Id2 deficiency promotes metastasis in a mouse model of ocular cancer

Abstract

The inhibitor of DNA binding 2 (Id2) basic helix-loop-helix protein interacts genetically and physically with the pocket proteins (Rb, p107 and p130) and has been implicated as an oncogene. In other studies, however, Id2 has been shown to function as a tumor suppressor. Here, we studied the role of Id2 in a well characterized model of ocular cancer in which the three pocket proteins are inactivated by generating mice lacking one or both Id2 alleles. Id2 deficiency had no impact on tumorigenesis in the eye. Unexpectedly, however, Id2 loss significantly increased the rate of metastasis. Liver metastases in Id2 heterozygotes demonstrated significant decrease of Id2 expression and loss of the remaining Id2 allele, strongly suggesting that Id2 inactivation specifically was required for metastasis in this model. These findings provide new insights into the role of Id2 in metastasis.

Fig. 1

Histopathologic photomicrographs of intraocular primary tumors demonstrating a typical small tumor (arrow) (a), medium tumor (b), and large tumor (c). Original magnification 4×. d Box-and-whiskers plots showing a primary intraocular tumor area as a percentage of the total area of the eye. Males and females are analyzed separately as indicated. The central box represents the values from the 25 to 75th percentile. The middle line represents the median. A line extends from the minimum to the maximum value. P values were calculated using the Mann–Whitney nonparametric method

Fig. 2

Liver metastasis in a Tyr-TAg/Id2^+/− mouse. a Low magnification external photograph. b High magnification external photograph, showing multiple foci of metastatic lesions on the liver surface. c Histopathologic photomicrograph showing multiple dark blue foci representing intrahepatic metastases. Original magnification 40×

Fig. 3

Kaplan-Meier survival plots comparing metastasis-free survival as a function of Id2 genotype. Id2 wild type = Tyr-TAg/Id2^+/+ mice. Id2 deficient = Tyr-TAg/Id2^+/− + Tyr-TAg/Id2^−/− mice. A sub-analysis of males only was performed as indicated. P values were calculated using the log rank test

Fig. 4

Analysis of Id2 mRNA expression and Id2 gDNA content in normal liver and liver metastases by real-time PCR. a Id2 mRNA expression in normal liver and liver metastasis from Id2^+/− mice normalized to β-actin relative to Id2^+/+ normal liver. b Id2 gDNA content normal liver and liver metastasis from Id2^+/− mice normalized to β-actin relative to Id2^+/+ normal liver. Each bar graph represents mean and SEM for samples performed in triplicate. Specimens from Id2^−/− and Id2^+/+ mice served as negative and positive controls, respectively. The asterisk indicates that there were no metastatic tumors in Id2^+/+ mice