Upregulation of PI3K/AKT/PTEN pathway is correlated with glucose and glutamine metabolic dysfunction during tamoxifen resistance development in MCF-7 cells

Abstract

Tamoxifen resistance is emerging as a big challenge in endocrine therapy of luminal A breast cancer patients. In this study, we aimed to determine the molecular changes of PI3K/AKT/PTEN signaling pathway during tamoxifen-resistance development using gradually increased doses of tamoxifen in one model, while fixing tamoxifen treatment dose at 35 μM for several times in the second model. An upregulation of AKT/PI3K genes was noticed at 30 μM tamoxifen concentration in cells treated with a gradual increase of tamoxifen doses. In the second model, significant upregulation of AKT1 was seen in cells treated with 35 μM tamoxifen for three times. All genes studied showed a significant increase in expression in resistant cells treated with 50 µM and 35 µM six times tamoxifen. These genes’ upregulation was accompanied by PTEN and GSK3 ß genes’ down-regulation, and it was in correlation to the changes in the metabolic rate of glucose in tamoxifen-resistant models. A significant increase in glucose consumption rate from culture media was observed in tamoxifen resistant cells with the highest consumption rate reported in the first day of culturing. Increased glucose consumption rates were also correlated with GLUL significant gene expression and non-significant change in c-MYC gene expression that may lead to increased endogenous glutamine synthesis. As a result, several molecular and metabolic changes precede acquired tamoxifen resistance could be used as resistance biomarkers or targets to reverse tamoxifen resistance.

Changes in Lactate Production, Lactate Dehydrogenase Genes Expression and DNA Methylation in Response to Tamoxifen Resistance Development in MCF-7 Cell Line

“…Gradual increase in TAM doses was accompanied with downregulation of LDHA gene expression and a significant increase in LDHB gene expression, parallel with demethylation of certain CpG sites in the promoter region. Interestingly, these changes in LDHA and LDHB gene expression were in parallel with our reported finding that there was no c-MYC gene overexpression, with a significant increase in glutamine production during TamR development [20]. Statistical significance was calculated by one-way ANOVA followed by Tukey post hoc test in GraphPad prism 8.0 software, considering the statistical significance as follows: * significant at P ≤ 0.05; results were expressed as mean ± SD (n = 3 runs for each sample).…”

“…Gradual increase in TAM doses was accompanied with downregulation of LDHA gene expression and a significant increase in LDHB gene expression, parallel with demethylation of certain CpG sites in the promoter region. Interestingly, these changes in LDHA and LDHB gene expression were in parallel with our reported finding that there was no c-MYC gene overexpression, with a significant increase in glutamine production during TamR development [20].…”

“…Morphological changes of breast cancer cell line MCF-7 were observed during the process of tamoxifen resistance development as reported previously [20] and are shown in Figure 1. Cells treated with 30 µM tamoxifen started to lose their epithelial-like shape and became round.…”

“…Different methods of tamoxifen resistance development in the MCF-7 cell line could affect gene expression differently [20]. Using the gradual increase in tamoxifen concentrations reported in this study, we showed that the increase in LDHB gene expression but not LDHA was correlated with lactate concentration increase in the media during the process of TamR development.…”

Endocrine resistance in breast cancer: from molecular mechanisms to therapeutic strategies

“…Endocrine-resistant cells were shown to exhibit higher glucose and glutamine uptake that is, in part, mediated by c-Myc overexpression whose inhibition reduced the uptake of glucose and glutamine leading to decreased cell growth [177]. Furthermore, glucose consumption rate was found to be higher in MCF-7 TamR cells which correlates with upregulation of PI3K/AKT/PTEN axis [178]. The ESR1 Y537S mutant-carrying cells were shown to exhibit elevated tricarboxylic acid (TCA) cycle activity, glucose independence and reliance on glutamine as a carbon source [179].…”