Since hepatocellular carcinoma (HCC) is a typical hypervascular malignant tumor with poor prognosis, targeting angiogenesis is an important therapeutic strategy for advanced HCC. Involvement of bone morphologic protein 9 (BMP9), a transforming growth factor-beta superfamily member, has recently been reported in the development of liver diseases and angiogenesis. Here, we aimed to elucidate the role of BMP9 signaling in promoting HCC angiogenesis and to assess the antiangiogenic effect of BMP receptor inhibitors in HCC. By analyzing HCC tissue gene expression profiles, we found that BMP9 expression was significantly correlated with angiogenesis-associated genes, including HIF-1α and VEGFR2. In vitro, BMP9 induced HCC cell HIF-1α/VEGFA expression and VEGFA secretion. Silencing of the inhibitor of DNA-binding protein 1 (ID1), a transcription factor targeted by BMP9 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression and VEGFA secretion, resulting in decreased human umbilical vein endothelial cell (HUVEC) lumen formation. BMP receptor inhibitors, which inhibit BMP9-ID1 signaling, suppressed BMP9-induced HIF-1α/VEGFA expression, VEGFA secretion, and HUVEC lumen formation. In vivo, the BMP receptor inhibitor LDN-212854 successfully inhibited HCC tumor growth and angiogenesis by inhibiting BMP9-ID1 signaling. In summary, BMP9-ID1 signaling promotes HCC angiogenesis by activating HIF-1α/VEGFA expression. Thus, targeting BMP9-ID1 signaling could be a pivotal therapeutic option for advanced HCC.

Alpha-fetoprotein (AFP) is an oncofetal protein that is elevated in a subset of hepatocellular carcinoma (HCC) with poor prognosis, but the molecular target activated in AFP-positive HCC remains elusive. Here, we demonstrated that the transcription factor forkhead box M1 (FOXM1) is upregulated in AFP-positive HCC. We found that FOXM1 expression was highly elevated in approximately 40% of HCC cases, and FOXM1-high HCC was associated with high serum AFP levels, a high frequency of microscopic portal vein invasion, and poor prognosis. A transcriptome and pathway analysis revealed the activation of the mitotic cell cycle and the inactivation of mature hepatocyte metabolism function in FOXM1-high HCC. The knockdown of FOXM1 reduced AFP expression and induced G2/M cell cycle arrest. We further identified that the proteasome inhibitor carfilzomib attenuated FOXM1 protein expression and suppressed cell proliferation in AFP-positive HCC cells. Carfilzomib in combination with vascular endothelial growth factor receptor 2 (VEGFR2) blockade significantly prolonged survival by suppressing AFP-positive HCC growth in a subcutaneous tumor xenotransplantation model. These data indicated that FOXM1 plays a pivotal role in the proliferation of AFP-positive liver cancer cells. Carfilzomib can effectively inhibit FOXM1 expression to inhibit tumor growth and could be a novel therapeutic option in patients with AFP-positive HCC who receive anti-VEGFR2 antibodies.

Hepatocellular carcinoma (HCC) is a highly lethal malignant neoplasm, and the involvement of bone morphogenetic protein 9 (BMP9) has been implicated in the pathogenesis of liver diseases and HCC. Our goal was to investigate the role of BMP9 signaling in regulating N6-methyladenosine (m⁶A) methylation and cell cycle progression, and evaluate the therapeutic potential of BMP receptor inhibitors for HCC treatment. We observed that elevated levels of BMP9 expression in tumor tissues or serum samples from HCC patients were associated with a poorer prognosis. Through in vitro experiments utilizing the m⁶A dot blotting assay, we ascertained that BMP9 reduced the global RNA m⁶A methylation level in Huh7 and Hep3B cells, thereby facilitating their cell cycle progression. This effect was mediated by an increase in the expression of the inhibitor of DNA-binding protein 1 (ID1). Additionally, using methylated RNA immunoprecipitation qPCR(MeRIP-qPCR), we showed that the BMP9-ID1 pathway promoted CyclinD1 expression by decreasing the m⁶A methylation level in the 5′ UTR of mRNA. This occurred through the upregulation of the fat mass and obesity-associated protein (FTO) in Huh7 and Hep3B cells. In our in vivo mouse xenograft models, we demonstrated that blocking the BMP receptor with LDN-212854 effectively suppressed HCC growth and induced global RNA m⁶A methylation. Overall, our findings indicate that the BMP9-ID1 pathway promotes HCC cell proliferation by down-regulating the m⁶A methylation level in the 5′ UTR of CyclinD1 mRNA. Targeting the BMP9-ID1 pathway holds promise as a potential therapeutic strategy for treating HCC.

Bone morphogenetic proteins (BMPs) are a group of growth factors belonging to the transforming growth factor β(TGF-β) family. While initially recognized for their role in bone formation, BMPs have emerged as significant players in liver diseases. Among BMPs with various physiological activities, this comprehensive review aims to delve into the involvement of BMP9 specifically in liver diseases and provide insights into the complex BMP signaling pathway. Through an enhanced understanding of BMP9, we anticipate the discovery of new therapeutic options and potential strategies for managing liver diseases.

Accumulating evidence suggests an association between Helicobacter pylori infection and liver diseases; however, it remains unclear whether H. pylori or its extracellular products directly influence hepatocellular carcinoma (HCC) biology. Here, we examined the effects of viable H. pylori, sterile H. pylori-conditioned supernatant (Hp-CS), and isolated H. pylori-derived outer membrane vesicles (Hp-OMVs) on HCC cells. H. pylori adhered to and invaded Huh7 and Hepa1-6 cells, increased Cyclin D1 expression, and enhanced the growth of tumors derived from bacteria-exposed Hepa1-6 cells in immunocompetent C57BL/6J mice. Hp-CS increased HCC cell metabolic activity, colony formation, migration, and invasion. Hp-OMVs were characterized by transmission electron microscopy and nanoparticle tracking analysis. Exposure to Hp-OMVs increased HCC cell metabolic activity, colony formation, cell-cycle progression, and tumor growth in a syngeneic mouse model. Hp-OMVs also increased PI3K, AKT, and mTOR phosphorylation and elevated Cyclin D1 expression. The PI3K inhibitor LY294002 attenuated these molecular changes and reduced Hp-OMV-induced cell-cycle progression and colony formation. These findings show that H. pylori and its extracellular products promote proliferation-associated phenotypes in experimental HCC models. Hp-OMVs are biologically active extracellular components of H. pylori, and pharmacological inhibition supports the involvement of PI3K/AKT/mTOR signaling and Cyclin D1 upregulation in their growth-promoting effects.