ABSTRACT
Obesity, oxidative stress, and inflammation are risk factors for hepatocellular carcinoma (HCC). We examined, in mice, the effects of Krüppel-like factor 9 (KLF9) knockout on: adiposity, hepatic and systemic oxidative stress, and hepatic expression of pro-inflammatory and NOX/DUOX family genes, in a high-fat diet (HFD) context. Male and female Klf9+/+ (wild type, WT) and Klf9-/- (knockout, KO) mice were fed HFD (beginning at age 35 days) for 12 weeks, after which liver and adipose tissues were obtained, and serum adiponectin and leptin levels, liver fat content, and markers of oxidative stress evaluated. Klf9-/- mice of either sex did not exhibit significant alterations in weight gain, adipocyte size, adipokine levels, or liver fat content when compared to WT counterparts. However, Klf9-/- mice of both sexes had increased liver weight/size (hepatomegaly). This was accompanied by increased hepatic oxidative stress as indicated by decreased GSH/GSSG ratio and increased homocysteine, 3-nitrotyrosine, 3-chlorotyrosine, and 4HNE content. Decreased GSH to GSSG ratio and a trend toward increased homocysteine levels were observed in the corresponding Klf9-/- mouse serum. Gene expression analysis showed a heightened pro-inflammatory state in livers from Klf9-/- mice. KLF9 suppresses hepatic oxidative stress and inflammation, thus identifying potential mechanisms for KLF9 suppression of HCC and perhaps cancers of other tissues.
ABSTRACT
UNLABELLED: Expression of the transcription factor Krüppel-like factor 9 (KLF9) is frequently reduced in colorectal cancers, although a tumor suppressive role has not been established. To determine if KLF9 suppresses intestinal adenoma formation, we generated mice of distinct Klf9 genotypes in the background of the Apc (Min/+) mouse and compared their adenoma burdens at 16 weeks of age. While small intestine adenoma burden remained unchanged among Klf9 genotypes, male and female Apc(Min/+)/Klf9(-/-) and Apc(Min/+)/Klf9(+/-) mice exhibited significantly more colon adenomas than their Apc(Min/+)/Klf9(+/+) counterparts. Microarray analysis showed significant increases in the expression of interferon-induced genes in the colon mucosa of female Apc (Min/+)/Klf9(+/-) and Apc(Min/+)/Klf9(-/-) compared to Apc(Min/+)/Klf9(+/+) mice, prior to overt adenoma occurrence. Gene upregulation was confirmed by qPCR of colon mucosa and by siRNA knockdown of KLF9 in human HT29 colorectal cancer cells. Increases in expression of these genes were further augmented by supplementation with Interferon ß1. Circulating levels of the cytokine, interferon-stimulated gene 15 (ISG15) were increased in Apc(Min/+)/Klf9(+/-) and Apc(Min/+)/Klf9(-/-) mice relative to Apc(Min/+)/Klf9(+/+). Additionally, colon mucosal levels of ISG15 were increased in Apc(Min/+)/Klf9(+/-) mice. Chromatin immunoprecipitation demonstrated KLF9 recruitment to the ISG15 promoter. Lastly, treatment with ISG15 suppressed apoptosis in HT29 cells, in the presence and absence of 5-fluorouracil (5FU). Results show KLF9 to be a haploinsufficient suppressor of colon tumorigenesis in Apc(Min/+) mice in part, by repression of ISG15 and the latter's antiapoptotic function. SUMMARY: Krüppel-like factor 9 (KLF9) is a haploinsufficient tumor suppressor in the ApcMin/+ mouse colon by suppressing expression of ISG15, an apoptosis-inhibiting cytokine.
