Id2 mediates tumor initiation, proliferation, and angiogenesis in Rb mutant mice

Abstract

The inhibitor of differentiation Id2 is a target of the retinoblastoma (Rb) protein during mouse embryogenesis. In Rb(+/-) mice, LOH at the wild-type Rb allele initiates pituitary adenocarcinoma, a tumor derived from embryonic melanotropes. Here we identify a critical role for Id2 in initiation, growth, and angiogenesis of pituitary tumors from Rb(+/-) mice. We show that proliferation and differentiation are intimately coupled in Rb(+/-) pituitary cells before tumor initiation. In Id2-null pituitaries, premature activation of basic helix-loop-helix-mediated transcription and expression of the cdk inhibitor p27(Kip1) impairs the proliferation of melanotropes and tumor initiation. Without Id2, Rb(+/-) mice have fewer early tumor lesions and a markedly decreased proliferation rate of the tumor foci. Expression of Id2 by pituitary tumor cells promotes growth and angiogenesis by functioning as a master regulator of vascular endothelial growth factor (VEGF). In human neuroblastoma, the N-Myc-driven expression of Id2 is sufficient and necessary for expression of VEGF. These results establish that aberrant Id2 activity directs initiation and progression of embryonal cancer.

FIG. 1.

Loss of the Id2 gene delays tumorigenesis in Rb^+/− mice. (A) Kaplan-Meyer analysis of Rb^+/− (n = 22) and Id2^−/−; Rb^+/− (n = 29) mice (P = 0.0001). (B) Morphology of the pituitary glands in Rb^+/− and Id2^−/−; Rb^+/− mice at 200 days of postnatal life. Neoplastic lesions of the pituitary were macroscopically evident in all nine Rb^+/− mice examined but only in one of seven Id2^−/−; Rb^+/− mice. Arrows indicate the pituitary gland. Representative pituitaries are shown. (C) H&E staining of paraffin sections of pituitary glands from Rb^+/− and Id2^−/−; Rb^+/− mice at 200 days of postnatal life. AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe; T, tumor. Arrowheads indicate distinct tumor foci in the intermediate lobe of Id2^−/−; Rb^+/− pituitary. Magnification, ×10.

FIG. 2.

Premature differentiation and cell cycle arrest in the intermediate lobe of E15.5 Rb^+/− pituitaries lacking Id2. (A) Decreased proliferation in Id2^−/−; Rb^+/− embryonic pituitary. Paraffin sections from embryos were immunostained for BrdU. Magnification, ×40. (B) Quantification of BrdU-positive cells was performed in the intermediate lobe of the developing pituitary from four E15.5 Rb^+/− (black bar) and five Id2^−/−; Rb^+/− (white bar) embryos. Error bars indicate standard deviation. *, P = 0.005. (C) Id2 is expressed in the intermediate lobe of E15.5 Rb^+/− pituitaries. Expression of MASH-1 transcription factor (D) and POMC (E) is elevated in the Id2^−/−; Rb^+/− pituitary intermediate lobe. Magnification, ×20. (F) The intermediate lobe of Id2^−/−; Rb^+/− pituitaries shows increased immunoreactivity for the cdk inhibitor p27^Kip1. Magnification, ×20. Each staining was performed with five Id2^−/−; Rb^+/− and four Rb^+/− embryos. Representative samples are shown. AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe.

FIG. 3.

Loss of the Id2 gene impairs tumor initiation and tumor cell proliferation in Rb^+/− pituitaries. (A) H&E staining of sagittal sections of pituitary glands from Rb^+/− and Id2^−/−; Rb^+/− mice at 90 days of postnatal life. Arrowheads indicate distinct tumor foci in the intermediate lobe of pituitary glands. Magnification, ×10. (B) H&E staining of paraffin sections of pituitary glands from 90-day-old Rb^+/− and Id2^−/−; Rb^+/− mice showing magnified views of representative tumor foci. Magnification, ×40. (C) Rb^+/− and Id2^−/−; Rb^+/− mice were injected with BrdU 1 h before being sacrificed. Formalin-fixed sections were immunostained for BrdU. Arrowheads indicate the area of tumor lesions. AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe. (D) Quantification of BrdU-positive cells in four 3-month-old Rb^+/− and four 4-month-old Id2^−/−; Rb^+/− mice was done by counting cells in tumor foci from nine sections of each pituitary. Error bars indicate standard deviation. *, P = 0.027.

FIG. 4.

Deregulated Id2 in pituitary tumors from Rb^+/− mice and primary human retinoblastomas. (A) Total protein lysates were prepared from pituitaries of mice on postnatal day 230. Two wild-type (WT) controls are shown. The levels of Id1, Id2, Id3, and cyclin D1 in samples from Rb^+/− and Id2^−/−; Rb^+/− mice were determined by Western blot analysis. Antibodies specific for each Id protein were used. The positive control is the neuroblastoma cell line NGP, which expresses a high level of Id2 and detectable levels of Id1 and Id3. Note that the Id1 and Id3 blots were overexposed to exclude expression in pituitary tumors. (B) Immunostaining using Id2 antibody of a formalin-fixed advanced pituitary tumor section from Rb^+/− (left panel) and Id2^−/−; Rb^+/− mice (right panel) demonstrates elevated levels of Id2 in tumor cells (magnification, ×36) whereas endothelial cells in the blood vessels are negative (inset magnification, ×90). (C) Sections from primary human retinoblastoma were immunostained for Id2 (magnification, ×36). (D) Immunoblot for expression of cyclin E and cyclin A. Membranes were probed with α-tubulin to equalize for loading.

FIG. 5.

Loss of Id2 impairs the angiogenic response in Rb^+/− pituitary tumors by decreasing the expression of VEGF. (A) Micrographs of advanced pituitary tumors dissected from Rb^+/− and Id2^−/−; Rb^+/− mice after perfusion with orange latex as described in Materials and Methods. Two representative tumors from each genotype are shown. Highly vascularized tumors with an obvious capillary network are present in Rb^+/− mice. A marked defect in vascularization characterizes the small tumors of Id2^−/−; Rb^+/− mice. (B) Immunostaining using PECAM antibody in formalin-fixed pituitary tumor sections. Magnification, ×20. (C) TSP-1 immunostaining demonstrates similar expression in pituitary tumors from Rb^+/− and Id2^−/−; Rb^+/− mice. (D) Defect of VEGF expression in tumors from Id2^−/−; Rb^+/− mice. Paraffin sections from matched advanced tumors were immunostained for VEGF. (E) Early expression of VEGF in tumors from Rb^+/− mice. Pituitary sections from three 3-month-old Rb^+/− and three 4-month-old Id2^−/−; Rb^+/− mice were immunostained for VEGF. Note that at this stage Rb^+/− tumors are still avascular. (F) Impaired expression of HIF-1α in tumors from Id2^−/−; Rb^+/− mice. Pituitary sections from matched advanced tumors were immunostained for HIF-1α. N, necrosis. Magnification, ×40.

FIG. 6.

Id2 enhances the expression of VEGF. (A) REF52 fibroblasts were infected with Ad-Id2 or Ad-GFP at a MOI of 100. Protein lysates and total RNA were prepared at the indicated times and analyzed for Id2 protein expression and VEGF mRNA. 28S rRNA indicates equal loading of samples. (B) Increased expression of VEGF is detected by Northern blot analysis of SH-N neuroblastoma cells that express Id2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as a loading control. (C) Immunoblot for expression of Id2 and VEGF in SH-N cells. (D) Western blot analysis of IMR32-Flag vector and IMR32-Flag-Id2 clones for expression of ectopic Id2 (Flag-Id2), endogenous Id2 (Id2), VEGF, and α-tubulin. (E) Early downregulation of Id2 and VEGF in LAN-1 cells treated with retinoic acid (RA). Cells were treated with 2 μM retinoic acid, and lysates were prepared at the indicated times and assayed by Western blot analysis for N-Myc, Id2, VEGF, cyclin A, and p27^Kip1. (F) Inhibition of VEGF expression by retinoic acid treatment is rescued by adenovirus expression of Id2. LAN-1 cells were infected with Ad-GFP and Ad-Id2 at a MOI of 50 and immediately treated with retinoic acid. Protein lysates were prepared 48 h later and analyzed by Western blotting for VEGF and α-tubulin. (G) Specific silencing of Id2 using siRNA impairs the expression of VEGF. LAN-1 cells were transfected with Id2 siRNA expression vectors. The immunoblot shows expression of Id2, VEGF, Id1, and α-tubulin. (H) Id2 silencing inhibits proliferation of neuroblastoma tumor cells. Triplicate cultures of LAN-1 cells were transfected with Id2 siRNA expression vectors and selected in puromycin, and colonies were counted after 21 days.