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We have previously shown that FHL2 cooperates with ß-catenin in the activation of Wnt target genes. Using mouse embryonic fibroblasts (MEF) derived from FHL2-null and wild type mice, we found that spontaneously immortalized FHL2-null variants harbor very low levels of D-type cyclins, reduced phosphorylation of Rb and decreased proliferative capacity. These cells retain wild-type p53 and p19ARF and are resistant to ß-catenin- and Ras-induced transformation. Furthermore, the inhibition of FHL2 by siRNA reduces colony formation in human transformed cell lines.

To investigate the role of FHL2 in tumorigenesis, we crossed FHL2-deficient mice with ApcD14/+ mice in which exon 14 of the adenomatous polyposis coli (APC) gene is deleted. Like the well-known Apcmin/+ mice, ApcD14/+ mice develop adenomas and carcinomas in the intestine and colon. By contrast, FHL2-null mice develop normally and have no detectable defect in the intestinal tract. We sacrified mice at 11 months and found 55% decrease in polyp number in ApcD14/+FHL2+/- mice (p < 0.01) and 68% decrease in ApcD14/+FHL2-/- mice (p < 0.005) compared to ApcD14/+FHL2+/+ littermates (ApcD14/+FHL2+/+ mice, 24.6 ± 12 polyps, n = 7; ApcD14/+FHL2+/- mice, 11.2 ± 9 polyps, n = 13; ApcD14/+FHL2-/- mice, 7.9 ± 5 polyps, n = 12). The size of polyps and proliferation rates measured by Ki67 staining were similar in ApcD14/+FHL2-/- animals compared to ApcD14/+FHL2+/+ mice. These results suggest that FHL2 deficiency suppresses tumor development at early stages. We then examined the intestinal tract of mice at 3 months, when few adenomas develop in the intestine of ApcD14/+ mice (maximum 4). Only 40% of ApcD14/+ mice nullyzygous for FHL2 (4/10) developed polyps against 73% of FHL2+/+ mice (8/11), confirming the role of FHL2 at early steps of intestinal tumorigenesis.

Activation of Wnt/ß-catenin signaling in polyps from both ApcD14/+FHL2+/+ and ApcD14/+FHL2-/- mice was evidenced by nuclear localization of ß-catenin and activated expression of its target genes c-myc, cyclin D1 and axin 2. Interestingly immunostaining patterns of phosphorylated Erk1/2 were altered in intestinal epithelia of ApcD14/+FHL2-/- mice compared to ApcD14/+FHL2+/+ mice. Additionally, expression of the Btc gene, a target of the MAP kinase pathway, was significantly activated in polyps from ApcD14/+FHL2+/+ animals, but not from ApcD14/+FHL2-/- mice. These results suggest that deficiency in FHL2 may pertub transmission of the MAP kinase signal in intestinal epithelia, resulting in inhibition of development of polyps at early stages.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA