DESARROLLO EMOCIONAL

The PI3K pathway is one of the major signalling transduction pathways, that receives several upstream inputs from epidermal growth factor (EGF), tumour growth factor (TGF) and epidermal growth factor receptor (EGFR) to name a few. It is a major effector pathway of the GTPase protein RAS, that is frequently altered in several malignancies. The RAS/RAF/ERK pathway interacts with the PI3K pathway at several levels: from RAS activating PI3K (Rodriguez-Viciana et al, 1996), to PI3K activating C-RAF (Wandzioch et al, 2004) and then ERK phosphorylating TSC2 (Ma et al, 2005). There are three classes of PI3K, with each having their own substrate specificities; the class IA PI3K will be discussed here as this class is the most widely implicated in its contribution in cancer. The class IA PI3Ks catalyse the phosphorylation of inositol-containing lipids, namely phosphatidylinositols (PtdIns). The class I PI3K proteins are comprised of a catalytic p110 subunit (PIK3CA) and a regulatory p85 subunit (PIK3R), which mediates the activation and localization of the enzyme. The regulatory subunits of class IA PI3Ks are encoded by one of three genes (a, b, and g), which can also undergo alternative splicing. The direct binding of the enzyme to the phosphotyrosine residues of the growth factor receptors leads to the allosteric activation of the catalytic subunit and resulting phosphorylation of phosphatidylinositol 4,5- bisphosphate (PIP2) to the active second messenger PIP3. This results in the activation of PDK1 at the plasma membrane. The p85 regulatory subunit is a phosphoprotein substrate of many cytoplasmic and

receptor tyrosine kinases; p85 associates with active tyrosine kinases either through direct interaction through its SH2 domain or indirectly through intermediate phosphoproteins such as insulin receptor substrates, IRS1 and IRS2 (Vivanco and Sawyer, 2002).

AKT is a major effector of the PI3K pathway and regulates a number of downstream targets to control growth and survival. At the membrane, PDK1 phosphorylates AKT on T308, which results in its partial activation. Phosphorylation of AKT at S473 by mTORC2 results in its full activation. DNA-PKcs can also activate AKT at S473 (Stronach et al, 2011). AKT has three isoforms which share significant homology and also substrate specificities such as PRAS40, but also have distinct substrates. AKT regulates cell growth and proliferation through its effects on TSC1/TSC2 complex and mTORC signalling and phosphorylation of p21 and p27. mTOR phosphorylation by AKT leads to the phosphorylation of its subsequent targets, S6K1 and 4E-BP-1 (Hemmings and Restuccia, 2012). AKT promotes cell survival though negative regulation of pro-apoptotic proteins such as Bad, or inhibition of pro-apoptotic signals generated from downstream transcription factors such as FoxO (Zhang et al, 2011). AKT also phosphorylates GSK3, leading to the de-repression of cell-cycle activating molecules including MYC and cyclin D1 (Helman and Meltzer, 2003). AKT is also known to contribute to other phenotypes such as cell invasion and migration through its phosphorylation of vimentin and paladin. Phosphatase and tensin homologue (PTEN) is a lipid phosphatase that antagonizes the PI3K pathways by the removal of the D3 phosphate from PIP3 thereby limiting or terminating the PI3K signalling in cells (Cantley and Neel, 1999).

In HGSOC the PI3K/Ras pathway is altered in about 45% of cases as detailed in figure 9, with frequent amplifications in PIK3CA, RAS and AKT2.

Figure 9: Altered pathways in HGSOC. Retinoblastoma (RB) and PI3K/RASA pathways are identified as being frequently altered in HGSOC. n=316. Figure taken with permission from the Cancer Genome Atlas Research Network, 2011.

Data from TCGA suggests that the PI3K pathway is altered in about 34% of HGSOC patients, however if accounted for downstream mTOR targets, then the pathway is altered in approximately 63% of cases, highlighting the importance of this pathway in promoting survival/evading apoptosis and promoting resistance to treatment. The mTOR kinase intercepts signals from several pathways, including the MAPK/ERK pathway in addition to the PI3K pathway. There are two mTOR complexes, complex 1 (mTORC1) and complex 2 (mTORC2). Phosphorylation of mTORC1 promotes mRNA translation and oncogenic protein synthesis and there are reports suggesting that hyperactivation of mTOR results in the selective translation of pro-survival (surviving, Mcl1), angiogenesis (VEGF-A) and DNA repair response (BRCA1, 53BP1) proteins (Musa and Schneider, 2015). mTORC2 is thought to play an important role in activating AKT through phosphorylation (Sarbassov et al, 2005). Although there are mTORC1 inhibitors such as rapamycin, studies indicate that perhaps inhibition of both complexes are required for therapeutic efficacy as without inhibition of mTORC2, AKT is still activated, stimulating pro-survival oncogenic signalling and thus would be capable of overcoming the effects of mTORC1 inhibition.

In PDAC, the mutations in PI3K frequently observed in other tumour types are not present, however PTEN is lost in approximately 75% of cases (Asano et al, 2004). Additionally, it has been suggested that the activation of the PI3K pathway is necessary and sufficient to maintain oncogenic RAS-transformed xenograft tumours, after the elimination of RAS (Lim and Counter, 2005), which has great significance in PDAC, given its reliance on RAS in driving tumourigenesis in the majority of PDAC cases. AKT2, a

PI3K effector is also amplified in 10-20% of PDAC, further demonstrating the importance of this pathway in PDAC. Furthermore, Eser et al, have demonstrated that inhibition of PI3K pathway effectively blocks carcinogenesis and tumour progression in KRASG12D _{driven PDAC (Eser et al, 2013).}

Many sarcomas have also been shown to have activated growth factor signalling pathways, presumably through activating mutations in growth factor receptors. Interaction with the p85 subunit of PI3K leads to the activation of the p110 catalytic subunit, triggering the signalling cascade, leading to the activation of AKT as mentioned above. Additionally, one of the most frequent somatic mutations are in the PIK3CA gene in sarcomas with complex karyotypes, making it an attractive pathway for therapeutic intervention (Kim et al, 2012). This was further supported by observation that inhibiting PI3K pathway using BKM120 as a single agent delayed tumour growth in a mouse model of high-grade soft-tissue sarcoma and had increased benefits in combination with doxorubicin, although not statistically significant (Kim et al, 2012). Furthermore, the complexity of this pathway and cross-talk with other pathways was further demonstrated when studies suggested that BKM120 acted in synergy with a MEK1/2 inhibitor, trametinib or an IGF1R inhibitor, NVP-AEW541 or an mTORC1 inhibitor rapamycin in vitro in STS cell line models (Anderson et al, 2015).