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_mfor 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

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

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 Ca(2+ )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

Psoriasin (S100A7) expression is altered during skin tumorigenesis

BACKGROUND: Psoriasin (S100A7) expression has previously been associated with psoriasiform hyperplasia as well as with tumor progression in breast cancer. Its expression profile for different stages of skin lesions is unknown. The aim of this study was to determine the relationship between psoriasin (S100A7) and tumor progression in skin. METHODS: Psoriasin was assessed by immunohistochemistry and levels of expression determined by semi-quantitative scoring in skin biopsies from 50 patients. The cohort included normal skin, actinic keratosis, squamous carcinoma in-situ, invasive squamous cell carcinoma, and basal cell carcinoma. RESULTS: In normal skin, psoriasin was rarely detected in epidermis but was expressed in underlying adnexae. In abnormal epidermis psoriasin was frequently expressed in abnormal keratinocytes in actinic keratosis, in-situ and invasive squamous cell carcinoma, but was rarely observed in the basal epidermal layer or in superficial or invasive basal cell carcinoma. The highest levels of expression were seen within squamous carcinoma in-situ. Significantly reduced levels of expression were observed in both unmatched (p = 0.0001) and matched (p < 0.004) invasive squamous cell carcinoma. Psoriasin expression within abnormal squamous lesions correlated with mitotic count (r = 0.54, p = 0.0036), however no significant relation was found with the intensity of dermal inflammatory cell infiltrates assessed within each pathology. CONCLUSION: These results suggest that altered psoriasin expression occurs in abnormal epidermis and that downregulation may be related to the onset of invasion in squamous cell carcinoma in skin

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

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

Abstract 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 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.</p