a Total RNA was extracted, reverse transcribed, and the products used as template for qPCR amplification of PDK4 and b-actin. The average ratio is shown; error bars indicate one standard deviation (n = 3 biological replicates). b Whole cell lysates were prepared, and PDK4 and actin protein were visualized by western blotting, first for PDK4 (upper panel), then for actin (lower panel). The PDK4 band is indicated by a red arrow; background bands are indicated by black arrows. The actin band is indicated by a blue arrow
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Fig1: Glucose deprivation results in increased PDK4 protein without a change in mRNA. MCF-7L and TamR-MCF-7 cells were treated for 2 days in media containing 4.5 mM glucose or 4.5 mM galactose. a Total RNA was extracted, reverse transcribed, and the products used as template for qPCR amplification of PDK4 and b-actin. The average ratio is shown; error bars indicate one standard deviation (n = 3 biological replicates). b Whole cell lysates were prepared, and PDK4 and actin protein were visualized by western blotting, first for PDK4 (upper panel), then for actin (lower panel). The PDK4 band is indicated by a red arrow; background bands are indicated by black arrows. The actin band is indicated by a blue arrow

Mentions: MCF-7L and TamR-MCF-7 were treated as described, and whole cell lysates were prepared in RIPA buffer supplemented with protease inhibitor cocktail, phenylmethanesulfonylfluoride, and sodium fluoride (all purchased from Sigma). Lysates were sonicated and cleared by high speed centrifugation. Protein concentration in the supernatants was determined by Bradford assay (BioRad), and equal quantities of protein were diluted in 6x-SDS-PAGE buffer and boiled. Lysate equivalent to 40 μg protein were resolved on 10 % SDS polyacrylamide gel (TGX gels, BioRad), then transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5 % nonfat dry milk in phosphate buffered saline with 1 % Tween-20 (PBS-T; Tween-20 purchased from Sigma). Membranes were incubated overnight at 4 degrees with anti-PDK4 antibody (Abgent Cat. No. AP7041B) diluted at 1:1000 in blocking solution. The membrane was washed thrice with PBS-T, then incubated for 1 h with horseradish peroxidase conjugated secondary antibody diluted 1:1000 in blocking solution. The membrane was washed thrice with PBS-T, then incubated with enhanced chemiluminescence reagents for 2 min (SuperSignal West Pico; Pierce). Antibody interactions were visualized by exposing the membrane to film. The membrane was then washed with PBS-T and, using the procedure described above, probed for actin to ensure equal loading (Sigma clone AC-40). The images in Fig. 1b were generated by scanning films with a flatbed scanner.Fig. 1


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)

Glucose deprivation results in increased PDK4 protein without a change in mRNA. MCF-7L and TamR-MCF-7 cells were treated for 2 days in media containing 4.5 mM glucose or 4.5 mM galactose. a Total RNA was extracted, reverse transcribed, and the products used as template for qPCR amplification of PDK4 and b-actin. The average ratio is shown; error bars indicate one standard deviation (n = 3 biological replicates). b Whole cell lysates were prepared, and PDK4 and actin protein were visualized by western blotting, first for PDK4 (upper panel), then for actin (lower panel). The PDK4 band is indicated by a red arrow; background bands are indicated by black arrows. The actin band is indicated by a blue arrow
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4641142&req=5

Fig1: Glucose deprivation results in increased PDK4 protein without a change in mRNA. MCF-7L and TamR-MCF-7 cells were treated for 2 days in media containing 4.5 mM glucose or 4.5 mM galactose. a Total RNA was extracted, reverse transcribed, and the products used as template for qPCR amplification of PDK4 and b-actin. The average ratio is shown; error bars indicate one standard deviation (n = 3 biological replicates). b Whole cell lysates were prepared, and PDK4 and actin protein were visualized by western blotting, first for PDK4 (upper panel), then for actin (lower panel). The PDK4 band is indicated by a red arrow; background bands are indicated by black arrows. The actin band is indicated by a blue arrow
Mentions: MCF-7L and TamR-MCF-7 were treated as described, and whole cell lysates were prepared in RIPA buffer supplemented with protease inhibitor cocktail, phenylmethanesulfonylfluoride, and sodium fluoride (all purchased from Sigma). Lysates were sonicated and cleared by high speed centrifugation. Protein concentration in the supernatants was determined by Bradford assay (BioRad), and equal quantities of protein were diluted in 6x-SDS-PAGE buffer and boiled. Lysate equivalent to 40 μg protein were resolved on 10 % SDS polyacrylamide gel (TGX gels, BioRad), then transferred to polyvinylidene fluoride membrane. The membrane was blocked with 5 % nonfat dry milk in phosphate buffered saline with 1 % Tween-20 (PBS-T; Tween-20 purchased from Sigma). Membranes were incubated overnight at 4 degrees with anti-PDK4 antibody (Abgent Cat. No. AP7041B) diluted at 1:1000 in blocking solution. The membrane was washed thrice with PBS-T, then incubated for 1 h with horseradish peroxidase conjugated secondary antibody diluted 1:1000 in blocking solution. The membrane was washed thrice with PBS-T, then incubated with enhanced chemiluminescence reagents for 2 min (SuperSignal West Pico; Pierce). Antibody interactions were visualized by exposing the membrane to film. The membrane was then washed with PBS-T and, using the procedure described above, probed for actin to ensure equal loading (Sigma clone AC-40). The images in Fig. 1b were generated by scanning films with a flatbed scanner.Fig. 1

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