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. 2009 Feb 15;124(4):793-8.
doi: 10.1002/ijc.23775.

Identification of Fat4 as a candidate tumor suppressor gene in breast cancers

Chao Qi  1 , Yiwei Tony Zhu, Liping Hu, Yi-Jun Zhu
Affiliations

Identification of Fat4 as a candidate tumor suppressor gene in breast cancers

Chao Qi et al. Int J Cancer. .

Abstract

Fat, a candidate tumor suppressor in Drosophila, is a component of Hippo signaling pathway involved in controlling organ size. We found that a approximately 3 Mbp deletion in mouse chromosome 3 caused tumorigenesis of a non-tumorigenic mammary epithelial cell line. The expression of Fat4 gene, one member of the Fat family, in the deleted region was inactivated, which resulted from promoter methylation of another Fat4 allele following the deletion of one Fat4 allele. Re-expression of Fat4 in Fat4-deficient tumor cells suppressed the tumorigenecity whereas suppression of Fat4 expression in the non-tumorigenic mammary epithelial cell line induced tumorigenesis. We also found that Fat4 expression was lost in a large fraction of human breast tumor cell lines and primary tumors. Loss of Fat4 expression in breast tumors was associated with human Fat4 promoter methylation. Together, these findings suggest that Fat4 is a strong candidate for a breast tumor suppressor gene.

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Figures

Fig. 1
Fig. 1
(a). Confirmation of ~3Mbp deletion on mouse chromosome 3. The 5’LTR and 3’LTR of the recombinant retroviral vector was located at 37,747kb and 40,623 kb on chromosome 3, respectively. PCR using primers located in the two regions as indicated was performed to confirm the deletion, which generated a 8.3-kb band (lane 1). M: Lamda DNA /HindIII Marker. (b). The histology of the tumor with deletion on chromosome 3. The tumor was harvested from a nude moue, sectioned and stained with H&E. The tumor was poorly differentiated with occasional gland formation.
Fig. 1
Fig. 1
(a). Confirmation of ~3Mbp deletion on mouse chromosome 3. The 5’LTR and 3’LTR of the recombinant retroviral vector was located at 37,747kb and 40,623 kb on chromosome 3, respectively. PCR using primers located in the two regions as indicated was performed to confirm the deletion, which generated a 8.3-kb band (lane 1). M: Lamda DNA /HindIII Marker. (b). The histology of the tumor with deletion on chromosome 3. The tumor was harvested from a nude moue, sectioned and stained with H&E. The tumor was poorly differentiated with occasional gland formation.
Fig. 2
Fig. 2
(a). Loss of Fat4 expression in the tumor and increased expression of Fat4 gene in tumor cells with the treatment of 5-aza-2’-deoxycytidine(5-Aza-dC). Quantitative real-time RT-PCR was performed to determine the Fat4 mRNA levels, which were normalized to control β-actin mRNA levels. Tumor cells were treated with 5 mM 5-aza-2’-deoxycytidine(5-Aza-dC) for three days. (b). The methylation status of the CpG island in NOG8 cells and tumor cells. The ~2.0 kb CpG island of Fat4 promoter region is shown with the analyzed region (370bp) indicated by arrows. This region which contains 22 CpG sites was directly sequenced after being amplified from sodium bisulfite-treated genomic DNA. A closed circle indicates a methylated CpG and an open circle indicates an unmethylated CpG.
Fig. 2
Fig. 2
(a). Loss of Fat4 expression in the tumor and increased expression of Fat4 gene in tumor cells with the treatment of 5-aza-2’-deoxycytidine(5-Aza-dC). Quantitative real-time RT-PCR was performed to determine the Fat4 mRNA levels, which were normalized to control β-actin mRNA levels. Tumor cells were treated with 5 mM 5-aza-2’-deoxycytidine(5-Aza-dC) for three days. (b). The methylation status of the CpG island in NOG8 cells and tumor cells. The ~2.0 kb CpG island of Fat4 promoter region is shown with the analyzed region (370bp) indicated by arrows. This region which contains 22 CpG sites was directly sequenced after being amplified from sodium bisulfite-treated genomic DNA. A closed circle indicates a methylated CpG and an open circle indicates an unmethylated CpG.
Fig. 3
Fig. 3
Expression of Fat4 gene inhibited the tumor growth in nude mice. (a). The mRNA expression of Fat4 gene as revealed by RT-PCR. Tumor Clone 3 and 4 expressed Fat4 while Clone 1 and 2 did not express Fat4. (b). The curve of tumor growth in nude mice. Two clones of tumor cells not expressing Fat4 or two clones of tumor cells expressing Fat4 (1×106 cells from each clone) were injected into nude mice, respectively. The experiment was performed in triplicate and the average tumor sizes were calculated. The tumors from clones expressing Fat4 were much smaller than that from tumor cell line not expressing Fat4.
Fig. 3
Fig. 3
Expression of Fat4 gene inhibited the tumor growth in nude mice. (a). The mRNA expression of Fat4 gene as revealed by RT-PCR. Tumor Clone 3 and 4 expressed Fat4 while Clone 1 and 2 did not express Fat4. (b). The curve of tumor growth in nude mice. Two clones of tumor cells not expressing Fat4 or two clones of tumor cells expressing Fat4 (1×106 cells from each clone) were injected into nude mice, respectively. The experiment was performed in triplicate and the average tumor sizes were calculated. The tumors from clones expressing Fat4 were much smaller than that from tumor cell line not expressing Fat4.
Fig. 4
Fig. 4
Inhibition of Fat4 led to tumorigenesis. (a). Representative photo of tumor formation 60 days after injection of NOG8 cells (106) expressing Fat4-shRNA or control RNA into nude mice. Note the tumor on the right flank, corresponding to NOG8 expressing Fat4-shRNA, and no tumor formation on the left flank, corresponding to NOG8 expressing control shRNA. (b). Inhibition of Fat4 mRNA by shRNA. Quantitative RT-PCR was performed with total RNA prepared from NOG8 cells expressing control shRNA (Lane 1) or Fat4-shRNA (Lane 2), and tumors developed from NOG8 cells expressing Fat4- shRNA (Lane 3). The Fat4 mRNA levels were normalized to control β-actin mRNA levels.
Fig. 4
Fig. 4
Inhibition of Fat4 led to tumorigenesis. (a). Representative photo of tumor formation 60 days after injection of NOG8 cells (106) expressing Fat4-shRNA or control RNA into nude mice. Note the tumor on the right flank, corresponding to NOG8 expressing Fat4-shRNA, and no tumor formation on the left flank, corresponding to NOG8 expressing control shRNA. (b). Inhibition of Fat4 mRNA by shRNA. Quantitative RT-PCR was performed with total RNA prepared from NOG8 cells expressing control shRNA (Lane 1) or Fat4-shRNA (Lane 2), and tumors developed from NOG8 cells expressing Fat4- shRNA (Lane 3). The Fat4 mRNA levels were normalized to control β-actin mRNA levels.
Fig. 5
Fig. 5
Increased YAP transcriptional activities in tumor cells not expressing Fat4 and NOG8 cells expressing Fat4-shRNA. Plasmid Gal4-YAP expressing fusion protein between YAP and Gal4 DNA binding domain or plasmid PM expressing Gal4 DNA binding domain was cotransfected with GAL-TKLUC vector into cells as indicated. The activity of luciferase from transfection with Gal4-YAP was divided by that from transfection with PM. Lane 1, NOG8; Lane 2, tumor cells not expressing Fat4; Lane 3, NOG8 expressing control shRNA; Lane 4, NOG8 expressing Fat4-shRNA.
Fig. 6
Fig. 6
(a). Loss of Fat4 expression in a fraction of breast cancers. Total RNA was made from 6 breast tumor cell lines and 5 primary breast tumors. RT-PCR was performed. BT-20, ZR-75-1, BT474, and primary tumor 1 to 3 did not express Fat4. (b). Schematic depiction of the CpG island in the human Fat4 promoter region. The CpG island spanning ~2.7 kb was located in the promoter region and first exon of Fat4 gene. Location of bisulfite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as while and gray arrows, respectively. Shown are results of bisulfite genomic sequencing of the fragment containing 10 CpG sites from BT-20 and MCF-7 cells, with an open circle representing an unmethylated CpG site and a close circle representing a methylated CpG site. (c). Methylation of Fat4 promoter in breast cancers. Methylation-specific PCR was performed. BT20, ZR-75-1, and primary tumor 1 contained only methylated alleles. BT474, and primary tumor 2 and 3 contained both unmethylated and methylated alleles. MCF-7, MDA-MB-157, MDA-MB-231, and primary tumor 4 and 5 contained only unmethylated alleles. The water control did not show evidence of contamination.
Fig. 6
Fig. 6
(a). Loss of Fat4 expression in a fraction of breast cancers. Total RNA was made from 6 breast tumor cell lines and 5 primary breast tumors. RT-PCR was performed. BT-20, ZR-75-1, BT474, and primary tumor 1 to 3 did not express Fat4. (b). Schematic depiction of the CpG island in the human Fat4 promoter region. The CpG island spanning ~2.7 kb was located in the promoter region and first exon of Fat4 gene. Location of bisulfite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as while and gray arrows, respectively. Shown are results of bisulfite genomic sequencing of the fragment containing 10 CpG sites from BT-20 and MCF-7 cells, with an open circle representing an unmethylated CpG site and a close circle representing a methylated CpG site. (c). Methylation of Fat4 promoter in breast cancers. Methylation-specific PCR was performed. BT20, ZR-75-1, and primary tumor 1 contained only methylated alleles. BT474, and primary tumor 2 and 3 contained both unmethylated and methylated alleles. MCF-7, MDA-MB-157, MDA-MB-231, and primary tumor 4 and 5 contained only unmethylated alleles. The water control did not show evidence of contamination.
Fig. 6
Fig. 6
(a). Loss of Fat4 expression in a fraction of breast cancers. Total RNA was made from 6 breast tumor cell lines and 5 primary breast tumors. RT-PCR was performed. BT-20, ZR-75-1, BT474, and primary tumor 1 to 3 did not express Fat4. (b). Schematic depiction of the CpG island in the human Fat4 promoter region. The CpG island spanning ~2.7 kb was located in the promoter region and first exon of Fat4 gene. Location of bisulfite genomic sequencing PCR primers and methylation-specific PCR primers are indicated as while and gray arrows, respectively. Shown are results of bisulfite genomic sequencing of the fragment containing 10 CpG sites from BT-20 and MCF-7 cells, with an open circle representing an unmethylated CpG site and a close circle representing a methylated CpG site. (c). Methylation of Fat4 promoter in breast cancers. Methylation-specific PCR was performed. BT20, ZR-75-1, and primary tumor 1 contained only methylated alleles. BT474, and primary tumor 2 and 3 contained both unmethylated and methylated alleles. MCF-7, MDA-MB-157, MDA-MB-231, and primary tumor 4 and 5 contained only unmethylated alleles. The water control did not show evidence of contamination.
Fig. 7
Fig. 7
Homology of the cytoplasmic region at the C-terminus between the mouse Fat4 (mFat4) and the D. melanogaster Fat (dFat). Pluses represent similar amino acids. Minuses represent space inserted for optimum alignment.
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