Deconjugation of Nedd8 from Cul1 is directly regulated by Skp1-F-box and substrate, and the COP9 signalosome inhibits deneddylated SCF by a noncatalytic mechanism.

COP9 signalosome (CSN) mediates deconjugation of the ubiquitin-like protein Nedd8 from the cullin subunits of SCF and other cullin-RING ubiquitin ligases (CRLs). This process is essential to maintain the proper activity of CRLs in cells. Here, we report a detailed kinetic characterization of CSN-mediated deconjugation of Nedd8 from SCF. CSN is an efficient enzyme, with a k(cat) of ~1 s(-1) and K(m) for neddylated Cul1-Rbx1 of ~200 nm, yielding a k(cat)/K(m) near the anticipated diffusion-controlled limit. Assembly with an F-box-Skp1 complex markedly inhibited deneddylation, although the magnitude varied considerably, with Fbw7-Skp1 inhibiting by ~5-fold but Skp2-Cks1-Skp1 by only ~15%. Deneddylation of both SCF(Fbw7) and SCF(Skp2-Cks1) was further inhibited ~2.5-fold by the addition of substrate. Combined, the inhibition by Fbw7-Skp1 plus its substrate cyclin E was greater than 10-fold. Unexpectedly, our results also uncover significant product inhibition by deconjugated Cul1, which results from the ability of Cul1 to bind tightly to CSN. Reciprocally, CSN inhibits the ubiquitin ligase activity of deneddylated Cul1. We propose a model in which assembled CRL complexes engaged with substrate are normally refractory to deneddylation. Upon consumption of substrate and subsequent deneddylation, CSN can remain stably bound to the CRL and hold it in low state of reduced activity.

Control of cullin-ring ubiquitin ligase activity by nedd8.

The Cullin-RING ubiquitin ligase (CRL) family, which may number as many as 350 different enzymes, has an enormous impact on cellular regulation. CRL enzymes regulate cell biology by conjugating ubiquitin onto target proteins that are involved in a multitude of processes. In most cases this leads to degradation of the target, but in some cases CRL-dependent ubiquitination acts as a switch to activate or repress target function. The ubiquitin ligase activity of CRLs is controlled by cycles of attachment and removal of the ubiquitin-like protein Nedd8. Conjugation of Nedd8 onto the cullin subunit of CRLs promotes assembly of an intact CRL complex and switches on ubiquitin ligase activity. Conversely, removal of Nedd8 switches off ubiquitin ligase activity and initiates CRL disassembly. Continuous maintenance of CRL function in vivo requires the activities of both the Nedd8-conjugating and deconjugating enzymes, pointing to a critical role of complex dynamics in CRL function. Here, we review how the Nedd8 cycle controls CRL activity and how perturbations of this cycle can lead to disease.

The S100A7-c-Jun activation domain binding protein 1 pathway enhances prosurvival pathways in breast cancer.

S100A7 is among the most highly expressed genes in preinvasive breast cancer, is a marker of poor survival when expressed in invasive disease, and promotes breast tumor progression in experimental models. To explore the mechanism of action, we examined the role of S100A7 in cell survival and found that overexpression of S100A7 in MDA-MB-231 cell lines promotes survival under conditions of anchorage-independent growth. This effect is paralleled by increased activity of nuclear factor-kappaB (3-fold) and phospho-Akt (4-fold), which are known to mediate prosurvival pathways. S100A7 and phospho-Akt are also correlated in breast tumors examined by immunohistochemistry (n = 142; P < 0.0001; r = 0.34). To explore the underlying mechanism, we examined the role of a putative c-Jun activation domain-binding protein 1 (Jab1)-binding domain within S100A7 using a panel of MDA-MB-231 breast cell lines stably transfected with either S100A7 or S100A7 mutated at the Jab1 domain. Structural analysis by three-dimensional protein modeling, immunoprecipitation, and yeast two-hybrid assay and functional analysis using transfected reporter gene and Western blot assays revealed that the in vitro effects of S100A7 on phospho-Akt and the nuclear factor-kappaB pathway are dependent on the Jab1-binding site and the interaction with Jab1. Enhanced epidermal growth factor receptor signaling was also found to correlate with the increased phospho-Akt. Furthermore, the Jab1-binding domain is also necessary for the enhanced tumorigenicity conferred by S100A7 expression in murine xenograft tumors in vivo. We conclude that the S100A7-Jab1 pathway acts to enhance survival under conditions of cellular stress, such as anoikis, which may promote progression of breast cancer.

BRCA1 and c-Myc associate to transcriptionally repress psoriasin, a DNA damage-inducible gene.

Evidence is accumulating to suggest that some of the diverse functions associated with BRCA1 may relate to its ability to transcriptionally regulate key downstream target genes. Here, we identify S100A7 (psoriasin), S100A8, and S100A9, members of the S100A family of calcium-binding proteins, as novel BRCA1-repressed targets. We show that functional BRCA1 is required for repression of these family members and that a BRCA1 disease-associated mutation abrogates BRCA1-mediated repression of psoriasin. Furthermore, we show that BRCA1 and c-Myc form a complex on the psoriasin promoter and that BRCA1-mediated repression of psoriasin is dependent on functional c-Myc. Finally, we show that psoriasin expression is induced by the topoisomerase IIalpha poison, etoposide, in the absence of functional BRCA1 and increased psoriasin expression enhances cellular sensitivity to this chemotherapeutic agent. Therefore, we identified a novel transcriptional mechanism that is likely to contribute to BRCA1-mediated resistance to etoposide.

Psoriasin interacts with Jab1 and influences breast cancer progression.

Psoriasin (S100A7) is expressed at low levels in normal breast epithelial cells but is highly expressed in preinvasive ductal carcinoma in situ. Persistent psoriasin expression occurs in some invasive carcinomas and is associated with poor prognostic factors. Whereas there is evidence that secreted psoriasin can act as a chemotactic factor for CD-4-positive lymphocytes in psoriatic skin lesions, an intracellular biological function is unknown. We have found that psoriasin physically interacts with Jab1 (c-jun activation-domain binding protein 1) in the yeast two-hybrid assay and confirmed this by coimmunoprecipitation assay in breast cancer cells. Psoriasin-transfected breast cancer cells showed increased nuclear Jab1 and demonstrated several features consistent with an alteration in Jab1 activity including an increase in activator protein-1 (AP-1) activity, increased expression of AP-1 and HIF-1-dependent genes, and reduced expression of the cell-cycle inhibitor p27(Kip1). Psoriasin overexpression was also associated with alteration of cellular functions that are associated with increased malignancy, including increased growth, decreased adhesion, and increased invasiveness in vitro, as well as increased tumorigenicity in vivo in nude mice. We conclude that intracellular psoriasin influences breast cancer progression and that this may occur through stimulation of Jab1 activity.

Expression analysis of the mouse S100A7/psoriasin gene in skin inflammation and mammary tumorigenesis.

BACKGROUND: The human psoriasin (S100A7) gene has been implicated in inflammation and tumor progression. Implementation of a mouse model would facilitate further investigation of its function, however little is known of the murine psoriasin gene. In this study we have cloned the cDNA and characterized the expression of the potential murine ortholog of human S100A7/psoriasin in skin inflammation and mammary tumorigenesis. METHODS: On the basis of chromosomal location, phylogenetic analysis, amino acid sequence similarity, conservation of a putative Jab1-binding motif, and similarities of the patterns of mouse S100A7/psoriasin gene expression (measured by RT-PCR and in-situ hybridization) with those of human S100A7/psoriasin, we propose that mouse S100A7/psoriasin is the murine ortholog of human psoriasin/S100A7. RESULTS: Although mouse S100A7/psoriasin is poorly conserved relative to other S100 family members, its pattern of expression parallels that of the human psoriasin gene. In murine skin S100A7/psoriasin was significantly upregulated in relation to inflammation. In murine mammary gland expression is also upregulated in mammary tumors, where it is localized to areas of squamous differentiation. This mirrors the context of expression in human tumor types where both squamous and glandular differentiation occur, including cervical and lung carcinomas. Additionally, mouse S100A7/psoriasin possesses a putative Jab1 binding motif that mediates many downstream functions of the human S100A7 gene. CONCLUSION: These observations and results support the hypothesis that the mouse S100A7 gene is structurally and functionally similar to human S100A7 and may offer a relevant model system for studying its normal biological function and putative role in tumor progression.

Psoriasin (S100A7) expression is associated with poor outcome in estrogen receptor-negative invasive breast cancer.

PURPOSE: Psoriasin (S100A7) is highly expressed in preinvasive ductal carcinoma in situ of the breast and persistent expression occurs in some invasive carcinomas. This study explores the clinical significance of psoriasin in relation to patient survival in invasive breast cancer. EXPERIMENTAL DESIGN: We examined psoriasin expression by immunohistochemistry in a cohort of 122 estrogen receptor-negative invasive ductal carcinomas. RESULTS: Psoriasin expression was observed in 64 of 122 cases (52%) but was not correlated with other prognostic factors (including progesterone receptor, stage, size, grade, and nodal status) within this cohort. However, in univariate analysis, psoriasin expression (nuclear and cytoplasmic) was associated with a shorter time to progression (P < 0.04) and poor survival (P < 0.03). In multivariate analysis, cytoplasmic psoriasin also emerged as independent indicator of time to progression (hazard ratio, 1.86; 95% confidence interval, 1.02-3.39; P = 0.044) and survival (hazard ratio, 2.12; 95% confidence interval, 1.06-4.23; P = 0.033). CONCLUSIONS: These results suggest that psoriasin expression may be associated with a worse prognosis in estrogen receptor-negative invasive ductal carcinomas and raise the possibility that psoriasin expression may also be an indicator of risk of progression in ductal carcinoma in situ.

S100A7 and the progression of breast cancer.

The S100 gene family comprises more than 20 members whose protein sequences encompass at least one EF-hand Ca2+ binding motif. The expression of individual family members is not ubiquitous for all tissues and there appears to be an element of tissue-specific expression. Molecular analysis of breast tumors has revealed that several S100s, including S100A2, S100A4 and S100A7, exhibit altered expression levels during breast tumorigenesis and/or progression. Subsequent studies have started to describe a functional role for these S100 proteins as well as their mechanism of action and the biochemical pathways they modify. The present review outlines what is known about S100A7 in breast cancer and summarizes the need to better understand the importance of this protein in breast cancer.

S100A7 (psoriasin) expression is associated with aggressive features and alteration of Jab1 in ductal carcinoma in situ of the breast.

INTRODUCTION: The S100A7 (psoriasin) gene is highly expressed in ductal carcinoma in situ (DCIS) of the breast and can be downregulated in invasive carcinoma. Persistent S100A7 expression in invasive carcinoma is associated with a worse prognosis, and this effect may be mediated in part through interaction with the multifunctional cell signaling protein Jab1. METHODS: In order to investigate the relationship between S100A7 and progression from DCIS to invasive carcinoma, we studied S100A7 expression in 136 patients with DCIS (including 46 patients with associated invasive carcinoma) by immunohistochemistry. RESULTS: S100A7 expression was present in 63 out of 136 (46%) of DCIS lesions and was associated with estrogen receptor negative status (P = 0.0002), higher nuclear grade (P < 0.0001), necrosis (P < 0.0001) and inflammation (P < 0.0001). S100A7 status was no different between DCIS with and DCIS without an invasive component, but higher levels of S100A7 were present in DCIS associated with invasive carcinoma (P < 0.004). Analysis of a subset of cases showed that S100A7 expression was also associated with an increase in nuclear Jab1 (n = 43; P = 0.0019) and reduced p27kip1 (n = 47; P = 0.0168). In cases of DCIS associated with invasive carcinoma, there was also a significant reduction in S100A7 between in situ and invasive components (n = 46; P < 0.0001). In pure DCIS cases treated by local excision, there was no difference in frequency of S100A7 expression between patients with recurrence of DCIS (n = 9) and those without (n = 36). CONCLUSION: The findings reported here suggest that, although S100A7 may not be a marker for recurrence of DCIS, it is associated with poor prognostic markers in DCIS and may influence progression of breast carcinoma through its interaction with and influence on Jab1.

RanBPM interacts with psoriasin in vitro and their expression correlates with specific clinical features in vivo in breast cancer.

BACKGROUND: Psoriasin has been identified as a gene that is highly expressed in pre-invasive breast cancer, but is often downregulated with breast cancer progression. It is currently unknown whether psoriasin influences epithelial cell malignancy directly or by affecting the surrounding environment. However the protein is found in the nucleus, cytoplasm as well as extracellularly. In the present study we have sought to identify potential psoriasin-binding proteins and to describe their expression profile in breast tumors. METHODS: The yeast two-hybrid method was used to identify potential binding partners for psoriasin. The interaction of psoriasin with RanBPM was confirmed in-vitro by co-immunoprecipitation. The expression of RanBPM and psoriasin was measured by RT-PCR in a series of breast cell lines, breast tumors and primary lymphocytes. RESULTS: We have identified RanBPM as an interacting protein by the yeast two-hybrid assay and confirmed this interaction in-vitro by co-immunoprecipitation. RT-PCR analysis of RanBPM mRNA expression in cell lines (n = 13) shows that RanBPM is widely expressed in different cell types and that expression is higher in tumor than in normal breast epithelial cell lines. RanBPM expression can also be induced in peripheral blood mononuclear cells by treatment with PHA. RanBPM mRNA is also frequently expressed in invasive breast carcinomas (n = 64) and a higher psoriasin/RanBPM ratio is associated with both ER negative (p < 0.0001) and PR negative status (p < 0.001), and inflammatory cell infiltrates (p < 0.0001) within the tumor. CONCLUSIONS: These findings support the hypothesis that psoriasin may interact with RanBPM and this may influence both epithelial and stromal cells and thus contribute to breast tumor progression.

S100 proteins and their influence on pro-survival pathways in cancer.

The S100 gene family is composed of at least 20 members that share a common structure defined in part by the Ca2+ binding EF-hand motif. These genes which are expressed in a discriminate fashion in specific cells and tissues, have been described to have either an intracellular or extracellular function, or both. S100 proteins are implicated in the immune response, differentiation, cytoskeleton dynamics, enzyme activity, Ca2+ homeostasis and growth. A potential role for S100 proteins in neoplasia stems from these activities and from the observation that several S100 proteins have altered levels of expression in different stages and types of cancer. While the precise role and importance of S100 proteins in the development and promotion of cancer is poorly understood, it appears that the binding of Ca2+ is essential for exposing amino acid residues that are important in forming protein-protein interactions with effector molecules. The identity of some of these effector molecules has also now begun to emerge, and with this the elucidation of the signaling pathways that are modulated by these proteins. Some of these interactions are consistent with the diverse functions noted above. Others suggest that, many S100s may also promote cancer progression through specific roles in cell survival and apoptosis pathways. This review summarizes these findings and their implications.