The Proline-rich inositol polyphosphate 5-Phosphatase (PIPP) is a negative regulator of phosphoinositide 3-Kinase/akt signalling in breast cancer

In response to cell stimulation, phosphoinositide 3-kinase (PI3K) is recruited to the plasma membrane where it transiently generates the second messenger lipid PtdIns(3,4,5)P3, which in turn recruits and activates specific proteins such as Akt to regulate a plethora of intracellular signalling cascades to promote cell proliferation, polarisation and survival. Negative regulation of the PI3K/Akt signalling pathway is facilitated by the inositol polyphosphate 3-phosphatase PTEN, or by members of the inositol polyphosphate 5-phosphatase family, which both degrade PtdIns(3,4,5)P3. The proline rich inositol polyphosphate 5-phosphatase (PIPP) is a relatively uncharacterised member of the 5-phosphatase family which hydrolyses PtdIns(3,4,5)P3 in vivo to negatively regulate Akt signalling in the neuronal-like PC12 cell line (Ooms et al., 2006). The PI3K/Akt signalling pathway is strongly implicated in breast cancer, and published gene array studies have suggested that PIPP mRNA expression is positively associated estrogen receptor positive breast cancers and improved prognosis (van 't Veer et al., 2002). Furthermore, two regions which flank the PIPP gene are associated with loss of heterozygosity in ~45% of breast cancer (Allione et al., 1998). Expression of the PIPP protein in primary human breast tumours has not been reported. In this study, we have shown that RNAi-mediated PIPP knockdown in mouse fibroblasts and the aggressive human breast cancer cell line, MDA-MB-231, results in enhanced anchorage-independent cell growth, increased cell proliferation under conditions of serum-starvation, decreased cell death in response to induction of apoptosis, aberrant polarity and increased in vivo tumour growth in xenograft studies. Immunoblot analysis of the PtdIns(3,4,5)P3 effector, Akt, demonstrated enhanced phosphorylation of Akt in both unstimulated and EGF-stimulated conditions. Increased phosphorylation of Akt was also reflected in increased levels of phosphorylated GSK3β and S6K (two Akt effectors) under EGF-stimulated and serum-starved conditions respectively. Collectively, this data suggests that PIPP negatively regulates PI3K/Akt signalling in mouse fibroblasts and human breast cancer cells. To examine PIPP protein expression in primary human breast tumour samples, PIPP-specific polyclonal and monoclonal antibodies were generated and characterised. It was identified that PIPP protein expression was lost in ~66% of all primary human breast cancers studied. Furthermore, PIPP was lost in ~78% of the clinically aggressive triple negative subtype of breast cancers. Immunohistochemistry analysis of normal breast tissue revealed that the PIPP protein exhibits both a cytoplasmic and nuclear localisation in luminal mammary epithelial cells. Interestingly, both immunohistochemistry and immunofluorescence analysis of the PIPP protein in PIPP-positive triple negative breast cancers and breast cancer cell lines demonstrated loss of PIPP from the cytoplasm. Therefore, both the loss and mislocalisation of PIPP may play a role in breast tumorigenesis. In summary, the evidence provided in this thesis suggests that PIPP may be a putative tumour suppressor protein that is frequently lost or mislocalised in aggressive triple-negative breast tumours. We propose a model whereby PIPP negatively regulates PI3K/Akt signalling at the plasma membrane in quiescent cells, and upon loss of PIPP, or mislocalisation of PIPP to the nucleus, increased PtdIns(3,4,5)P3-mediated signalling may lead to hyperproliferation, aberrant polarisation, anchorage-independent cell growth and increased cell survival, ultimately promoting breast cancer development.