Interaction of tumor cells and astrocytes promotes breast cancer brain metastases through TGF-β2/ANGPTL4 axes

Figures

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  Fig. 1. ACM upregulates ANGPTL4 expression in TNBC cells. a Volcano plots for differential ACM-responsive genes. The log-fold change relative to P5D is plotted on the x-axis (the downregulated genes represented by green points, the upregulated genes represented by red points) and the negative log10 p-value is plotted on the y-axis. The black, dashed line represents the p-value cutoff (0.01, Student’s t-test) and log FC cutoff (±1.0). b Heatmap of differential ACM-responsive gene expression. Heatmap of gene expression alterations seen across all probe sets, with triplicate samples per culture condition. Each column represents a single culture and each row represents a single gene. Red indicates overexpressed probe sets, whereas green indicates underexpressed probe sets. The top cluster diagram indicates the hierarchical clustering order of the individual samples analyzed. The right cluster diagram represents differentially expressed probes with p < 0.01 and FC > 2.0. As shown, ANGPTL4 is one of the top overexpressed genes. c qPCR of ANGPTL4 transcripts in ACM-conditioned MDA-MB-231 cells. *p < 0.05 compared with P5D and +p < 0.05 compared with P10AD by the two-sided Student’s t-test, n =…

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  Fig. 2. ANGPTL4 contributes to the brain metastases of MDA-MB-231 cells. a ANGPTL4 s6hRNA decreased mRNA expression level of ANGPTL4 in MDA-MB-231 cells. MDA-MB-231 cells were transfected with ANGPTL4 shRNA-1 and shRNA-2, respectively, or non-targeting control shRNA and further used for qPCR analysis for ANGPTL4 expression. **p < 0.01 compared with the control group by the two-sided Student’s t-test. b Effects of ANGPTL4 knockdown on ANGPTL4 protein expression in MDA-MB-231 cells. MDA-MB-231 cells transfected with A4shRNA-1 (A4shRNA) or control shRNA (ConshRNA) were used for ELISA analysis of ANGPTL4 expression. *p < 0.05 and **p < 0.01 compared with the control group by the two-sided Student’s t-test. c, d Role of ANGPTL4 in brain lesions of MDA-MB-231 cells. A4shRNA/ACM (n = 7) and ConshRNA/ACM MDA-MB-231 (n = 11) cells were injected into the left ventricle of nude mice. Tumor lesions were measured by bioluminescent imaging. c Representative brain metastasis tumor images. d Total brain flux at 21 days post injection. *p = 0.029 by the two-sided Student’s t-test comparing A4shRNA/ACM (mean 2.51e + 6, SD 2.56e + 6) with ConshRNA/ACM (mean 5.35e + 7, SD 1.29e + 8)

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  Fig. 3. Astrocyte-secreted TGF-β2 induces ANGPTL4 expression in TNBC cells. a Rat and human astrocytes produce TGF-β2. TGF-β2 was quantified by ELISA analysis in both rat and human ACM. b TGF-β2-induced ANGPTL4 expression in MDA-MB-231 and MDA-MB-468 cells. Cells were treated with vehicle or TGF-β2 (5 ng/ml) and ANGPTL4 expression was quantified in both mRNA and protein by qPCR and ELISA, respectively. **p < 0.01 and ***p < 0.005 compared with vehicle by the two-sided Student’s t-test, n = 3. c Astrocyte-derived TGF-β2 was involved in the ANGPTL4 induction in TNBC cells. ACM with or without TGF-β2 depletion was used to culture MDA-MB-231 and MDA-MB-468 cells, and analyzed by ELISA to quantify ANGPTL4 expression. *p < 0.05 and **p < 0.01 compared with ACM by the two-sided Student’s t-test. n = 3. ACM: rat astrocyte-conditioned medium; hACM: human astrocyte-conditioned medium

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  Fig. 4. TGF-β2 induces ANGPTL4 expression through the TGFβR/SMAD signaling pathway. a TGFβR mediated ANGPTL4 expression in TNBC cells. TNBC cells were treated with vehicle or SB431542 or SB525334, and then stimulated with 5 ng/ml TGF-β2. The resultant supernatants and cell lysates were analyzed by ELISA to quantity ANGPTL4 expression. #p < 0.01 compared with basal, *p < 0.05 compared with vehicle by the two-sided Student’s t-test, n = 3. b TGF-β2 activated SMAD3. Immunoblot (IB) was performed to examine the phosphorylation of SMAD3 upon TGF-β2 (5 ng/ml) induction. c TGF-β2 attributed to SMAD3/SMAD4 complex formation in MDA-MB-231 cells. Immunoprecipitation (IP) followed by IB experiments was performed to examine the SMAD3/SMAD4 complex in MDA-MB-231 cells upon TGF-β2 (5 ng/ml) induction. d SMAD3 and SMAD4 mediated TGF-β2-induced ANGPTL4 expression in MDA-MB-231 cells. The expression of SMAD3 and SMAD4 in MDA-MB-231 cells were knocked down by siRNAs (left panel), and the resultant supernatants and the cell lysates were analyzed by ELISA to quantity ANGPTL4 expression (right panel). *p < 0.05 compared with control siRNA-treated cells by the two-sided Student’s t-test. n = 3

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  Fig. 5. TNBC cell-derived IL-1β and TNF-α enhance the expression of TGF-β2 in astrocytes. a Astrocyte-derived TGF-β2 was increased by tumor cell-conditioned medium (TCM) from TNBC cells. Quantity of basal TGF-β2 expressions in TCM (left panel); TCM was added to culture astrocytes, and the resultant culture supernatants were analyzed by ELISA to quantify TGF-β2 expression (right panel). Astrocytes cultured in serum-free breast cancer cell line media (SFM) that was not preconditioned by tumor cells were used as control. *p < 0.05 compared with astrocytes in TCM with SFM; +p < 0.05 compared with MCF-10A in TCM and #p < 0.05 compared with MCF-7 in TCM by the two-sided Student’s t-test. n = 3. b Mean centralized the mRNA of IL-1β and TNF-α in three subtypes of breast cancer. Data from TCGA 719 breast cancer cohort. ***p < 0.005 compared with luminal and Her2 + breast cancers by the two-sided Student’s t-test. c TNBC cells produced IL-1β and TNF-α. TCM from different cells was analyzed by ELISA to quantity the levels of IL-1β (left) and TNF-α (right), respectively. *p < 0.05 compared with MCF-10A in TCM and +p < 0.05 compared with MCF-7 in TCM by the two-sided Student’s t-test. n = 3. d TNBC…

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  Fig. 6. Working hypothesis. Invading tumor cells derived IL-1β and TNF-α that upregulated the expression of TGF-β2 in astrocytes. The TGF-β2 then through TGF-βR and SMAD signaling induced the expression of ANGPTL4 in tumor cells to promote brain lesions of the tumor cells