Cancer-associated Fibroblasts Release Exosomal CBFB That Dictate an Aggressive Bone Metastasis Phenotype in Breast Cancer

Abstract Background: Breast cancer up to date remains the one of the most prevalent female malignancies in the world. Better prognostic and therapeutic biomarkers are urgently required for these patients. Circulating exosomes are shown to participate in tumorigenesis including distance metastasis and of prognostic/therapeutic potential. Methods: Sera from control health, primary breast cancer, and bone metastatic breast cancer patients were collected. The Exosome were isolated from collected sera and culture medium from previously steps, and a standard procedure was performed. We utilized MDA-MB-436-derived xenograft mouse model to demonstrate that silencing CBFB (core binding factor subunit β) significantly reduced bone-metastasis in association with reduced expression of OPN, IL-6, Runx2 and OPN as well as reduced exosomes containing CBFB.Results: We found that circulating exosomes (Exos), from bone metastatic patients with breast cancer, were enriched with CBFB. Fibroblasts co-cultured with Exos showed increased α-SMA, vimentin expression and increased secretion of IL-6 and OPN; non-metastatic breast cancer cells co-cultured with Exos exhibited increased markers including vimentin, snail1, CXCR4 and Runx2. Subsequent analysis revealed that these Exos were enriched with bone metastasis associated maker CBFB. Gene-silencing experiments metastatic MDA-MB-436 and MDA-MB-157 cells, demonstrated that CBFB significantly reduced metastatic potential, reflected by the suppression of vimentin, CXCR4, snail1 and Runx2, CD44 and OPN. In contrary, CBFB-overexpression resulted in the increased metastasis associated genes in non-metastatic T47D and MCF7 cells. The CBFB-enriched exosomes derived from MDA-MB-436 enhanced metastatic phenotypes of low metastatic potential breast cancer cell lines. Conclusion: We demonstrated the essential roles of CBFB in the promotion of bone metastasis in breast cancer cells. The suppression of CBFB led to the decreased tumor burden and bone metastasis in association with decreased markers of bone metastasis including CXCR4, Snail, CD44, OPN, Runx2 and IL-6.

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Cancer-Associated Fibroblasts Release Exosomal CBFB That Dictate an Aggressive Bone Metastasis Phenotype in Breast Cancer

10.21203/rs.3.rs-135095/v1 ◽

2020 ◽

Author(s):

Chih-Ming Su ◽

Yu-Hsin Lai ◽

Oluwaseun Adebayo Bamodu ◽

Kuang-Tai Kuo ◽

Iat-Hang Fong ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Bone Metastasis ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Therapeutic Potential ◽

Standard Procedure ◽

Metastatic Breast ◽

Metastatic Potential ◽

Breast Cancer Cell Lines

Abstract BackgroundBreast cancer up to date remains the one of the most prevalent female malignancies in the world. Better prognostic and therapeutic biomarkers are urgently required for these patients. Circulating exosomes are shown to participate in tumorigenesis including distance metastasis and of prognostic/therapeutic potential. MethodsSera from control health, primary breast cancer, and bone metastatic breast cancer patients were collected. The Exosome were isolated from collected sera and culture medium from previously steps, and a standard procedure was performed. We utilized MDA-MB-436-derived xenograft mouse model to demonstrate that silencing CBFB (core binding factor subunit β) significantly reduced bone-metastasis in association with reduced expression of OPN, IL-6, Runx2 and OPN as well as reduced exosomes containing CBFB.ResultsWe found that circulating exosomes (Exos), from bone metastatic patients with breast cancer, were enriched with CBFB. Fibroblasts co-cultured with Exos showed increased α-SMA, vimentin expression and increased secretion of IL-6 and OPN; non-metastatic breast cancer cells co-cultured with Exos exhibited increased markers including vimentin, snail1, CXCR4 and Runx2. Subsequent analysis revealed that these Exos were enriched with bone metastasis associated maker CBFB. Gene-silencing experiments metastatic MDA-MB-436 and MDA-MB-157 cells, demonstrated that CBFB significantly reduced metastatic potential, reflected by the suppression of vimentin, CXCR4, snail1 and Runx2, CD44 and OPN. In contrary, CBFB-overexpression resulted in the increased metastasis associated genes in non-metastatic T47D and MCF7 cells. The CBFB-enriched exosomes derived from MDA-MB-436 enhanced metastatic phenotypes of low metastatic potential breast cancer cell lines. ConclusionWe demonstrated the essential roles of CBFB in the promotion of bone metastasis in breast cancer cells. The suppression of CBFB led to the decreased tumor burden and bone metastasis in association with decreased markers of bone metastasis including CXCR4, Snail, CD44, OPN, Runx2 and IL-6.

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Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo

International Journal of Molecular Sciences ◽

10.3390/ijms21228444 ◽

2020 ◽

Vol 21 (22) ◽

pp. 8444

Author(s):

Ga-Eun Lim ◽

Jee Young Sung ◽

Suyeun Yu ◽

Younmi Kim ◽

Jaegal Shim ◽

...

Keyword(s):

Breast Cancer ◽

Metastatic Breast Cancer ◽

Er Stress ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Metastatic Breast ◽

Breast Cancer Cell Lines ◽

Syngeneic Mouse Model ◽

Apoptosis Protein ◽

4T1 Cells

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

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