Cross-talk between ER and HER2 regulates c-MYC-mediated glutamine metabolism in aromatase inhibitor resistant breast cancer cells - PubMed (original) (raw)

Cross-talk between ER and HER2 regulates c-MYC-mediated glutamine metabolism in aromatase inhibitor resistant breast cancer cells

Zhike Chen et al. J Steroid Biochem Mol Biol. 2015 May.

Abstract

Resistance to endocrine therapies in hormone receptor (HR)-positive breast cancer is a significant clinical problem for a considerable number of patients. The oncogenic transcription factor c-MYC (hereafter referred to as MYC), which regulates glutamine metabolism in cancer cells, has been linked to endocrine resistance. We were interested in whether MYC-mediated glutamine metabolism is also associated with aromatase inhibitor (AI) resistant breast cancer. We studied the expression and regulation of MYC and the effects of inhibition of MYC expression in both AI sensitive and resistant breast cancer cells. Considering the role of MYC in glutamine metabolism, we evaluated the contribution of glutamine to the proliferation of AI sensitive and resistant cells, and performed RNA-sequencing to investigate mechanisms of MYC-mediated glutamine utilization in AI resistance. We found that glutamine metabolism was independent of estrogen but still required estrogen receptor (ER) in AI resistant breast cancer cells. The expression of MYC oncogene was up-regulated through the cross-talk between ER and human epidermal growth factor receptor 2 (HER2) in AI resistant breast cancer cells. Moreover, the glutamine transporter solute carrier family (SLC) 1A5 was significantly up-regulated in AI resistant breast cancer cells. ER down-regulator fulvestrant inhibited MYC, SLC1A5, glutaminase (GLS) and glutamine consumption in AI resistant breast cancer cells. Inhibition of MYC, SLC1A5 and GLS decreased AI resistant breast cancer cell proliferation. Our study has uncovered that MYC expression is up-regulated by the cross-talk between ER and HER2 in AI resistant breast cancer cells. MYC-mediated glutamine metabolism is associated with AI resistance of breast cancer.

Keywords: Aromatase inhibitor resistance; Breast cancer; ER; Glutamine; HER2; c-MYC.

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Figures

Fig. 1

Fig. 1. Up-regulation of c-MYC in AI resistant breast cancer cells

A. Real time PCR analysis of MYC expression in MCF7aro (hormone stripped for 72 hr) and LTEDaro cells treated with DMSO, testosterone (1nM) or testosterone (1nM) plus fulvestrant (100nM) for 6 hr. Values were normalized to ACTB and plotted relative to the expression of the MCF7aro cells treated with DMSO. Data are means ± SE (n=3). B. Western blotting analysis of c-MYC expression in whole-cell lysates from MCF7aro or LTEDaro cells transfected with a nontargeting control (Ctrl) siRNA or a siRNA targeting c-MYC for 72 hr. C. Cell proliferation of MCF7aro or LTEDaro cells transfected with a nontargeting control (Ctrl) siRNA or a siRNA targeting c-MYC. MTT was measured at the indicated time points. Data are means ± SE (n=5).

Fig. 2

Fig. 2. Cross-talk between ER and HER2 up-regulates c-MYC in AI resistant breast cancer cells

A. Western blotting analysis of the expression of c-MYC, ER and HER2, and the phosphorylation of MAPK and AKT in MCF7aro and LTEDaro cells cultured in normal medium. The quantification of c-MYC, p-MAPK, MAPK was normalized to the loading control GAPDH, and values were calculated relative to the expression of the MCF7aro cells. B. c-MYC protein levels in whole-cell lysates from LTEDaro cells transfected with a nontargeting control (Ctrl) siRNA or a siRNA targeting HER2 for 72 hr. The quantification of c-MYC, p-MAPK, MAPK was normalized to GAPDH, and values were calculated relative to the expression of the LTEDaro cells treated with control siRNA. C. c-MYC protein levels in whole-cell lysates from LTEDaro cells transfected with a nontargeting control (Ctrl) siRNA or a siRNA targeting MAPK for 72 hr. The quantification of c-MYC, p-MAPK, MAPK was normalized to GAPDH, and values were calculated relative to the expression of the LTEDaro cells treated with control siRNA. D. c-MYC protein levels in whole-cell lysates from LTEDaro cells treated with DMSO or the AKT inhibitor MK-2206 (1μM) for 24 hr. The quantification of c-MYC was normalized to GAPDH, and values were calculated relative to the expression of the LTEDaro cells treated with DMSO.

Fig. 3

Fig. 3. Glutamine consumption is independent of estrogen but requires ER in AI resistant breast cancer cells

A. Cell growth responses of MCF7aro cells (hormone stripped for 72 hr) to glutamine or glucose deprivation in the presence or absence of testosterone. MTT was measured at day 6. Data are means ± SE (n=6). B. Cell growth responses of LTEDaro cells to glutamine or glucose deprivation in the presence or absence of testosterone. MTT was measured at day 6. Data are means ± SE (n=6). C. Cell growth, glutamine consumption and ammonia production in MCF7aro cells (hormone stripped for 72 hr) treated with DMSO, testosterone (1nM), testosterone (1nM) plus letrozole (200nM), or testosterone (1nM) plus fulvestrant (100nM). Cell number, glutamine and ammonia concentrations were measured at day 3. Glutamine consumption was calculated by subtracting the concentration of the sample from the concentration of the medium control. Ammonia production was calculated by subtracting the concentration of the medium control from the concentration of the sample. Data are means ± SE. D. Cell growth, glutamine consumption and ammonia production in LTEDaro cells treated with DMSO, testosterone (1nM), testosterone (1nM) plus letrozole (200nM), or testosterone (1nM) plus fulvestrant (100nM). Cell number, glutamine and ammonia concentrations were measured at day 3. Glutamine consumption was calculated by subtracting the concentration of the sample from the concentration of the medium control. Ammonia production was calculated by subtracting the concentration of the medium control from the concentration of the sample. Data are means ± SE.

Fig. 4

Fig. 4. Fulvestrant suppresses glutamine-mediated cell proliferation and expression of genes in lipid and glucose metabolism in AI resistant breast cancer cells

A. The dendrogram and heat map show the hierarchical cluster analysis of gene-expression data from LTEDaro cells cultured in media without glutamine, with glutamine, or with both glutamine and fulvestrant for 24 hr. Gene-expression profiles were obtained from RNA-sequencing data. Three groups of genes were analyzed: cell proliferation, cell cycle and lipid biosynthesis. Columns represent individual genes, and rows represent individual samples. Each cell in the matrix represents the expression level of a gene in an individual sample. The scale bar indicates the level of expression; red indicates a high level of expression, and blue a low level of expression. B. Cell proliferation of LTEDaro cells cultured in media without glutamine, with glutamine, or with both glutamine and fulvestrant. MTT was measured at day 7. Data are means ± SE (n=6). Asterisk (*) denotes P <0.05 (t test). C. The heat map shows the gene-expression analysis of data from LTEDaro cells cultured in media without glutamine (Gln−), with glutamine (Gln+), or with both glutamine and fulvestrant (Gln/Fulvestrant) for 24 hr. Gene-expression profiles were obtained from RNA-sequencing data. Columns represent individual genes that are involved in glucose metabolism, and rows represent fold changes of Gln/Fulvestrant vs. Gln+ (left) or Gln+ vs. Gln− (right). The scale bar indicates the level of expression; red indicates a high level of expression, and blue a low level of expression.

Fig. 5

Fig. 5. Fulvestrant inhibits c-Myc, SLC1A5 and GLS in AI resistant breast cancer cells

A. The dendrogram and heat map show the hierarchical cluster analysis of gene-expression data from LTEDaro cells cultured in media without glutamine, with glutamine, or with both glutamine and fulvestrant for 24 hr. Gene-expression profiles were obtained from RNA-sequencing data. Two groups of genes were analyzed: ESR1 activation and MYC activation. Columns represent individual genes, and rows represent individual samples. Each cell in the matrix represents the expression level of a gene in an individual sample. The scale bar indicates the level of expression; red indicates a high level of expression, and blue a low level of expression. B. Western blotting analysis of ERα and c-MYC expression in whole-cell lysates from LTEDaro cells treated with fulvestrant (100nM) at the indicated time points. C. Real time PCR analysis of SLC1A5 expression in MCF7aro (hormone stripped for 72 hr) and LTEDaro cells treated with DMSO, testosterone (1nM) or testosterone (1nM) plus fulvestrant (100nM) for 6 hr. Values were normalized to ACTB and plotted relative to the expression of the MCF7aro cells treated with DMSO. Data are means ± SE (n=3). D. Glutamine consumption in LTEDaro cells treated with DMSO or the SLC1A5 inhibitor GPNA (0.5mM). Glutamine concentrations were measured at day 3. Glutamine consumption was calculated by subtracting the concentration of the sample from the concentration of the medium control. Data are means ± SE. E. Cell proliferation of LTEDaro cells treated with DMSO or the SLC1A5 inhibitor GPNA (0.5mM). MTT was measured at day 4. Data are means ± SE (n=6). F. Real time PCR analysis of GLS expression in MCF7aro (hormone stripped for 72 hr) and LTEDaro cells treated with DMSO, testosterone (1nM) or testosterone (1nM) plus fulvestrant (100nM) for 6 hr. Values were normalized to ACTB and plotted relative to the expression of the MCF7aro cells treated with DMSO. Data are means ± SE (n=3). G. Glutamine consumption in LTEDaro cells treated with DMSO or the GLS inhibitor compound 968 (10μM). Glutamine concentrations were measured at day 3. Glutamine consumption was calculated by subtracting the concentration of the sample from the concentration of the medium control. Data are means ± SE. H. Cell proliferation of LTEDaro cells treated with DMSO or the GLS inhibitor compound 968 (10μM). MTT was measured at day 4. Data are means ± SE (n=6). Asterisk (*) denotes P <0.05 (t test).

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