br Fig ASC stimulated BC cells exhibit the EMT
Fig. 5. ASC-stimulated BC Nifurtimox exhibit the EMT phenotype in vitro and possess the pulmonary metastasis potential in vivo. (A-B) The mRNA levels of the indicated EMT-related genes were examined in monocultured 4T1, miR20bup 4T1 cells, and cells cocultured with ASCs for 3 days were examined. The results were normalized, and the density value of the 4T1(#) or miR20b-4T1(#) was set to 1. The cells were treated with SCF and a miR20b inhibitor was used as a positive control. (C) Tumor metastasis analysis. Representative images of the lung tissue samples and micrographs of the HE staining. (D) Relative metastasis in lung nodules was analyzed in each group of nude mice (n = 5 per group). (E-F) Western blot analysis of the expression levels of the indicated EMT-related proteins in tumor xenografts. *P < .05, **P < .01, ***P < .001.
cocultured with ASCs. The results indicate that coculture of 4T1 cells with ASCs resulted in increased expression levels of N-cadherin, vi-mentin and Twist and decreased expression levels of E-cadherin
(Fig. 5A–B). These changes can be mimicked by treatment with SCF and a miR20b inhibitor, but the expressions levels of the EMT-related genes were reduced in the 4T1 cells overexpressing miR20b that were
Fig. 6. Overview of the proposed pathways of ASC-induced metastasis of breast cancer cells.
cocultured with ASCs (Fig. 5A–B).
To investigate ASC-educated BC cell metastasis in vivo, we per-formed subcutaneous coinjection of ASCs with untreated 4T1 cells or with 4T1 cells overexpressing miR20b into nude mice. The results showed that ASC-stimulated BC cells spread out to form more nodules in the lung compared to the untreated BC cells (Fig. 5C–D). In contrast, interfering with miR20b expression in 4T1 cells inhibited pulmonary metastasis (Fig. 5C–D). Similarly, upregulating miR20b in 4T1 cells reduced the numbers of ASC-induced metastasis nodules in the lung (Fig. 5C–D). Furthermore, the results were confirmed by detecting the expressions levels of E-cadherin, N-cadherin, vimentin and Twist using western blot of xenograft tumor tissue samples (Fig. 5E–F). Taken to-gether, our findings demonstrated that ASCs can induce pulmonary metastasis of BC cells in vivo through the inhibition of miR20b.
Breast cancer metastasis after surgery is associated with the wound healing signaling pathways that can increase local immunosuppression, exert direct eﬀects on the tumor microenvironment and promote distant metastasis . ASCs with autologous fat grafts are used to repair the loss of breast tissue after breast cancer surgery, however, our previous study has shown that ASCs can promote tumorigenesis and metastasis in breast cancer . The precise mechanisms that govern tumor me-tastasis remain to be elucidated. In this study, we found that c-Kit-po-sitive ASCs can produce high levels of SCF to influence BC cells and promote their EMT and metastasis through the inhibition of miR20b biogenesis (Fig. 6). Mechanistically, we demonstrated that SCF-depen-dent induction of miR20b downregulation is mediated by the MAPK-p38/E2F1 pathway that can activate HIF-1α/VEGFA and induce EMT and metastasis of BC cells.
SCF plays a critical role in guiding cell migration during develop-ment. In addition, SCF activates tissue-resident mast cells to generate a tumor-promoting angiogenic microenvironment . The tumor-pro-moting eﬀect of SCF are considered to be caused by KitL stimulation of c-Kit+ tumor cells by triggering downstream activation of the MAPK pathway , of which the p38 and ERK proteins are generally involved in survival, proliferation, and cell cycle progression . E2F1, as a transcriptional factor, plays a critical role in cell cycle progression .
E2F1 activity is mediated by interaction with p38, which modifies its Ser403 and Thr433 residues . Our results clearly confirm that E2F1 is regulated by the p-c-Kit/ MAPK-p38 pathway to mediate the ASC-induced progression of breast cancer cells.
Recent studies have shown the importance of miRNA in regulating cancer progression . miR20b has been reported as a negative reg-ulator of breast cancer angiogenesis and metastasis . However, only a few studies have elucidated the mechanisms by which miR20b in-teracts with ASCs in the modulation of target gene expression and function in BC cell migration, invasion and metastasis. Previous studies have indicated that posttranscriptional expression of miR20b is trig-gered by E2F1 during the regulation of myoblast proliferation and diﬀerentiation . In addition, several cancer metastasis-associated genes, including HIF-1α and VEGFA, have been reported to be targeted by miR20b in breast cancer cells . In this study, we demonstrated that miR20b biogenesis in BC cells can be inhibited by ASC-released SCF through the downstream c-Kit/MAPK-p38/E2F1 signaling cascade and that miR20b acts as a tumor suppressor miRNA by inhibiting BC cell migration and invasion, particularly in EMT and metastasis. Moreover, bioinformatic approaches suggested that miR20b may target HIF-1α/VEGFA, and our study has shown a positive correlation be-tween ASC-released SCF and HIF-1α/VEGFA; additionally, HIF-1α can be recruited to the VEGFA promoter in BC cells following ASC treat-ment to inhibit the miR20b-dependent attenuation of the HIF-1α nu-clear accumulation during the ASC-induced BC cell migration and in-vasion. Previous studies have shown hypoxia can induce cytoskeletal remodeling and angiogenesis through HIF-1α/VEGFA to drive EMT and metastasis [19,20], and our results demonstrated that ASC-released SCF induced miR-20b downregulation in BC cells and might induce EMT and lung metastasis through the activation of the HIF-1α/VEGFA to up-regulate the N-cadherin, vimentin and Twist transcriptions in vitro and vivo.