Targeting AKT Kinase in Hydroxytamoxifen-Resistant Breast Cancer Cells

More than 650,000 people die each year from breast cancer, making it a particularly significant disease worldwide. The development of about 70% of breast tumors depends on steroid hormones, namely, estrogens. Estrogens trigger signaling pathways that support tumor growth and progression. Hydroxytamoxifen (HT) halting estrogen-induced tumor growth is among the most effective drugs in current anticancer therapy. The purpose of this work was to investigate approaches to overcome breast cancer cell resistance to hydroxytamoxifen. Cells with resistance to the antiestrogen hydroxytamoxifen were obtained by long-term incubation of parental MCF7 cells with this drug. Estrogen receptor α (ERα) expression and progesterone receptor (PR) expression were analyzed by immunoblotting. The resistant MCF7/HT cells were found not to lose ERα expression. These cells were found to have slightly reduced ERα activity when compared to parental MCF7 cells. The expression of PR, one of the ERα targets, was downregulated in hydroxytamoxifen-resistant cells. AKT kinase belongs to the PI3K/AKT/mTOR signaling pathway, and its activity is associated with resistance. Three types of AKT inhibitors were evaluated, including AKT inhibitor IV (6-(2-benzothiazolyl)-1-ethyl-2-[2-(methylphenylamino)ethenyl]-3-phenyl-1H-benzimidazolium, monoiodide), 10-DEBC (2-chloro-N,N-diethyl-10H-phenoxazine-10-butanamine, monohydrochloride), and luminespib (HSP90 inhibitor, 5-[2,4-dihydroxy-5-(1-methylethyl)phenyl]-N-ethyl-4-[4-(4-morpholinylmethyl)phenyl]-3-isoxazolecarboxamide). All three compounds showed high antiproliferative activity against hydroxytamoxifen-resistant cells. The IC50 value of 10-DEBC was 4.2 µM, and this was when the AKT inhibitor IV was more active with IC50 value of 390 nM. The HSP90 inhibitor luminespib, which reduces AKT expression, showed the highest activity against parental and hydroxytamoxifen-resistant breast cancer cells, with IC50 values of 14 and 18 nM, respectively. Thus, the hydroxytamoxifen-resistant cells were found to partially retain hormone signaling and to be sensitive to selective AKT inhibitors. The best effects were discovered for HSP90-AKT blocker luminespib, with an IC50 value of about 20 nM

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months.Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells.Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression