Ceshi Chen

The Fbw7 Tumor Suppressor Targets KLF5 for Ubiquitin-Mediated Degradation and Suppresses Breast Cell Proliferation

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 GSK3beta-mediated KLF5 phosphorylation-dependent 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.

WW domain-containing E3 ubiquitin protein ligase 1 targets p63 transcription factor for ubiquitin-mediated proteasomal degradation and regulates apoptosis

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 ΔNp63 and TAp63. Increasing evidence suggests that TAp63 sensitizes cells to apoptosis but ΔNp63 has an opposite function. In this study, we show that WWP1 binds, ubiquitinates, and destructs both ΔNp63α and TAp63α. The protein–protein interaction occurs between the PY motif of p63 and the WW domains of WWP1. The knockdown of WWP1 by siRNA increases the endogenous ΔNp63α 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 TAp63α, 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.

Genetic and Expression Aberrations of E3 Ubiquitin Ligases in Human Breast Cancer

Recent studies revealed that E3 ubiquitin ligases play important roles in breast carcinogenesis. Clinical research studies have found that (epi)-genetic (deletion, amplification, mutation, and promoter methylation) and expression aberration of E3s are frequent in human breast cancer. Furthermore, many studies have suggested that many E3s are either oncogenes or tumor suppressor genes in breast cancer. In this review, we provide a comprehensive summary of E3s, which have genetic and/or expression aberration in breast cancer. Most cancer-related E3s regulate the cell cycle, p53, transcription, DNA repair, cell signaling, or apoptosis. An understanding of the oncogenic potential of the E3s may facilitate identifying and developing individual E3s as diagnosis markers and drug targets in breast cancer. (Mol Cancer Res 2006;4(10):695–707)

YAP Promotes Breast Cell Proliferation and Survival Partially through Stabilizing the KLF5 Transcription Factor

The Yes-associated protein (YAP), an oncoprotein in the Hippo tumor suppressor pathway, regulates tumorigenesis and has been found in a variety of tumors, including breast, ovarian, and hepatocellular cancers. Although YAP functions through its WW domains, the YAP WW domain-binding partners have not yet been completely determined. With this study, we demonstrate that YAP functions partially through its binding to KLF5, a transcription factor that promotes breast cell proliferation and survival. YAP interacted with the KLF5 PY motif through its WW domains, preventing the E3 ubiquitin ligase WWP1 from ubiquitinating KLF5. Overexpression of the wild-type YAP but not the WW domain-mutated YAP up-regulated KLF5 protein levels and mRNA expression levels of KLF5 downstream target genes, including FGFBP1 (alias FGF-BP) and ITGB2. In addition, knockdown of YAP decreased expression levels of KLF5, FGF-BP, and ITGB2. Depletion of either YAP or KLF5 decreased breast cell proliferation and survival in MCF10A and SW527 breast cell lines, and stable knockdown of either YAP or KLF5 suppressed SW527 xenograft growth in mice. The YAP upstream kinase LATS1 suppressed the KLF5-FGF-BP axis, as well as cell growth through YAP signaling. Both YAP and KLF5 are coexpressed in estrogen receptor ERα-negative breast cell lines. These findings suggest that KLF5 could be an important transcription factor partner for YAP and may contribute to the Hippo pathway.

KLF5 Promotes Breast Cell Survival Partially through Fibroblast Growth Factor-binding Protein 1-pERK-mediated Dual Specificity MKP-1 Protein Phosphorylation and Stabilization

Krüpple-like transcription factor 5 (KLF5) is a zinc-finger transcription factor promoting cell survival and tumorigenesis in multiple cancers. A high expression level of KLF5 has been shown to be associated with shorter breast cancer patient survival. However, the role of KLF5 and mechanism of KLF5 actions in breast cancer remain unclear. In this study, we found that KLF5 knockdown by small interfering RNA in two breast cell lines, MCF10A and BT20, induces apoptosis. Interestingly, a pro-survival phosphatase, dual specificity mitogen-activated protein kinase phosphatase 1 (MKP-1), is down-regulated by KLF5 ablation. Consistently, KLF5 overexpression increases the MKP-1 protein expression in Hs578T and MCF7. We further found that MKP-1 is essential and sufficient for KLF5 to promote breast cell survival. However, MKP-1 is not a KLF5 direct transcription target because the MKP-1 mRNA level is not regulated by KLF5. By cycloheximide chase assays, we found that KLF5 decreases MKP-1 protein degradation via activating the ERK signaling. Inhibition of pERK by the pharmacological inhibitor U0126 specifically blocks KLF5-induced MKP-1 phosphorylation and stabilization. Additionally, constitutive activation of ERK by constitutively activated MEK1 rescues the KLF5 depletion-induced MKP-1 down-regulation. Consistently, the phosphorylation-deficient MKP-1 mutant cannot be stabilized by KLF5. Finally, the activation of ERK by KLF5 is very likely through the KLF5 direct target gene FGF-BP in breast cells. These findings suggest that KLF5 is a pro-survival factor that promotes breast cell survival partially through pERK-mediated MKP-1 phosphorylation and stabilization. The KLF5-FGF-BP-pERK-MKP-1 signaling axis may provide new therapeutic targets for invasive breast cancer.

TAZ antagonizes the WWP1-mediated KLF5 degradation and promotes breast cell proliferation and tumorigenesis

Krüppel-like factor 5 (KLF5) is a PY motif-containing transcription factor promoting breast cell proliferation. The KLF5 protein is rapidly degraded through the proteasome after ubiquitination by E3 ubiquitin ligases, such as WWP1 and SCF(Fbw7). In this study, we demonstrated that a transcriptional co-activator with the PDZ-binding motif (TAZ) upregulated the KLF5 expression through antagonizing the WWP1-, but not Fbw7-, mediated KLF5 ubiquitination and degradation. TAZ interacted with KLF5 through the WW domain of TAZ and the PY motif of KLF5, which is the binding site for WWP1. TAZ inhibited WWP1-KLF5 protein interaction and WWP1-mediated KLF5 ubiquitination and degradation in a WW domain-dependent manner. Overexpression of TAZ upregulated the protein levels of KLF5 and FGF-BP, which is a well-established KLF5 target gene. In addition, depletion of TAZ in both 184A1 and HCC1937 breast cells downregulated protein levels of KLF5 and FGF-BP and inhibited cell growth. Furthermore, stable depletion of either TAZ or KLF5 significantly suppressed HCC1937 xenograft growth in immunodeficient mice. Knockdown of LATS1, a TAZ upstream inhibitory kinase, up-regulated the protein levels of KLF5 and FGF-BP in 184A1 and promoted cell growth through TAZ. Finally, both KLF5 and TAZ were co-expressed in a subset of estrogen receptor α-negative breast cell lines. These results, for the first time, suggest that TAZ promotes breast cell growth partially through protecting KLF5 from WWP1-mediated degradation and enhancing KLF5s activities.

Krüppel-like factor 5 promotes breast cell proliferation partially through upregulating the transcription of fibroblast growth factor binding protein 1

The Krüppel-like factor 5 (KLF5) is a zinc-finger transcription factor promoting cell proliferation, cell-cycle progression and survival. A high expression level of KLF5 mRNA has been shown to be associated with shorter breast cancer patient survival. However, the mechanism of KLF5 action in breast cancer is still not clear. In this study, we found that both KLF5 and its downstream gene fibroblast growth factor binding protein 1 (FGF-BP) are co-expressed in breast cell lines and primary tumors. Manipulation of the KLF5 expression can positively regulate the FGF-BP mRNA and protein levels in multiple breast cell lines. In addition, the secreted FGF-BP protein in the conditional medium is also regulated by KLF5. Furthermore, we demonstrated that KLF5 binds and activates the FGF-BP promoter through a GC box by luciferase reporter, oligo pull down and chromatin immunoprecipitation (ChIP) assays. When FGF-BP is depleted by siRNA, KLF5 fails to promote cell proliferation in MCF10A, SW527 and TSU-Pr1. We further demonstrated that overexpression or addition of FGF-BP rescues the KLF5-knockdown-induced growth arrest in MCF10A cells. Finally, KLF5 significantly promotes MCF7 breast cancer cell xenograft growth in athymic nude mice. These findings suggest that KLF5 may promote breast cancer cell proliferation at least partially through directly activating the FGF-BP mRNA transcription. Understanding the mechanism of KLF5 action in breast cancer may result in useful diagnostic and therapeutic targets.

WW domain containing E3 ubiquitin protein ligase 1 targets the full-length ErbB4 for ubiquitin-mediated degradation in breast cancer

ErbB4, a member of the epidermal growth factor receptor family, plays a role in normal breast and breast cancer development by regulating mammary epithelial cell proliferation, survival and differentiation. In this study, we show that WWP1, a C2-WW-HECT type E3 ubiquitin ligase, binds, ubiquitinates and destructs ErbB4-CYT1, but much less efficiently for CYT2, isoforms (both JMa and JMb). The protein–protein interaction occurs primarily between the first and third WW domains of WWP1 and the second PY motif of ErbB4. Knockdown of WWP1 by two different small interfering RNAs increases the endogenous ErbB4 protein levels in both MCF7 and T47D breast cancer cell lines. In addition, overexpression of the wild type, but not the catalytic inactive WWP1, dramatically decreases the endogenous ErbB4 protein levels in MCF7. Importantly, we found that WWP1 negatively regulates the heregulin-β1-stimulated ErbB4 activity as measured by the serum response element report assay and the BRCA1 mRNA expression. After a systematic screening of all WWP1 family members by small interfering RNA, we found that AIP4/Itch and HECW1/NEDL1 also negatively regulate the ErbB4 protein expression in T47D. Interestingly, the protein expression levels of both WWP1 and ErbB4 are higher in estrogen receptor-α-positive than in estrogen receptor-α-negative breast cancer cell lines. These data suggest that WWP1 and its family members suppress the ErbB4 expression and function in breast cancer.

MiRNA-296-3p-ICAM-1 axis promotes metastasis of prostate cancer by possible enhancing survival of natural killer cell-resistant circulating tumour cells.

Natural killer (NK) cells are important in host to eliminate circulating tumour cells (CTCs) in turn preventing the development of tumour cells into metastasis but the mechanisms are very poorly defined. Here we find that the expression level of miR-296-3p is much lower in the non-metastatic human prostate cancer (PCa) cell line P69 than that in the highly metastatic cell line M12, which is derived from P69. We demonstrate that miR-296-3p directly targets and inhibits the expression of intercellular adhesion molecule 1 (ICAM-1) in the malignant M12. The data from clinical tissue microarrays also show that miR-296-3p is frequently upregulated and ICAM-1 is reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested in vivo by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly identified miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis.

Identification of valid reference genes for the normalization of RT-qPCR expression studies in human breast cancer cell lines treated with and without transient transfection.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.