Integrated RNA-seq and DNase-seq analyses identify phenotype-specific BMP4 signaling in breast cancer.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) plays an important role in cancer pathogenesis. In breast cancer, it reduces proliferation and increases migration in a cell line-dependent manner. To characterize the transcriptional mediators of these phenotypes, we performed RNA-seq and DNase-seq analyses after BMP4 treatment in MDA-MB-231 and T-47D breast cancer cells that respond to BMP4 with enhanced migration and decreased cell growth, respectively. RESULTS: The RNA-seq data revealed gene expression changes that were consistent with the in vitro phenotypes of the cell lines, particularly in MDA-MB-231, where migration-related processes were enriched. These results were confirmed when enrichment of BMP4-induced open chromatin regions was analyzed. Interestingly, the chromatin in transcription start sites of differentially expressed genes was already open in unstimulated cells, thus enabling rapid recruitment of transcription factors to the promoters as a response to stimulation. Further analysis and functional validation identified MBD2, CBFB, and HIF1A as downstream regulators of BMP4 signaling. Silencing of these transcription factors revealed that MBD2 was a consistent activator of target genes in both cell lines, CBFB an activator in cells with reduced proliferation phenotype, and HIF1A a repressor in cells with induced migration phenotype. CONCLUSIONS: Integrating RNA-seq and DNase-seq data showed that the phenotypic responses to BMP4 in breast cancer cell lines are reflected in transcriptomic and chromatin levels. We identified and experimentally validated downstream regulators of BMP4 signaling that relate to the different in vitro phenotypes and thus demonstrate that the downstream BMP4 response is regulated in a cell type-specific manner.

The impact of bone morphogenetic protein 4 (BMP4) on breast cancer metastasis in a mouse xenograft model.

Bone morphogenetic protein 4 (BMP4) is a key regulator of cell proliferation and differentiation. In breast cancer cells, BMP4 has been shown to reduce proliferation in vitro and interestingly, in some cases, also to induce migration and invasion. Here we investigated whether BMP4 influences breast cancer metastasis formation by using a xenograft mouse model. MDA-MB-231 breast cancer cells were injected intracardially into mice and metastasis formation was monitored using bioluminescence imaging. Mice treated with BMP4 developed metastases slightly earlier as compared to control animals but the overall number of metastases was similar in both groups (13 in the BMP4 group vs. 12 in controls). In BMP4-treated mice, bone metastases were more common (10 vs. 7) but adrenal gland metastases were less frequent (1 vs. 5) than in controls. Immunostaining revealed no differences in signaling activation, proliferation rate, blood vessel formation, EMT markers or the number of cancer-associated fibroblasts between the treatment groups. In conclusion, BMP4 caused a trend towards accelerated metastasis formation, especially in bone. More work is needed to uncover the long-term effects of BMP4 and the clinical relevance of these findings.

The impact of lobular and ductal breast cancer histology on the metastatic behavior and long term survival of breast cancer patients.

The aim of the study was to evaluate the long-term survival of patients with invasive lobular carcinomas (ILC) and invasive ductal carcinomas (IDC) and the metastatic behavior of these two disease entities. Originally, all consecutive patients with pure lobular invasive breast cancers diagnosed between 1990 and 1999 in the area served by the Tampere University Hospital and their matched IDC controls were identified and re-evaluated histopathologically in this follow-up study, resulting in a total of 243 ILCs and 243 IDCs. Data on recurrences and survival were collected until the end of year 2009. Statistical analyses including Kaplan-Meier method, log-rank test, Fisher's exact test and Cox regression analysis were performed with the PASW Statistics 18.0 computer program. P-values of <0.05 were considered statistically significant. Within the mean follow-up time of 10.04 years, locoregional recurrences were significantly more common among the ILCs than IDCs (35 vs. 20, p = 0.04), but no differences in the total number of distant recurrences or bilaterality were observed. However, when the first distant recurrence sites were studied, ILC patients had significantly less lung metastases (p = 0.04), but more skin metastases (p = 0.04). During the whole follow-up period IDCs metastasized significantly more frequently to the lungs (p = 0.002), whereas gastrointestinal metastases were more common among ILCs (p = 0.02). Although the known favorable prognostic factors (hormone receptor positivity, low grade, low s-phase) were more common for the ILCs, the disease-free survival, the overall survival and the survival after recurrence did not differ between the groups. However, the Cox-regression model showed significantly worse survival for ILCs after adjusting for age, TNM-status, grade and ER-positivity (p = 0.004). In conclusion, ILC and IDC differ in respect for visceral metastases. Despite the known favorable prognostic factors and originally favorable survival, patients with lobular histology appear to have a worse survival in the multivariate analysis after a prolonged follow-up.

Bone morphogenetic protein 7 expression associates with bone metastasis in breast carcinomas.

BACKGROUND: We recently showed that bone morphogenetic protein 7 (BMP7) is overexpressed in primary breast tumors. Here we explored the clinical significance of BMP7 expression in breast cancer. MATERIALS AND METHODS: This study included 483 breast cancer patients with complete clinicopathological information and up to 15 years of follow-up. Samples contained 241 lobular carcinomas, 242 ductal carcinomas, and 40 local recurrences. BMP7 protein expression was determined using immunohistochemistry. RESULTS: BMP7 was expressed in 47% of the primary tumor samples and 13% of the local recurrences. The primary tumors expressed BMP7 more often than the corresponding local recurrences (P = 0.004). BMP7 expression was dependent on the tumor subtype; 57% of the lobular carcinomas but only 37% of the ductal carcinomas were BMP7 positive (P = 0.0001). BMP7 expression was associated with accelerated bone metastasis formation (P = 0.040), especially in ductal carcinomas (P = 0.033), and multivariate analysis confirmed that BMP7 is an independent prognostic indicator for early bone metastasis development (P = 0.032). CONCLUSION: BMP7 is clearly associated with bone metastasis formation and thus might have clinical utility in identification of patients with increased risk of bone metastasis. This is the first time that bone inducing factor BMP7 has been linked to the bone metastasis process in breast cancer.