Heterozygosity for Pten promotes tumorigenesis in a mouse model of medulloblastoma

Abstract

Background: Recent publications have described an important role for cross talk between PI-3 kinase and sonic hedgehog signaling pathways in the pathogenesis of medulloblastoma.

Methodology/principal findings: We crossed mice with constitutive activation of Smoothened, SmoA1, with Pten deficient mice. Both constitutive and conditional Pten deficiency doubled the incidence of mice with symptoms of medulloblastoma and resulted in decreased survival. Analysis revealed a clear separation of gene signatures, with up-regulation of genes in the PI-3 kinase signaling pathway, including downstream activation of angiogenesis in SmoA1+/-; Pten +/- medulloblastomas. Western blotting and immunohistochemistry confirmed reduced or absent Pten, Akt activation, and increased angiogenesis in Pten deficient tumors. Down-regulated genes included genes in the sonic hedgehog pathway and tumor suppressor genes. SmoA1+/-; Pten +/+ medulloblastomas appeared classic in histology with increased proliferation and diffuse staining for apoptosis. In contrast, Pten deficient tumors exhibited extensive nodularity with neuronal differentiation separated by focal areas of intense staining for proliferation and virtually absent apoptosis. Examination of human medulloblastomas revealed low to absent PTEN expression in over half of the tumors. Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent PTEN expression.

Conclusions/significance: This suggests that PTEN expression is a marker of favorable prognosis and mouse models with activation of PI-3 kinase pathways may be important tools for preclinical evaluation of promising agents for the treatment of medulloblastoma.

Figure 1. Compound SmoA1, Pten heterozygotic mice exhibit an increased incidence of medulloblastoma and reduced survival.

SmoA1 +/+ mice were crossed with Pten +/− mice and followed for symptoms. (A) Observed tumor incidence in Pten deficient versus control mice over the course of 1 year. (B) Kaplan-Meier survival analysis of SmoA1 +; Pten +/− (n = 43) and SmoA1 +; Pten (LoxP/-); Nestin-cre + (n = 8) mice, compared to SmoA1 +; Pten +/+ (n = 127) and SmoA1 +; Pten (LoxP/-); Nestin-cre - (n = 7) (p<0.0001, Log-rank) controls. (C) Mice with global Pten deficiency as well as those with conditional, partial knock-out of Pten developed symptoms earlier than controls (p<0.000001 and <0.005, respectively).

Figure 2. Pten deficiency activates PI-3 kinase signaling and drives medulloblastoma histology from classic to extensive nodularity.

(A) SmoA1 +; Pten +/+ medulloblastomas demonstrated classic histology; SmoA1 +; Pten +/− tumors were extensively nodular in histology. Pten deficient tumors exhibited focal regions of intense staining for activated Akt (white arrow) and virtually absent staining for Pten. Only regions around blood vessels (black arrow) stained positive for Pten in SmoA1 +/−; Pten +/− tumors. (B) Western blotting revealed decreased expression of Pten and increased activation of Akt in SmoA1 +/−; Pten +/− medulloblastomas (n = 3), compared to controls (n = 7). (C) Total RNA was extracted from SmoA1 +/−; Pten +/+ (n = 4) and SmoA1 +/−; Pten +/− (n = 8) medulloblastomas and hybridized to Illumina mouse microarray chips. Expression of genes involved in PI-3 kinase signaling was up-regulated in Pten deficient tumors. Red pixels represent increased expression and green pixels represent decreased expression. Each column corresponds to mRNA extracted from an individual mouse medulloblastoma. The blue bar at the top of each heatmap indicates Pten +/+ and the yellow bar indicates Pten +/− tumors.

Figure 3. SmoA1 +; Pten +/− medulloblastomas down-regulate expression of targets of sonic hedgehog (Shh) signaling.

(A) Analysis of microarrays revealed that Shh pathway genes were down-regulated in SmoA1 +; Pten +/− mouse tumors (n = 8) versus controls (n = 4). Gli2, a downstream target of Smoothened, was attenuated 4.2-fold. (B) Using real-time, RT-PCR we confirmed down-regulated expression of mRNA for downstream targets of Smoothened. Relative expression of Gli1, Gli2, N-myc, and cyclin-D1 was decreased 5.6, 7.6, 4.3, and 2.8-fold (*, p<0.05 for all transcripts), respectively in SmoA1 +; Pten +/− (white bars) medulloblastomas. Error bars, standard error of the mean. (C)We verified strong nuclear staining for Gli2, the major downstream target of activated Shh signaling, in SmoA1 +; Pten +/+ medulloblastomas. Conversely, staining for Gli2 was both cytoplasmic and weaker in the nucleus in the nodules of SmoA1 +; Pten +/− medulloblastomas.

Figure 4. Pten +/− mouse medulloblastomas display increased angiogenesis.

(A) Pten deficient medulloblastomas (n = 8) exhibited higher expression of genes involved in angiogenesis versus controls (n = 4). (B) Real-time, RT-PCR confirmed increased expression of Vegfa mRNA in SmoA1 +; Pten +/− (n = 7) compared to SmoA1 +; Pten +/+ (n = 4) tumors (p = 0.005). Error bars, standard error of the mean. (C) Increased CD31-positive staining of endothelial cells in Pten +/− (n = 4) (small black arrow), compared to Pten +/+ (n = 4) medulloblastomas. SmoA1 +; Pten +/− tumors (n = 4) also exhibited increased CD34-positive staining and staining of larger-bore blood vessels (large black arrow) than SmoA1 +; Pten +/+ medulloblastomas (n = 4).

Figure 5. Pten +/− medulloblastomas exhibit down-regulation of genes involved in cell cycle control, with associated increased proliferation and reduced apoptosis.

(A) Lower expression of genes involved in cell cycle control (Table S3) in tumors from SmoA1 +/−; Pten +/− (n = 8) versus control (n = 4) mice. (B) Focal areas of intense staining for PCNA and virtually absent staining for Cleaved Caspase-3 in Pten +/− tumors (n = 5), compared to Pten +/+ medulloblastomas (n = 7).

Figure 6. Loss of PTEN expression is an indicator of poor prognosis in patients with medulloblastoma.

(A) PTEN expression was scored from 0 to 2+ in human medulloblastoma (n = 111) tumors. (B) Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent (score = 0) PTEN expression (n = 16), compared to survival of patients with detectable PTEN (n = 26) expression (score = 1–2) ( Table 1 ) by IHC (p<0.0005).