Abstract
Pregnancy increases breast cancer risk for all women for at least 5 years after parturition. During weaning and involution, the breast microenvironment becomes tumor promotional. Exosomes provide cell-cell communication during physiologic processes such as lactation, but also in breast cancer. We determined whether molecules in milk exosomes from healthy lactating women modulate the development and progression of breast cancer. Thirteen nursing women provided three (transitional, mature, and wean) milk samples. Exosomes were extracted and MCF7 and MCF10A breast cells labeled. The expression of six proteins linked to breast cancer was measured. On the basis of the findings, TGFβ2 concentration in exosome samples was measured, breast cells incubated with the exosomes and effect (epithelial-mesenchymal transition, EMT) on EMT-related proteins [E-cadherin, α-smooth muscle actin (α-SMA), filamentous (F)-actin and vimentin] measured. Human milk exosomes entered benign and malignant breast cells. The greatest change in wean milk protein was in TGFβ2 (P = 0.01). Exosomes with a high (but not low) level of TGFβ2 led to EMT in both cancer and benign cells, based on (i) change in cell morphology, actin cytoskeleton, and loss of cell-cell junction structure and (ii) increased α-SMA and vimentin and decreased E-cadherin. TGFβ2 is significantly upregulated in breast milk exosomes during weaning/early involution. Breast milk exosomes containing high levels of TGFβ2 induce changes in both benign and malignant breast epithelial cells, consistent with the development and progression of breast cancer, suggesting a role for high TGFβ2-expressing breast milk exosomes in influencing breast cancer risk. Clin Cancer Res; 22(17); 4517-24. ©2016 AACR.
Highlights
Pregnancy increases breast cancer riskPregnancy-associated breast cancers (PABCs) diagnosed after childbirth are generally aggressive, with a poor prognosis
Exosomes with a high level of TGFb2 led to epithelial–mesenchymal transition (EMT) in both cancer and benign cells, based on (i) change in cell morphology, actin cytoskeleton, and loss of cell– cell junction structure and (ii) increased a-smooth muscle actin (a-smooth muscle actin (SMA)) and vimentin and decreased E-cadherin
TGFb2 is significantly upregulated in breast milk exosomes during weaning/early involution
Summary
Milk exosomes from healthy lactating women enter breast cancer and benign breast cells We used confocal microscopy to confirm that, when cocultured, human milk exosomes get inside of both MCF7 and MCF10A breast cells (Fig. 1). Confocal image of MCF7 (A–C) and MCF10a (D–F) cells cocultured with milk exosomes demonstrating GFP binding to CD63, a protein used to. The actin cytoskeletal structure dramatically changes during EMT, which is correlated with a migration phenotype of the transformed cells During this transition, the cells lose cell–cell junctions. TGFb2 stimulation markedly changed the actin cytoskeletal structure showing stress fibers and cell–cell junction structures with the loss of E-cadherin (Pos control, Fig. 5A and B). During this transition, there is a notable increase in a-SMA and vimentin expression. A similar actin cytoskeletal change and loss of E-cadherin expression were evident after high (TGFbH) but not low (TGFbL) TGFb2 exosome treatment. There was a > 60% decrease in expression in the positive control and in the TGFb2H-treated group, whereas the decrease after treatment with TGFb2L was 21% (Supplementary Fig. S1)
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