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Identification of differential phosphorylation and sub-cellular localization of the metastasis suppressor, NDRG1
Kyung Chan Park1, Sharleen V Menezes1, Danuta S Kalinowski1
1Molecular Pharmacology and Pathology Program, Discipline of Pathology and Bosch Institute, Medical Foundation Building (K25), The University of Sydney, Sydney, New South Wales 2006, Australia.
Abstract:
The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), exhibits pleiotropic activity, inhibiting metastasis of various tumor-types, while being correlated with metastasis in others. Notably, NDRG1 phosphorylation and cleavage are associated with its function, although it is unclear if these modifications occur universally, or selectively, in different cancer cell-types and if it contributes to its pleiotropy. Considering the suggested DNA repair role of nuclear NDRG1, the effects of the above post-translational modifications on its nuclear localization was examined. Herein, the full-length (FL) and truncated (T) NDRG1 isoforms were detected using a C-terminus-directed antibody, while only the FL isoform was identified using an N-terminus-directed antibody. For the first time, we demonstrate that the expression of the NDRG1 FL and T forms occurs in all cancer cell-types examined, as does its phosphorylation (p-NDRG1) at Ser330 and Thr346. The FL isoform localized highly in the nucleus compared to the T isoform. Moreover, p-NDRG1 (Ser330) was also markedly localized in the nucleus, while p-NDRG1 (Thr346) was predominantly cytoplasmic in all cell-types. These results indicate the N-terminus region and phosphorylation at Ser330 could be crucial for NDRG1 nuclear localization and function. PTEN silencing indicated that p-NDRG1 (Thr346) could be regulated differentially in different tumor cell-types, indicating PTEN may be involved in the mechanism(s) underlying the pleiotropic activity of NDRG1. Finally, therapeutics of the di-2-pyridylketone thiosemicarbazone class increased nuclear NDRG1 isoforms (FL and T) detected by the C-terminus-directed antibody in HepG2 cells, while having no significant effect in PC3 cells, indicating differential activity depending on the cell-type.
Insights
N-myc downstream regulated gene-1 (NDRG1) phosphorylation and isoforms are present in all cancer cells. Nuclear localization of NDRG1, influenced by its N-terminus and Ser330 phosphorylation, is key to its function.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- N-myc downstream regulated gene-1 (NDRG1) is a metastasis suppressor with varied roles across tumor types.
- NDRG1's pleiotropic activity is linked to post-translational modifications like phosphorylation and cleavage, but their universal or selective occurrence and impact on function remain unclear.
- Nuclear NDRG1 is implicated in DNA repair, necessitating an understanding of how modifications affect its subcellular localization.
Purpose of the Study:
- To investigate the expression and localization of NDRG1 full-length (FL) and truncated (T) isoforms across various cancer cell types.
- To determine the impact of phosphorylation at Ser330 and Thr346 on NDRG1 localization and potential roles in its pleiotropy.
- To examine the effect of PTEN silencing and specific therapeutics on NDRG1 expression and localization.
Main Methods:
- Detection of NDRG1 FL and T isoforms using N-terminus and C-terminus specific antibodies.
- Analysis of NDRG1 phosphorylation at Ser330 and Thr346 via Western blotting.
- Assessment of subcellular localization (nuclear vs. cytoplasmic) of NDRG1 isoforms and phosphoisomers.
- PTEN silencing and treatment with di-2-pyridylketone thiosemicarbazone class therapeutics.
Main Results:
- Both NDRG1 FL and T isoforms, along with phosphorylation at Ser330 and Thr346 (p-NDRG1), are expressed in all examined cancer cell types.
- NDRG1 FL isoform shows high nuclear localization; p-NDRG1 (Ser330) is also nuclear, while p-NDRG1 (Thr346) is predominantly cytoplasmic.
- PTEN silencing suggests differential regulation of p-NDRG1 (Thr346), hinting at PTEN's role in NDRG1's pleiotropic activity.
- Therapeutics increased nuclear NDRG1 in HepG2 cells but not PC3 cells, indicating cell-type-specific responses.
Conclusions:
- The N-terminus and Ser330 phosphorylation are critical for NDRG1 nuclear localization and function.
- Differential regulation of p-NDRG1 (Thr346) by PTEN may contribute to NDRG1's pleiotropic effects in various cancers.
- The therapeutic efficacy of di-2-pyridylketone thiosemicarbazones on nuclear NDRG1 varies significantly between cancer cell types.
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