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Updated: Jun 20, 2026
Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene
Published on: December 31, 2014
Identification of transforming activity of free fatty acid receptor 2 by retroviral expression screening
Hisashi Hatanaka1, Mamiko Tsukui, Shuji Takada
1Division of Functional Genomics, Jichi Medical University, Tochigi, Japan.
Abstract:
Gallbladder cancer (GBC) is a highly fatal malignancy in humans. Genetic alterations in KRAS or TP53 as well as overexpression of ERBB2 have been shown to contribute to the development of certain types of GBC. However, many cases of GBC do not harbor such genetic changes, with other transforming events awaiting discovery. We here tried to identify novel cancer-promoting genes in GBC, with the use of a retroviral cDNA expression library. A retroviral cDNA expression library was constructed from a surgically resected clinical specimen of GBC, and was used to infect 3T3 fibroblasts in a focus formation assay. cDNA incorporated into the transformed foci was rescued by PCR. One such cDNA was found to encode free fatty acid receptor 2 (FFAR2), a G protein-coupled receptor for short-chain fatty acids. The oncogenic potential of FFAR2 was confirmed both in vitro with the focus formation assay and by evaluation of cell growth in soft agar as well as in vivo with a tumorigenicity assay in nude mice. The isolated FFAR2 cDNA had no sequence alterations, suggesting that upregulation of FFAR2 expression may contribute to malignant transformation. Indeed, all of quantitative RT-PCR, in situ hybridization, and immunohistochemical analyses showed that the amount of FFAR2 mRNA and its protein product was increased in digestive tract cancer specimens. Furthermore, short-chain fatty acids potentiated the mitogenic action of FFAR2 in 3T3 cells. Our data thus, for the first time, implicate FFAR2 in carcinogenesis of the digestive tract.
Insights
Researchers identified free fatty acid receptor 2 (FFAR2) as a novel gene promoting gallbladder cancer (GBC) and other digestive tract cancers. Upregulated FFAR2 expression contributes to malignant transformation and tumor growth.
Area of Science:
- Oncology
- Molecular Biology
- Gastroenterology
Background:
- Gallbladder cancer (GBC) is a fatal malignancy with known genetic drivers like KRAS, TP53, and ERBB2.
- Many GBC cases lack these common genetic alterations, indicating a need to discover novel oncogenes.
- Understanding these additional genetic events is crucial for advancing GBC treatment strategies.
Purpose of the Study:
- To identify novel cancer-promoting genes in gallbladder cancer (GBC) using a functional genomics approach.
- To investigate the role of newly identified genes in the malignant transformation and progression of GBC.
- To explore the potential involvement of identified genes in other digestive tract cancers.
Main Methods:
- Construction of a retroviral cDNA expression library from clinical GBC specimens.
- Infection of 3T3 fibroblasts with the library, followed by focus formation assays to identify transformed cells.
- Rescue and sequencing of cDNA from transformed foci to identify candidate oncogenes.
- In vitro assays (focus formation, soft agar) and in vivo tumorigenicity assays in nude mice to confirm oncogenic potential.
- Quantitative RT-PCR, in situ hybridization, and immunohistochemistry to analyze FFAR2 expression in cancer tissues.
Main Results:
- A novel gene, free fatty acid receptor 2 (FFAR2), was identified as a potential oncogene in GBC.
- FFAR2 demonstrated oncogenic potential through in vitro focus formation, soft agar growth, and in vivo tumor formation.
- FFAR2 mRNA and protein levels were significantly upregulated in GBC and other digestive tract cancer specimens.
- Short-chain fatty acids were found to potentiate the mitogenic effects of FFAR2.
Conclusions:
- Free fatty acid receptor 2 (FFAR2) is implicated as a novel cancer-promoting gene in gallbladder cancer.
- Upregulation of FFAR2 expression contributes to malignant transformation and tumorigenesis in the digestive tract.
- FFAR2 represents a potential therapeutic target for gallbladder and other digestive tract cancers.
