Final Report Summary - PI3K/ONCOGENESIS (The role of p110alpha isoform of PI 3-kinase in oncogenesis and cellular senescence) We applied for the Marie Curie postdoctoral fellowship in order to investigate:- the role of the p110a PI3K isoform in cell survival, cellular senescence and its interaction with other signalling pathways, including it's negative regulator PTEN.- the signal transduction pathways activated by the oncogenic mutant p110aH1047R.-the role of the oncogenic mutant p110aH1047R in tumour development and cancer.The results of this work and the findings derived have major implications for the understanding of the role of p110a in human cancer and therefore for the development of rational therapies for the treatment of cancers in which PI3K is over activated.Role of p110a in cell survival (published on PNAS):The PI3K pathway is one of the most frequently mutated pathways in human cancer. A better understanding of the role of the individual class I PI3K isoforms in cell survival and proliferation is important for the development of therapeutics targeting the PI3K pathway, especially to determine whether inhibition of multiple PI3K isoforms is essential to block cell proliferation and survival.In collaboration with Dr Lazaros Foukas, a postdoctoral fellow in Bart Vanhaesebroeck's lab, I have characterised the contribution of p110a isoform of PI3K to cell survival and proliferation both in mouse hematopoietic progenitor cells (HPC) and mouse embryonic fibroblasts (MEFs). This work has been recently published in PNAS (107(25):11381-6). We demonstrated that a small fraction of total class I PI3K activity is sufficient to sustain cell survival and proliferation. Persistent inhibition of selected PI3K isoforms can allow the remaining isoform(s) to couple to upstream signaling pathways in which they are not normally engaged. Such functional redundancy of class IA PI3K isoforms upon sustained PI3K inhibition has implications for the development and use of PI3K inhibitors in cancer.Role of p110a in PTEN induced tumourgenesis (under preparation):The tumour suppressor PTEN is a negative regulator of the PI3K signalling, that is found to be mutated or lost in many types of human cancer. Mice lacking one copy of PTEN (PTEN+/-) develop a broad spectrum of tumours comparable with the spectrum of its mutation in human cancer. Inactivation of the lipid phosphatase PTEN leads to high intracellular PIP3 levels, and, as a consequence, overactivation of different pathways controlling cell proliferation, survival and migration. The ability of PTEN to oppose the PI3K pathway through its lipid phophatase activity has been regarded as the main mechanism by which PTEN suppresses tumourigenesis. However, growing evidence support the idea that either protein phosphatase, or more recently, phosphatase independent activities of PTEN may be crucial for its role as a tumour suppressor. Despite all the data available, we still have scant information regarding the role of the different PI3K isoforms in PTEN driven tumourigenesis and the contribution of lipid phospahatase independent functions of PTEN to carcinogenesis.In this study we use a genetic approach to gain more information regarding the role of p110a and p110b isoforms of PI3K in PTEN induced tumourigenesis. We found that even though there is no significant difference in the overall survival of the experimental PTEN+/- p110aD933A/WT (in which we inactivate one copy of p110a) and control PTEN+/- mice, the development of some pathologies or tumour types such as thyroid tumours, glomerulonephritis (a renal disease characterised by inflammation of the glomeruli) or phaeochromocytoma (a neuroendocrine tumor of the medulla of the adrenal glands), is clearly affected after hitting one copy of p110a in PTEN+/- mice.We had similar results after crossing PTEN+/- and p110bD933A/WT mice (in which we inactivate one copy of p110b), where even though there is no significant difference in the overall survival of the experimental PTEN+/- p110bD933A/WT and control PTEN+/- animals, the development of prostate cancer is clearly affected after hitting one copy of p110b, as previously reported with another genetic model.Interestingly, the levels of phosphatidylinositol-3,4,5-trisphosphate (PIP3), the lipid product of PI3K, in healthy prostate tissues show an apparent lack of correlation between PIP3 levels and tumour development, with low PIP3 in PTEN+/- p110aD933A/WT mice which are not protected from prostate cancer; and high PIP3 in PTEN+/- p110bD933A/WT mice, which develop prostate cancer with lower frequency. This lack of correlation is also obvious when looking at the signalling downstream PI3K in PTEN+/- p110aD933A/WT lymphomas, in which in spite of low levels of PIP3, the activation status of AKT and of some of its downstream targets do not seem to be affected These findings have a major impact in the treatment of tumours in which the PI3K pathway is on. Some of these tumours may not be responsive to inhibition of PI3K. Also, we show that P-AKT may not be a good readout to assess inhibition of the PI3K.Role of p110aH1047R in oncogenesis:p110a is the only PI3K isoform that is found mutated in human cancer. One of the most frequent mutations is H1047R in the kinase domain, which increases the kinase activity and promotes a possible facilitation of the binding of p110a to membranes. This mutation is known to be oncogenic both in vitro and in vivo. However we still have scant information regarding the mechanism of action of p110aH1047R from a molecular and cellular point of view and regarding its role as a tumour promoter.In order to investigate the role of p110aH1047R in normal cell biology and oncogenesis, we generated a mouse ubiquitously expressing the mutant protein. We found that p110aH1047R induces embryonic lethality in the heterozygous state. p110aH1047R/WT embryos die around E9.5. The mutant embryos, which show an increase in the PI3K-AKT signalling pathway, are consistently smaller in size compared with wild-type littermates and show developmental abnormalities like absence of body twisting and closure of the neural tube at E9.5. We found that mutant embryos present an increase in apoptosis and p53 levels. However, we have found that p110aH1047R induced embryonic lethality cannot be rescued in p53 knockout background. In collaboration with Shankar Srinivas from University of Oxford, we are now characterising the mutant embryos in detail, focusing in the primary cilia which may explain some of the developmental defects we observe.Our PIK3CA mutant allele is a hypomorphic allele, thanks to the targeting of a neomycin (Neo) cassette within PIK3CA intron 18. Owing to transcriptional interference, the presence of the selection cassette reduces p110a expression levels compared to the wild-type gene. Removal of the frt-flanked selection cassette (after breeding our chimeras with Flp-recombinase transgenic females) brings back p110a protein levels to normal leading to embryonic lethality: we have normal levels of p110a, but a more active protein due to the oncogenic H1047R mutation. On the contrary, embryos and mice carrying the Neo selection cassette, and therefore a more active protein but whose levels are decrease compare to wild-type, are viable and fertile.We have generated an inducible oncogenic p110a mouse (p110aH1047R+cassette/WT; FlpERT2) by crossing the p110aH1047R+cassette/WT hypomorphic mouse with an inducible FLPeERT2 transgenic mouse. We can activate p110aH1047R in these mice by tamoxifen administration (tamoxifen activates FLPeERT2 to induce the recombination of the Neo cassette leading to an increase in mutant p110a protein levels in adult tissues). After treating 8 weeks old oncogenic (p110aH1047R+cassette/WT; FlpERT2) and control mice (FlpERT2) with tamoxifen, we are currently checking them for tumour development. Analysis by PCR shows that the recombination of the Neo cassette occurs globally in a broad panel of tissues. We are now assessing the activation of the P3K in these tissues by western blot analysis. Interestingly, the activation of p110aH1047R in adult mice leads to lethality at different times after induction by still unknown causes. The tissues and organs of the oncogenic sick mice are being fixed and kept in order to perform a histopathological analysis that helps us undestand the impact of global activation of p110aH1047R in normal physiology and tumour development.Cell based studies using both E8.5 p110aH1047R/WT mutant MEFs and E13.5 p110aH1047R+cassette/WT; FlpERT2 inducible MEFs are currently been performed in order to understand the role of this oncogene in cell biology. This work is revealing very new and exciting data regarding the effect of oncogenic p110a in normal cell biology and will definitely provide a better fundamental understanding of PI3K signalling revealing potential new targets for intervention in cancer. Related documents Final Report - PI3K/ONCOGENESIS (The role of p110alpha isoform of PI 3-kinase in oncogenesis and cellular senescence)
