Neuroendocrine tumors (NET) form a heterogeneous group of malignancies. The phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K-Akt-mTOR) signaling pathway has been demonstrated to play a major role in NET by regulating cell growth, proliferation, cell survival and protein synthesis ). Furthermore, alterations in genes regulating this pathway are reported in pancreatic NET (PNET). Furthermore, elevated mTOR expression and activity is associated with a higher proliferative capacity and worse prognosis. mTOR proves to be an interesting target for therapy of NET with mTOR-inhibiting rapamycin and analogs (rapalogs) such as everolimus. mTOR acts as the catalytic subunit of two functionally distinct complexes, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Although effectively blocking mTORC1, rapalogs only have a limited, dose-dependent action on the mTORC2. Recent phase III trials with everolimus show an improved progression-free survival in monotherapy in progressive advanced pancreatic NET and in combination with long-acting octreotide in advanced carcinoid tumors. However, adaptive resistance to mTOR inhibition with rapalogs is described. This adaptive resistance may be caused by induction of activating phosphorylation of Akt, upstream of mTOR in the PI3K-Akt-mTOR pathway. The effect of rapalogs on mTOR signaling may be circumvented through increased activity of mTORC2 and this may lead to resistance to rapalogs.
The first aim (WP 1) of the project is to investigate the resistance mechanisms that play a key role in adaptation to everolimus treatment in PNET. To accomplish this, in vitro transcription and phosphorylation of the components of the PI3K-Akt-mTOR pathway will be studied in sensitive and secondary resistant PNET cell lines. For the transcription studies, gene expression microarrays will be performed on RNA extracted from the cell lines that are sensitive and the ones with induced (secondary) resistance. The role of Akt phosphorylation by reduced inhibition of S6K1-IRS-IGF axis and other possible unknown feedback loops will be evaluated with the western blotting.
The second aim (WP 2) of the project is to determine whether DNA methylation of genes and promoter regions associated with the mTOR pathway play a role in adaptive resistance to everolimus. Therefore the DNA methylation status of sensitive and secondary resistant PNET cell lines will be studied using Illumina's Infinium methylation 450k beadchip microarrays. These methylation microarray data will be integrated with the transcription microarray data to identify functional methylation pattern changes. In patient material, important epigenetic changes, identified in the cell lines, will be quantified with pyrosequencing and will be correlated with gene expression, studied with real time PCR. Furthermore, this will be correlated to resistance to everolimus in a retrospective study with the goal of describing predictive biomarkers for response to therapy with everolimus.
The third aim (WP 3) of the project is to evaluate if dual inhibition of mTOR and interesting therapeutic targets (such as IGF, PI3K, mTORC2, EGFR), which will be identified in the first parts of this research project, might overcome acquired resistance to everolimus. The role of the transcription and phosphorylation of the PI3K-Akt-mTOR pathway during dual inhibition will be studied in vitro in sensitive and secondary resistant PNET cell lines. An in vivo experiment using an orthotopic PNET cancer model, comparing dual inhibition to, respectively, placebo, mTOR inhibition alone and inhibition of the identified therapeutic targets alone will be conducted. Response will be evaluated using microPET/CT. Ex vivo studies using immunohistochemistry, real time PCR and western blotting will be used to the dual inhibition to activation of the PI3K-Akt-mTOR pathway.