Sequestration of E12/E47 and suppression of p27KIP1 play a role in Id2-induced proliferation and tumorigenesis

Abstract

Id2 is a member of the helix-loop-helix (HLH) family of transcription regulators known to antagonize basic HLH transcription factors and proteins of the retinoblastoma tumor suppressor family and is implicated in the regulation of proliferation, differentiation, apoptosis and carcinogenesis. To investigate its proposed role in tumorigenesis, Id2 or deletion mutants were re-expressed in Id2(-/-) dermal fibroblasts. Ectopic expression of Id2 or mutants containing the central HLH domain increased S-phase cells, cell proliferation in low and normal serum and induced tumorigenesis when grafted or subcutaneously injected into athymic mice. Similar to their downregulation in human tumors, the expression of cyclin-dependent kinase inhibitors p27(KIP1) and p15(INK4b) was decreased by Id2; the former by downregulation of its promoter by the Id2 HLH domain-mediated sequestration of E12/E47. Re-expression of p27(KIP1) in Id2-overexpressing cells reverted the hyperproliferative and tumorigenic phenotype, implicating Id2 as an oncogene working through p27(KIP1). These results tie together the previously observed misregulation of Id2 with a novel mechanism for tumorigenesis.

Fig. 1.

Re-expression of Id2 in Id2^−/− fibroblasts increases proliferation. (A, left) PCR genotyping with Id2- and neomycin-specific primers. (A, middle) RT–PCR of Id2^−/− transduced with LHCX or Id2/LHCX. (A, right) Immunoblot of cells transduced with Id2/LHCX or LHCX. (B, left) Id2^−/− transduced with Id2/LHCX or LHCX were plated and viable cells counted. (B, right) Fluorescence-activated cell sorting (FACS) analysis of Id2/LHCX or LHCX cells. (C) Bromodeoxyuridine (BrdU) incorporation. (D) Cell counts in 1% serum. For all experiments, ** or *** represent P < 0.01 or P < 0.001 compared with controls; results are the means ± SDs of three biological replicates of a representative experiment; essentially the same results were obtained in three independent experiments. Technical replicates (e.g. multiple cell counts) were also performed for each of the biological replicates but were not incorporated in the statistics for the three independent experiments.

Fig. 2.

Re-expression of Id2 in Id2^−/− fibroblasts induces neoplastic transformation and tumors in skin grafts and after subcutaneous injection into athymic mice. (A) Foci quantitation in Id2/LHCX and LHCX fibroblasts. (B) Cells (1 × 10⁵) were plated, fed every 3 days and counted on the days indicated. (C) Fibroblasts were grafted onto athymic mice with human keratinocytes. Tumor volume, pictures of grafted mice and histology are shown. (D) Cells were injected subcutaneously and tumor volume measured at indicated times.*, **, *** represent P < 0.05, P < 0.01, or P < 0.001 compared with controls; results are the means ± SD of three biological replicates of a representative experiment; essentially the same results were obtained in three independent experiments.

Fig. 3.

Re-expression of Id2 in Id2^−/− fibroblasts causes transcriptional downregulation of p27^KIP1 via E12/E47 sequestration. (A) Immunoblot analysis of INK and CIP/KIP cell cycle regulators, Id1 and Id3 (p16^INK4a was undetectable). (B) Downregulation of p27^KIP1 RNA by Id2 and effects on Id1 and Id3 RNA as determined by RT–PCR. (C) Immunoblot analysis of cells in low and normal serum (top) or at high density (bottom). (D) Luciferase assays of cells cotransfected with Renilla vector and p27^KIP1 promoter–luciferase (top left); Chromatin immunoprecipitation analysis (bottom left); EMSA (top right) showing binding of E12/E47 to E-box #4 (bottom right). *** represent P < 0.001 compared with controls; results are the means ± SD of three biological replicates of a representative experiment; essentially the same results were obtained in three independent experiments.

Fig. 4.

The Id2 HLH domain is responsible for increased S phase and tumorigenesis. (A) Full-length Id2 and mutants cloned into LHCX. (B) Id2^−/− cells transduced with Id2 or mutants were subjected to RT–PCR (top) or immunoprecipitation of labeled proteins with anti-Id2-C20 or anti-Flag (bottom). (C) FACS of Id2 or mutant-expressing cells. (D) Subcutaneous athymic mouse tumor growth. **, *** represent P < 0.01, or P < 0.001 compared with controls; results are the means ± SD of three biological replicates of a representative experiment; essentially the same results were obtained in three independent experiments.

Fig. 5.

Re-expression of p27^KIP1 in Id2/LHCX cells reverts the hyperproliferative phenotype. RT–PCR (A) and FACS (B) of cells transiently transfected with Id2 or p27^KIP1. (C) RT–PCR of primary human fibroblasts transiently transfected with Id2/LHCX or mutants. (D) Cells in (C) were cotransfected with pRLTK Renilla vector and the p27^KIP1 proximal promoter–luciferase and subjected to promoter assays.