The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. Rapamycin and its derivatives that specifically inhibit mTOR are now being actively evaluated in clinical trials. Recently, the inhibition of mTOR has been shown to reverse Akt-dependent prostate intraepithelial neoplasia. However, many cancer cells are resistant to rapamycin and its derivatives. The mechanism of this resistance remains a subject of major therapeutic significance. Here we report that the inhibition of mTOR by rapamycin triggers the activation of two survival signaling pathways that may contribute to drug resistance. Treatment of human lung cancer cells with rapamycin suppressed the phosphorylation of p70S6 kinase and 4E-BP1, indicating an inhibition of mTOR signaling. Paradoxically, rapamycin also concurrently increased the phosphorylation of both Akt and eIF4E. The rapamycin-induced phosphorylation of Akt and eIF4E was suppressed by the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002, suggesting the requirement of PI3K in this process. The activated Akt and eIF4E seem to attenuate rapamycin's growth-inhibitory effects, serving as a negative feedback mechanism. In support of this model, rapamycin combined with LY294002 exhibited enhanced inhibitory effects on the growth and colony formation of cancer cells. Thus, our study provides a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor. (Cancer Res 2005; 65(16): 7052-8)

Celecoxib appears to induce apoptosis in human NSCLC through the extrinsic death receptor pathway.

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.

ABSTRACT

The transcription factor KLF5 is highly expressed in basal-like breast cancer and promotes breast cancer cell proliferation, survival, migration and tumour growth. Here we show that, in breast cancer cells, KLF5 is stabilized by the deubiquitinase (DUB) BAP1. With a genome-wide siRNA library screen of DUBs, we identify BAP1 as a bona fide KLF5 DUB. BAP1 interacts directly with KLF5 and stabilizes KLF5 via deubiquitination. KLF5 is in the BAP1/HCF-1 complex, and this newly identified complex promotes cell cycle progression partially by inhibiting p27 gene expression. Furthermore, BAP1 knockdown inhibits tumorigenicity and lung metastasis, which can be rescued partially by ectopic expression of KLF5. Collectively, our findings not only identify BAP1 as the DUB for KLF5, but also reveal a critical mechanism that regulates KLF5 expression in breast cancer. Our findings indicate that BAP1 could be a potential therapeutic target for breast and other cancers.

WWP1 E3 ubiquitin ligase has previously been shown to be frequently amplified and overexpressed in prostate and breast cancers. However, the mechanism of WWP1 action is still largely unknown. p63, a member of the p53 family of transcription factors, has an important function in tumor development by regulating apoptosis. Using alternative promoters, p63 can be expressed as DNp63 and TAp63. Increasing evidence suggests that TAp63 sensitizes cells to apoptosis but DNp63 has an opposite function. In this study, we show that WWP1 binds, ubiquitinates, and destructs both DNp63a and TAp63a. The proteinprotein interaction occurs between the PY motif of p63 and the WW domains of WWP1. The knockdown of WWP1 by siRNA increases the endogenous DNp63a level in the MCF10A and 184B5 immortalized breast epithelial cell lines and confers resistance to doxorubicin-induced apoptosis. On the other hand, the knockdown of WWP1 increases the endogenous level of TAp63a, induces apoptosis, and increases sensitivity to doxorubicin and cisplatin in the HCT116 colon cancer cell line in a p53-independent manner. Finally, we found that DNA damage chemotherapeutic drugs induce WWP1 mRNA and protein expression in a p53-dependent manner. These data suggest that WWP1 may have a context-dependent role in regulating cell survival through targeting different p63 proteins for degradation.

The amplification of the q21 band of chromosome 8 (8q21) occurs in a large percentage of breast cancers. WWP1, an HECT domain-containing ubiquitin E3 ligase located in the 8q21 region, negatively regulates the TGF-b tumor suppressor pathway. To characterize the role of WWP1 in breast cancer, we analyzed WWP1 gene dosage and expression level as well as WWP1's function. A copy number gain of WWP1 was found in 51% (18/35) of breast cancer cell lines and in 41% (17/41) of primary breast tumors. Expression of WWP1 mRNA was analyzed with real-time RT-PCR, Northern blot, and Western blot. WWP1 mRNA is upregulated in 58% (19/33) of breast cancer cell lines, and overexpression of WWP1 is significantly correlated with a gene copy number gain. In a panel of cDNA from primary breast tumors and normal tissues, expression of WWP1 in tumors is significantly higher than that in normal tissues. Functionally, RNAi-mediated WWP1 knockdown significantly induced cell growth arrest and apoptosis in the MCF7 and HCC1500 breast cancer cell lines. Consistently, WWP1 inhibition activated caspases. Forced overexpression of WWP1 by the lentiviral system in 2 immortalized breast epithelial cell lines MCF10A and 184B5 promoted cell proliferation. These results suggest that genomic aberrations of WWP1 may contribute to the pathogenesis of breast cancer. ' 2007 Wiley-Liss, Inc.Key words: WWP1; breast cancer; gene amplification; overexpression; apoptosis A copy number gain or loss is a frequent genetic alteration in solid tumors including breast cancer. A copy number gain of 8q21 was detected in a high percentage of familial and sporadic breast tumors.1-3 Although several 8q21 genes, such as TPD52 and E2F5, have been reported to be amplified and overexpressed in breast cancer, 4,5 their roles have not been firmly established in breast tumorigenesis, and the underlying target genes of 8q21 amplification in breast cancer remain to be elucidated.Ubiquitin proteasome pathway (UPP)-mediated protein degradation plays an important role in breast cancer cell proliferation, apoptosis and carcinogenesis.6 Several oncogenic ubiquitin E3 ligases have been identified as diagnosis markers or potential drug targets in human breast cancer.7 For example, Skp2, an F-box protein in the SCF ubiquitin ligase complex, targets the CDK inhibitor p27 kip for degradation and is overexpressed in a subset of breast carcinomas. 8 The ring finger E3 ligase EFP mediates estrogen-induced cell growth and facilitates 14-3-3r tumor suppressor ubiquitination and proteolysis.9 EFP mRNA and protein were reported to be overexpressed and significantly correlated with poor prognosis of breast cancer patients. 10WW domain-containing protein 1 (WWP1) is an HECT (homologous to the E6-associated protein carboxyl terminus) domain-containing E3 ubiquitin ligase. Growing evidence suggests that WWP1 negatively regulates the TGF-b tumor suppressor pathway by mediating the ubiquitination and degradation of TGFb receptor 1 (TbR1), In our previous study, we found that WWP1 is amplified and overe...

Fbw7 is a tumor suppressor frequently inactivated in cancers. The KLF5 transcription factor promotes breast cell proliferation and tumorigenesis through upregulating FGF-BP. The KLF5 protein degrades rapidly through the ubiquitin proteasome pathway. Here, we show that the Skp1-CUL1-Fbw7 E3 ubiquitin ligase complex (SCF Fbw7 ) targets KLF5 for ubiquitin-mediated degradation in a GSK3β-mediated KLF5 phosphorylationdependent manner. Mutation of the critical S303 residue in the KLF5 Cdc4 phospho-degrons motif ( 303 SPPSS)abolishes the protein interaction, ubiquitination, and degradation by Fbw7. Inactivation of endogenous Fbw7 remarkably increases the endogenous KLF5 protein abundances. Endogenous Fbw7 suppresses the FGF-BP gene expression and breast cell proliferation through targeting KLF5 for degradation. These findings suggest that Fbw7 inhibits breast cell proliferation at least partially through targeting KLF5 for proteolysis. This new regulatory mechanism of KLF5 degradation may result in useful diagnostic and therapeutic targets for breast cancer and other cancers. Cancer Res; 70(11); 4728-38. ©2010 AACR.