PTEN controls tumor-induced angiogenesis

Abstract

Mutations of the tumor suppressor PTEN, a phosphatase with specificity for 3-phosphorylated inositol phospholipids, accompany progression of brain tumors from benign to the most malignant forms. Tumor progression, particularly in aggressive and malignant tumors, is associated with the induction of angiogenesis, a process termed the angiogenic switch. Therefore, we tested whether PTEN regulates tumor progression by modulating angiogenesis. U87MG glioma cells stably reconstituted with PTEN cDNA were tested for growth in a nude mouse orthotopic brain tumor model. We observed that the reconstitution of wild-type PTEN had no effect on in vitro proliferation but dramatically decreased tumor growth in vivo and prolonged survival in mice implanted intracranially with these tumor cells. PTEN reconstitution diminished phosphorylation of AKT within the PTEN-reconstituted tumor, induced thrombospondin 1 expression, and suppressed angiogenic activity. These effects were not observed in tumors reconstituted with a lipid phosphatase inactive G129E mutant of PTEN, a result that provides evidence that the lipid phosphatase activity of PTEN regulates the angiogenic response in vivo. These data provide evidence that PTEN regulates tumor-induced angiogenesis and the progression of gliomas to a malignant phenotype via the regulation of phosphoinositide-dependent signals.

Figure 1

Stable expression of PTEN and PTEN mutants in U87MG cells regulates AKT. (A) Cell lysates from the U87MG (U87) cell line and U87 cells infected with a retroviral vector encoding PTEN (pBabe-Puro-PTEN) or mutants of PTEN (pBabe-Puro-PTEN-G129E or R130 M) were resolved by SDS/PAGE, and equal amounts of proteins were loaded per lane, immunoblotted with antisera to PTEN, phospho-AKT, and total AKT, and visualized by enhanced chemiluminescence. The basal levels of PTEN (Top), phosphorylated AKT (Ser-473) (Middle), and total AKT (Bottom) are shown. The status of the PTEN gene in each stable cell line was designated as: WT.E1 and WT.C7, two separate clones expressing WT PTEN; R130 M and G129E are mutated PTEN, R130 M is inert as both a protein and a lipid phosphatase. The G129E PTEN can dephosphorylate acidic phosphopeptides, but cannot dephosphorylate lipid substrate, PtdIns(3,4,5)P₃. The U87MG (U87) cell line is the parental cell line isolated from a human glioblastoma multiforme patient. (B) Comparison of in vitro growth of U87MG cells transduced with mutants of PTEN. Equal number of cells (1 × 10⁵) were incubated in RPMI + 10% FBS for different times, and cell numbers were quantitated by direct cell counting.

Figure 2

Effects of PTEN on growth of U87MG cells in vivo. (A) Cell growth in vivo. To determine the rate of cell growth in vivo, equal amounts of cells (5 × 10⁶) from each cell line were implanted at the right ventral flank by s.c. injection. The formation and growth of the s.c. tumor was monitored, and the volume of the tumor was determined by a three-dimensional measurement at the times indicated (day 0, the date of implantation, no tumor is detected). Data were analyzed by Student's t test, and differences were significant comparing the PTEN deficient (U87MG, R130 M, G129E) to the WT PTEN (WT.E1, WT.C7), n = 5, number of mice; P < 0.0001. (B) Stereophotography of s.c. tumor sites in mice implanted with the parental U87 tumor, PTEN minus (Left) versus WT PTEN reconstituted tumor cells (Right). These tumors represent 25 and 42 days after implantation for PTEN minus versus WT PTEN reconstituted tumors, respectively. (Magnification, ×40.) (C) Immunoblot of cryostat tissue sections from s.c. tumor for the expression pattern of PTEN, AKT, and phosphorylated AKT. Frozen tissue sections were solubilized in Laemmli sample buffer, total protein was quantitated, and equal protein was loaded on SDS/PAGE. The data shown are representative of tissue analysis from five animals per experimental group.

Figure 3

PTEN suppresses angiogenesis. Immunohistochemical analysis of staining with CD31 antibody to evaluate the angiogenesis response within the parental U87MG tumor (A) and PTEN reconstituted tumors (B), implanted into the s.c. tissue. In PTEN minus and tumors expressing mutants of PTEN, there are more new vessels formed (angiogenesis) (Upper, arrow indicated) than in WT PTEN reconstituted tumor (Lower), indicating the PTEN has direct influence on angiogenesis during tumor growth. (C) MVD counts were performed on tumor tissue stained with anti-CD31 antibody as described (32) to determine the effect of expression of PTEN and specific PTEN mutants (G129E or R130 M) on tumor-induced angiogenesis. Bars represent SD, five animals per group. Statistical analysis by Student's t test demonstrateed significant difference between MVD of PTEN null and PTEN catalytic mutants as compared with WT PTEN reconstituted tumors, n = 5, number of mice; P < 0.001. (D) PTEN regulates the expression of TSP-1 in U87MG cells. RNase protection assay was used to measure levels of TSP-1 mRNA in WT PTEN expressing U87 cells or cells transduced with a mutant catalytically dead PTEN (G129R). U87MG cells were infected with retrovirus encoding WT PTEN (WT), the catalytically dead, G129R mutant (GR), or empty vector retrovirus (−) and selected for 10 days in puromycin. RNA was harvested and RNase protection assays were carried out by using probes for TSP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A probe for GAPDH was used as a normalization control. (E) Thrombospondin immunoblot analysis. U87MG transduced with WT PTEN (WT) or a catalytic mutant PTEN (G129R) in an ecdysone inducible expression system (36) were induced (48 h) with 0.5 μM muristirone or assayed without induction to determine the effect of PTEN expression on the induction of TSP-1 by Western blotting. Supernates from cells were prepared and proteins were resolved on SDS/PAGE and probed with anti-TSP-1 antibody. There is clear up-regulation of TSP-1 in WT PTEN-transduced U87 cultures compared with U87 cells expressing the lipid phosphatase-deficient G129R mutant PTEN.

Figure 4

Effects of PTEN reconstitution on survival in an orthotopic brain tumor model. Equivalent number of parental U87 cells or U87 cells reconstituted with WT or mutant alleles of PTEN (1 × 10⁶ cells) were implanted in right frontal lobe of nude mice. Stereophotography of whole brains from mice implanted with U87MG tumor cells (day 25) (A) or PTEN reconstituted (day 42) (B). The implantation site is shown by position of arrow in the WT PTEN reconstituted tumor (B). (Magnification: ×20.) (C) Survival plots for mice implanted with PTEN minus or parental U87 cells transduced with mutants of PTEN as shown. Survival data represents 15 animals per experimental group. P < 0.0001 for difference observed between the PTEN+ and PTEN− groups for survival. (D) Immunohistochemical analysis for anti-CD31 staining (brown staining) for microvessels within a U87MG parental PTEN-deficient brain tumor on day 22 after stereotactic implantation. (E) Anti-CD31 staining of microvessels within a WT PTEN reconstituted U87MG brain tumor evaluated on day 42 after implantation. (F) MVD counts were performed on brain tumor tissue stained with anti-CD31 antibody as described (32) to determine the effect of expression of PTEN and specific PTEN mutants (G129E or R130 M) on tumor-induced angiogenesis. Bars represent SD, four animals per group. Statistical analysis by n = 5 (number of mice), P < 0.01.