Loss of Id2 Potentiates the Tumorigenic Effect of Rb Inactivation in a Mouse Model of Retinoblastoma

Abstract

Purpose

In some cancers, the oncogenic consequences of inactivating the retinoblastoma protein (Rb) appear to be mediated by unrestrained activity of the inhibitor of DNA binding protein Id2. The role of Id2 has not yet been investigated in the prototype cancer Rb-defective cancer, retinoblastoma itself. This study investigated whether loss of Id2 modified the effects of Rb inactivation in a mouse model of retinoblastoma.

Methods

Id2 was analyzed in cultured cells using qPCR, Western blot, and colony formation assays. LHβ-Tag transgenic mice were crossed with Id2 heterozygotes to obtain mice with all three Id2 genotypes. Intraocular tumors were assessed for size, degree of differentiation, mitotic index, and tumor vascular density at 15 weeks of age.

Results

Retinoblastoma cell lines expressed low levels of Id2 mRNA and protein. Depletion of Id2 in Rb-inactivated cells increased clonogenic activity. Id2-deficient tumors in vivo were significantly larger, less differentiated, and more vascularized than Id2-wild-type tumors (P = 0.02, P = 0.01, P = 0.0001, respectively). There was a dosage effect for loss of each Id2 allele with respect to differentiation and vascular density.

Conclusions

Id2 suppresses rather than promotes tumor progression in this mouse model of retinoblastoma. Id2 can act as either an oncogene or a tumor suppressor depending on context.

Introduction

Inhibitor of DNA binding (Id) proteins, four of which have been identified in mammals (Id1 to Id4), are dominant-negative inhibitors of basic helix-loop-helix (bHLH) transcription factors and have roles in cell proliferation, differentiation, angiogenesis, and tumorigenesis.^1–4 In some cancers, Id2 appears to act as an oncogene.⁵ In neuroblastoma, Id2 is an effector of the N-myc oncogene.⁶ The Rb family of pocket proteins (Rb1, p107, and p130) can bind and inhibit Id2, and it has been suggested that the oncogenic consequences of Rb loss are due, at least in part, to the unrestrained activity of Id2.^7–10

On the other hand, Id2 is dispensable for tumorigenesis in other myc-induced tumors, such as lymphoma and epidermal neoplasia.^11,12 Recent studies suggest that Id2 can actually suppress the development of tumors in the intestine and mammary gland.^13,14 Thus, the role of Id2 appears to be tissue specific and context dependent.¹⁵

Since Id2 has been implicated as a mediator of oncogenesis in Rb-deficient tumors, we wished to determine the role of Id2 in retinoblastoma itself. Id2 mRNA and protein were expressed at very low levels in retinoblastoma cells, and loss of Id2 in a transgenic model of retinoblastoma led to increased tumor size, loss of differentiation, and increased tumor vascular density. These results indicate that Id2 acts as a tumor suppressor, rather than an oncogene, in this model of retinoblastoma. These findings provide new insights into retinoblastoma tumor biology, and they reinforce further the concept that Id2 can act as either an oncogene or a tumor suppressor depending on context.

Materials and Methods

Cell Culture Studies

Y79 (ATCC HTB-18), WERI-RB-1 (ATCC HTB-169), and CHLA223 (gift of A. Linn Murphree, Childrens Hospital Los Angeles, Los Angeles, California, USA) retinoblastoma cells were measured for Id2 expression by realtime qPCR and Western blot analyses. Mel202 uveal melanoma cells (gift of B. Ksander, Harvard University, Boston, Massachusetts, USA) and U2OS osteosarcoma cells (ATCC HTB-96) were used as positive controls. Real-time PCR was performed using the IQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, California, USA) and qPCR was performed by monitoring the increase of SYBR Green fluorescence in realtime using i-Cycler (Bio-Rad Laboratories, Hercules, California, USA). Custom primers were synthetized by Integrated DNA Technologies (IDT, Coralville, Iowa, USA): UBC_F 5′-ATTTGGGTCGCGGTTCTTG-3′, UBC_R 5′-TGCCTTGACATTCTCGATGGT-3′, ID2_F5′-ATATCAGCATCCTGTCCTTGCAG-3′, ID2_R 5′-GAAATCATGAACACCGCTTATTCAG-3′. At least one primer of each pair crossed exon-exon boundaries to prevent amplification of genomic DNA, and primer quality (lack of primer-dimer amplification) was confirmed by melting curve analysis. Relative quantitation of the gene expression was performed using the standard curve method (user bulletin 2 of the ABI Prism 7700 Sequence Detection System, Applied Biosystems, Foster City, California, USA). In each experiment the relative amount of target gene mRNA was calculated from the standard curve and normalized to the relative amount of reference gene RNA (UBC RNA), which was obtained from a similar standard curve. Western blot was performed using an antibody that recognized Id2 (1:200; C-20; Santa Cruz Biotechnology, Santa Cruz, California, USA). Colony formation assays were performed with Rb-inactivated U2OS cells¹⁶ using Id2 sens and antisense⁷ (gift of A. Iavarone, Columbia University, New York, New York, USA), full-length Rb¹⁷ and RbΔ10¹⁸ (gift of J. Bartek, Danish Cancer Society, Copenhagen, Denmark) expression vectors, as described previously.^19,20 Transfections (24h) were done using Effectene (Qiagen, Valencia, California, USA).

Generation of LHβ-Tag/Id2 Mice, Genotyping and Histopathology

Animal experiments were approved by the Washington University Animal Studies Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. LHβ-Tag mice (gift of D.M. Albert, University of Wisconsin, Madison, Wisconsin, USA) were generated as previously described²¹ and maintained on a CB6F1 (C57BL/6 × BALB/c) background. These mice have retina-specific expression of large T antigen, which blocks Rb, as well as the other two pocket proteins, p107 and p130.²¹ By 6–8 weeks of age, these animals develop multifocal, bilateral retinal tumors with histopathologic features closely resembling human retinoblastoma.²¹ LHβ-Tag mice were crossed with Id2 heterozygotes on the 129/Sv background²² (gift of Y. Yokota, Fukui Medical University, Fukui, Japan), which were subsequently interbred to generate LHβ-Tag mice with all three Id2 genotypes (129/Sv background ranged from 50–75% for LHβ-Tag/Id2 mice and corresponding control mice). All mice were genotyped for SV40 Tag and Id2 by PCR analysis of tail biopsies as previously described.^21,22 Mice were euthanized at 15 weeks of age. It was not possible to evaluate mice beyond this age because they began to develop advanced eye tumors that required euthanasia to avoid discomfort. Eyes were fixed in formalin and embedded in paraffin (ten LHβ-Tag/Id2^+/+, ten LHβ-Tag/Id2^+/-, and four LHβ-Tag/Id2^-/-, with each eye measured in triplicate). Four micron sections were stained with hematoxylin and eosin and examined in a masked fashion.

Analysis of Ocular Tumors

Tumor diameter was measured as the ratio of the largest linear basal tumor diameter to the largest linear retinal diameter, measured on histopathologic sections through the region of maximum tumor diameter. This technique allowed us to normalize for minor differences in eye size and sectioning. For these measurements, photomicrographic images were taken of coronal sections through the largest diameter of the tumor and uploaded into ImageJ software for analysis (http://rsb.info.nih.gov/ij/). The degree of tumor differentiation was measured as the number of photoreceptor rosettes per 40× field.²³ The mitotic index was measured as the number of mitotic figures per 40× field. Vascular density was measured as the number of intratumoral vessels per 40× field. At least ten 40× fields were examined. Measurements were normalized for tumor area.

Statistical Analysis

Results were analyzed for statistical significance by two-tailed t-test using MedCalc software version 9.5.1.0 (http://medcalcsoftware.com/medcalc.php); P < 0.05 was considered statistically significant. Since the variability from eye to eye is similar to that from animal to animal, it is customary in this inbred line to consider each eye as an independent “experiment.” Thus, the error bars and p values were calculated from the individual eyes in each group.

Results

Id2 mRNA and protein were expressed at very low levels in human retinoblastoma cells (Figure 1A and 1B). Depletion of Id2 in Rb-inactivated U2OS cells using an antisense expression vector led to a significant increase in anchorage-independent growth and colony formation compared to the cells transfected with Id2 sense or Rb expression constructs (Figure 1C). This clonogenic activity was blocked by co-expression of a constitutively active Rb allele (RbΔ10) (Figure 1C).

Figure 1.

Id2 mRNA and protein expression in retinoblastoma cell lines. (A) Real-time qPCR; (B) Western blot; (C) Colony formation assays in Rb-inactivated U2OS cells using the indicated expression constructs. Mel202 and U2OS cell lines were used as positive controls. Error bars represent standard error.

LHβ-Tag transgenic retinoblastoma mice were crossed with Id2 heterozygotes to create mice with all three Id2 genotypes.^21,22 Mice of all three Id2 genotypes developed intraocular retinoblastomas by 15 weeks of age (Figure 2). The mean diameter of the LHβ-Id2^-/- tumors was significantly greater than that of the LHβ-Id2^+/+ tumors (P = 0.02) and LHβ-Id2^+/- tumors (P = 0.02) (Figure 3A). There was no significant difference in diameter between the LHβ-Id2^+/+ and LHβ-Id2^+/- tumors (P = 0.9). The degree of tumor differentiation of LHβ-Id2^+/+ tumors was significantly greater than the LHβ-Id2^-/- tumors (P = 0.01) and LHβ-Id2^+/- tumors (P = 0.02) (Figure 3B). The vascular density of the LHβ-Id2^-/- tumors was significantly greater than the LHβ-Id2^+/+ tumors (P = 0.0001) and LHβ-Id2^+/- tumors (P = 0.0001) (Figure 3C). The mitotic index was not significantly different between Id2 deficient and Id2 wild-type tumors (data not shown). Metastasis was not detected at necropsy in any of the animals at the age of 15 weeks, and it was not possible to keep the animals alive longer due to concerns about discomfort associated with the development of large eye tumors.

Figure 2.

Histopathology of LHβ-Tag retinoblastomas with three Id2 genotypes, as indicated. Images were obtained at 10× magnification (left panels) and 40× magnification (right panels). Arrows in right panels indicate photoreceptor rosettes.

Figure 3.

Histomorphometric analysis of LHβ-Tag retinoblastomas with three Id2 genotypes, as indicated: (A) Tumor size; (B) Photoreceptor differentiation; (C) Vascular density. Error bars represent standard error. The error bars and p values were calculated from individual eyes in each group (ten LHβ-Tag/Id2^+/+, ten LHβ-Tag/Id2^+/-, and four LHβ-Tag/Id2^-/-).

Discussion

Since Rb can negatively regulate Id2, and loss of Rb can promote tumorigenesis by unleashing oncogenic activities of Id2,^7–10 we predicted initially that deletion of Id2 in LHβ-Tag mice would delay or prevent the development of retinoblastoma. Instead, all Id2-deficient LHβ-Tag mice developed retinoblastomas, and these tumors were significantly larger, less differentiated, and more vascularized than Id2 wild-type tumors. It is unlikely that the observed phenotypic differences were due to differences in genetic backgrounds since the same background was present in LHβ-Id2^+/+, LHβ-Id2^+/-, and LHβ-Id2^-/- mice.

Id2 is highly expressed in the developing and adult retina,²⁰ and it is down-regulated in human retinoblastomas.²⁴ These observations, coupled with our findings reported herein, suggest that Id2 may function as a tumor suppressor in the retina and that loss of Id2 may be an important step in retinoblastoma progression. The fact that Id2 deficiency alone does not lead to retinal tumors indicates that Id2 loss is not an initiating event but, rather, an event that occurs during tumor progression. This possibility is consistent with the “M3” concept described by Gallie and co-workers.²⁵ Once tumor formation has occurred, as a result of Rb inactivation, the subsequent loss of Id2 appears to convey a selective growth advantage by increasing tumor vascularity and decreasing the limits on growth associated with differentiation. A similar role for Id2 has recently been shown in hepatocellular carcinoma.²⁶ Retinoblastoma joins a growing list of cancers in which Id2 appears to function as a tumor suppressor, including breast,¹³ intestinal,¹⁴ prostate,²⁷ and hepatocellular carcinoma.²⁶ Further, we recently showed that Id2 suppresses tumor progression in uveal (ocular) melanoma, where inactivation of Id2 leads to more aggressive, dedifferentiated, stem-like cancer cells.²⁰

The observed association between Id2 loss and increased tumor vascularization was of particular interest. Intratumoral vascularity induced by vascular endothelial growth factor (VEGF)-mediated angiogenesis is prevalent in human retinoblastoma.²⁸ Indeed, increased intratumoral vascularity is associated with increased tumor aggressiveness.²⁹ Rapidly growing retinoblastomas develop regions of hypoxia that induce expression of VEGF, hypoxia inducible factor 1-alpha (HIF1A), and other proteins that can promote angiogenesis and tumor progression.³⁰ Id2 inhibits VEGF and HIF1A, and loss of Id2 is associated with increased tumor aggressiveness by allowing increased VEGF expression.²⁶ Thus, Id2 may function as a tumor suppressor in retinoblastoma, at least in part, by inhibiting angiogenesis. These findings provide new insights into the pathogenesis of retinoblastoma and may lead to new therapeutic targets in the treatment of retinoblastoma.