Maspin (mammary serine protease inhibitor or SerpinB5) acts as a tumor suppressor when overexpressed in aggressive cancer cell lines. However, its role in human cancer is controversial. Maspin expression has been associated with a poor prognosis in some studies, whereas in others, with favorable outcome. The clinical data suggest, however, that nuclearlocalized maspin is associated with improved survival. We hypothesized that the tumor suppressor activity of maspin may require nuclear localization, and that the discordance between clinical and experimental reports is a consequence of the variable subcellular distribution of maspin. Furthermore, we surmized that nuclear maspin could function as a tumor suppressor through the regulation of genes involved in tumor growth and invasion. Maspin or maspin fused to a nuclear export signal were expressed in metastatic human breast and epidermoid carcinoma cell lines. We found that pan-cellular localized maspin inhibited in vivo tumor growth and metastasis when assessed in xenograft chicken embryo and murine mammary fat pad injection models. However, when maspin was excluded from the nucleus via a nuclear exclusion signal, it no longer functioned as a metastasis suppressor. Using chromatin immunoprecipitation, we show that nuclear maspin was enriched at the promoter of colony-stimulating factor-1 (CSF-1) and associated with diminished levels of CSF-1 mRNA. Our findings demonstrate that the nuclear localization of maspin is required for its tumor and metastasis suppressor functions in vivo, and suggest that its mechanism of action involves, in part, direct association of maspin with target genes. KEYWORDS: breast cancer; chromatin immunoprecipitation; maspin; metastasis; nuclear localization Maspin (mammary serine protease inhibitor or SerpinB5), a member of the serpin family of serine protease inhibitors, was identified in 1994 as a tumor and metastasis suppressor. 1Overexpression of maspin inhibits cell motility and invasion in vitro, and decreases tumor formation, metastasis and angiogenesis in vivo.2 Initially, secreted maspin was thought to inhibit migration and increase cell adhesion via interaction with b1-integrin and other extracellular matrix components. 2 However, the molecular mechanism of such activity has yet to be convincingly demonstrated, and remains the subject of debate. Indeed, the very existence of extracellular maspin has recently been questioned. 3 Maspin has also been detected in the nucleus. 2 Maspin expression has been characterized in different cancers, particularly in breast cancer. Several studies showed that maspin mRNA is reduced in primary tumors and undetectable in metastases. 4,5 Conversely, others have shown that increased maspin is associated with poor prognosis. 6,7 These conflicting observations might be explained by distinct subcellular localization of maspin in cancer cells. Indeed, recent studies indicate that nuclear maspin associates with well-differentiated phenotype and improved survival, whereas cytoplasmic maspin is associated...

Introduction Breast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. So far, these epigenetic contributions to breast cancer metastasis have not been well characterized, and there is only a limited understanding of the functional mechanisms affected by such epigenetic alterations. Furthermore, no genome-wide assessments have been undertaken to identify altered DNA methylation patterns in the context of metastasis and their effects on specific functional pathways or gene networks.

BackgroundWe have previously identified genome-wide DNA methylation changes in a cell line model of breast cancer metastasis. These complex epigenetic changes that we observed, along with concurrent karyotype analyses, have led us to hypothesize that complex genomic alterations in cancer cells (deletions, translocations and ploidy) are superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes observed in breast cancer metastasis.Methodology/Principal FindingsWe undertook simultaneous high-resolution, whole-genome analyses of MDA-MB-468GFP and MDA-MB-468GFP-LN human breast cancer cell lines (an isogenic, paired lymphatic metastasis cell line model) using Affymetrix gene expression (U133), promoter (1.0R), and SNP/CNV (SNP 6.0) microarray platforms to correlate data from gene expression, epigenetic (DNA methylation), and combination copy number variant/single nucleotide polymorphism microarrays. Using Partek Software and Ingenuity Pathway Analysis we integrated datasets from these three platforms and detected multiple hypomethylation and hypermethylation events. Many of these epigenetic alterations correlated with gene expression changes. In addition, gene dosage events correlated with the karyotypic differences observed between the cell lines and were reflected in specific promoter methylation patterns. Gene subsets were identified that correlated hyper (and hypo) methylation with the loss (or gain) of gene expression and in parallel, with gene dosage losses and gains, respectively. Individual gene targets from these subsets were also validated for their methylation, expression and copy number status, and susceptible gene pathways were identified that may indicate how selective advantage drives the processes of tumourigenesis and metastasis.Conclusions/SignificanceOur approach allows more precisely profiling of functionally relevant epigenetic signatures that are associated with cancer progression and metastasis.

The zinc-binding protein metallothionein (MT) is associated with resistance to apoptosis. We examined whether MT regulates the zinc-dependent antiapoptotic transcription factor nuclear factor B (NF-B), which is up-regulated under many conditions that lead to elevated MT expression. NF-B protein levels and NF-B-dependent reporter gene activity were examined in clonal MT(ϩ) (MT-WT) and MT(Ϫ) (MT-KO) fibroblastic cell lines. The amount of cellular NF-B p65 protein in MT-KO was less than 20% of the amount in MT-WT cells, in accord with increased sensitivity of MT-KO cells to apoptosis. NF-B p65 mRNA levels, and NF-B p50 subunit and IB␣ protein levels, were unchanged. NF-B activity assessed by expression of a transfected NF-B reporter construct was less than half that observed in MT-KO cells. Decreased nuclear localization of NF-B p65 in MT-KO clones was not responsible for differences in activity. In fact, MT-KO cells had higher nuclear levels of NF-B p65 than did MT-WT cells, despite a lower cellular NF-B level and function, suggesting that metallothionein mediated the specific activity of NF-B. Reconstitution of MT by stable incorporation of an MT-1 expression vector in MT-KO cells resulted in increased NF-B p65 (but not IB␣ or NF-B p50), increased NF-B-dependent reporter activity, and increased resistance to apoptosis. These data support the hypothesis that metallothionein positively regulates the cellular level and activity of NF-B.

Metallothioneins (MTs) mediate resistance to metal and non-metal toxicants. To differentiate the role of MTs from other protective factors, resistance to zinc (Zn), cadmium (Cd), tertbutyl hydroperoxide (tBH), and cisplatin (CDDP) was compared in renal cell lines from wild type (MT-WT) and MT-1/MT-2 knockout (MT-KO) mice. MT-WT cells were more resistant to tBH than MT-KO cells but, unexpectedly, were more sensitive to Zn, Cd, and CDDP. Thus, basal expression of MT conferred resistance to tBH, but not to Cd or CDDP. Pretreatment with Zn increased MT expression and enhanced resistance to Cd and CDDP only in MT-WT cells, indicating a critical role for MT in this form of resistance. By contrast, Zn-pretreatment increased resistance to subsequent Zn exposure, but did not alter resistance to tBH, regardless of MT-status. Therefore, Zn-induced resistance to subsequent exposure to Zn (but not to Cd or CDDP) was mediated by non-MT factors, and neither Zn-induced MT nor other factors affected tBH sensitivity. Furthermore, antisense down-regulation of MT in human HeLa cells reduced basal MT levels and resistance to TBH, but not to Cd or CDDP. Therefore, basal MT alone can mediate resistance to TBH (but not to Cd or CDDP) in mouse and human cells. These data suggest that MT can mediate resistance to toxicants by different mechanisms, some of which correlate with the cellular content of MT protein. Moreover, resistance to some agents (Cd and CDDP) can be enhanced by inducing MT. Resistance to other agents (tBH) requires only basal (non-induced) MT levels.