Abstract
Metastatic outcomes depend on the interactions of metastatic cells with a specific organ microenvironment. Our previous studies have shown that triple-negative breast cancer (TNBC) MDA-MB-231 cells passaged in astrocyte-conditioned medium (ACM) show proclivity to form brain metastases, but the underlying mechanism is unknown. The combination of microarray analysis, qPCR, and ELISA assay were carried out to demonstrate the ACM-induced expression of angiopoietin-like 4 (ANGPTL4) in TNBC cells. A stable ANGPTL4-knockdown MDA-MB-231 cell line was generated by ANGPTL4 short-hairpin RNA (shRNA) and inoculated into mice via left ventricular injection to evaluate the role of ANGPTL4 in brain metastasis formation. The approaches of siRNA, neutralizing antibodies, inhibitors, and immunoprecipitation were used to demonstrate the involved signaling molecules. We first found that ACM-conditioned TNBC cells upregulated the expression of ANGPTL4, a secreted glycoprotein whose effect on tumor progression is known to be tumor microenvironment- and tumor-type dependent. Knockdown of ANGPTL4 in TNBC MDA-MB-231 cells with shRNA decreased ACM-induced tumor cell metastatic growth in the brain and attributed to survival in a mouse model. Furthermore, we identified that astrocytes produced transforming growth factor-beta 2 (TGF-β2), which in part is responsible for upregulation of ANGPTL4 expression in TNBC through induction of SMAD signaling. Moreover, we identified that tumor cells communicate with astrocytes, where tumor cell-derived interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) increased the expression of TGF-β2 in astrocytes. Collectively, these findings indicate that the invading TNBC cells interact with astrocytes in the brain microenvironment that facilitates brain metastases of TNBC cells through a TGF-β2/ANGPTL4 axis. This provides groundwork to target ANGPTL4 as a treatment for breast cancer brain metastases.
Highlights
The incidence of breast cancer brain metastasis (BM) has increased in recent years, in the subpopulation of triple-negative breast cancer (TNBC).[1,2,3] TNBC, which accounts for 15–25% of all breast cancers,[4,5] has problematic diagnosis because patients with TNBC do not respond to hormone or HER2-targeted therapies due to the lack of expression of ER, PR, and HER2.4 the exact pathologic process of BM in the TNBC subset is poorly understood up to now
The most intriguing finding from our earlier studies is that after passaging the MDA-MB-231 cells in astrocyte-conditioned medium (ACM) for five passages, the invasion competence of the tumor cell showed a sudden and rapid increase, which led to an increase in vitro invasion when compared with the cells passaged 1–4 times in ACM.[17]
To gain insight into the mechanism of the BM, we investigated whether the ACM affected gene expression by microarray expression analysis using MDA-MB-231/P5A, MDAMB-231/P5D, and MDA-MB-231/P10AD
Summary
The incidence of breast cancer brain metastasis (BM) has increased in recent years, in the subpopulation of triple-negative breast cancer (TNBC) (estrogen receptor -/progesterone receptor -/human epidermal growth factor receptor 2 -, ER-/PR-/Her2-).[1,2,3] TNBC, which accounts for 15–25% of all breast cancers,[4,5] has problematic diagnosis because patients with TNBC do not respond to hormone or HER2-targeted therapies due to the lack of expression of ER, PR, and HER2.4 the exact pathologic process of BM in the TNBC subset is poorly understood up to now These facts emphasize the clinical importance of BM in TNBC and provide an explanation for why patients with TNBC have the worst outcomes out of all breast cancer patients.
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