Cyclin D1 is a mediator of gastrointestinal stromal tumor KIT-independence.

Oncogenic KIT or PDGFRA tyrosine kinase mutations are compelling therapeutic targets in most gastrointestinal stromal tumors (GISTs), and the KIT inhibitor, imatinib, is therefore standard of care for patients with metastatic GIST. However, some GISTs lose expression of KIT oncoproteins, and therefore become KIT-independent and are consequently resistant to KIT-inhibitor drugs. We identified distinctive biologic features in KIT-independent, imatinib-resistant GISTs as a step towards identifying drug targets in these poorly understood tumors. We developed isogenic GIST lines in which the parental forms were KIT oncoprotein-dependent, whereas sublines had loss of KIT oncoprotein expression, accompanied by markedly downregulated expression of the GIST biomarker, protein kinase C-theta (PRKCQ). Biologic mechanisms unique to KIT-independent GISTs were identified by transcriptome sequencing, qRT-PCR, immunoblotting, protein interaction studies, knockdown and expression assays, and dual-luciferase assays. Transcriptome sequencing showed that cyclin D1 expression was extremely low in two of three parental KIT-dependent GIST lines, whereas cyclin D1 expression was high in each of the KIT-independent GIST sublines. Cyclin D1 inhibition in KIT-independent GISTs had anti-proliferative and pro-apoptotic effects, associated with Rb activation and p27 upregulation. PRKCQ, but not KIT, was a negative regulator of cyclin D1 expression, whereas JUN and Hippo pathway effectors YAP and TAZ were positive regulators of cyclin D1 expression. PRKCQ, JUN, and the Hippo pathway coordinately regulate GIST cyclin D1 expression. These findings highlight the roles of PRKCQ, JUN, Hippo, and cyclin D1 as oncogenic mediators in GISTs that have converted, during TKI-therapy, to a KIT-independent state. Inhibitors of these pathways could be effective therapeutically for these now untreatable tumors.

Sustained Mutant KIT Activation in the Golgi Complex Is Mediated by PKC-θ in Gastrointestinal Stromal Tumors.

PURPOSE: Tumorigenesis of gastrointestinal stromal tumors (GIST) is driven by gain-of-function mutations in the KIT gene, which result in overexpression of activated mutant KIT proteins (MT-KIT). However, the mechanism of MT-KIT overexpression is poorly understood. EXPERIMENTAL DESIGN: By protein expression analysis and immunofluorescent microscopic analysis, we determine the stability and localization of MT-KIT in four GIST cell lines with different mutations and HeLa cells transfected with mutant KIT model vectors. We also used 154 human GIST tissues to analyze the relationship between the expression of PKC-θ and MT-KITs, and correlations between PKC-θ overexpression and clinicopathological parameters. RESULTS: We report that four different MT-KIT proteins are intrinsically less stable than wild-type KIT due to proteasome-mediated degradation and abnormally localized to the endoplasmic reticulum (ER) or the Golgi complex. By screening a MT-KIT-stabilizing factor, we find that PKC-θ is strongly and exclusively expressed in GISTs and interacts with intracellular MT-KIT to promote its stabilization by increased retention in the Golgi complex. In addition, Western blotting analysis using 50 GIST samples shows strong correlation between PKC-θ and MT-KIT expression (correlation coefficient = 0.682, P < 0.000001). Immunohistochemical analysis using 154 GISTs further demonstrates that PKC-θ overexpression significantly correlates with several clinicopathological parameters such as high tumor grade, frequent recurrence/metastasis, and poor patient survival. CONCLUSIONS: Our findings suggest that sustained MT-KIT overexpression through PKC-θ-mediated stabilization in the Golgi contributes to GIST progression and provides a rationale for anti-PKC-θ therapy in GISTs. Clin Cancer Res; 23(3); 845-56. ©2016 AACR.