Pro- and anti-tumourigenic effects of Bone morphogenetic protein 9 signaling in hepatocellular carcinoma

Bone morphogenetic protein 9 (BMP-9) is a hepatic cytokine that belongs to the transforming growth factor b superfamily. Recent studies have shown that the role of the BMP-9 pathway in hepatocellular carcinoma (HCC) is rather controversial. BMP-9 was described to promote cell proliferation and epithelial-to-mesenchymal transition (EMT) in some studies, whereas in others it acted antiproliferative and decreased mesenchymal markers in HCC cells.

HCC is still the third leading cause of cancer death worldwide. Therefore it is important to identify new therapeutic targets to improve our current diagnostic and therapeutic strategies. The aim of this study was therefore to identify the role of BMP-9 signaling in HCC.

Basal expression of BMP-9 signaling pathway molecules such as ALK1, ALK2, Activin A Receptor Type 2A (ACVR2A), Activin A Receptor Type 2B (ACVR2B), BMPRII (BMPR2), endoglin (ENG), Smad1 (SMAD1), was assessed in two human HCC cell lines (HLE and Hep3B) by RT-qPCR and Western blot. Smad1 phosphorylation (Ser463/465), cell proliferation and cell migration in the absence or presence of BMP-9 (5 ng/mL) was assessed by Western blot, proliferation assay and wound healing (scratch) assays, respectively. Public databases such as The Cancer Genome Atlas (TCGA), The Cancer Proteome Atlas (TCPA), Tumor Immune Estimation Resource (TIMER), Cancer Cell Line Encyclopedia (CCLE) and Hepamine were used to extensively analyze available expression data for BMP-9 signaling pathway molecules. A small set of freshly collected samples from 4 HCC and 1 CCC patient were additionally analyzed for validation of the in silico findings.

Data from TCGA show that the BMP-9 signaling pathway is strongly associated with 48 different signaling proteins related to relevant pathways in cancer development such as p38 mitogen activated kinases, interferon gamma, p53 and angiogenesis pathways. BMP-9 signaling pathway genes are coexpressed with relevant cancer-associated pathway genes related to cell proliferation, migration, angiogenesis, and extracellular matrix remodelling in HCC patients. Co-expression analysis showed that 69 and 44 genes are exclusively jointly coexpressed with ALK1/SMAD1 and ALK2/BMPR2 in HCC patients, respectively. CCLE data showed a differential drug resistance in HCC cell lines depending on their BMP-9 in vitro response. Finally, BMP-9 stimulation promoted HLE but inhibited Hep3B cell proliferation and migration which seems to be the result of the presence of diverse sets of receptors/co-factors in the two lines. Alk1 signalling resulted in a malignant cellular phenotype whereas signalling via Alk2 leads to stabilizing (benign) responses.

Taken together, these results suggest that the BMP-9/Alk1 signaling pathway, which is upregulated in HCC, promotes tumour development whereas non-malignant hepatocytes (and some HCC cell lines) express no Alk1 and respond to BMP-9 via the Alk2 pathway which stabilizes the differentiated parenchymal phenotype of the cells (= non-tumourigenic). Thereby characterization of the individual tumour of a patient regarding its Alk1 levels should help to predict further outcome like aggressiveness and possible responsiveness to BMP-9 targeted therapies.