Vladimir S. Spiegelman

The many faces of β -TrCP E3 ubiquitin ligases: reflections in the magic mirror of cancer

Beta-transducin repeats-containing proteins (β-TrCP) serve as the substrate recognition subunits for the SCFβ-TrCP E3 ubiquitin ligases. These ligases ubiquitinate specifically phosphorylated substrates and play a pivotal role in the regulation of cell division and various signal transduction pathways, which, in turn, are essential for many aspects of tumorigenesis. We review the functions of the SCFβ-TrCP ligases in the light of their relevance to cell growth, survival and transformation. Mechanisms underlying β-TrCP regulation and their aberration in human and animal cancer as well as prospective of targeting β-TrCP as a means of anticancer therapy are also discussed.

S100A4 accelerates tumorigenesis and invasion of human prostate cancer through the transcriptional regulation of matrix metalloproteinase 9

We previously showed that the calcium-binding protein S100A4 is overexpressed during the progression of prostate cancer (CaP) in humans and in the TRAMP (transgenic adenocarcinoma of the mouse prostate) mouse model. We tested a hypothesis that the S100A4 gene plays a role in the invasiveness of human CaP and may be associated with its metastatic spread. We observed that siRNA-mediated suppression of the S100A4 gene significantly reduced the proliferative and invasive capability of the highly invasive CaP cells PC-3. We evaluated the mechanism through which the S100A4 gene controls invasiveness of cells by using a macroarray containing 96 well characterized metastatic genes. We found that matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor (TIMP-1) were highly responsive to S100A4 gene suppression. Furthermore, S100A4 suppression significantly reduced the expression and proteolytic activity of MMP-9. By employing an MMP-9-promoter reporter, we observed a significant reduction in the transcriptional activation of the MMP-9 gene in S100A4-siRNA-transfected cells. Cells overexpressing the S100A4 gene (when transfected with pcDNA3.1-S100A4 plasmid) also significantly expressed MMP-9 and TIMP-1 genes with increased proteolytic activity of MMP-9 concomitant to increased transcriptional activation of the MMP-9 gene. S100A4-siRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. Our data demonstrate that the S100A4 gene controls the invasive potential of human CaP cells through regulation of MMP-9 and that this association may contribute to metastasis of CaP cells. We suggest that S100A4 could be used as a biomarker for CaP progression and a novel therapeutic or chemopreventive target for human CaP treatment.

CRD-BP mediates stabilization of βTrCP1 and c- myc mRNA in response to β-catenin signalling

Although constitutive activation of β-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the β-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that β-catenin stabilizes the mRNA encoding the F-box protein βTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of β-catenin/Tcf transcription factor. CRD-BP binds to the coding region of βTrCP1 mRNA. Overexpression of CRD-BP stabilizes βTrCP1 mRNA and elevates βTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)βTrCP E3 ubiquitin ligase and in accelerated turnover of its substrates including IκB and β-catenin. CRD-BP is essential for the induction of both βTrCP1 and c-Myc by β-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active β-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of βTrCP1, in the activation of dimeric transcription factor NF-κB and in the suppression of apoptosis in these cancers.

Wnt/β-Catenin Signaling Induces the Expression and Activity of βTrCP Ubiquitin Ligase Receptor

Abstract β-transducing repeat-containing protein (βTrCP) targets the ubiquitination and subsequent degradation of both β-catenin and IκB, thereby playing an important role in β-catenin/Tcf and NF-κB-dependent signaling. Here evidence is presented that β-catenin/Tcf signaling elevates the expression of βTrCP mRNA and protein in a Tcf-dependent manner, which does not require βTrCP transcription. Induction of βTrCP expression by the β-catenin/Tcf pathway results in an accelerated degradation of the wild-type β-catenin, suggesting that the negative feedback loop regulation may control the β-catenin/Tcf pathway. This signaling also upregulated NF-κB transactivation without affecting the activity of IκB kinase, thereby establishing that the maintenance of the βTrCP level is important for coordination between β-catenin/Tcf and NF-κB signaling.

Increased expression of p50-NF-κB and constitutive activation of NF-κB transcription factors during mouse skin carcinogenesis

To elucidate the possible role of NF-κB in mouse skin carcinogenesis we studied the expression of p50 (NF-κB1), p52 (NF-κB2), p65 (RelA) and IκB-α inhibitor as well as κB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IκB-α protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in κB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive κB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IκB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different κB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related κB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.

Green tea polyphenol EGCG suppresses cigarette smoke condensate-induced NF-κB activation in normal human bronchial epithelial cells

Cigarette smoke is a powerful inducer of inflammatory responses resulting in disruption of major cellular pathways with transcriptional and genomic alterations driving the cells towards carcinogenesis. Cell culture and animal model studies indicate that (−)-epigallocatechin-3-gallate (EGCG), the major polyphenol present in green tea, possesses potent anti-inflammatory and antiproliferative activity capable of selectively inhibiting cell growth and inducing apoptosis in cancer cells without adversely affecting normal cells. Here, we demonstrate that EGCG pretreatment (20–80 μM) of normal human bronchial epithelial cells (NHBE) resulted in significant inhibition of cigarette smoke condensate (CSC)-induced cell proliferation. Nuclear factor-κB (NF-κB) controls the transcription of genes involved in immune and inflammatory responses. In most cells, NF-κB prevents apoptosis by mediating cell survival signals. Pretreatment of NHBE cells with EGCG suppressed CSC-induced phosphorylation of IκBα, and activation and nuclear translocation of NF-κB/p65. NHBE cells transfected with a luciferase reporter plasmid containing an NF-κB-inducible promoter sequence showed an increased reporter activity after CSC exposure that was specifically inhibited by EGCG pretreatment. Immunoblot analysis showed that pretreatment of NHBE cells with EGCG resulted in a significant downregulation of NF-κB-regulated proteins cyclin D1, MMP-9, IL-8 and iNOS. EGCG pretreatment further inhibited CSC-induced phosphorylation of ERK1/2, JNK and p38 MAPKs and resulted in a decreased expression of PI3K, AKT and mTOR signaling molecules. Taken together, our data indicate that EGCG can suppress NF-κB activation as well as other pro-survival pathways such as PI3K/AKT/mTOR and MAPKs in NHBE cells, which may contribute to its ability to suppress inflammation, proliferation and angiogenesis induced by cigarette smoke.

Gli2 Is Targeted for Ubiquitination and Degradation by β-TrCP Ubiquitin Ligase

The Hedgehog (Hh) signaling pathway plays a crucial role in embryogenesis and has been linked to the development of several human malignancies. The transcription factor Gli2 plays a key role in the transduction of Hh signals by modulating transcription of some Hh target genes, yet the mechanisms that control Gli2 protein expression are largely unknown. Here we report that β-transducin repeat-containing protein (β-TrCP) E3 ubiquitin ligase is required for Gli2 degradation. β-TrCP2 directly binds wild type Gli2 and promotes its ubiquitination. Single amino acid substitution in Gli2 putative binding site inhibits its interaction with β-TrCP2, its ubiquitination, and stabilizes the Gli2 protein. Stable Gli2 mutant is expressed in higher levels and is more potent in the activation of Gli-dependent transcription as compared with wild type Gli2. We also found that GLI2 protein is expressed highly in prostate cancer cell lines and primary tumors, whereas the level of GLI2 mRNA is not appreciably different in normal and neoplastic prostate. These data identify β-TrCP2 as a pivotal regulator of Gli2 expression and point to an important role for posttranslational modulation of GLI2 protein levels in Hh pathway-associated human prostate cancer.

Negative Regulation of Prolactin Receptor Stability and Signaling Mediated by SCFβ-TrCP E3 Ubiquitin Ligase

ABSTRACT Ubiquitin-dependent degradation of hormone receptors is emerging as a key mechanism that regulates the magnitude and duration of hormonal effects on cells and tissues. The pituitary hormone prolactin (PRL) is involved in regulating cell differentiation, proliferation, and survival. PRL engages its receptor (PRLR) to initiate various signaling cascades, including the phosphorylation and activation of Stat5. We found that PRL promotes interaction between PRLR and the F-box protein β-TrCP2, which functions as a substrate recognition subunit of the SCFβ-TrCP E3 ubiquitin ligase. This interaction requires PRLR phosphorylation and the integrity of serine 349 within a conserved motif, which is similar to conserved motifs present in other substrates of SCFβ-TrCP. The PRLRS349A mutant is resistant to ubiquitination and is more stable than its wild-type counterpart. Phosphorylated PRLR undergoes ubiquitination by SCFβ-TrCP in vitro. Knockdown of β-TrCP expression inhibits the ubiquitination and degradation of PRLR and promotes PRL-dependent phosphorylation of Stat5 as well as Stat5-dependent transcription in cells. Furthermore, the activation of Stat5 and the stimulation of cell growth by PRL are augmented in cells expressing the PRLRS349A mutant. These data indicate that PRLR is a novel SCFβ-TrCP substrate and implicate β-TrCP as an important negative regulator of PRL signaling and cellular responses to this hormone.

Oncogenic beta-catenin signaling networks in colorectal cancer.

β-catenin has two distinct functions, namely, maintaining cell-to-cell adhesion and mediating the Wnt/β-catenin signal transduction pathway, which plays pivotal roles in embryogenesis and in malignant transformation of cells. The oncogenic properties of Wnt/β-catenin signaling stem from alteration in phosphorylation-dependent protein degradation and subcellular localization of β-catenin from cell membrane to the nucleus, where it binds to T-cell factor (Tcf) to form a bipartite transcription factor. The β-catenin/Tcf complex facilitates transcription of target genes that encode effectors for activation of cell proliferation and invasion and inhibition of apoptosis, leading to colorectal cancer development. In addition, in the tumor invasion front, stabilized and activated β-catenin interacts with other molecular pathways to facilitate tumor progression. This review highlights the β-catenin-dependent oncogenic signaling network involved in the multi-step process of colorectal tumorigenesis. Wnt signaling evidently regulates stem cells, leading them to differentiate or self-renew. We address roles of oncogenic β-catenin signaling in the microenvironment of the tumor-host interface that determine the individual tumor’s malignant potential and in regulation of putative cancer stem or progenitor cells that represent plausible targets for cancer eradication.

MicroRNA-340-mediated degradation of microphthalmia-associated transcription factor mRNA is inhibited by the coding region determinant-binding protein.

Alternative cleavage and polyadenylation generate multiple transcript variants of mRNA isoforms with different length of 3′-untranslated region (UTR). Alternative cleavage and polyadenylation enable differential post-transcriptional regulation of transcripts via the availability of different cis-acting elements in 3′-UTRs. Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and melanogenesis. It has also been implicated in melanoma development. Here we show that melanoma cells favor the expression of MITF mRNA with shorter 3′-UTR. This isoform of mRNA is regulated by microRNA, miR-340. miR-340 interacts with two of its target sites on the 3′-UTR of MITF mRNA, causing mRNA degradation and decreased expression and activity of MITF. On the other hand, the RNA-binding protein coding region determinant-binding protein, shown to be highly expressed in melanoma, directly binds to the 3′-UTR of MITF mRNA and prevents the binding of miR-340 to its target sites, resulting in stabilization of the MITF transcript and elevated expression and transcriptional activity of MITF. This interplay between RNA-binding protein and miRNA describes the important mechanism of regulation of MITF in melanocytes and malignant melanomas.