Sin3a acts through a multi-gene module to regulate invasion in Drosophila and human tumors.

Chromatin remodeling proteins regulate multiple aspects of cell homeostasis, making them ideal candidates for misregulation in transformed cells. Here, we explore Sin3A, a member of the Sin3 family of proteins linked to tumorigenesis that are thought to regulate gene expression through their role as histone deacetylases (HDACs). We identified Drosophila Sin3a as an important mediator of oncogenic Ret receptor in a fly model of Multiple Endocrine Neoplasia Type 2. Reducing Drosophila Sin3a activity led to metastasis-like behavior and, in the presence of Diap1, secondary tumors distant from the site of origin. Genetic and Chip-Seq analyses identified previously undescribed Sin3a targets including genes involved in cell motility and actin dynamics, as well as signaling pathways including Src, Jnk and Rho. A key Sin3a oncogenic target, PP1B, regulates stability of ß-Catenin/Armadillo: the outcome is to oppose T-cell factor (TCF) function and Wg/Wnt pathway signaling in both fly and mammalian cancer cells. Reducing Sin3A strongly increased the invasive behavior of A549 human lung adenocarcinoma cells. We show that Sin3A is downregulated in a variety of human tumors and that Src, JNK, RhoA and PP1B/ß-Catenin are regulated in a manner analogous to our Drosophila models. Our data suggest that Sin3A influences a specific step of tumorigenesis by regulating a module of genes involved in cell invasion. Tumor progression may commonly rely on such 'modules of invasion' under the control of broad transcriptional regulators.

In vivo regulation of p21 by the Kruppel-like factor 6 tumor-suppressor gene in mouse liver and human hepatocellular carcinoma.

Kruppel-like factor (KLF) 6 is a tumor-suppressor gene functionally inactivated by loss of heterozygosity, somatic mutation and/or alternative splicing that generates a dominant-negative splice form, KLF6-SV1. Wild-type KLF6 (wtKLF6) expression is decreased in many human malignancies, which correlates with reduced patient survival. Additionally, loss of the KLF6 locus in the absence of somatic mutation in the remaining allele occurs in a number of human cancers, raising the possibility that haploinsufficiency of the KLF6 gene alone contributes to cellular growth dysregulation and tumorigenesis. Our earlier studies identified the cyclin-dependent kinase inhibitor p21 as a transcriptional target of the KLF6 gene in cultured cells, but not in vivo. To address this issue, we have generated two genetic mouse models to define the in vivo role of KLF6 in regulating cell proliferation and p21 expression. Transgenic overexpression of KLF6 in the liver resulted in a runted phenotype with decreased body and liver size, with evidence of decreased hepatocyte proliferation, increased p21 and reduced proliferating cell nuclear antigen expression. In contrast, mice with targeted deletion of one KLF6 allele (KLF6+/-) display increased liver mass with reduced p21 expression, compared to wild type littermates. Moreover, in primary hepatocellular carcinoma samples, there is a significant correlation between wtKLF6 and p21 mRNA expression. Combined, these data suggest that haploinsufficiency of the KLF6 gene may regulate cellular proliferation in vivo through decreased transcriptional activation of the cyclin-dependent kinase inhibitor p21.

E-cadherin is a novel transcriptional target of the KLF6 tumor suppressor.

The tumor suppressor KLF6 is a member of the Krüppel-like family of transcription factors, which has been implicated in the pathogenesis of several human carcinomas. Uncovering the transcriptional targets relevant for its tumorigenic properties, including cellular proliferation and invasion, will be essential to understanding possible mechanisms by which KLF6 and its antagonistic splice form, KLF6-SV1, regulate this development. To begin defining possible metastatic-related pathways, we analysed the effect of KLF6 dysregulation on a recognized suppressor of cellular invasion, E-cadherin. Targeted KLF6 reduction in an ovarian cancer cell line, SKOV-3, resulted in a 50% reduction of E-cadherin expression (P<0.01) and conversely, KLF6-SV1 silencing upregulated E-cadherin approximately fivefold (P<0.0001). These changes resulted from KLF6 directly transactivating the E-cadherin promoter as demonstrated by luciferase promoter assay and chromatin immunoprecipitation (ChIP). KLF6-mediated changes in E-cadherin levels were accompanied by downstream changes in both the subcellular localization of beta-catenin and c-myc expression levels. Moreover, and consistent with these experimental findings, patient-derived epithelial ovarian tumors with low KLF6 and high KLF6-SV1 expression ratios had significantly decreased E-cadherin expression (P<0.0001). These combined findings highlight the E-cadherin pathway as a novel and functionally important mediator by which changes in KLF6 and KLF6-SV1 can directly alter ovarian tumor invasion and metastasis.

KLF6, a candidate tumor suppressor gene mutated in prostate cancer.

Kruppel-like factor 6 (KLF6) is a zinc finger transcription factor of unknown function. Here, we show that the KLF6 gene is mutated in a subset of human prostate cancer. Loss-of-heterozygosity analysis revealed that one KLF6 allele is deleted in 77% (17 of 22) of primary prostate tumors. Sequence analysis of the retained KLF6 allele revealed mutations in 71% of these tumors. Functional studies confirm that whereas wild-type KLF6 up-regulates p21 (WAF1/CIP1) in a p53-independent manner and significantly reduces cell proliferation, tumor-derived KLF6 mutants do not. Our data suggest that KLF6 is a tumor suppressor gene involved in human prostate cancer.

The apolipoprotein(a) gene is regulated by sex hormones and acute-phase inducers in YAC transgenic mice.

High plasma concentrations of apolipoprotein (a) (apo(a)) have been implicated as a major independent risk factor for atherosclerosis in humans. Apo(a) is a large, evolutionarily new gene (present primarily in primates) for which considerable controversy exists concerning the factors that regulate its expression. To investigate the in vivo regulation of apo(a), we have created several lines of YAC transgenic mice containing a 110-kb human apo(a) gene surrounded by greater than 60 kb of 5' and 3' flanking DNA. Studies in humans have suggested that acute-phase inducers increase and sex steroids decrease apo(a) concentrations, but these results are controversial. Analysis of the YAC transgenic mice conclusively supports the hypothesized role of sex steroids and refutes the suggested role of acute-phase inducers in regulating the apo(a) gene.