The human serine/threonine NIMA-related protein kinase family comprises eleven members, named Nek1 to Nek11. Of these, Nek1, Nek4, Nek8, Nek10 and Nek11 are implicated in the DNA damage response (DDR) with Nek11 playing a central role in the G2/M checkpoint. In response to DNA damage, Chk1 is activated by the ATM/ATR kinases. Chk1 then phosphorylates and activates Nek11 before both Chk1 and Nek11 phosphorylate Cdc25A. This promotes binding of SCFB-TrCP and subsequent degradation of Cdc25A resulting in cell cycle arrest at G2/M. Nek11 protein expression is also increased in colorectal cancers. This study focuses on the role of Nek11 in colorectal cancer cells to examine whether targeting Nek11 in combination with DNA damaging agents may have a clinical benefit for colorectal cancer patients. Using RNAi to deplete Nek11 in HCT116 colorectal cancer cells we show that Nek11 is required for the G2/M arrest in response to ionizing radiation and chemotherapeutically relevant drugs, oxaliplatin and irinotecan. Furthermore, depletion of Nek11 alone and in combination with IR results in apoptosis and loss of cell survival. Nek11 exists as several closely related but distinct isoforms in colorectal cancer cells: Nek11 Long (L), Nek11 Short (S), Nek11C and Nek11D. We reveal that depletion of Nek11S results in a more substantial abrogation of the G2/M checkpoint, as compared to depletion of the Nek11L or D isoforms. Furthermore, through the use of stable cell lines, we show Nek11 isoforms exhibit distinct localisation patterns and all isoforms are able to undergo nucleocytoplasmic shuttling, regulated by the C-terminal domain. Excitingly, we observe Nek11 localisation at sites of DNA damage foci in response to IR exposure. Overall, our findings reveal an essential role for Nek11 at the G2/M checkpoint in HCT116 cells contributing not only to their arrest but also their survival after DNA damage. Hence, Nek11 could be an exciting target for the development of novel anti-cancer drugs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647110 |
| Date | January 2015 |
| Creators | Sabir, Sarah Ruby |
| Contributors | Fry, Andrew; Jones, George |
| Publisher | University of Leicester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/2381/32226 |
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