Ramsay, Alison Kay
Validation of the MEK5 and ERK5 pathway as targets for therapy in prostate cancer and analysis of the ERK5 signalling complex.
MD thesis, University of Glasgow.
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Extracellular signal-regulated protein kinase 5 (ERK5) is a member of the mitogen activated protein (MAP) kinase family which is specifically activated by mitogen/extracellular signal regulated kinase kinase-5 (MEK5). Over recent years, abnormal MEK5/ERK5 signalling has been shown to be important in prostate carcinogenesis with increased levels of ERK5 immunoreactivity being associated with Gleason sum score (p<0.0001), bone metastases (p=0.0044) and locally advanced disease at diagnosis (p=0.0023). In addition PC3 cells over-expressing ERK5 displayed enhanced proliferation, migration and invasion. Taken together, these data suggest MEK5/ERK5 pathway to be biological important in prostate cancer and a potential target in invasive prostate cancer.
Using siRNA to target ERK5 expression, I found that reduced ERK5 expression significantly inhibited cellular proliferation, motility and invasion in prostate cancer PC3 cells when compared to the controls, (p<0.005). Our group has previously reported upregulated ERK5 expression in primary human prostate cancer specimens. In this study, I was able to validate these results and demonstrate moderate-strong levels of cytoplasmic staining in 63% cases of PIN/PIA. High levels of cytoplasmic (55%) and nucleur (73%) immunoreactivity was also shown in a range of metastatic prostate tumours (n=11).
A number of similarities and interactions between ERK5 and ERK1/2 have recently been identified and there is suggestion that ERK5 may in fact regulate some of the cellular functions originally attributed to ERK1/2. Potential ‘cross-talk’ between ERK5 and ERK1/2 signalling was investigated using siRNA for each individual isoform of ERK1/2. ERK1 knockdown resulted in increased ERK5 activation in addition to prolonged ERK2 phosphorylation. Proliferation studies were also performed in PC3 cells, the results of which support published data that ERK1 acts as a negative regulator and ERK2 as a positive regulator of cell proliferation.
ERK5 has been shown to regulate the activity of several transcription factors and recent evidence suggests that ERK5 may be heat shock protein (HSP) 90 dependent. To further investigate the ERK5 signalling network and its interacting proteins, I performed mass spectrometry-based quantitative proteomics using SILAC labelled cells. Results from this study support the theory that HSP90 does associate with ERK5 however contrary to published data my results show that it is not involved in ERK5 activation.
Our results validate the importance of the MEK5-ERK5 signalling pathway as a potential target for therapy in prostate cancer and highlight a novel functional and biochemical relationship between ERK1 and HSP 90 with ERK5 signalling.
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