ENO1 (α-enolase) expression is significantly correlated with reduced survival and poor prognosis in many cancer types, including lung cancer. However, the function of ENO1 in carcinogenesis remains elusive. In this study, we found that high expression of ENO1 is present in metastatic lung cancer cell lines and malignant tumors and is associated with poor overall survival of patients with lung cancer. Knockdown of ENO1 decreased cancer cell proliferation and invasiveness, whereas overexpression of ENO1 enhanced these processes. Moreover, ENO1 expression promoted tumor growth in orthotopic models and enhanced lung tumor metastasis in tail-vein injection models. These effects were mediated by upregulation of mesenchymal markers N-cadherin and vimentin and the epithelial-to-mesenchymal transition regulator SLUG, along with concurrent downregulation of E-cadherin. Mechanistically, ENO1 interacted with hepatocyte growth factor receptor (HGFR) and activated HGFR and Wnt signaling via increased phosphorylation of HGFR and the Wnt coreceptor LRP5/6. Activation of these signaling axes decreased GSK3β activity via Src–PI3K–AKT signaling and inactivation of the β-catenin destruction complex to ultimately upregulate SLUG and β-catenin. In addition, we generated a chimeric anti-ENO1 mAb (chENO1-22) that can decrease cancer cell proliferation and invasion. chENO1-22 attenuated cancer cell invasion by inhibiting ENO1-mediated GSK3β inactivation to promote SLUG protein ubiquitination and degradation. Moreover, chENO1-22 prevented lung tumor metastasis and prolonged survival in animal models. Taken together, these findings illuminate the molecular mechanisms underlying the function of ENO1 in lung cancer metastasis and support the therapeutic potential of a novel antibody targeting ENO1 for treating lung cancer.
Significance:
This study shows that ENO1 promotes lung cancer metastasis via HGFR and WNT signaling and introduces a novel anti-ENO1 antibody for potential therapeutic use in lung cancer.
CCR6 is a G protein-coupled receptor (GPCR) that recognizes a single chemokine ligand, CCL20 and is primarily expressed by leukocytes. Upon ligand binding, CCR6 activates Gαi heterotrimeric G proteins to induce various potential cellular outcomes through context-specific cell signaling. It is well known that differential phosphorylation of Ser and Thr residues in the C-terminal domains or intracellular loops of GPCRs can generate barcodes that regulate GPCR function by regulating the recruitment of β-arrestins. In this study, we demonstrate that ligand binding to CCR6 induces receptor phosphorylation at Ser/Thr residues in the C-terminal tail, rather than intracellular loops. Using mutagenesis experiments, we determined that distinct clusters of Ser/Thr residues in the C-terminal domain differentially regulate CCL20-induced signaling and cellular response. Substituting the Thr360/Ser361/Thr363 cluster or the Ser370/Ser371 cluster with Ala residues modulated cellular response upon CCL20 stimulation. Notably, receptor internalization, chemotaxis, F-actin distribution, transient ERK1/2 activation, and β-arrestin 2 recruitment were oppositely affected by mutating the two clusters, suggesting that phosphorylation of CCR6 C-terminal Ser/Thr residues directs the cell signaling response upon receptor activation. Moreover, activated CCR6 weakly recruited β-arrestin 1 in comparison with β-arrestin 2, and the two arrestin proteins seemed to play overlapping but distinct roles in mediating CCL20/CCR6-induced cellular responses. Taken together, the effects of site-specific Ser/Thr phosphorylation on CCR6 demonstrate the existence of barcodes on the protein that dictate the activation of different cell signaling profiles and lead to distinct biological outcomes.
Unstable GTP cyclohydrolase I (GCH) mutations in dopa-responsive dystonia (DRD) can exert a dominant-negative effect in the HeLa cell model, but in a batch of cells this effect could not be shown. Through differential display, we found a higher Hsc70 expression in the non-dominant-negative cells. We further demonstrated that ectopic expression of Hsp40/Hsp70 stabilized the GCH mutant G201E. Moreover, Hsp90 inhibitor geldanamycin destroyed the wild-type GCH level, and heat shock increased the synthesis of GCH protein. Therefore, the dominant-negative effect produced by unstable proteins would be susceptible to the status of molecular chaperones, which could be the modifying genes and therapeutic targets for DRD and other genetic diseases.
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