The canonical Wnt signaling pathway controls normal embryonic development, cellular proliferation and growth, and its aberrant activity results in human carcinogenesis. The core component in regulation of this pathway is β-catenin, but molecular regulation mechanisms of β-catenin stability are not completely known. Here, our recent studies have shown that KCTD1 strongly inhibits TCF/LEF reporter activity. Moreover, KCTD1 interacted with β-catenin both in vivo by co-immunoprecipitation as well as in vitro through GST pull-down assays. We further mapped the interaction regions to the 1-9 armadillo repeats of β-catenin and the BTB domain of KCTD1, especially Position Ala-30 and His-33. Immunofluorescence analysis indicated that KCTD1 promotes the cytoplasmic accumulation of β-catenin. Furthermore, protein stability assays revealed that KCTD1 enhances the ubiquitination/degradation of β-catenin in a concentration-dependent manner in HeLa cells. And the degradation of β-catenin mediated by KCTD1 was alleviated by the proteasome inhibitor, MG132. In addition, KCTD1-mediated β-catenin degradation was dependent on casein kinase 1 (CK1)- and glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation and enhanced by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). Moreover, KCTD1 suppressed the expression of endogenous Wnt downstream genes and transcription factor AP-2α. Finally, we found that Wnt pathway member APC and tumor suppressor p53 influence KCTD1-mediated downregulation of β-catenin. These results suggest that KCTD1 functions as a novel inhibitor of Wnt signaling pathway.

The role of Eps8 in human breast cancer was studied, and we found that Eps8 was overexpressed in >60% of human breast cancer samples compared with adjacent normal breast tissues by immunohistochemical analysis. Eps8 was highly expressed in the highly invasive breast cancer cell line MDA-MB‑231 compared with the weakly invasive breast cancer cell lines MCF7 and MDA-MB‑468. MCF7 cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced MCF7 breast cancer cell growth and survival as assessed by MTT analysis, cell viability and liquid colony formation, whereas the lentiviral expression of Eps8 shRNA in MDA-MB‑231 cells resulted in a significant reduction in cellular growth and proliferation in vitro and in vivo. Furthermore, Eps8 knockdown inhibited breast cancer cell migration in wound healing assays, decreased the number and size of EGF-induced filopodia and increased the sensitivity of breast cancer cells to cisplatin analyzed by MTT assays. Eps8 knockdown decreased the levels of phosphorylated extracellular signal-regulated protein kinase (ERK) and MMP9 but increased p53. Moreover, Eps8 knockdown suppressed a partial EMT-like transition and showed a significant increase in E-cadherin and decrease in N-cadherin and vimentin. These results suggest that Eps8 is overexpressed in human breast cancers, possibly by regulating ERK signaling, MMP9, p53 and EMT-like transition to affect breast cancer cell growth, migration and invasion. Therefore, Eps8 might represent a novel potential target in human breast cancer therapy.

Transcription factor AP-2α is involved in many types of human cancers, but its role in hepatocellular carcinogenesis is largely unknown. In this study, we found that expression of AP-2α was low in 40% of human hepatocellular cancers compared with adjacent normal tissues by immunohistochemical analysis. Moreover, AP-2α expression was low or absent in hepatocellular cancer cell lines (HepG2, Hep3B, SMMC-7721 and MHHC 97-H). Human liver cancer cell lines SMMC-7721 and Hep3B stably overexpressing AP-2α were established by lentiviral infection and puromycin screening, and the ectopic expression of AP-2α was able to inhibit hepatocellular cancer cell growth and proliferation by cell viability, MTT assay and liquid colony formation in vitro and in vivo. Furthermore, AP-2α overexpression decreased liver cancer cell migration and invasion as assessed by wound healing and Transwell assays, increasing the sensitivity of liver cancer cells to cisplatin analyzed by MTT assays. Also AP-2α overexpression suppressed the sphere formation and renewed the ability of cancer stem cells. Finally, we found that AP-2α is epigenetically modified and modulates the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), β-catenin, p53, EMT, and CD133 expression in liver cancer cell lines. These results suggested that AP-2α expression is low in human hepatocellular cancers by regulating multiple signaling to affect hepatocellular cancer cell growth and migration. Therefore, AP-2α might represent a novel potential target in human hepatocellular cancer therapy.

Transcription factor AP-2β mediates the transcription of a number of genes implicated in mammalian development, cell proliferation, and carcinogenesis. Although the expression pattern of AP-2β has been analyzed in cervical cancer cell lines, the functions and molecular mechanism of AP-2β are unknown. Here, we found that AP-2β significantly inhibits TCF/LEF reporter activity. Moreover, AP-2β and β-catenin interact both in vitro through GST pull-down assays and in vivo by co-immunoprecipitation. We further identified the interaction regions to the DNA-binding domain of AP-2β and the 1-9 Armadillo repeats of β-catenin. Moreover, AP-2β binds with β-TrCP and promotes the degradation of endogenous β-catenin via the proteasomal degradation pathway. Immunohistochemistry analysis revealed a negative correlation between the two proteins in cervical cancer tissues and cell lines. Finally, functional analysis showed that AP-2β suppresses cervical cancer cell growth in vitro and in vivo by inhibiting the expression of Wnt downstream genes. Taken together, these findings demonstrated that AP-2β functions as a novel inhibitor of the Wnt/β-catenin signaling pathway in cervical cancer.