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Tumor metastasis remains an obstacle in cancer treatment and is responsible for most cancer-related deaths. Nm23-H1 is one of the first metastasis suppressor proteins discovered with the ability to inhibit metastasis of many cancers including breast, colon, and liver cancer. Although loss of Nm23-H1 is observed in aggressive cancers and correlated with metastatic potential, little is known regarding the mechanisms that regulate its cellular level. Here, we examined the mechanisms that control Nm23-H1 expression in breast cancer cells. Initial studies in aggressive MDA-MB-231 cells (expressing low Nm23-H1) and less invasive MCF-7 cells (expressing high Nm23-H1) revealed that mRNA levels correlated with protein expression, suggesting that transcriptional mechanisms may control Nm23-H1 expression. Truncational analysis of the Nm23-H1 promoter revealed a proximal and minimal promoter that harbor putative binding sites for transcription factors including CTCF and EGR1. CTCF and EGR1 induced Nm23-H1 expression and reduced cell migration of MDA-MB-231 cells. Moreover, CTCF and EGR1 were recruited to the Nm23-H1 promoter in MCF-7 cells and their expression correlated with Nm23-H1 levels. This study indicates that loss of Nm23-H1 in aggressive breast cancer is apparently caused by downregulation of CTCF and EGR1, which potentially drive Nm23-H1 expression to promote a less invasive phenotype.

Upregulation of transmembrane protein 97 (TMEM97) has been associated with progression and poor outcome in multiple human cancers, including breast cancer. Recent studies suggest that TMEM97 may be involved in the activation of the Wnt/β-catenin pathway. However, the molecular mechanism of TMEM97 action on Wnt/β-catenin signaling is completely unclear. In the current study, TMEM97 was identified as an LRP6-interacting protein. TMEM97 could interact with LRP6 intracellular domain and enhance LRP6-mediated Wnt signaling in a CK1δ/ε-dependent manner. The binding of TMEM97 to LRP6 facilitated the recruitment of CK1δ/ε to LRP6 complex, resulting in LRP6 phosphorylation at Ser 1490 and the stabilization of β-catenin. In breast cancer cells, knockout of TMEM97 attenuated the Wnt/β-catenin signaling cascade via regulating LRP6 phosphorylation, leading to a decrease in the expression of Wnt target genes AXIN2, LEF1, and survivin. TMEM97 deficiency also suppressed cell viability, proliferation, colony formation, migration, invasion, and stemness properties in breast cancer cells. Importantly, TMEM97 knockout suppressed tumor growth through downregulating the Wnt/β-catenin signaling pathway in a breast cancer xenograft model. Taken together, our results revealed that TMEM97 is a positive modulator of canonical Wnt signaling. TMEM97-mediated Wnt signaling is implicated in the tumorigenesis of breast cancer, and its targeted inhibition may be a promising therapeutic strategy for breast cancer.

The sulfur-coordinated organoiridium(III) complexes pbtIrSS and ppyIrSS, which contain C,N and S,S (dithione) chelating ligands, were found to inhibit breast cancer tumorigenesis and metastasis by targeting Wnt/β-catenin signaling for the first time. Treatment with pbtIrSS and ppyIrSS induces the degradation of LRP6, thereby decreasing the protein levels of DVL2, β-catenin and activated β-catenin, resulting in downregulation of Wnt target genes CD44 and survivin. Additionally, pbtIrSS and ppyIrSS can suppress cell migration and invasion of breast cancer cells. Furthermore, both complexes show the ability to inhibit sphere formation and mediate the stemness properties of breast cancer cells. Importantly, pbtIrSS exerts potent anti-tumor and anti-metastasis effects in mouse xenograft models through the blockage of Wnt/β-catenin signaling. Taken together, our results indicate that pbtIrSS has great potential to be developed as a breast cancer therapeutic agent with a novel mechanism.

Background CK1 is involved in regulating Wnt/β-catenin signaling and represents a promising target for the treatment of breast cancer. A purine derivative longdaysin has recently been identified as a novel modulator of cellular circadian rhythms through targeting the protein kinases CK1δ, CK1α, and ERK2. However, the antitumor activity of longdaysin and its underlying mechanisms remain unclear. Methods The inhibitory effect of longdaysin on Wnt/β-catenin signaling was investigated using the SuperTOPFlash reporter system. The levels of phosphorylated LRP6, total LRP6, DVL2, active β-catenin, and total β-catenin were examined by Western blot. The expression of Wnt target genes was determined using real-time PCR. The ability of colony formation of breast cancer cells was measured by colony formation assay. The effects of longdaysin on cancer cell migration and invasion were assessed using transwell assays. The effect of longdaysin on cancer stem cells was tested by sphere formation assay. The in vivo antitumor effect of longdaysin was evaluated using MDA-MB-231 breast cancer xenografts. Results Longdaysin suppressed Wnt/β-catenin signaling through inhibition of CK1δ and CK1ε in HEK293T cells. In breast cancer Hs578T and MDA-MB-231 cells, micromolar concentrations of longdaysin attenuated the phosphorylation of LRP6 and DVL2 and reduced the expression of active β-catenin and total β-catenin, leading to the downregulation of Wnt target genes Axin2, DKK1, LEF1, and Survivin. Furthermore, longdaysin inhibited the colony formation, migration, invasion, and sphere formation of breast cancer cells. In MDA-MB-231 breast cancer xenografts, treatment with longdaysin suppressed tumor growth in association with inhibition of Wnt/β-catenin signaling. Conclusion Longdaysin is a novel inhibitor of the Wnt/β-catenin signaling pathway. It exerts antitumor effect through blocking CK1δ/ε-dependent Wnt signaling.

Background Gigantol is a bibenzyl compound derived from several medicinal orchids. This biologically active compound has been shown to have promising therapeutic potential against cancer cells, but its mechanism of action remains unclear. Methods The inhibitory effect of gigantol on Wnt/β-catenin signaling was evaluated with the SuperTOPFlash reporter system. The levels of phosphorylated low-density lipoprotein receptor related protein 6 (LRP6), total LRP6 and cytosolic β-catenin were determined by Western blot analysis. The expression of Wnt target genes was analyzed using real-time PCR. Cell viability was measured with a MTT assay. The effect of gigantol on cell migration was examined using scratch wound-healing and transwell migration assays. Results Gigantol decreased the level of phosphorylated LRP6 and cytosolic β-catenin in HEK293 cells. In breast cancer MDA-MB-231 and MDA-MB-468 cells, treatment with gigantol reduced the level of phosphorylated LRP6, total LRP6 and cytosolic β-catenin in a dose-dependent manner, resulting in a decrease in the expression of Wnt target genes Axin2 and Survivin. We further demonstrated that gigantol suppressed the viability and migratory capacity of breast cancer cells. Conclusion Gigantol is a novel inhibitor of the Wnt/β-catenin pathway. It inhibits Wnt/β-catenin signaling through downregulation of phosphorylated LRP6 and cytosolic β-catenin in breast cancer cells.