PTEN, a lipid phosphatase, is one of the most frequently mutated tumour suppressors in human cancer. Several recent studies have highlighted the importance of ubiquitylation in regulating PTEN tumour-suppressor function, but the enzymatic machinery required for PTEN ubiquitylation is not clear. In this study, by using a tandem affinity-purification approach, we have identified WWP2 (also known as atrophin-1-interacting protein 2, AIP-2) as a PTEN-interacting protein. WWP2 is an E3 ubiquitin ligase that belongs to the NEDD4-like protein family, which is involved in regulating transcription, embryonic stem-cell fate, cellular transport and T-cell activation processes. We show that WWP2 physically interacts with PTEN and mediates its degradation through a ubiquitylation-dependent pathway. Functionally, we show that WWP2 controls cellular apoptosis and is required for tumorigenicity of cells. Collectively, our results reveal a functional E3 ubiquitin ligase for PTEN that plays a vital role in tumour-cell survival.

Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors then can be used to establish short-term explant cultures or intracranial xenografts. The focus of this manuscript is to review the procedures associated with the establishment, maintenance and utilization of a primary GBM xenograft panel.

Positron emission tomography (PET) imaging with the amino acid tracer 6-18F-fluoro-l-3,4-dihydroxy-phenylalanine (18F-DOPA) may provide better spatial and functional information in human gliomas than CT or MRI alone. The l-type amino acid transporter 1 (LAT1) is responsible for membrane transport of large neutral amino acids in normal cells. This study assessed the relationship between LAT1 expression and 18F-DOPA uptake in human astrocytomas. Endogenous LAT1 expression was measured in established glioblastoma (GBM) cell lines and primary GBM xenografts using Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Uptake of 18F-DOPA was approximated in vitro using 3H-l-DOPA as an analog. Uptake of 3H-l-DOPA was assessed in cells expressing LAT1 shRNA or LAT1 siRNA and compared to non-targeted (NT) control shRNA or siRNA sequences, respectively. To demonstrate the clinical relevance of these findings, LAT1 immunofluorescence staining was compared with corresponding regions of 18F-DOPA PET uptake in patients with newly diagnosed astrocytomas. LAT1 mRNA and protein expression varies in GBM, and the extent of 3H-l-DOPA uptake was positively correlated with endogenous LAT1 expression. Stable shRNA-mediated LAT1 knockdown in T98 and GBM28 reduced 3H-l-DOPA uptake relative to NT shRNA by 57 (P < 0.0001) and 52 % (P < 0.001), respectively. Transient siRNA-mediated LAT1 knockdown in T98 reduced 3H-l-DOPA uptake relative to NT siRNA up to 68 % (P < 0.01). In clinical samples, LAT1 expression positively correlated with 18F-DOPA PET uptake (P = 0.04). Expression of LAT1 is strongly associated with 3H-l-DOPA uptake in vitro and 18F-DOPA uptake in patient biopsy samples. These results define LAT1 as a key determinant of 18F-DOPA accumulation in GBM.

PURPOSE The therapeutic benefit of temozolomide (TMZ) in glioblastoma (GBM) is limited by resistance. The goal of this study was to elucidate mechanisms of TMZ resistance in GBM. EXPERIMENTAL DESIGN We developed an in vivo GBM model of TMZ resistance and used paired parental and TMZ resistant tumors to define the mechanisms underlying the development of resistance and the influence of histone deacetylation (HDAC) inhibition. RESULTS Analysis of paired parental and resistant lines demonstrated upregulation of MGMT expression in 3 of the 5 resistant xenografts. While no significant change was detected in MGMT promoter methylation between parental and derivative resistant samples, chromatin immunoprecipitation demonstrated an association between MGMT upregulation and elevated acetylation of lysine 9 of histone H3 (H3K9-ac) and decreased di-methylation (H3K9-me2) in GBM12 and GBM14. In contrast, TMZ resistance development in GBM22 was not linked to MGMT expression and both parental and resistant lines had low H3K9-ac and high H3K9-me2 within the MGMT promoter. In the GBM12 TMZ resistant line, MGMT re-expression was accompanied by increased recruitment of SP1, C-JUN, NF-kB and p300 within the MGMT promoter. Interestingly, combined treatment of GBM12 flank xenografts with TMZ and the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) favored the evolution of TMZ resistance by MGMT over-expression as compared to treatment with TMZ alone. CONCLUSION This study demonstrates, for the first time, a unique mechanism of TMZ resistance development driven by chromatin mediated MGMT upregulation and highlights the potential for epigenetically directed therapies to influence the mechanisms of resistance development in GBM.