The cell survival pathway autophagy, sequesters and degrades long lived and damaged proteins, recycling for reuse as biosynthetic building blocks. Autophagy is often dysregulated in disease pathologies, evidences shows both loss or gain of autophagy function can provide survival advantage. Constitutive activation of the KRas pathway occurs in cancer, with mutant Ras inducing autophagy for survival. Microarray datasets reveal that KRas mutant cancers increased MAP4K3 mRNA, which encodes a serine/threonine specific protein kinase that regulates mTORC1 signalling, in response to nutrients. Since nutrient signalling and autophagy are closely linked, the potential role for MAP4K3 in autophagy regulation, through mTORC1 in KRas mutant colorectal cancer cells, was investigated. MAP4K3 protein expression was elevated in KRas mutant (HCT116) cells compared to isogenic KRas wild type (HKH2) cells. Although silencing of MAP4K3 using shRNA decreased cell proliferation and metabolic activity in both cell lines, MAP4K3 silencing specifically diminished basal autophagic flux in KRas mutant cells, revealing a potential role for MAP4K3 in the regulation of autophagy. Loss of MAP4K3 expression after shRNA silencing led to decreased hVps34 activity and LC3 processing, both of which are essential for autophagy activation and progression. Furthermore, inhibition/silencing of MAP4K3, revealed a feedback mechanism that led to increased ERK1/2 phosphorylation in both cell lines. Combined Silencing of MAP4K3 with the MEK inhibitor PD184352 resulted in preferential sensitisation of KRas mutant cells to apoptotic cell death. This thesis identifies the novel function of MAP4K3 in autophagy control, through control of the hVps34 complex, furthermore the autophagy pathway is up-regu lated in KRas mutant cells and its inhibition sensitises KRas mutant cells towards MEK inhibitors. Thus, therapeutic targeting of MAP4K3 could be combined with Ras pathway inhibition to selectively target KRas mutant cancers.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:676466 |
| Date | January 2014 |
| Creators | Fee, Emily |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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