Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Abstract

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Fig. 1.

SFK and FGFR-dependent phosphorylation of PTEN at tyrosine 240 in GBM cells. (A) GBM39 cells cultured ex vivo were treated with 100 nM dasatinib for 24 h, as shown before analysis by Western blotting. (B) Neurosphere cultures from GBM specimens were treated with dasatinib as indicated for 24 h before analysis by Western blotting. (C) GBM39 neurospheres were treated with 100 nM dasatinib and 1 μM PD173074 alone or in combination as shown. (D) HEK 293T cells were transfected with wild-type or Y240F PTEN and c-SRC constructs as shown. Inhibitors (Das, 100 nM dasatinib; PD, 50 nM PD173074) were added to cells 24 h after transfection, and cells were harvested after a further 24 h. PTEN was immunoprecipitated from lysates, and pan-phosphotyrosine or phosphorylation of Y240 were detected by Western blotting. (E) HEK 293T cells were transfected with PTEN and FGFR2 expression constructs as shown, and samples were processed as in D. (F) NIH 3T3 cells were serum-starved for 24 h before stimulation with 20 ng/mL bFGF and harvested over a time course after stimulation as indicated. (G) HEK 293T cells were cotransfected with constructs expressing FGFR2 and either empty vector control or FLAG-HA–tagged PTEN. Lysates were subject to immunoprecipitation with anti-HA antibodies, and coprecipitation of FGFR2 was detected by Western blotting.

Fig. 2.

Phosphorylation of Y240 does not affect PTEN control of PI3K/AKT signaling. (A) PTEN expression levels in U87MG cells after retroviral infection and selection compared with endogenous PTEN expression in GBM39, murine ink4a/arf^−/− astrocytes, and HEK 293T cells. (B) Cell cycle profiles of U87MG cells reconstituted with PTEN wild-type or Y240F as in A, measured by propidium iodide staining and flow cytometry to assess DNA content. (C) AKT phosphorylation in U87MG cells reconstituted with wild-type PTEN or alleles bearing tyrosine phosphorylation site mutations as shown. Asterisk denotes the endogenous mutant PTEN. (D) Astrocytes derived from ink4a/arf^−/−/pten^fl/fl mice after deletion of pten, expressing empty vector [pBabe-blasticidin (pBB)] or constitutively active SCR (pBB-SRC Y530F), were reconstituted with empty vector [pBabe-puro (pBP)] or pBP-wild-type PTEN or pBP-Y240F PTEN as shown. Cells were serum-starved for 24 h before harvesting. (E) Yeast expressing galactose-inducible p110α-CAAX were transformed with empty vector or PTEN alleles as shown and assessed for their ability to grow on galactose plates by spot assay as described in SI Appendix, SI Materials and Methods. (F) The catalytic activity of recombinant purified GST-tagged PTEN (wild-type or Y240F) proteins against soluble PI-3,4,5-P₃ was measured by Biomol Green assay as described in SI Appendix, SI Materials and Methods.

Fig. 5.

Implication of PTEN Y240 phosphorylation in resistance to erlotinib in vitro. (A) Ink4a/Arf/PTEN^−/− astrocytes expressing ΔEGFR were reconstituted with wild-type or Y240F PTEN alleles and after blasticidin selection were exposed to erlotinib for 72 h before measurement of cell viability by WST-1 assay. Error bars indicate SEM. (B) GBM39 cells were treated with erlotinib for 48 h in basal medium or in the presence of 20 ng/mL bFGF or 20 ng/mL EGF as shown, and relative cell viability was measured by WST1 assay. Decrease in viability is shown as the percentage change relative to untreated cells. (C) GBM39 cells collected after 48 h of erlotinib treatment were analyzed for cleaved PARP by Western blotting. (D) Acute changes in RTK signaling pathway components upon erlotinib treatment in the absence or presence of bFGF stimulation were assessed by Western blotting. Where indicated, 5 μM erlotinib was added to GBM39.

Fig. 3.

Phosphorylation of PTEN in GBM patient samples is correlated with FGFR3 expression and associated with shortened survival in EGFR-positive GBM. (A) Adjacent FFPE sections from s.c. xenograft tumors established from U87MG-ΔEGFR cells expressing either wild-type or Y240F PTEN were stained with antibodies specific for total PTEN and p-PTEN (Y240). Representative images from each tumor type are shown. (B) Paraffin-embedded tissue samples obtained after surgical resection of GBM patients were stained with antibodies specific for total and phospho-Y240 PTEN as indicated. Arrowheads indicate strong positivity for total PTEN in the vascular endothelium of a PTEN-negative tumor, characteristic of GBM (40× magnification). (C) FGFR3 transcript levels were measured by quantitative reverse transcriptase PCR using RNA isolated from GBM samples. (D) Kaplan-Meier survival curves showing overall survival of EGFR-positive GBM patients as a function of PTEN Y240 phosphorylation (SI Appendix, Table S2). (E) Representative images from GBM tissue microarray samples showing p-SFK, p-PTEN, and total PTEN staining.

Fig. 4.

Phosphorylation of PTEN is increased in EGFR TKI-resistant GBM. (A) Quantification and representative staining patterns of PTEN Y240 phosphorylation observed in patients as a function of response to EGFR TKIs. (B) Quantification and representative staining of biopsies from two GBMs that initially responded to EGFR TKI therapy but later acquired resistance coincident with increased phosphorylation of PTEN.