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A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells
Published on: July 3, 2013
Transcriptome analysis of a human colorectal cancer cell line shows molecular targets of insulin-like growth
Daniela Diehl1, Harald Lahm, Eckhard Wolf
1Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilian University, Munich, Germany.
Abstract:
Insulin-like growth factor II (IGF-II) is expressed commonly in colorectal tumors. IGF-binding protein-4 (IGFBP-4) counteracts the tumor promoting activities of IGF-II by binding this growth factor. We have shown previously that in LS1034 cells, which highly express IGF-II, overexpression of IGFBP-4 led to a strong reduction in proliferation, colony formation and invasive capacity. To investigate the effects of IGFBP-4 at the molecular level we analyzed growth parameters of LS1034 human colon cancer cells vs. cells expressing the murine IGFBP-4 (mIGFBP-4) and used a subtractive cDNA library approach in combination with cDNA array hybridization to detect changes in the mRNA expression profiles. The mRNA levels for several proteins that are known to affect important biological properties of neoplastic cells, such as proteolysis, proliferation and differentiation were altered by overexpression of IGFBP-4. Transcript levels for tumor markers, like the carcinoembryonic antigen-related cell adhesion molecule (CEACAM), were reduced by elevated mIGFBP-4. Changes at the mRNA level were confirmed by Western blotting for CST1 (proteolysis or protease inhibitor), COX-2 (cell motility) and CEACAM5 (tumor marker). Furthermore, the effect of mIGFBP-4 on apoptosis was investigated and no increase of apoptosis could be detected in the IGFBP-4 overexpressing LS1034 cells. Our data indicate that IGFBP-4 is involved in the regulation of gene products that are known or supposed to be important for the pathogenesis of colon cancer cells.
Insights
Insulin-like growth factor-binding protein-4 (IGFBP-4) inhibits colon cancer cell growth by altering gene expression. Overexpression of IGFBP-4 reduced proliferation and invasion, impacting key cancer-related proteins.
Area of Science:
- Molecular Biology
- Cancer Research
- Genomics
Background:
- Insulin-like growth factor II (IGF-II) promotes colorectal tumor growth.
- IGF-binding protein-4 (IGFBP-4) antagonizes IGF-II's pro-tumor effects.
- Previous studies showed IGFBP-4 reduces LS1034 colon cancer cell proliferation and invasion.
Purpose of the Study:
- To investigate the molecular mechanisms by which IGFBP-4 affects colon cancer cell growth.
- To analyze changes in gene expression profiles in response to IGFBP-4 overexpression.
- To confirm mRNA level changes at the protein level.
Main Methods:
- LS1034 human colon cancer cells were engineered to overexpress murine IGFBP-4 (mIGFBP-4).
- Subtractive cDNA library construction and cDNA array hybridization were used to profile mRNA expression.
- Western blotting was employed to validate changes in specific protein levels (CST1, COX-2, CEACAM5).
Main Results:
- Overexpression of mIGFBP-4 altered mRNA levels of proteins involved in proteolysis, proliferation, and differentiation.
- Transcript levels for tumor markers, including carcinoembryonic antigen-related cell adhesion molecule (CEACAM), were decreased.
- Protein expression changes for CST1, COX-2, and CEACAM5 were confirmed; no significant increase in apoptosis was observed.
Conclusions:
- IGFBP-4 plays a regulatory role in colon cancer pathogenesis.
- IGFBP-4 influences gene products critical for neoplastic cell behavior.
- IGFBP-4 may serve as a therapeutic target for colon cancer by modulating key cellular pathways.
