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. 2019;20(3):328-337.
doi: 10.1080/15384047.2018.1529101. Epub 2018 Oct 18.

FSTL1 enhances chemoresistance and maintains stemness in breast cancer cells via integrin β3/Wnt signaling under miR-137 regulation

Shaoqiang Cheng  1 , Yuanxi Huang  1 , Chun Lou  1 , Yanxia He  2 , Yue Zhang  2 , Qingyuan Zhang  2
Affiliations

FSTL1 enhances chemoresistance and maintains stemness in breast cancer cells via integrin β3/Wnt signaling under miR-137 regulation

Shaoqiang Cheng et al. Cancer Biol Ther. 2019.

Abstract

FSTL1 is a protein coding gene associated with cell signaling pathway regulation and the progression of a variety of disorders. In this study, we hypothesized that FSTL1 increases oncogenesis in breast cancer by enhancing stemness and chemoresistance. RT-PCR and IHC revealed significantly higher FSTL1 mRNA and protein levels in TNBC than in non-TNBC specimens and in breast cancer cell lines. We then found that FSTL1 levels were significantly increased in chemoresistant cells. LIVE/DEAD, MTT cell viability and colony formation assays did in fact demonstrate that FSTL1 is required for CDDP and DOX chemoresistance in breast cancer cell lines. FSTL1 overexpression caused significant elevation of stem cell biomarkers, as well as breast cancer cell proliferation. To determine whether the Wnt/β-catenin signaling pathway is involved in the observed effects of FSTL1, we assessed levels of pathway target. TOP/FOP flash, colony formation, and tumor sphere formation assays indicated that FSTL1 activates Wnt/β-catenin signaling through integrin β3. We then sought to identify a microRNA (miRNA) that regulates FSTL1 activity. Luciferase assays demonstrated that miR-137 reduces FSTL1 mRNA and protein levels. Ultimately, our findings indicate that there is an miR-137/FSTL1/integrin β3/Wnt/β-catenin signaling axis in breast cancer cells that regulates stemness and chemoresistance.

Keywords: FSTL1; Wnt signaling; breast cancer; chemoresistance; integrin β3; miR-137; stemness.

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Figures

Figure 1.
Figure 1.
FSTL1 expression is increased in TNBC tissues, TNBC cells, and TNBC cells of multiple drug resistance. (a) Representative immunostaining for FSTL1 expression in TNBC and non-TNBC tissues. (b) Relative FSTL1 mRNA expression in TNBC and non-TNBC tissues. p < 0.001. (c, d) Relative FSTL1 mRNA (c) and protein (d) expression in one normal mammary epithelial cell line MCF-10A and four breast cancer cell lines MDA-MB-231, MDA-MB-468, and HCC38. * p < 0.05. (e, f) Relative FSTL1 mRNA (e) and protein (f) expression in CDDP or DOX resistant MDA-MB-231 and MDA-MB-468 cells analyzed by RT-PCR and western blot, respectively. * p < 0.05.
Figure 2.
Figure 2.
FSTL1 enhances multiple drug resistance in breast cancer cells. (a) FSTL1 protein expression in MDA-MB-231, MDA-MB-468 and HCC38 cells transfected with control vector or FSTL1 plasmids by western blot. (b, c) Cell LIVE/DEAD assays in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells treated with CDDP (b) or DOX (c), respectively. *p < 0.05. (d, e) Cell viability in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells treated with different doses of CDDP (d) or DOX (e) indicated, respectively. (f, g) Colony formation in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells treated with different doses of CDDP (f) or DOX (g) indicated, respectively. *p < 0.05.
Figure 3.
Figure 3.
FSTL1 augments breast cancer cell stemness traits. (a) Tumor sphere formation assays in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells. Sphere numbers were quantified. *p < 0.05. (b) Protein expression of stem cell markers (CD133, Nanog, and SOX2) in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells. *p < 0.05. (c) Cell cycle analysis in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells. *p < 0.05. (d) MTT assays were performed to analyze cell proliferation in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells.
Figure 4.
Figure 4.
FSTL1 activates Wnt/β-catenin signaling through integrin β3. (a, b) mRNA (a) and protein (b) expression of Wnt/β-catenin signaling target genes (β-catenin, myc, and cyclin D1) in FSTL1 overexpression MDA-MB-231, MDA-MB-468 and HCC38 cells. *p < 0.05. (c) TOP/FOP ratios in FSTL1 overexpression MDA-MB-231, MDA-MB-468 cells and HCC38. *p < 0.05. (d) Western blots of integrin β3 and FSTL1 expression in integrin β3 knockdown MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. (e) TOP/FOP ratios in integrin β3 knockdown MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. *p < 0.05. (f) Relative colony formation in integrin β3 knockdown MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. *p < 0.05. (g) Number of tumor spheres in integrin β3 knockdown MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. *p < 0.05.
Figure 5.
Figure 5.
FSTL1 is targeted by miR-137. (a) Predicted binding site in the 3ʹUTR of the FSTL1 gene with miR-137. (b) Mutant of FSTL1 3ʹUTR reporter plasmid was constructed. (c) Luciferase reporter assays were performed in 293T cells with co-transfection of indicated wild-type or mutant 3ʹUTR FSTL1 constructs and miR-137 or NC (negative control). *p < 0.05. (d) Relative expressions of miR-137 in MDA-MB-231, MDA-MB-468 and HCC38 cells with miR-137 or NC transfection. **p < 0.01. (e, f) RT-PCR (e) and western blot (f) analyses of FSTL1 expression in MDA-MB-231, MDA-MB-468 and HCC38 cells with miR-137 or NC transfection. **p < 0.01.
Figure 6.
Figure 6.
FSTL1 is required for miR-137-regulated Wnt/β-catenin signaling. (a) Western blot of FSTL1 expression in miR-137 transfected MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. (b) Western blot assays of Wnt/β-catenin signaling target gene expression in miR-137 transfected MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. *p < 0.05. (c) TOP/FOP ratios in miR-137 transfected MDA-MB-231 and HCC38 cells with or without FSTL1 overexpression. *p < 0.05.
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The study was supported by Natural Science Foundation of Heilongjiang Province (ZD2016018) and National Natural Science Foundation of China (81673006).