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Updated: Aug 21, 2026
Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Published on: December 31, 2014
Decreased tumorigenicity of c-Myc-transformed fibroblasts expressing active USF2
Chungyoul Choe1, Nanyue Chen, Michèle Sawadogo
1Department of Molecular Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Abstract:
USF is a small family of basic helix-loop-helix leucine zipper (bHLH-zip) transcription factors with DNA binding specificities similar to that of the c-Myc oncoprotein. Evidence for a role of USF in growth control includes inhibition of c-Myc-dependent cellular transformation in vitro and loss of USF transcriptional activity in many cancer cell lines. However, a direct effect of USF on the tumorigenicity of an established cell line has never been demonstrated. Here, cell lines derived from rat embryo fibroblasts transformed by c-Ha-Ras and either c-Myc or E1A were used as model system to investigate the tumor suppression ability of USF. Overexpression of USF2 stimulated transcription and inhibited colony formation in c-Myc-transformed, but not E1A-transformed, fibroblasts. Stable clones expressing high USF2 levels were constructed from c-Myc-transformed fibroblasts. In two of these clones, overexpressed USF2 did not activate transcription, and there was no significant change in the transformed phenotype. In contrast, a clone that expressed transcriptionally active USF2 exhibited altered morphology and a strongly decreased ability to proliferate in semisolid medium. The ability of these cells to form tumors in nude mice was also decreased by a factor of more than 30 as compared to the parental cell line or cells overexpressing transcriptionally inactive USF2. Cotransfection assays with USF- or Myc-specific dominant-negative mutants indicated that active USF2 inhibited cellular transformation by preventing transcriptional repression by c-Myc.
Insights
Transcription factor USF2 (upstream transcription factor 2) suppresses tumor growth by inhibiting c-Myc activity. This study demonstrates USF2
Area of Science:
- Molecular Biology
- Cancer Research
- Transcription Factors
Background:
- USF (upstream transcription factor) is a family of bHLH-zip transcription factors with DNA binding similar to c-Myc.
- USF plays a role in growth control, inhibiting c-Myc-dependent transformation.
- USF transcriptional activity is lost in many cancer cell lines, but its direct effect on tumorigenicity is unproven.
Purpose of the Study:
- To investigate the tumor suppressor ability of USF.
- To determine the direct effect of USF on the tumorigenicity of established cell lines.
Main Methods:
- Used rat embryo fibroblasts transformed by c-Ha-Ras and either c-Myc or E1A as a model system.
- Overexpressed USF2 in transformed fibroblasts.
- Constructed stable clones with high USF2 levels from c-Myc-transformed fibroblasts.
- Assessed transcriptional activity, transformed phenotype, proliferation in soft agar, and tumor formation in nude mice.
- Utilized USF- or Myc-specific dominant-negative mutants in cotransfection assays.
Main Results:
- USF2 overexpression stimulated transcription and inhibited colony formation in c-Myc-transformed, but not E1A-transformed, fibroblasts.
- Transcriptionally active USF2 in c-Myc-transformed fibroblasts led to altered morphology, decreased proliferation, and a >30-fold reduction in tumor formation in nude mice.
- Inactive USF2 overexpression did not alter the transformed phenotype or tumorigenicity.
- Active USF2 inhibited cellular transformation by preventing c-Myc-mediated transcriptional repression.
Conclusions:
- Transcriptionally active USF2 exhibits tumor suppressor activity against c-Myc-driven transformation.
- USF2 directly inhibits the tumorigenicity of established cancer cell lines.
- USF2 functions, in part, by counteracting the transcriptional repression mediated by c-Myc.
