S3337

ING5 inhibits lung cancer invasion and epithelial-mesenchymal transition by inhibiting the WNT/β-catenin pathway

Background: ING5 is the last member of the Inhibitor of Growth (ING) candidate tumor suppressor family that has been implicated in multiple cellular functions, including cell cycle regulation, apoptosis, and chromatin remodeling. Our previous study showed that ING5 overexpression inhibits lung cancer aggressiveness and epithelial-mesenchymal transition (EMT), with unknown mechanisms. Methods: Western blotting was used to detect total and phosphorylated levels of β-catenin and EMT-related proteins. Immunofluorescent staining was used to observe E-cadherin expression. Proliferation and colony formation, wound healing, and Transwell migration and invasion assays were performed to study the proliferative and invasive abilities of cancer cells. Results: ING5 overexpression promotes phosphorylation of β-catenin at Ser33/37, leading to a decreased β-catenin protein level. Small hairpin RNA-mediated ING5 knockdown significantly increased the β-catenin level and inhibited phosphorylation of β-catenin S33/37. Treatment with the WNT/β-catenin inhibitor XAV939 inhibited ING5-knockdown promoted proliferation, colony formation, migration, and invasion of lung cancer A549 cells, with increased phosphorylation of β-catenin S33/37 and a decreased β-catenin level. XAV939 also impaired ING5-knockdown-induced EMT, as indicated by upregulated expression of the EMT marker E-cadherin, an epithelial marker; and decreased expression of N-cadherin, a mesenchymal marker, and EMT-related transcription factors, including Snail, Slug, Twist, and Smad3. Furthermore, XAV939 could inhibit the activation of both IL-6/STAT3 and PI3K/Akt signaling pathways. Conclusion: ING5 inhibits lung cancer invasion and EMT by inhibiting the WNT/β-catenin pathway.

BMX activates Wnt/β-catenin signaling pathway to promote cell proliferation and migration in breast cancer

Background: Breast cancer has become a dangerous killer for the female, which seriously threatened women's life, leading to huge pressures to society. The present study assessed the mechanism underlying the involvement of bone marrow tyrosine kinase on chromosome X (BMX) in breast cancer development.
Methods: The expression of BMX was examined by qPCR and immunohistochemistry. The effect of BMX on cell proliferation and migration was detected by Clone formation assay and Transwell assay. In vitro study, the correlation of BMX with Wnt/β-catenin pathway was explored by western blot and TOP/FOP flash assay.
Results: In the present study, we found that BMX was up-regulated in breast cancer, which was associated with the tumor differentiation and TNM stage. Oncogenic BMX enhanced the ability of breast cancer cell proliferation and migration. Furthermore, BMX could up-regulate the protein expression levels of p-β-catenin (Y142), p-β-catenin(Y654) and inhibit the expression level of p-β-catenin (S33/37), thus activating Wnt/β-catenin pathway in MCF-7 and MDA-MB-231 cells. In addition, we revealed that BMX promoted GSK3β phosphorylation, which suppressed the degradation of β-catenin.
Conclusions: In this study, we identified that BMX-activated Wnt/β-catenin signaling pathway, playing an oncogenic role in breast cancer, suggesting that BMX could become a potential treatment target of breast cancer.

p21-activated kinase 4 critically regulates melanogenesis via activation of the CREB/MITF and β-catenin/MITF pathways

p21-activated kinase 4 (PAK4) regulates a wide range of cellular events, including cytoskeletal remodeling, cell growth, and survival. Our previous study identified PAK4 as a key regulator of cAMP-response element-binding protein (CREB) that acts upstream of microphthalmia-associated transcription factor (MITF), a master transcription factor in melanogenesis. We therefore investigated the role of PAK4 in melanogenesis. Melanocytes express both PAK2 and PAK4 isoforms, but only RNA interference knockdown of PAK4 significantly influenced α-melanocyte-stimulating hormone (α-MSH)-induced melanogenesis in B16 melanoma cells. Consistent with this result, PAK4 inhibition by PF3758309, a potent ATP-competitive inhibitor of PAKs, suppressed not only α-MSH-induced melanogenesis in B16 melanoma and human epithelial melanocyte cells but also UVB-induced melanogenesis in the skin of melanin-possessing hairless mice (HRM-2) in a dose-dependent manner. Inhibition of PAK4 over several days markedly decreased the levels of CREB, MITF, and tyrosinase in both HRM-2 mice and B16 melanoma cells. Moreover, PAK4 knockdown and inhibition suppressed α-MSH-stimulated β-catenin phosphorylation at serine 675 (S675) but enhanced phosphorylation at S33/37, an indicator for ubiquitination-dependent proteolysis. Together, our results provide evidence that PAK4 promotes α-MSH/UVB-induced melanogenesis via the CREB and Wnt/β-catenin signaling pathways and suggest that PAK4 may be a potential therapeutic target in pigmentation disorders.

The MyoD family inhibitor domain-containing protein enhances the chemoresistance of cancer stem cells in the epithelial state by increasing β-catenin activity

Cancer cells with mesenchymal attributes potentially display chemoresistance. Cancer stem cells (CSCs), which are intrinsically resistant to most chemotherapy agents, exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal states. However, the drug response of CSCs in the epithelial and mesenchymal states has not been completely investigated. In this study, we found that epithelial-type (E-cadherin^high/CD133^high) CSCs displayed a higher sphere formation ability and chemoresistance than mesenchymal-type (E-cadherin^lowCD133^high) CSCs. Gene expression profiling of the CSC and non-CSC subpopulations with distinct epithelial-to-mesenchymal transition (EMT) states showed that MyoD family inhibitor domain-containing (MDFIC) was selectively upregulated in epithelial-type CSCs. Knockdown of MDFIC sensitized epithelial-type CSCs to chemotherapy agents. Ectopic expression of MDFIC increased the chemoresistance of mesenchymal-type CSCs. In a tissue microarray, high MDFIC expression was associated with poor prognosis of non-small cell lung cancer (NSCLC) patients. A mechanistic study showed that the MDFIC p32 isoform, which is located in the cytoplasm, interacted with the destruction complex, Axin/GSK-3/β-catenin. This interaction stabilized β-catenin by inhibiting β-catenin phosphorylation at S33/37 and increased the nuclear translocation and transcriptional activity of β-catenin. Knockdown of β-catenin decreased MDFIC-enhanced chemoresistance. These results suggested that the upregulation of MDFIC enhanced the chemoresistance of epithelial-type CSCs by elevating β-catenin activity. Thus, targeting MDFIC-regulated β-catenin signaling of epithelial-type CSCs may be a potential strategy to overcome chemoresistance in NSCLC.