Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased β-catenin and downregulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3β was significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling.

Gholam Azad M ，Hussaini M ，Russell TM ，Richardson V ，Kaya B ，Dharmasivam M ，Richardson DR ... -  《-》

Targeting the metastasis suppressor, NDRG1, using novel iron chelators: regulation of stress fiber-mediated tumor cell migration via modulation of the ROCK1/pMLC2 signaling pathway.

Sun J ，Zhang D ，Zheng Y ，Zhao Q ，Zheng M ，Kovacevic Z ，Richardson DR ... -  《-》

Hypoxia-induced NDRG1 C-terminal poly-phosphorylation impairs its tumor suppressor function in renal cell carcinoma.

Hypoxia is a critical factor driving tumor invasion and metastasis. N-myc Downstream Regulated Gene 1 (NDRG1), a known suppressor of invasion and metastasis in various cancers including clear cell renal cell carcinoma (ccRCC), remains poorly understood in the context of hypoxic regulation. Notably, the carboxy terminus (C-terminus) of NDRG1 contains multiple phosphorylation sites within a unique three-tandem-repeat sequence that responds to hypoxic conditions. However, the precise regulatory mechanisms and functional significance of phosphorylation in this region remain unexplored. Our research uncovered that hypoxia triggers poly-phosphorylation at the C-terminus of NDRG1, thereby facilitating epithelial-mesenchymal transition (EMT) in ccRCC. NDRG1 knockdown alone was sufficient to induce EMT, augmenting the invasive and metastatic capabilities of ccRCC cells. Specifically, hypoxia-induced phosphorylation at the C-terminus (sites 328/330 and 346/356/366, phosphorylated by SGK1) of NDRG1 enhances its SUMOylation and ubiquitination, leading to NDRG1 degradation. NDRG1 typically forms a complex with E-cadherin and β-catenin to suppress WNT signaling; however, this complex is disrupted by phosphorylation at the 346/356/366 sites. In vivo studies demonstrated that NDRG1 knockdown expedited tumor growth and pulmonary metastasis, but the re-expression of phosphorylation-deficient mutants, particularly at sites 328/330 and 346/356/366, significantly mitigated these tumor-promoting effects. Our study demonstrates that hypoxia-induced C-terminal poly-phosphorylation of NDRG1 promotes its degradation, activating the WNT pathway and driving ccRCC malignancy. These findings underscore the role of NDRG1 phosphorylation in ccRCC progression under hypoxic conditions and highlight potential therapeutic targets for intervention.

Guo Q ，Quan MY ，Zheng J ，Yu C ，Yang L ，Li J ，Weng Q ，Dhlamini Q ，Yang W ，Cai J ，Ao G ，Chen L ，Li JM ，Cheng X ，Jin X ，Xu Y ，Zhang JS ... -  《-》

Targeting the Metastasis Suppressor, N-Myc Downstream Regulated Gene-1, with Novel Di-2-Pyridylketone Thiosemicarbazones: Suppression of Tumor Cell Migration and Cell-Collagen Adhesion by Inhibiting Focal Adhesion Kinase/Paxillin Signaling.

Metastasis is a complex process that is regulated by multiple signaling pathways, with the focal adhesion kinase (FAK)/paxillin pathway playing a major role in the formation of focal adhesions and cell motility. N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor in many solid tumor types, including prostate and colon cancer. Considering the antimetastatic effect of NDRG1 and the crucial involvement of the FAK/paxillin pathway in cellular migration and cell-matrix adhesion, we assessed the effects of NDRG1 on this important oncogenic pathway. In the present study, NDRG1 overexpression and silencing models of HT29 colon cancer and DU145 prostate cancer cells were used to examine the activation of FAK/paxillin signaling and the formation of focal adhesions. The expression of NDRG1 resulted in a marked and significant decrease in the activating phosphorylation of FAK and paxillin, whereas silencing of NDRG1 resulted in an opposite effect. The expression of NDRG1 also inhibited the formation of focal adhesions as well as cell migration and cell-collagen adhesion. Incubation of cells with novel thiosemicarbazones, namely di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone and di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone, that upregulate NDRG1 also resulted in decreased phosphorylation of FAK and paxillin. The ability of these thiosemicarbazones to inhibit cell migration and metastasis could be mediated, at least in part, through the FAK/paxillin pathway.

Wangpu X ，Lu J ，Xi R ，Yue F ，Sahni S ，Park KC ，Menezes S ，Huang ML ，Zheng M ，Kovacevic Z ，Richardson DR ... -  《-》

Long non-coding RNA SNHG11 regulates the Wnt/β-catenin signaling pathway through rho/ROCK in trabecular meshwork cells.

Trabecular meshwork (TM) cell dysfunction is the leading cause of elevated intraocular pressure (IOP) and glaucoma. The long non-coding RNA (lncRNA) small nucleolar RNA host gene 11 (SNHG11) is associated with cell proliferation and apoptosis, but its biological functions and role in glaucoma pathogenesis remain unclear. In the present study, we investigated the role of SNHG11 in TM cells using immortalized human TM and glaucomatous human TM (GTM3 ) cells and an acute ocular hypertension mouse model. SNHG11 expression was depleted using siRNA targeting SNHG11. Transwell assays, quantitative real-time PCR analysis (qRT-PCR), western blotting, and CCK-8 assay were used to evaluate cell migration, apoptosis, autophagy, and proliferation. Wnt/β-catenin pathway activity was inferred from qRT-PCR, western blotting, immunofluorescence, and luciferase reporter and TOPFlash reporter assays. The expression of Rho kinases (ROCKs) was detected using qRT-PCR and western blotting. SNHG11 was downregulated in GTM3 cells and mice with acute ocular hypertension. In TM cells, SNHG11 knockdown inhibited cell proliferation and migration, activated autophagy, and apoptosis, repressing the Wnt/β-catenin signaling pathway, and activated Rho/ROCK. Wnt/β-catenin signaling pathway activity increased in TM cells treated with ROCK inhibitor. SNHG11 regulated Wnt/β-catenin signaling through Rho/ROCK by increasing GSK-3β expression and β-catenin phosphorylation at Ser33/37/Thr41 while decreasing β-catenin phosphorylation at Ser675. We demonstrate that the lncRNA SNHG11 regulates Wnt/β-catenin signaling through Rho/ROCK via β-catenin phosphorylation at Ser675 or GSK-3β-mediated phosphorylation at Ser33/37/Thr41, affecting cell proliferation, migration, apoptosis, and autophagy. Through its effects on Wnt/β-catenin signaling, SNHG11 is implicated in glaucoma pathogenesis and is a potential therapeutic target.

Liu L ，Yang X ，Zhang J ，Jiang W ，Hou T ，Zong Y ，Bai H ，Yang K ，Yang X ... -  《-》