5268

SERPINB5

serpin peptidase inhibitor, clade B (ovalbumin), member 5;SERPINB5;serpin peptidase inhibitor, clade B (ovalbumin), member 5

PI-5|peptidase inhibitor 5|protease inhibitor 5 (maspin)|serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 5|serpin B5

18q21.33

protein-coding

Maspin has been shown to inhibit tumor cell invasion and metastasis in breast tumor cells. Maspin expression was detected in normal breast and prostate epithelial cells, whereas tumor cells exhibited reduced or no expression. However, the regulatory mechanism of maspin expression remains unknown. We report here a rapid and robust induction of maspin expression in prostate cancer cells (LNCaP, DU145, and PC3) and breast tumor cells (MCF7) following wild type p53 expression from an adenovirus p53 expression vector (AdWTp53). p53 activates the maspin promoter by binding directly to the p53 consensus-binding site present in the maspin promoter. DNA-damaging agents and cytotoxic drugs induced endogenous maspin expression in cells containing the wild type p53. Maspin expression was refractory to the DNA-damaging agents in cells containing mutant p53. These results, combined with recent studies of the tumor metastasis suppressor gene KAI1 and plasminogen activator inhibitor 1 (PAI1), define a new category of molecular targets of p53 that have the potential to negatively regulate tumor invasion and/or metastasis.

Maspin (mammary serpin) is an inhibitor of serine proteases with tumor suppressor activity in breast cancer. Maspin was originally identified by subtractive hybridization in normal breast epithelial cells, but its expression decreased during tumor progression. The loss of maspin gene expression with increasing malignancy is by transcriptional regulation. Maspin is known to be involved in invasion and metastasis, interact with the p53 tumor suppressor pathway, and act as an inhibitor of angiogenesis. The immunohistochemical analysis and reverse-transcription polymerase chain reaction of maspin in normal human breast tissue and breast carcinoma indicated a stepwise reduction of maspin expression during the progression from ductal carcinoma in situ to invasive carcinoma to lymph node metastasis. The lack of maspin expression in breast cancer seems to be associated with a short disease-free survival and supports maspins function as an indicator for tumor aggressiveness and metastatic potential. New studies on the gene regulation of maspin provide evidence for promising potential of possible re-expression of maspin in tumor cells. The function of maspin as a tumor suppressor gene involved in tumor invasion, metastasis, and angiogenesis may not be limited to breast cancer.

OBJECTIVES: The suppressor gene maspin (Serpin B5) is a promising candidate for future treatment. We have examined the messenger RNA (mRNA) and protein expression of maspin in normal breast tissue, breast cancer primaries, brain metastases and breast cancer cell lines. Results were compared to hormone receptor expression and proliferation index. METHODS: Maspin mRNA expression was examined by real-time polymerase chain reaction in fresh frozen human samples and breast cancer cell lines MCF-7, T47-D and MDA-MB-231. Maspin protein, estrogen and progesterone receptor expression as well as Ki-67 proliferation index were detected by immunohistochemistry from 16 patients with breast cancer primaries and breast cancer brain metastases. RESULTS: In relation to normal breast tissue, maspin mRNA expression was decreased in primary tumors and again decreased in brain metastases. Normalized C(T) values were 1 (normal tissue), 0.3 (primary tumors) and 0.13 (brain metastases). Immunohistochemistry revealed same tendencies. In comparison to poorly invasive breast cancer cell lines, maspin mRNA expression was decreased in highly invasive and metastatic 231-parental cell lines. In contrast, maspin mRNA expression was increased in 231-brain, and it was not detectable in 231-bone. Patients with maspin-positive primary tumors showed longer survival. DISCUSSION: This finding adds maspin to the list of metastasis suppressor genes possibly involved in the formation of breast cancer brain metastases.

Maspin, a tumor suppressor (SERPINB5), inhibits cancer migration, invasion, and metastasis in vitro and in vivo. The tumor-suppressing effects of maspin depend in part on its ability to enhance cell adhesion to extracellular matrix. Although the molecular mechanism of maspin's action is still unclear, its functional domain is believed to be located at the reactive center loop (RCL). We have elucidated the role of maspin RCL on adhesion, migration, and invasion by transfecting the highly invasive human breast carcinoma MDA-MB-231 cell line with pcDNA3.1-His/FLAG containing wild-type maspin, ovalbumin, or maspin/ovalbumin RCL chimeric mutants in which maspin RCL is replaced by ovalbumin (MOM) and vice versa (OMO). MDA-MB-231 cells transfected with maspin- or OMO-containing recombinant expression plasmid manifested significant increase in adhesion to fibronectin and reduction in in vitro migration and invasion through Matrigel compared with mock transfection or cells transfected with ovalbumin or MOM. Proteomics analysis of maspin- or OMO-transfected MDA-MB-231 cells revealed reduction in contents of proteins known to promote cancer metastasis and those of ubiquitin-proteasome pathway, while those with tumor-suppressing properties were increased. Furthermore, MDA-MB-231 cells containing maspin or OMO transgene have significantly higher levels of ubiquitin and ubiquitinated conjugates, but reduced 20S proteasome chymotrypsin-like activity. These results clearly demonstrate that the tumor-suppressive properties of maspin reside in its RCL domain.

BACKGROUND & AIMS: Little is known about functions of microRNA (miR) passenger strands (miR*) or their roles in tumor development or progression. We screened for miRs and miR* with levels that were altered in metastatic colorectal cancer (CRC) cells and human tumor samples and investigated their targets and effects on cell function and tumor progression in mice. METHODS: We performed array-based profile analysis to identify miRs with levels that were increased more than 2-fold in metastatic (SW620) CRC cells compared with nonmetastatic (SW480) cells. Quantitative polymerase chain reaction and in situ hybridization analyses were used to measure miRNA levels in CRC cell lines and human tumor samples. We used miRNA duplex mimics or inhibitors to increase and decrease levels of miRNA in CRC cells and assessed their activities and ability to form metastatic xenograft tumors in nude mice. RESULTS: Levels of miR-221* and miR-224 were reduced in metastatic compared with nonmetastatic CRC cells; levels in human tumor samples correlated inversely with tumor stage and metastasis to lymph nodes as well as patient survival times. SW480 cells transfected with miR-221* or miR-224 inhibitors had increased motility in vitro compared with SW480 control cells and formed larger, more metastatic tumors when injected into mice. SW620 cells transfected with miR-221* or miR-224 mimics had reduced migration and motility in vitro and formed smaller tumors with fewer metastases in mice compared with control SW620 cells. We identified the 3 untranslated region of MBD2 messenger RNA as a target of miR-221* and miR-224. MBD2 silences the gene encoding maspin, a suppressor of metastasis. In CRC cells, we found that miR-221* and miR-224 increase the expression of maspin through MBD2 down-regulation. CONCLUSIONS: In metastatic CRC cells, reduced levels of miR-221* and miR-224 increase levels of MBD2, thereby decreasing expression of the metastasis suppressor maspin. Increased activities of miR-221* and miR-224 reduce growth and metastasis of CRC xenograft tumors in mice; these miRs might be developed as therapeutic reagents or biomarkers of CRC progression.

ETHNOPHARMACOLOGICAL RELEVANCE: A traditional Chinese medicine Scutellaria baicalensis is prescribed for the treatment of a variety of inflammatory diseases and tumors in clinic in China. However, the detailed mechanism of anti-metastasis effect of wogonin, a main active ingredient of Scutellaria baicalensis, remains elusive. AIM OF THE STUDY: The present study was designed to investigate the action and mechanism of wogonin on the mobility and invasion of human gallbladder carcinoma GBC-SD cells. MATERIALS AND METHODS: Viability, apoptosis, mRNA and protein expression of tumor cells were analyzed by MTT, Annexin V staining, real-time PCR and Western blot, respectively. The migration and invasion assay was used to evaluate the anti-metastasis effect of wogonin. Knockdown of maspin was performed by specific small interference RNA. RESULTS: Wogonin at the dose of 1-10 muM, which did not induce apoptosis, significantly inhibited the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells. In addition, the expressions of matrix metalloproteinase (MMP)-2, MMP-9 and phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2) but not phosphorylated Akt were dramatically suppressed by wogonin in a concentration-dependent manner. Furthermore, the metastasis suppressor maspin was confirmed as the downstream target of wogonin. Both maspin mRNA and protein were upregulated by wogonin. Interestingly, the knockdown of maspin resulted in almost completely blocking of wogonin-induced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2 as well as the mobility and invasion activity of GBC-SD cells. CONCLUSIONS: These findings suggest that wogonin inhibits cell mobility and invasion by upregulating the metastasis suppressor maspin. Together, these data provide novel insights into the chemoprotective effect of wogonin, a main active ingredient of Chinese medicine Scutellaria baicalensis.

The thrombin receptor protease activated receptor-1 (PAR-1) is overexpressed in metastatic melanoma cell lines and tumor specimens. Previously, we demonstrated a significant reduction in tumor growth and experimental lung metastasis after PAR-1 silencing via systemic delivery of siRNA encapsulated into nanoliposomes. Gene expression profiling identified a 40-fold increase in expression of Maspin in PAR-1-silenced metastatic melanoma cell lines. Maspin promoter activity was significantly increased after PAR-1 silencing, suggesting that PAR1 negatively regulates Maspin at the transcriptional level. ChIP analyses revealed that PAR-1 decreases binding of Ets-1 and c-Jun transcription factors to the Maspin promoter, both known to activate Maspin transcription. PAR-1 silencing did not affect Ets-1 or c-Jun expression; rather it resulted in increased expression of the chromatin remodeling complex CBP/p300, as well as decreased activity of the CBP/p300 inhibitor p38, resulting in increased binding of Ets-1 and c-Jun to the Maspin promoter and higher Maspin expression. Functionally, Maspin expression reduced the invasive capability of melanoma cells after PAR-1 silencing, which was abrogated after rescuing with PAR-1. Furthermore, tumor growth and experimental lung metastasis was significantly decreased after expressing Maspin in a metastatic melanoma cell line. Moreover, silencing Maspin in PAR-1-silenced cells reverted the inhibition of tumor growth and experimental lung metastasis. Herein, we demonstrate a mechanism by which PAR-1 negatively regulates the expression of the Maspin tumor-suppressor gene in the acquisition of the metastatic melanoma phenotype, thus attributing an alternative function to PAR-1 other than coagulation.

For most cancer cell types, the acquisition of metastatic activity leads to clinically incurable disease. Improvements in surgery and radiotherapy, and the development of new chemotherapeutic agents or their use in new combinations, have, so far, only incrementally improved patient survival. Despite the obvious importance of metastasis, the process remains incompletely characterized at the molecular and biochemical levels. tumor metastasis is a complex process and requires multiple cellular functions over time. From cellular invasion, extravasation from the primary tumor, intravasation to the secondary organs, to successful colonization, tumor cells utilize many cellular or biochemical mechanisms to complete the metastatic spread. During the process of metastasis, there are consistent changes in gene expression. Studies of genes that are reduced or silenced have yielded surprising insights into in vivo mechanisms of regulating tumor metastasis. This review describes a tumor suppressor gene, Maspin, which is often silenced in cancer cells and exhibits suppressing activity against tumor growth and metastasis. Maspin has been shown to be involved in processes that are important to both tumor growth and metastasis such as cell invasion, angiogenesis, and more recently apoptosis. Hence, many efforts have been devoted to deciphering the molecular mechanism of maspin. While some insights have come from the protease inhibitory effect of maspin, more perceptive results on how maspin may function in suppressing tumor metastasis have come from studies of gene manipulation, protein interactions and global protein profiling.

OBJECTIVE: Maspin, a 42-kDa protein, belongs to the serpin family of protease inhibitors and is known to have tumor-suppressor function. In this study, we investigated the interrelationship between clinicopathologic findings and maspin expression in oral squamous cell carcinoma (OSCC). METHODS: Using immunohistochemical techniques to examine the expression levels of maspin in OSCC, maspin expression in OSCC was detected in 46 (64.8%) of 71 cases. We also compared the clonicopathologic features of OSCC cases with maspin expression levels. Moreover, we examined expression of maspin in eight cell lines derived from OSCC using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: There was a significant correlation between decreased maspin expression and T-category (P < 0.01), lymph metastasis (P < 0.0001), and mode of invasion (P < 0.0001). Patients with positive maspin expression had a significantly better prognosis (P < 0.001). Lower expression of maspin was also seen in cell lines derived from grade 4D, which shows stronger invasive potential than other grades of OSCC. CONCLUSION: Maspin may be a useful marker to identify the potential for progression in OSCC.

OBJECTIVE: To investigate the correlation of Maspin expression with angiogenesis in ovarian cancer. METHODS: The expression of Maspin, VEGF, heparanase gene was detected by immunohistochemistry in formalin-fixed, paraffin-embedded specimens of normal ovaries (n=31) and epithelial ovarian cancers (n=35). Correlations of Maspin, VEGF and heparanase expression with clinicopathological parameters of ovarian tumors were statistically analyzed. Microvessel density (MVD) was counted by endothelial cells immunostained with anti-CD34 monoclonal antibody. RESULTS: The expression of Maspin, VEGF, heparanase protein were up-regulated in ovarian cancer cells compared with control group (P < 0.05). Significant association was observed in Maspin protein expression and clinical parameters such as non-serous ovarian carcinoma, lower tumor stage and non-ascites (P < 0. 05). VEGF protein expression correlated with ascites positively (P < 0.05). Heparanase protein expression was identified obviously to correlate with lymph node involvement and ascites (P < 0.05). MVD has significant correlation with lymph node involvement and tumor stage (P < 0.05). Maspin protein expression correlated negatively with VEGF and MVD(P < 0.05), but had no relationship with heparanase (P > 0.05). Significant direct association was demonstrated between VEGF protein expression and MVD (P < 0.05), but VEGF protein expression had no correlation with heparanase. CONCLUSION: Maspin might have inhibitory effects on invasion and metastasis of epithelian ovarian cancer.

BACKGROUND: Early metastasis and infiltration are survival limiting characteristics of pancreatic ductal adenocarcinoma (PDAC). Thus, PDAC is likely to harbor alterations in metastasis suppressor genes that may provide novel diagnostic and therapeutic opportunities. This study investigates a panel of metastasis suppressor genes in correlation to PDAC phenotype and examines promoter methylation for regulatory influence on metastasis suppressor gene expression and for its potential as a diagnostic tool. METHODS: metastatic and invasive potential of 16 PDAC cell lines were quantified in an orthotopic mouse model and mRNA expression of 11 metastasis suppressor genes determined by quantitative RT-PCR. Analysis for promoter methylation was performed using methylation specific PCR and bisulfite sequencing PCR. Protein expression was determined by Western blot. RESULTS: In general, higher metastasis suppressor gene mRNA expression was not consistent with less aggressive phenotypes of PDAC. Instead, mRNA overexpression of several metastasis suppressor genes was found in PDAC cell lines vs. normal pancreatic RNA. Of the investigated metastasis suppressor genes, only higher AKAP12 mRNA expression was correlated with decreased metastasis (P < 0.05) and invasion scores (P < 0.01) while higher SERPINB5 mRNA expression was correlated with increased metastasis scores (P < 0.05). Both genes promoters showed methylation, but only increased SERPINB5 methylation was associated with loss of mRNA and protein expression (P < 0.05). SERPINB5 methylation was also directly correlated to decreased metastasis scores (P < 0.05). CONCLUSIONS: AKAP12 mRNA expression was correlated to attenuated invasive and metastatic potential and may be associated with less aggressive phenotypes of PDAC while no such evidence was obtained for the remaining metastasis suppressor genes. Increased SERPINB5 mRNA expression was correlated to increased metastasis and mRNA expression was regulated by methylation. Thus, SERPINB5 methylation was directly correlated to metastasis scores and may provide a diagnostic tool for PDAC.

Inflammation enhances tumour promotion through NF-kappaB-dependent mechanisms. NF-kappaB was also proposed to promote metastatogenesis through epithelial-mesenchymal transition. Yet a mechanistic link between inflammation and metastasis is missing. We identified a role for IkappaB kinase alpha (IKKalpha), activated by receptor activator of NF-kappaB (RANK/TNFRSF11A), in mammary epithelial proliferation during pregnancy. Owing to similarities between mammary and prostate epithelia, we examined IKKalpha involvement in prostate cancer and its progression. Here we show that a mutation that prevents IKKalpha activation slows down CaP growth and inhibits metastatogenesis in TRAMP mice, which express SV40 T antigen in the prostate epithelium. Decreased metastasis correlated with elevated expression of the metastasis suppressor Maspin, the ablation of which restored metastatic activity. IKKalpha activation by RANK ligand (RANKL/TNFSF11) inhibits Maspin expression in prostate epithelial cells, whereas repression of Maspin transcription requires nuclear translocation of active IKKalpha. The amount of active nuclear IKKalpha in mouse and human prostate cancer correlates with metastatic progression, reduced Maspin expression and infiltration of prostate tumours with RANKL-expressing inflammatory cells. We propose that tumour-infiltrating RANKL-expressing cells lead to nuclear IKKalpha activation and inhibition of Maspin transcription, thereby promoting the metastatic phenotype.

Maspin is a serine protease inhibitor with anti-tumor activity, including inhibition of tumor growth, angiogenesis, invasion, motility, and metastasis. Normal mammary and prostate cells express maspin at high levels. In contrast, breast and prostate cancer tissue samples and cell lines exhibit reduced or no expression of the maspin transcript. Previously we have demonstrated that introduction of an intact chromosome 18 into the bone-derived metastatic prostate cancer cell line, PC-3, resulted in reduced in vitro growth and in vivo metastatic potential. The goal of this study was to determine whether maspin is the tumor/metastasis suppressor on chromosome 18 responsible for this phenotype. To investigate whether maspin, when produced at endogenous levels, is capable of inhibiting metastasis to bone we transfected a bacterial artificial chromosome (BAC) genomic clone containing the maspin gene into PC-3 cells that aggressively metastasize to bone in an animal model. The BAC transfected PC-3 cells exhibited an in vitro phenotype consistent with maspin acting as a tumor suppressor. Analysis of the PC-3 maspin transfectants in an in vivo bone metastasis assay resulted in significant reduction of the number and severity of skeletal metastasis, compared with parental PC-3 cells. However, maspin had no effect on the ability of PC-3 cells to metastasize to extra-skeletal sites in this model. These results indicate that maspin expression likely plays a role in reducing the tumor cell's ability to seed to bone or in inhibition of growth in the bone microenvironment. However, it does not affect the ability to metastasize to distant sites.