BMP4-Induced Suppression of Breast Cancer Metastasis Is Associated with Inhibition of Cholesterol Biosynthesis

Abstract

We reported previously that in preclinical models, BMP4 is a potent inhibitor of breast cancer metastasis and that high BMP4 protein levels predict favourable patient outcomes. Here, we analysed a breast cancer xenograft with or without enforced expression of BMP4 to gain insight into the mechanisms by which BMP4 suppresses metastasis. Transcriptomic analysis of cancer cells recovered from primary tumours and phosphoproteomic analyses of cancer cells exposed to recombinant BMP4 revealed that BMP4 inhibits cholesterol biosynthesis, with many genes in this biosynthetic pathway being downregulated by BMP4. The treatment of mice bearing low-BMP4 xenografts with a cholesterol-lowering statin partially mimicked the anti-metastatic activity of BMP4. Analysis of a cohort of primary breast cancers revealed a reduced relapse rate for patients on statin therapy if their tumours exhibited low BMP4 levels. These findings indicate that BMP4 may represent a predictive biomarker for the benefit of additional statin therapy in breast cancer patients.

Keywords: BMP4; breast cancer; cholesterol biosynthesis; metastasis; statins.

Figure 1

RNA sequencing analysis reveals BMP4 target genes that may regulate breast cancer metastasis. (a) Volcano plots highlighting significantly up- and downregulated genes by BMP4 in 231-HM tumours. Tumour cells were recovered from primary tumours following disaggregation and flow cytometry. Statistical analysis was completed using limma 3.42.2 package in R. (b) Western blotting validation of changes in protein levels of BMP4 target genes that were identified in RNA sequencing analysis (annotated in red in panel (a)). Proteins were extracted from resected 231-HM primary tumours with modified expression of BMP4. HSP90 was used as loading control. VC, vector control.

Figure 2

Mass spectrometric analysis of 231-HM tumour cells treated with recombinant human BMP4 in vitro. (a) Volcano plot highlighting significantly up- and downregulated phosphopeptides following 45 min of exposure to recombinant BMP4 (20 ng/mL). Proteins that are implicated in cholesterol biosynthesis are annotated in red. (b) Volcano plot highlighting significantly up- and downregulated total proteins following 24 h of exposure to recombinant BMP4 (40 ng/mL). (c) Comparison of BMP4-induced transcriptomic changes in vivo (Y axis) to BMP4-induced total proteomic changes (X axis) in vitro. Statistical analysis in (a,b) was completed using limma package in R.

Figure 3

BMP4 negatively regulates cholesterol biosynthesis genes in 231-HM xenograft tumours. (a) Dot plots highlighting most significantly regulated Hallmark and C2 pathways in BMP4-expressing 231-HM xenograft tumours. Pathways that relate to cholesterol biosynthesis are annotated in red. Statistical analysis was completed using clusterProfiler 4.8.3 package in R. (b) Enrichment plots of BMP4-induced gene expression changes that relate to Hallmark cholesterol homeostasis pathway and Reactome cholesterol biosynthesis pathway. Data visualisation was completed using enrichplot 1.20.3 package in R. (c) Heatmap of BMP4-induced gene expression changes in Hallmark cholesterol homeostasis pathway. Data visualisation was completed using ComplexHeatmap 2.16.0 package in R.

Figure 4

Validation of suppression of cholesterol biosynthesis by BMP4 in vivo and in vitro. (a) Quantitation of free (annotated in blue) and total (annotated in grey) cholesterol levels in 231-HM primary tumours. n ≥ 9/group, mean ± SEM. (b) RT-qPCR analysis of expression of cholesterol biosynthesis genes in 231-HM cells cultured in 0.5% serum. n = 3/group, mean ± SEM. (c,d) Expression of genes that relate to cholesterol biosynthesis both in vitro and from primary tumours in vivo. In (d), RNA was extracted either from in vitro cultured 231-HM cells or from resected 231-HM xenograft tumours. n = 3/group, mean ± SEM. (e) Constitutively active SREBP2 (N-terminal; nSREBP2) promoted activity of sterol regulatory element (SRE) that regulates expression of cholesterol biosynthesis genes in HEK293T cells. (f) Expression of cholesterol biosynthesis genes in MDA-MB-231HM cells with constitutively active nSREBP2 and with or without exogeneous BMP4 expression. n = 3/group, mean ± SEM. Statistical analysis was completed using Student’s t test. ns, not significant; *, p < 0.05; **, p < 0.01.

Figure 5

Statin therapy suppresses the metastasis of 231-HM xenograft tumours and is associated with a lower risk of breast cancer relapse in patients. (a) The effect of lovastatin on the growth of primary 231-HM tumours. 231-HM cells (1 × 106) were injected into the mammary glands of NSG mice. The mice were treated with lovastatin (10 mg/kg; 5 days/week) once the tumours became palpable following injection. n = 15/group, mean ± SEM. (b) The weights of resected 231-HM tumours. The tumours were resected on day 18 after tumour cell inoculation, when the average tumour volume reached approximately 400 mm3. n = 15/group, mean ± SEM. (c) Representative images of TurboGFP-tagged metastatic lesions in the lungs and livers visualised ex vivo using the Maestro imaging system. The mice were euthanised 15 days after tumour resection. (d) The normalised metastatic burden in the lungs (left) and livers (right) at the endpoint. n = 15/group, mean ± SEM. (e–j) The prognostic value of statin use in a cohort of 407 breast cancer patients. Relapse-free survival (e) and distant relapse free survival (h) in all patients in the cohort. (f,i): patients whose tumours were low for BMP4. (g,j): patients whose tumours were high for BMP4. Statistical analysis was completed using the survival: 3.1-11 package in R. ns, not significant; ***, p < 0.001.