Abstract A166: PTEN inactivation in gastrointestinal stromal tumors (GIST): Possible relevance for treatment of imatinib-resistant disease.
Authors: Quattrone Anna , Wozniak Agnieszka , Dewaele Barbara , Floris Giuseppe , Vanspauwen Vanessa , Schoffski Patrick , Sciot Raf , Debiec-Rychter Maria 
Published: June 2012

Background: Inactivation of PTEN occurs in numerous tumor types and has been implicated as a resistance mechanism to receptor tyrosine kinase (RTK)-targeted therapies in human cancer. The role of PTEN inactivation in GIST in the acquisition of late resistance to imatinib (IM), a RTK inhibitor, remains elusive.

Aim: We investigated the incidence and the mechanisms of PTEN inactivation in a cohort of GIST specimens, and the impact of PI3K inhibition on KIT signaling in IM-sensitive vs. IM-resistant GIST cell lines.

Methods: Molecular characterization of PTEN was performed on 65 (23 primary, 10 recurrent/metastatic, and 32 IM-resistant) GIST by CGH array/FISH, mutational analysis, PTEN promoter methylation and QPCR analysis. The PTEN expression and KIT downstream signaling were assessed by Western blotting (WB). The effect of PTEN knock-down by siRNA on KIT signaling was examined in KIT-positive (GIST48) and KIT-negative (KUL-5) GIST cell lines. The impact of NVP-BEZ235 (BEZ), a dual PI3K/mTOR inhibitor, was evaluated in vitro, alone and in combination with IM on IM-sensitive (GIST882, GIST882LY) and IM-resistant (GIST48, GIST48B) cell lines. GIST882LY is a sub-line with bi-allelic PTEN loss. GIST48 has an IM-resistant KIT-D820A mutation, whereas GIST48B has lost its former dependence on KIT oncoprotein. Biological consequences of PI3K inhibition were determined by WB for KIT signaling pathways and by cell proliferation and apoptosis assays.

Results: On genomic level PTEN was affected mainly by mono-allelic loss (9%, 16% and 36% of primary, recurrent/metastatic and IM-resistant tumors, respectively). PTEN mutations or PTEN promoter hypermethylation were not found. By QPCR, a positive correlation was observed between low PTEN expression and its genomic loss (p&lt;0.01). Reduced PTEN expression was confirmed in GIST with mono-allelic PTEN by WB. In vitro, siRNA silencing of PTEN in GIST cell lines resulted in up-regulation of PI3K/AKT/S6 and ERK1/2 pathways, which was partially reverted by PI3K inhibition. By WB, both GIST882 and GIST882LY were sensitive to IM and BEZ. However, GIST 882LY required higher dose of IM (250nM) and BEZ (50nM) to show the same inhibitory effects on AKT and S6 phosphorylation in comparison with GIST882 (100nM and 25nM, respectively). Inhibition of PI3K pathway resulted in up-regulation of ERK in these models. In contrast, combination of BEZ (25nM) and IM (50nM) induced a synergistic effect on the inhibition of both pathways. IM did not show effects on GIST48 or GIST48B. Conversely, both cell lines were sensitive to BEZ (at 200nM and 100nM, respectively). Antiproliferative effects of BEZ were strong in GIST882, GIST882LY and GIST48 cell lines in a dose and time dependent manner (IC50's of 25, 50 and 75nM after 72 h, respectively); these effects were potentiated in combination with IM. In contrast, BEZ alone (up to 100nM) or in combination with IM (up to 2000nM) did not inhibit proliferation of the KIT-negative GIST48B cell line. No induction of apoptosis was observed in any of our models or conditions.

Conclusions: Downregulation of PTEN occurs frequently in IM-resistant GIST in parallel with PI3K/AKT pathway hyperactivation. Inhibitors of the PI3K/AKT pathway represent a potential treatment option for GIST and additional research efforts are required to fully explore this possible treatment strategy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A166.