Bacterial extracellular vesicles affect endocrine therapy in MCF7 cells

Abstract

Background: : The microbiome is important in the development and progression of breast cancer. This study investigated the effects of microbiome derived from Klebsiella on endocrine therapy of breast cancer using MCF7 cells. The bacterial extracellular vesicles (EVs) that affect endocrine therapy were established through experiments focused on tamoxifen efficacy.

Methods: : The microbiomes of breast cancer patients and healthy controls were analyzed using next-generation sequencing. Among microbiome, Klebsiella was selected as the experimental material for the effect on endocrine therapy in MCF7 cells. MCF7 cells were incubated with tamoxifen in the absence/presence of bacterial EVs derived from Klebsiella pneumoniae and analyzed by quantitative real-time polymerase chain reaction and Western blot.

Results: : Microbiome derived from Klebsiella is abundant in breast cancer patients especially luminal A subtype compared to healthy controls. The addition of EVs derived from K pneumoniae enhances the anti-hormonal effects of tamoxifen in MCF7 cells. The increased efficacy of tamoxifen is mediated via Cyclin E2 and p-ERK.

Conclusion: : Based on experiments, the EVs derived from K pneumoniae are important in hormone therapy on MCF7 cells. This result provides new insight into breast cancer mechanisms and hormone therapy using Klebsiella found in the microbiome.

Figure 1

Heat map shows the relative abundance of different phyla and genera of the microbiomes of breast cancer patients and healthy controls. The upper heat map shows the difference in phyla and the lower heat map shows the difference in genera.

Figure 2

Relationship between hormone-positive breast cancer and Klebsiella. (A) Klebsiella is abundant in the microbiomes of luminal A subtype breast cancer patients. Klebsiella was 5.58-fold more abundant in breast cancer patients, especially in luminal A subtype patients. (B) Klebsiella pneumonia EVs were extracted by mass culture and confirmed by transmission electron microscopy. The average diameter of a K pneumoniae EV was approximately 30 nm. (C) Frequency of Klebsiella microbiome in healthy control and patient groups. Patients were divided into 2 groups according to the frequency of the Klebsiella microbiome. (D) Comparison of alpha diversity between the healthy control group and patient group. (E) Comparison of beta diversity between the healthy control group and patient group. EVs = extracellular vesicles.

Figure 3

Treatment of MCF7 cells with K pneumoniae EVs or tamoxifen. Relative cell survival percentages following treatment with K pneumoniae EVs. The cells were treated with distilled water (CTL) or K pneumoniae EVs at 100 ng/ml or 1 μg/ml, DMSO (CTL) with 10 μM Tam, 10 μM Tam plus 100 ng/ml K pneumoniae EVs for 72 h. Surviving cells were counted in a Neubauer chamber. The percentages of viable cells are indicated over each bar. Relative cell survival percentages are shown as percentages of untreated viable cells. ∗∗P < 0.01. CTL = control, EVs = extracellular vesicles, Tam = tamoxifen.

Figure 4

Cyclin E2 and p-ERK mediate Klebsiella pneumoniea EV-induced beneficial effect of tamoxifen on MCF7 cells. (A) and (B) Expression of p-AKT1/2/3, p-ERK, p21, and β-actin following treatment with K pneumoniae EVs or tamoxifen. MCF7 cells were treated with DMSO (CTL) and 10 μM tamoxifen, 100 ng/ml K pneumoniae EVs (K.p), or 10 μM tamoxifen and 100 ng/ml K pneumoniae EVs for 72 h. The expression of p-AKT1/2/3, p-ERK, p21, and β-actin was detected by immunoblotting. Lane 1, control; lane 2, 100 ng/ml K pneumoniae EVs; lane 3, 10 μM tamoxifen; and lane 4, 10 μM tamoxifen plus 100 ng/ml K pneumoniae EVs. (C) and (D) Expression of cyclin A1, A2, B1, B2, D1, D2, E1, E2, p21, p27, and TNF following treatment with K pneumoniae EVs or tamoxifen. Expression of cyclin D1 (CCND1), cyclin D2 (CCND2), cyclin E1 (CCNE1), cyclin E2 (CCNE2), cyclin A1 (CCNA1), cyclin A2 (CCNA2), cyclin B1 (CCNB1), cyclin B2 (CCNB2), p21, p27, and TNF was compared between the tamoxifen and control-treated group (Tam + CTL) and the tamoxifen and K pneumoniae EV-treated group (Tam + K.p). When cells were co-treated with K pneumoniae EVs and tamoxifen, expression of cyclin E2 was decreased. ∗∗∗P < 0.001. CTL = control, EVs = extracellular vesicles.