Adiponectin inhibits Wnt co-receptor, Lrp6, phosphorylation and ß-catenin signaling.

Adiponectin is a pleiotropic adipokine implicated in obesity, metabolic syndrome and cardiovascular disease. Recent studies have identified adiponectin as a negative regulator of tissue fibrosis. Wnt/ß-catenin signaling has also been implicated in metabolic syndrome and can promote tissue fibrosis, but the extent to which adiponectin cross-regulates Wnt/ß-catenin signaling is unknown. Using primary human dermal fibroblasts and recombinant purified proteins, we show that adiponectin can limit ß-catenin accumulation and downstream gene activation by inhibiting Lrp6 phosphorylation, a key activation step in canonical Wnt signaling. Inhibition of Wnt3a-mediated Lrp6 phospho-activation is relatively rapid (e.g., by 30 min), and is not dependent on established adiponectin G-protein coupled receptors, AdipoR1 and R2, suggesting a more direct relationship to Lrp6 signaling. In contrast, the ability of adiponectin to limit Wnt-induced and baseline collagen production in fibroblasts requires AdipoR1/R2. These results suggest the possibility that the pleiotropic effects of adiponectin may be mediated through distinct cell surface receptor complexes. Accordingly, we propose that the anti-fibrotic activity of adiponectin may be mediated through AdipoR1/R2 receptors, while the ability of adiponectin to inhibit Lrp6 phospho-activation may be relevant to other recently established roles for Lrp6 signaling in glucose metabolism and metabolic syndrome.

Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFß signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFß-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

Snail represses the splicing regulator epithelial splicing regulatory protein 1 to promote epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT), a tightly regulated process that is critical for development, is frequently re-activated during cancer metastasis and recurrence. We reported previously that CD44 isoform switching is critical for EMT and showed that the splicing factor ESRP1 inhibits CD44 isoform switching during EMT. However, the mechanism by which ESRP1 is regulated during EMT has not been fully understood. Here we show that the transcription repressor Snail binds to E-boxes in the ESRP1 promoter, causing repression of the ESRP1 gene. Biochemically, we define the mechanism by which ESRP1 regulates CD44 alternative splicing: ESRP1 binds to the intronic region flanking a CD44 variable exon and causes increased variable exon inclusion. We further show that ectopically expressing ESRP1 inhibits Snail-induced EMT, suggesting that down-regulation of ESRP1 is required for function by Snail in EMT. Together, these data reveal how the transcription factor Snail mediates EMT through regulation of a splicing factor.

CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression.

Epithelial-mesenchymal transition (EMT) is a tightly regulated process that is critical for embryogenesis but is abnormally activated during cancer metastasis and recurrence. Here we show that a switch in CD44 alternative splicing is required for EMT. Using both in vitro and in vivo systems, we have demonstrated a shift in CD44 expression from variant isoforms (CD44v) to the standard isoform (CD44s) during EMT. This isoform switch to CD44s was essential for cells to undergo EMT and was required for the formation of breast tumors that display EMT characteristics in mice. Mechanistically, the splicing factor epithelial splicing regulatory protein 1 (ESRP1) controlled the CD44 isoform switch and was critical for regulating the EMT phenotype. Additionally, the CD44s isoform activated Akt signaling, providing a mechanistic link to a key pathway that drives EMT. Finally, CD44s expression was upregulated in high-grade human breast tumors and was correlated with the level of the mesenchymal marker N-cadherin in these tumors. Together, our data suggest that regulation of CD44 alternative splicing causally contributes to EMT and breast cancer progression.