During epithelial homeostasis, stem cells divide to produce progenitor cells, which not only proliferate to generate the cell mass but also respond to cellular signaling to transition from a proliferative state to a differentiation state. Such a transition involves functional alterations of transcriptional factors, yet the underlying molecular mechanisms are poorly understood. Recent studies have implicated Kruppel-like factors (KLFs) including KLF5 in the renewal and maintenance of stem/progenitor cells. Here we demonstrate that the pro-proliferative factor KLF5 becomes anti-proliferative upon TGF␤-mediated acetylation in an in vitro model of epithelial homeostasis. In the HaCaT epidermal cell line treated with or without TGF␤, we found that KLF5 was not only essential for cell proliferation, it was also indispensable for TGF␤-induced anti-proliferation in these cells. KLF5 inhibited the expression of p15 (CDKN2B), a cell cycle inhibitor, without TGF␤, but became a coactivator in TGF␤-induced p15 expression in the same cells. Mechanistically, TGF␤ recruited acetylase p300 to acetylate KLF5, and acetylation in turn altered the binding of KLF5 to p15 promoter, resulting in the reversal of KLF5 function. These studies not only demonstrate that a basic transcription factor can be both pro-proliferation and anti-proliferation in epithelial homeostasis, they also present a unique mechanism for how transcriptional regulation changes during the transition from proliferation to inhibition of proliferation. Furthermore, they establish KLF5 as an essential cofactor for TGF␤ signaling.Epithelia constitute the surface and lining of many solid tissues and have essential functions in different tissues. They are maintained through epithelial homeostasis, which involves the proliferation of stem/progenitor cells and the differentiation of their daughter cells. Epithelial homeostasis constantly occurs in vivo, and its disruption causes different diseases including cancer (1). At the molecular level, signaling from stroma compartment in a tissue such as transforming growth factor ␤ (TGF␤) 2 signaling regulates epithelial homeostasis through a transcriptional network, but the molecular details are not well understood.The basic transcription factor KLF5 is ubiquitously expressed in many tissues including the breast, colon, intestine, lung, prostate, etc (2-5). KLF5 is highly expressed in proliferating epithelial cells such as immortal but untransformed epithelial cell lines and proliferating primary cultures of epithelial cells, which mostly represent progenitor cells (2, 3, 6, 7). In normal intestine, KLF5 is expressed at a higher level in basal rapidly proliferating cells, but at a lower level in mature and differentiated cells (8) and knock-out of one KLF5 allele significantly reduced the size of villi in mouse intestine (8). In vivo overexpression of KLF5 in epidermis causes hyperplasia of basal cells but lack of mature skin (9), further indicating a proproliferative role of KLF5 in epithelial homeostasis. Recently a combinatio...

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KLF5 is a transcription factor that plays important roles in multiple physical and pathological processes, including cell growth, cell cycle regulation, and angiogenesis. To better characterize KLF5 function in bladder carcinogenesis, we established stable TSU-Pr1 cell clones expressing different levels of KLF5. These clones were then characterized for cell growth, cell cycle progression, tumorigenesis, and alteration in gene expression. Overexpression of KLF5 promoted tumorigenesis of the TSU-Pr1 cancer cells in mice. Consistently, KLF5 increased G1 to S phase transition, which was accompanied by the upregulation of cyclin D1, phosphorylation of MAPK and Akt, and reduced protein levels for CDK inhibitors p27 and p15. Microarray analysis combined with expression verification in different cell systems identified a number of additional genes that are potentially regulated by KLF5, including HBP17, ITGA6, and RAIG1. These findings suggest that the KLF5 transcription factor plays an oncogenic role in the TSU-Pr1 bladder cancer cell line through the regulation of a subset of genes. ' 2005 Wiley-Liss, Inc.

The transcription factor KLF5 plays an important role in human carcinogenesis. In epithelial cells, the KLF5 protein is tightly regulated by the ubiquitin-proteasome pathway. To better understand the mechanisms for the regulation of KLF5 protein, we identified and characterized an E3 ubiquitin ligase for KLF5, i.e. WWP1. We found that WWP1 formed a protein complex with KLF5 in vivo and in vitro. Furthermore, WWP1 mediated the ubiquitination and degradation of KLF5, and the catalytic cysteine residue of WWP1 is essential for its function. A PY motif in a transactivation domain of KLF5 is necessary for its interaction with WWP1. Finally, WWP1 was amplified and overexpressed in some cancer cell lines from the prostate and breast, which negatively regulated the function of KLF5 in gene regulation. These findings not only established WWP1 as an E3 ubiquitin ligase for KLF5, they also further implicated the KLF5 pathway in human carcinogenesis.

Deletion of chromosome 6q14-q22 is common in multiple human cancers including prostate cancer, and chromosome 6 transferred into cancer cells induces senescence and reduces cell growth, tumorigenicity and metastasis, indicating the existence of one or more tumor-suppressor genes in 6q. To identify the 6q tumor-suppressor gene, we first narrowed the common region of deletion to a 2.5 Mb interval at 6q14-15. Of the 11 genes located in this minimal deletion region and expressed in normal prostates, only snoRNA U50 was mutated, demonstrated transcriptional downregulation and inhibited colony formation in prostate cancer cells. The mutation, a homozygous 2 bp (TT) deletion, was found in two of 30 prostate cancer cell lines/xenografts and nine of 89 localized prostate cancers (eleven of 119 or 9% cancers). Two of 89 (2%) patients with prostate cancer also showed the same mutation in their germline DNA, but none of 104 cancer-free control men did. The homozygous deletion abolished U50 function in a colony formation assay. Analysis of 1371 prostate cancer cases and 1371 matched control men from a case-control study nested in a prospective cohort showed that, although a germline heterozygous genotype of the deletion was detected in both patients and controls at similar frequencies, the homozygosity of the deletion was significantly associated with clinically significant prostate cancer (odds ratio 2.9; 95% confidence interval 1.17-7.21). These findings establish snoRNA U50 as a reasonable candidate for the 6q tumor-suppressor gene in prostate cancer and likely in other types of cancers.

Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.

Deletion of chromosome 6q is frequent in breast cancer, and the deletion often involves a region in 6q14-q16. At present, however, the underlying tumor suppressor gene has not been established. Based on a recent study identifying snoRNA U50 as a candidate for the 6q14-16 tumor suppressor gene in prostate cancer, we investigated whether U50 is also involved in breast cancer. PCR-based approaches showed that U50 underwent frequent genomic deletion and transcriptional downregulation in cell lines derived from breast cancer. Mutation screening identified the same 2-bp deletion of U50 as in prostate cancer in both cell lines and primary tumors from breast cancer, and the deletion was both somatic and in germline. Genotyping of a cohort of breast cancer cases and controls for the mutation demonstrated that, while homozygous genotype of the mutation was rare, its heterozygous genotype occurred more frequently in women with breast cancer. Functionally, re-expression of U50 resulted in the inhibition of colony formation in breast cancer cell lines. These results suggest that noncoding snoRNA U50 plays a role in the development and/or progression of breast cancer.

KLF5 plays important roles in a variety of cellular processes including proliferation and differentiation. Recently KLF5 was shown to reverse its function in proliferative and p15 regulation upon transforming growth factor-␤ (TGF␤)-mediated acetylation. To understand how KLF5 acetylation functions in TGF␤-induced p15 transcription, we characterized the interactions of KLF5 with other transcription factors and promoter DNA elements in the context of TGF␤. KLF5 interacted with Smad2-4 and Miz-1 in a TGF␤-independent manner, but interacted with Myc only when TGF␤ was activated, and at least some of the interactions had an additive effect on TGF␤-induced p15 transcription. Oligo pulldown assays showed that binding of Myc to the Inr element was KLF5-dependent, and TGF␤ could enhance the binding when more KLF5 was available. Furthermore, TGF␤ induced an interaction between KLF5 and the p300 acetylase, and acetylation of KLF5 was necessary for Smad4 to associate with p300. Failure in KLF5 acetylation not only prevented p300-assembled Smad4-KLF5 complex formation on p15 promoter but also affected the binding of Smad4 and FOXO3 on the p15 promoter in vivo. These findings suggest that without TGF␤, some KLF5 associates with Smads in the nucleus and other KLF5 associates with Miz-1 on the p15 promoter to repress its transcription. Activation of TGF␤ recruits p300 to the KLF5-Smad complex to acetylate KLF5, and the complex with acetylated KLF5 binds to the Smad binding element and alters the binding of other factors to p15 promoter to induce its transcription. Transforming growth factor-␤ (TGF␤)2 is the most potent and widespread growth-inhibitory cytokine known in mammals (1, 2). TGF␤ signaling involves receptor binding, Smad modification and nuclear translocation, and the formation of Smad transcriptional complexes on gene promoters, regulating the expression of a large number of genes in different biological processes (3, 4). The same TGF␤ signaling has different regulatory roles in different contexts (1, 2, 5, 6), most likely through different transcriptional cofactors preexisting in different types of cells including proliferating epithelial cells that differentiate upon TGF␤ activation.The cyclin-dependent kinase inhibitor p15 (also named CDKN2B and p15INK4b ) is a well documented tumor suppressor in many types of cancers (7,8). The cytostatic effect of p15 is based on its inhibition of cyclin-dependent kinases CDK4 and CDK6, which arrests the cell cycle in the G 1 phase. p15 is an important effector of TGF␤ and is dramatically induced upon TGF␤ treatment in normal epithelial cells (9). Transcriptional induction of p15 by TGF␤ involves the formation of a Smad transcriptional complex containing Miz-1 (10), Myc (11), Sp1 (12) and FOXO (5). On the other hand, acetylase p300 is a well established co-activator of the TGF␤ pathway (13,14). In epithelial cells, TGF␤ signaling also recruits acetylase p300 to the Smad complex to acetylate some transcription factors and mediate TGF␤-induced transcription (6, 15).Human Kruppel-l...