Glycogen synthase kinase 3 beta (GSK3β) is highly inactivated in epithelial cancers and is known to inhibit tumor migration and invasion. The zinc-finger-containing transcriptional repressor, Slug, represses E-cadherin transcription and enhances epithelial-mesenchymal transition (EMT). In this study, we find that the GSK3β-pSer9 level is associated with the expression of Slug in non-small cell lung cancer (NSCLC). GSK3β-mediated phosphorylation of Slug facilitates Slug protein turnover. Proteomic analysis reveals that the C-terminus of Hsc70-interacting protein (CHIP) interacts with wild-type Slug (wtSlug). Knockdown of CHIP stabilizes the wtSlug protein and reduces Slug ubiquitylation and degradation. In contrast, nonphosphorylatable Slug-4SA is not degraded by CHIP. The accumulation of nondegradable Slug may further lead to the repression of E-cadherin expression and promote cancer cell migration, invasion, and metastasis. Our findings provide evidence of a de novo GSK3β-CHIP-Slug pathway that may be involved in the progression of metastasis in lung cancer.

Tumor angiogenesis is a critical process during cancer progression that modulates tumor growth and metastasis. Here, we identified an anti-angiogenic microRNA, miR-320, which is decreased in oral squamous cell carcinoma (OSCC) cell lines and tumor tissues from OSCC patients, down-regulated in blood vessels and inversely correlated with vascularity in OSCC tissues. Neuropilin 1 (NRP1), an important regulator of angiogenesis, was found to be a target of miR-320. The 3'-untranslated region of NRP1 mRNA contains multiple miR-320 binding sites, and its expression was regulated by miR-320. By administering either miR-320 precursor or antagonist, we found that miR-320 suppressed the migration, adhesion and tube formation of vascular endothelial cells. Knockdown of NRP1 abolished antagomiR-320-induced cell migration. Additionally, miR-320 expression was regulated by hypoxia in growth factor-deficient conditions by the hypoxia-inducible factor 1-alpha. Furthermore, lentivirus carrying the miR-320 precursor suppressed the tumorigenicity of OSCC cells and tumor angiogenesis in vivo. Taken together, these data show that miR-320 regulates the function of vascular endothelial cells by targeting NRP1 and has the potential to be developed as an anti-angiogenic or anti-cancer drug.

Comparison of protein stability in eukaryotic cells has been achieved by cycloheximide, which is an inhibitor of protein biosynthesis due to its prevention in translational elongation. It is broadly used in cell biology in terms of determining the half-life of a given protein and has gained much popularity in cancer research. Here we present a full cycloheximide chase assay in our laboratory using a lung adenocarcinoma cell line, CL1-5, as a model.

a-Catulin is an oncoprotein that helps sustain proliferation by preventing cellular senescence. Here, we report that a-catulin also drives malignant invasion and metastasis. a-Catulin was upregulated in highly invasive nonsmall cell lung cancer (NSCLC) cell lines, where its ectopic expression or short-hairpin RNA-mediated attenuation enhanced or limited invasion or metastasis, respectively. a-Catulin interacted with integrin-linked kinase (ILK), a serine/threonine protein kinase implicated in cancer cell proliferation, antiapoptosis, invasion, and angiogenesis. Attenuation of ILK or a-catulin reciprocally blocked cell migration and invasion induced by the other protein. Mechanistic investigations revealed that a-catulin activated Akt-NF-kB signaling downstream of ILK, which in turn led to increased expression of fibronectin and integrin avb3. Pharmacologic or antibodymediated blockade of NF-kB or avb3 was sufficient to inhibit a-catulin-induced cell migration and invasion. Clinically, high levels of expression of a-catulin and ILK were associated with poor overall survival in patients with NSCLC. Taken together, our study shows that a-catulin plays a critical role in cancer metastasis by activating the ILK-mediated Akt-NF-kB-avb3 signaling axis. Cancer Res; 73(1); 428-38. Ó2012 AACR.