Probes for WNT5A

WNTs are lipid-modified proteins that control multiple functions in development and disease via short- and long-range signaling. However, it is unclear how these hydrophobic molecules spread over long distances in the mammalian brain. Here we show that WNT5A is produced by the choroid plexus (ChP) of the developing hindbrain, but not the telencephalon, in both mouse and human. Since the ChP produces and secretes the cerebrospinal fluid (CSF), we examine the presence of WNT5A in the CSF and find that it is associated with lipoprotein particles rather than exosomes. Moreover, since the CSF flows along the apical surface of hindbrain progenitors not expressing Wnt5a, we examined whether deletion of Wnt5a in the ChP controls their function and find that cerebellar morphogenesis is impaired. Our study thus identifies the CSF as a route and lipoprotein particles as a vehicle for long-range transport of biologically active WNT in the central nervous system.

Obesity increases the risk of hepatocellular carcinoma (HCC), but precise identification and characterization of druggable oncogenic pathways that contribute to the progression of NAFLD to HCC, and hence to the increased incidence and aggressiveness of HCC in obese individuals is lacking. In this regard, we demonstrate that the Indian Hedgehog (Ihh) signaling pathway is upregulated in the fatty livers of mice consuming a high fat diet, and furthermore sustained in HCC tumors specifically within the context of a NAFLD microenvironment. Using a diet-induced mouse model of HCC wherein only obese mice develop HCC, targeted ablation of hepatocyte-secreted Ihh results in a decreased tumor burden and lower grade tumors. Ihh activation regulates the transdifferentiation of ciliated stellate cells and proliferation of Epcam+ ductal cells to promote fibrosis. Mechanistically, increased expression of hitherto uncharacterized effectors of Hh pathway, namely Myc and Tgf-β2 is critical to the observed physiology. This pro-tumorigenic response is driven by increased expression of Wnt5a to effect a poorly-differentiated and invasive tumor phenotype. Wnt5a secreted from activated stellate cells act on Ror2-expressing hepatocytes. We further demonstrate that Wnt5a expression is also elevated in poorly-differentiated HCC cells, suggesting that these ligands are also able to function in an autocrine positive feedback manner to sustain poorly-differentiated tumors. Taken together, our study provides a mechanistic understanding for how Ihh signaling promotes HCC tumorigenesis specifically in obese mice. We propose that therapeutic targeting of the Hh pathway offers benefit for patients with dietary / NAFLD-driven steatotic HCC.

Abstract

OBJECTIVE:

Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, can confer aggressive properties to cancer cells by secreting multiple factors. Their phenotypes are stably maintained, but the mechanisms are not fully understood. We aimed to show the critical role of epigenetic changes in CAFs in maintaining their tumour-promoting capacity and to show the validity of the epigenomic approach in identifying therapeutic targets from CAFs to starve cancer cells.

DESIGN:

Twelve pairs of primary gastric CAFs and their corresponding non-CAFs (NCAFs) were established from surgical specimens. Genome-wide DNA methylation and H3K27me3 analyses were conducted by BeadArray 450K and ChIP-on-Chip, respectively. Functions of potential a therapeutic target were analysed by inhibiting it, and prognostic impact was assessed in a database.

RESULTS:

CAFs had diverse and distinct DNA methylation and H3K27me3 patterns compared with NCAFs. Loss of H3K27me3, but not DNA methylation, in CAFs was enriched for genes involved in stem cell niche, cell growth, tissue development and stromal-epithelial interactions, such as WNT5A, GREM1, NOG and IGF2. Among these, we revealed that WNT5A, which had been considered to be derived from cancer cells, was highly expressed in cancer stromal fibroblasts, and was associated with poor prognosis. Inhibition of secreted WNT5A from CAFs suppressed cancer cell growth and migration.

CONCLUSIONS:

H3K27me3 plays a crucial role in defining tumour-promoting capacities of CAFs, and multiple stem cell niche factors were secreted from CAFs due to loss of H3K27me3. The validity of the epigenetic approach to uncover therapeutic targets for cancer-starving therapy was demonstrated.