A functional genomics approach for the identification of putative tumor suppressor genes: Dickkopf-1 as suppressor of HeLa cell transformation.
We described previously the isolation and characterization of two non-tumorigenic revertants from the HeLa cervical carcinoma cell line, and demonstrated that loss of the transformed phenotype in these cells was the result of dominant somatic mutations. The goal of the present study was to use cDNA microarrays to identify candidate tumor suppressors among the set of genes whose increased expression correlated with loss of tumorigenicity in both revertants. Among the genes with significantly increased expression levels in both HA and HF revertants we identified Insulin Growth Factor Binding Protein-3 (IGFBP-3) and the Dickkopf-1 (DKK-1) genes. Both of these genes encode secreted proteins implicated in the modulation cell growth and differentiation, and IGFBP-3 was shown previously to have tumor suppressing activity. To test the hypothesis that increased expression of IGFBP-3 or the DKK-1 genes could have contributed to the suppression of tumorigenicity in the revertants, we expressed IGFBP-3 or DKK-1 in HeLa cells, and assessed their effects on anchorage dependent and independent growth, and tumor formation in athymic nude mice. Ectopic expression of IGFBP-3 or DKK-1 resulted in significantly decreased growth in soft agar. HeLa cells expressing ectopic IGFBP-3 or DKK-1 showed statistically significant differences in the kinetics of tumor formation. In any tumors that arose in animals injected with the IGFBP-3 expressing cells, there was a complete loss of IGFBP-3 activity, as measured by binding to IGF-1 and IGF-2 proteins. All tumors that arose after injection of cells expressing DKK-1, invariably showed almost a complete loss of ectopic DKK-1 expression. The observations that loss of DKK-1 expression or IGFBP-3 activity was required for tumorigenicity suggested that both proteins encode putative tumor suppressor genes. We also show that while DKK-1 expression does not affect cell growth in vitro, the protein does sensitize cells to apoptosis. We also demonstrated that effect of DKK-1 was not due to inhibition of beta-catenin/TCF4-regulated transcription. Taken together, our results indicate that somatic cell genetics combining with gene expression profiling may be a useful approach for the identification of functional suppressors of malignant cell growth.
Mikheev
Andrei M
AM
Program in Cancer Biology, Division of Public Health, Fred Hutchinson Cancer Research Center, Seattle, WA 98104-2092, USA.
Mikheeva
Svetlana A
SA
Liu
Binrong
B
Cohen
Pinchas
P
Zarbl
Helmut
H
D000818:Animals; D017209:Apoptosis; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002455:Cell Division; D002471:Cell Transformation, Neoplastic; D003598:Cytoskeletal Proteins / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D005260:Female; D016147:Genes, Tumor Suppressor / Q000502:physiology*; D023281:Genomics; D006367:HeLa Cells; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics*; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008819:Mice, Nude; D009419:Nerve Tissue Proteins; D020411:Oligonucleotide Array Sequence Analysis; D011506:Proteins / Q000235:genetics*; D051820:TCF Transcription Factors; D015534:Trans-Activators / Q000502:physiology; D000073940:Transcription Factor 4; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors / Q000502:physiology; D051176:beta Catenin
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; C495270:CTNNB1 protein, human; C495267:CTNNB1 protein, mouse; D003598:Cytoskeletal Proteins; C501126:DKK1 protein, human; D004268:DNA-Binding Proteins; C501127:Dkk1 protein, mouse; D018972:Insulin-Like Growth Factor Binding Protein 3; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D011506:Proteins; D051820:TCF Transcription Factors; C544894:TCF7L2 protein, human; C090743:Tcf4 protein, mouse; C544895:Tcf7l2 protein, mouse; D015534:Trans-Activators; D000073940:Transcription Factor 4; D058118:Transcription Factor 7-Like 2 Protein; D014157:Transcription Factors; D051176:beta Catenin