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Title: Selected genes involved in GIST evolution from indolent to advanced tumours
Other Titles: Een selectie van genen betrokken bij de evolutie van indolente naar progressieve GIST
Authors: Quattrone, Anna
Issue Date: 12-Jun-2015
Abstract: Gastrointestinal stromal tumours (GIST) are the most frequent mesenchymal neoplasms of the digestive system. The annual incidence of clinically relevant GIST is around 13 cases per million inhabitants, whereas small incidental forms (<1 cm in size), referred to as micro-GIST, are found in at least 30% of the elderly population. Interstitial Cells of Cajal (ICC), innervated cells which regulate the peristalsis of the bowel, are thought to be the GIST cell of origin, as they are both dependent on KIT tyrosine kinase receptor and share similar morphology and immunophenotype. Approximately 85% of GIST harbour gain-of-function mutations in KIT or PDGFRA genes, which represent initiating event in GIST development, triggering constitutive stimulation of the RAS/RAF/MAPK, PI3K/AKT/mTOR and JAK/STAT signalling cascades. These pathways are crucial for GIST cell proliferation, survival and migration. Nevertheless, although KIT/PDGFRA oncogenic mutations are necessary they are not sufficient for GIST malignant transformation. As a matter of fact, micro-GIST carry the same type of KIT/PDGFRA mutations, but they rarely progress into clinically significant GIST. This indicates that GIST oncogenic evolution is possibly supported by further genetic and epigenetic changes, and might be sustained by additional environmental stimuli. The biological mechanisms and the different factors involved in this multi-step malignant transformation are still largely unknown. Bearing in mind that first and second line treatment of aggressive GIST with tyrosine kinase inhibitors, such as imatinib or sunitinib, are rarely completely successful, a better understanding of GIST pathogenesis becomes necessary for the discovery of new target molecules, and the development of more effective treatments.
In this thesis we have performed a comprehensive cytogenetic, molecular and functional characterization of GIST. In particular, we investigated the relevance of two selected genes, CCK2R and PTEN, and their contribution to GIST development and progression.
CCK2R belongs to the superfamily of G protein-coupled receptors. It is expressed in the gastrointestinal tract, where it mediates the action of gastrin; it regulates gastric acid secretion as well as growth and differentiation of the gastric epithelium. After gastrin stimulation, CCK2R is able to activate important downstream signalling, e.g. PLC and PKC, RAS/MAPK and PI3K/AKT pathways, which promote cell survival, proliferation and invasion. Interestingly, activated CCK2R displays growth-promoting effects on pre-neoplastic gastrointestinal cells. Expression of CCK2R has been demonstrated in different type of cancers, while it was not found in the corresponding normal tissues. These observations suggest a potential role of CCK2R in tumour pathogenesis.
In this thesis, we have shown that CCK2R is commonly expressed in GIST of gastric origin. Moreover, we observed higher level of CCK2R in GIST characterized by low malignant potential in comparison with high-risk/malignant ones, irrespective of the tumour genotype. Notably, we have found expression of CCK2R also in cases of incidental ICC hyperplasia and gastric micro-GIST. In addition, by in vivo studies using human GIST xenografts, we have demonstrated that gastrin-dependent stimulation of CCK2R leads to increased cell proliferation and tumour volume. In fact, we observed hyper-activation of PKC-θ and KIT kinases, and subsequently over-activation of major downstream KIT signalling pathways (AKT and MAPK). These findings suggest a promoting role of gastrin and CCK2R in early stages of GIST evolution, particularly in tumours that arise in the gastric microenvironment.
On the other hand, in this thesis we have found partial or total loss of PTEN expression in advanced malignant GIST, especially in tumour that showed resistance to imatinib therapy. This is in line with the common inactivation of crucial tumour suppressor genes in the late stages of GIST evolution. Furthermore, we have demonstrated that PTEN deficiency was primarily due to monoallelic loss of the PTEN gene. PTEN is a phosphatase that negatively regulates the PI3K/AKT signalling cascade. After in vitro silencing of PTEN in GIST cells, we have observed up-regulation of both the PI3K/AKT and the MAPK pathways, which are connected to each other by a complex network of cross-talks and feedback loops. Interestingly, we have found residual survival signalling after imatinib treatment and paradoxal over-activation of MAPK after dual PI3K/mTOR inhibition with NVPBEZ235. Combined treatment successfully counteracted the effects of PTEN silencing on AKT and MAPK activation in imatinib-sensitive GIST cell lines, whereas substantial MAPK hyper-phosphorylation was still observed in imatinib-resistant cells. In this cell context concomitant inhibition of the PI3K/AKT and RAS/MAPK pathways should be tested. Our results highlight the importance of PTEN deficiency in GIST progression. Moreover, PTEN represents an important biomarker to properly select GIST patients for appropriate therapies.
In conclusion, CCK2R expression and PTEN inactivation promote GIST progression at different stages, providing selective advantages to the tumour cells. Accumulation of aberrations during GIST malignant transformation leads to tumour heterogeneity, which influences the responses to treatment. A greater knowledge of crucial genes involved in GIST evolution will lead to an earlier diagnosis, better prognosis and more effective therapies.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Laboratory for Genetics of Malignant Disorders
Translational Cell & Tissue Research

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