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.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1444-2) contains supplementary material, which is available to authorized users.

Highlights

  • Therapies that target the estrogen receptor-α (ER) have resulted in significant improvement in clinical outcomes for breast cancer patients (Early Breast Cancer Trialists’ Collaborative G 2005; Strasser-Weippl et al 2013)

  • While the pathway targeted quantitative PCR (qPCR) arrays revealed a two fold relative difference in Pyruvate dehydrogenase kinase-isoform 4 (PDK4), using single gene qPCR with quantitation based on a five point standard curve, we found that PDK4 mRNA was five times more abundant in TamR-MCF-7 relative to their parental cells, with no difference in 18s rRNA (Fig. 3a)

  • The data presented above point to altered regulation of pyruvate dehydrogenase as mediating breast cancer cell sensitivity to estrogen receptor ligands such as tamoxifen and ICI, and reveal an important role for the serine threonine kinase PDK4 in acquired selective estrogen receptor modulators (SERMs) resistance

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Summary

Background

Therapies that target the estrogen receptor-α (ER) have resulted in significant improvement in clinical outcomes for breast cancer patients (Early Breast Cancer Trialists’ Collaborative G 2005; Strasser-Weippl et al 2013). Oncogenes and cell cycle regulatory proteins have been shown to regulate the metabolism of fuels in addition to cellular proliferation, with consequences for endocrine therapy in breast cancer cells (Shajahan-Haq et al 2014; Lee et al 2014; Wang et al 2006). Consistent with that notion, malignant breast cancer cells are metabolically coupled with tumor associated fibroblasts, with fibroblasts fermenting substrates and providing lactate for oxidation by cancer cells; this relationship promotes SERM resistance (Martinez-Outschoorn et al 2011). Taken together, these observations suggest that fuel metabolism is plastic in breast cancer cells, and that utilization of fuels impacts response to endocrine therapy. We conclude that regulatory circuits that control oxidation of carbon derived from glucose are altered during selection for antiestrogen resistance

Methods
Results
B C a a b control siRNA PDK4 siRNA abbcabc
Discussion
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