Psoriasin is aberrantly expressed in human breast cancer and is related to clinical outcomes

Psoriasin (S100A7) is a member of the S100 gene family that is involved in psoriasis, which can be induced in cultured squamous epithelial cells. Previously, this molecule has been shown to be over-expressed in human ductal carcinoma in situ and invasive breast cancers. This current study examine the levels of expression of this molecule in a group of human breast tumours, with particular emphasis on the relationship with the clinical outcomes using quantitative PCR and the distribution in tumours using immunohistochemistry. Psoriasin is primarily expressed in breast cancer cells, and at very low level in normal breast epithelial cells. Quantitative analysis of psoriasin mRNA has shown that breast tumour tissues exhibited a significantly high level of psoriasin compared with normal tissues (p=0.0026). The levels do not correlate with nodal status of breast tumours, however levels in grade 2 and grade 3 tumours were significantly higher compared with that in grade 1 tumours (p=0.07 and p=0.0015, vs grade 1 respectively). Lobular carcinoma also had higher levels of psoriasin compared with ductal tumours. The most interesting observation is that levels of psoriasin were significantly higher in patients who developed metastatic disease and in patients who died of breast cancer (p=0.02 and p<0.001, vs disease-free, respectively). It is concluded that aberrant expression of psoriasin is commonly seen in human breast cancer and that excessively high levels are correlated with the clinical outcomes.     

Psoriasin(S 100A7)蛋白的结构与功能

Psoriasin(S100A7)是一种分子量为22.9 kD的同源二聚体蛋白,属于S100蛋白家族成员。通过与钙离子结合,参与细胞内外重要的生命活动。其基因定位于染色体1q21.2-q22。Psoriasin(S100A7)蛋白最初是从银屑病异常增生的角化细胞中分离得到的,故称为Psoriasin,即银屑素。Psoriasin在异常增生的角化细胞、膀胱鳞状细胞癌和原位及浸润乳腺癌中表达上调。Psoriasin这种分泌和表达特征有可能作为膀胱鳞状细胞癌和乳腺癌等恶性肿瘤的候选标志物。     

Loss of ICAM-1 signaling induces psoriasin (S100A7) and MUC1 in mammary epithelial cells

Psoriasin (S100A7), a member of the S100 gene family, is highly expressed in high-grade comedo ductal carcinoma in situ (DCIS), with a higher risk of local recurrence. Psoriasin is, therefore, a potential biomarker for DCIS with a poor prognosis. High-grade DCIS is characterized by a high proliferation rate and crowded cells, consequently, lose contact with the extracellular matrix. The aim of this study was, therefore, to elucidate the involvement of adhesion signals in the regulation of psoriasin. Protein expression was evaluated by Western blotting, flow cytometry, and immunohistochemistry, and using breast carcinoma SAGE databases available from the CGAP website. Intercellular adhesion molecule 1 (ICAM-1) was down-regulated in MCF10A cells using short hairpin RNA. We found a significant negative correlation between the expression of ICAM-1 and psoriasin, and a positive correlation between psoriasin and MUC1 in normal and DCIS SAGE libraries. In a cluster analysis of 34 adhesion molecules and 20 S100 proteins, we showed that SAGE libraries expressing the S100 proteins—psoriasin, calgranulin-A, and calgranulin-B—clustered together. Interestingly, the expression of all the three proteins correlated strongly to the oncogenic MUC1. We confirmed the negative correlation between ICAM-1 and psoriasin/MUC1, when normal and breast cancer cells were cultured in suspension and on collagen, respectively. The down-regulation of ICAM-1 by short hairpin RNA in MCF10A cells led to the induction of psoriasin, calgranulin-A, calgranulin-B, and MUC1, and we demonstrated that these up-regulations were not ROS dependent. By blocking the phospholipase C (PLC)-IP3 pathway in these cells, we showed that the induction of psoriasin diminished. The results suggest that psoriasin is an intracellular calcium-dependent target of the PLC pathway. Our findings suggest that the down-regulation of ICAM-1 in mammary epithelial cells may contribute both to the high expression of psoriasin seen in some high-grade DCIS tumors and to the induction of MUC1.     

A putative role for psoriasin in breast tumor progression

Psoriasin (S100A7) was identifi;ed as a gene highly expressed in psoriatic keratinocytes and highly and more frequently expressed in ductal carcinoma in situ (DCIS) than in invasive breast carcinomas (IBC), suggesting a potential role in tumor progression. Psoriasin expression is associated with poor prognostic factors in both DCIS and IBC. Several putative functions have been proposed for psoriasin in various disease types, but none of these can fully explain its involvement in breast tumor progression. Here, we show that down-regulation of endogenous psoriasin expression via stable short hairpin RNAs in a human IBC cell line (MDA-MB-468) increases cell migration and invasion without influencing cell proliferation and survival in vitro but inhibits tumor growth in vivo. These seemingly paradoxical results are potentially explained by the dramatic up-regulation and down-regulation of matrix metalloproteinase-13 and vascular endothelial growth factor (VEGF), respectively, observed in cells with decreased psoriasin levels compared with controls. Correlating with this, high psoriasin expression in human IBC is associated with increased angiogenesis and worse clinical outcome, and psoriasin mRNA levels are coordinately regulated with VEGF and other genes related to hypoxia and mitochondrial reactive oxygen species (ROS). Based on these results, we propose that psoriasin may play a role in breast tumor progression by promoting angiogenesis and enhancing the selection for cells that overcome its anti-invasive function. This hypothesis may explain why psoriasin expression is highest in high-grade and/or estrogen receptor-negative tumors, as these are associated with increased hypoxia and ROS, a setting in which the angiogenic effects of psoriasin are most important.     

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.     

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.     

Estrogen receptor-β regulates psoriasin (S100A7) in human breast cancer

We have previously observed a paradoxical relationship of the psoriasin/S100A7 gene with estrogen response in-vitro in ERα positive cells but its association with ERα negative status in-vivo raising the possibility that S100A7 might be regulated by ERβ in breast cancer. Using doxycycline-inducible ERβ and ERα expressing MCF-7 cells the hypothesis that psoriasin/S100A7 is ERβ regulated was investigated To explore the relationship between psoriasin/S100A7 and ERβ expression in-vivo, we also assessed a cohort of 233 ERα negative breast tumors using tissue microarrays and immunohistochemistry. Psoriasin/S100A7 was increased by 17β-estradiol (E2) following ERβ induction, in several clones of ERβ over-expressing but not in the original MCF-7 cells, nor clones over-expressing ERα. The effect of E2 on psoriasin/S100A7 was inhibited by 4-hydroxytamoxifen and ICI 182780 but not with a selective ERα antagonist. An ERβ selective-agonist but not an ERα selective-agonist, induced psoriasin/S100A7. This induction still occurred after stable down-regulation of ERα using siRNA in ERβ inducible cells. E2 increased psoriasin/S100A7 mRNA but cycloheximide treatment inhibited this effect. A relationship between ERβ and psoriasin/S100A7 was observed in the p53 immunohistochemically negative subset of invasive breast tumors in-vivo (r = 0.225, p = 0.046, n = 79). In conclusion we demonstrate that E2 induction of psoriasin/S100A7 can be specifically regulated through ERβ in-vitro and associated with ERβ in-vivo. These data support the hypothesis that psoriasin/S100A7 is specifically regulated by ERβ activity and could be useful to guide future therapies targeting ERβ in certain phenotypic subsets of breast cancers in-vivo.     

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.     

S100A7 (Psoriasin), highly expressed in Ductal Carcinoma <Emphasis Type="Italic">In Situ</Emphasis>(DCIS), is regulated by IFN-gamma in mammary epithelial cells

Background  

The aim of the present work was to explore signal transduction pathways used in the regulation of S100A7 (psoriasin). Members of the S100 gene family participate in many important cellular functions. Psoriasin, S100A8 (calgranulin A) and S100A9 (calgranulin B) are expressed in ductal carcinoma in situ (DCIS), as well as in the hyperproliferative skin disease, psoriasis. In the latter condition, a disturbance in the STAT pathway has recently been reported. This pathway is implicated in the regulation of IFN-gamma, widely recognized as a key cytokine in psoriasis. IFN-gamma also exerts anti-tumor action in a number of tumor cell types, including breast cancer. We therefore examined the effect of IFN-gamma and STAT-signaling on the psoriasin expression.     

Jab1 is a target of EGFR signaling in ERα-negative breast cancer

Introduction

c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERα^-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERα^- phenotype.

Methods

MDA-MB-231 and MDA-MB-468 ERα^-/EGFR⁺ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry.

Results

EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERα^- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004).

Conclusion

Jab1 is a target of EGFR signaling in ERα^- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERα^- breast cancer.     

Jab1 is a target of EGFR signaling in ERalpha-negative breast cancer

Introduction

c-Jun activation domain-binding protein-1 (Jab1) is a multifunctional signaling protein that previously has been shown to be a master regulator of a poor prognostic gene signature in invasive breast cancer and to mediate the action of S100A7. Since epidermal growth factor receptor (EGFR), like S100A7, is often expressed in estrogen receptor-alpha-negative (ERα^-) breast cancer, we set out to investigate the role of Jab1 in mediating EGFR signaling, another facet of the ERα^- phenotype.

Methods

MDA-MB-231 and MDA-MB-468 ERα^-/EGFR⁺ cell lines were assessed for localization of Jab1 and levels of downstream genes by immunofluorescence and nuclear protein extract assay following treatment with epidermal growth factor (EGF) and extracellular signal-regulated kinase (ERK) pathway inhibitor. A cohort of 424 human breast tumors was also assessed by immunohistochemistry.

Results

EGF treatment of cell lines resulted in increased Jab1 nuclear expression. This effect was inhibited by the ERK pathway inhibitor, PD98059. EGF treatment was also associated with colocalization of pERK (phosphorylated ERK) and Jab1 as well as regulation of the Jab1 downstream target gene, p27. When Jab1 activity was knocked down, p27 levels were restored to pre-EGF treatment level. Analysis of EGFR and Jab1 expression in a cohort of invasive breast tumors by tissue microarray and immunohistochemistry confirmed a relationship between EGFR and increased nuclear Jab1 within the ERα^- subset (n = 154, P = 0.019). The same association was also confirmed for S100A7 and Jab1 (P = 0.036), and high Jab1 nuclear expression was most frequent in tumors that were positive for both EGFR and S100A7 (P = 0.004).

Conclusion

Jab1 is a target of EGFR signaling in ERα^- cell lines and breast tumors and therefore may be a common central factor and potential therapeutic target for important cell signaling pathways in ERα^- breast cancer.     

Triptolide inhibits matrix metalloproteinase-9 expression and invasion of breast cancer cells through the inhibition of NF-κB and AP-1 signaling pathways

Triptolide is a diterpenoid epoxide that is endogenously produced by the thunder god vine, Tripterygium wilfordii Hook F. Triptolide has demonstrated a variety of biological activities, including anticancer activities, in previous studies. Invasion and metastasis are the leading causes of mortality for patients with breast cancer, and the increased expression of matrix metalloproteinase-9 (MMP-9) has been shown to be associated with breast cancer invasion. Therefore, the aim of the present study was to investigate the effect of triptolide on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced cell invasion and MMP-9 expression in breast cancer cells. The expression of signal molecules was examined by western blotting, zymography and quantitative polymerase chain reaction; an electrophoretic mobility gel shift assay was also used, and cell invasiveness was measured by an in vitro Matrigel invasion assay. The MCF-7 human breast cancer cell line was treated with triptolide at the highest concentrations at which no marked cytotoxicity was evident. The results demonstrated that triptolide decreased the expression of MMP-9 through inhibition of the TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK) and the downregulation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity. In addition, a Transwell assay revealed that triptolide reduced the ability of MCF-7 cells to invade Matrigel. These data demonstrate that the anti-invasive effect of triptolide is associated with the inhibition of ERK signaling and NF-κB and AP-1 activation, and suggest that triptolide may be a promising drug for 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.     

Preparation and characterization of monoclonal antibodies against mouse Jab1/CSN5 protein

Jab1, also known as the fifth component of the COP9 signalosome complex (CSN5), directly interacts with and regulates the activity and stability of multiple intracellular regulatory molecules, such as c-Jun, p27, p53, Cullin, Smad4, and HIF1alpha. In addition, a high level of Jab1 is observed in a variety of human cancers and is sometimes correlated with a poor prognosis, suggesting that Jab1 contributes to cancer cell proliferation and survival and could be a novel target of cancer therapy. In this report, we generated five mouse monoclonal antibodies to a bacterially produced recombinant mouse Jab1 protein and examined their capabilities and limitations in commonly used assays, including enzyme linked immunosorbent assay (ELISA), Western blotting with denatured and native polyacrylamide gel electrophoresis (PAGE), immunoprecipitation, and immunofluorescence microscopy, finding the most suitable antibody for each application. Because these antibodies proved useful for immunohistochemical staining for Jab1 in fixed sections of human cancer samples, they should be useful in determining the expression and subcellular distribution of Jab1 in human tumors.     

Increased AP-1 activity in drug resistant human breast cancer MCF-7 cells

The expression, DNA binding, and transactivating activity of activator protein 1 (AP-1) was examined in a series of multidrug resistant (MDR) MCF-7 human breast cancer cells that have increasing levels of MDR1 gene expression. We observed an increase in the amount of both c-jun and c-fos mRNA in cells with 12-, 65-, or 200-fold higher resistance to adriamycin when compared to drug-sensitive MCF-7 wild type (WT) cells. Electrophoretic mobility shift assays (EMSA) demonstrated an increase in the DNA binding activity of an AP-1 complex in nuclear extracts from MDR MCF-7 cells when compared to extracts from WT cells. We observed a proportional increase in luciferase expression from a reporter vector containing consensus AP-1 binding sites in transiently transfected MDR cells when compared to WT cells, indicating that AP-1 mediated gene expression is increased in drug-resistant MCF-7 cells. Since the MDR1 promoter contains a putative AP-1 binding site, we used EMSA to examine AP-1 binding activity to an oligonucleotide probe that contained the relevant MDR1 promoter sequences (–123 to –108). Nuclear extracts from resistant MCF-7 cells displayed an increased level of DNA binding of Jun/Jun dimers to the probe, indicating that AP-1 was capable of binding to this promoter site. A luciferase reporter construct containing triplicate copies of the MDR1 promoter sequence was expressed at higher levels in transiently transfected MDR cells when compared to expression in WT cells. Co-transfection of WT cells with a c-jun expression vector and either of the AP-1 luciferase constructs demonstrated that c-jun could activate gene expression from both the consensus and the MDR1 AP-1 sites in a dose dependent manner. In addition, RT-PCR and western blot analysis showed that levels of MDR1 mRNA and Pgp were increased in c-jun transfected WT cells. Taken together, these data indicate that increased AP-1 activity may be an important mediator of MDR by regulating the expression of MDR1.