Maspin

Protease inhibitor 5 (PI5)

Gene: maps to 18q21.3 (Schneider S.S. et al., 1995). The PAI-2 gene maps to the same region. Ets-, AP-1- and hormonal-responsive elements (HRE) have been found in the maspin promoter (Zhang M. et al., 1997a).
mRNA: size: 3.0 kb.
Protein: a 375-amino acid, 42-kDa member of the serpin family of protease inhibitors (also including PAI-1 and PAI-2). Maspin was originally isolated from normal mammary epithelium and was shown to have tumor-suppressive activities.

Cell lines:
- Maspin mRNA was expressed in normal mammary epithelial strains, but not in MCF-7, MDA-MB-157, -231, 435, -436, -468, T-47D, ZR-75-1, BT-549, and Hs578T breast cancer cell (BCC) lines nor in breast fibroblasts (Zou Z. et al., 1994). Southern blot analysis of XbaI-restricted DNA from normal and tumor cells revealed no gross structural alterations of the maspin gene in the tumor cells. This suggests that the maspin gene is downregulated but not mutated in cancer cells.

- Transfection of MDA-MB-435 BCC with the maspin gene did not alter their growth properties, but reduced their ability to induce tumors and to invade through a basement membrane matrix in vitro (Zou Z. et al., 1994).

- Differential expression of maspin in normal and carcinoma-derived mammary epithelial cells was found to be regulated at the transcriptional level. Ets and Ap1 sites have been identified in the maspin promoter that are active in regulating maspin expression in normal mammary epithelial cells but inactive in tumor cells. The Ets site alone was sufficient to activate transcription in a heterologous promoter, whereas the Ap1 site cooperated with Ets in activation. The enhancing function by Ets and Ap1 elements was decreased in primary tumor cells (21NT) and was abolished in invasive tumor cells (MDA-MB-231). Thus, loss of maspin expression during tumor progression results at least in part from the absence of transactivation through the Ets and Ap1 sites (Zhang M. et al., 1997b).

- It was found that single-chain tissue plasminogen activator (sctPA) specifically interacts with the maspin reactive site loop peptide and forms a stable complex with recombinant maspin [rMaspin(i)]. Major effects of rMaspin(i) were observed on plasminogen activation by sctPA. First, rMaspin(i) activated free sctPA. Second, it inhibited sctPA preactivated by poly-D-lysine. Third, rMaspin(i) exerted a biphasic effect on the activity of sctPA preactivated by fibrinogen/gelatin, acting as a competitive inhibitor at low concentrations (< 0.5 microM) and as a stimulator at higher concentrations. Fourth, 38-kDa C-terminal truncated rMaspin(i) further stimulated fibrinogen/gelatin-associated sctPA. rMaspin(i) did not inhibit urokinase-type plasminogen activator, plasmin, chymotrypsin, trypsin, or elastase. The kinetic data were quantitatively consistent with a model in which two segregated domains of maspin interact with the catalytic and activating domains of sctPA. These complex interactions between maspin and sctPA in vitro suggest a mechanism by which maspin regulates plasminogen activation by sctPA bound to the epithelial cell surface (Sheng S. et al., 1998).

- Treatment of MDA-MB-435 BCC with recombinant maspin (rMaspin) was shown to result in the inhibition of BCC invasion in vitro, in the selective adhesion of BCC to a fibronectin matrix, and in the conversion from a fibroblastic to a more epithelial-like phenotype. The inhibition of the invasive process could be abrogated by a blocking antibody to the alpha₅beta₁ integrin (Seftor R.E.B. et al., 1998).

- Cultured normal human mammary epithelial cells (HMECs) were compared to 9 cultured human breast cancer cell (BCC) lines. HMECs expressed maspin mRNA and displayed a completely non-methylated maspin gene promoter with an open chromatin structure. In contrast, 7 of 9 breast cancer cell lines had no detectable maspin expression and 6 of these 7 maspin-negative breast cancer cell lines also displayed an aberrant pattern of cytosine methylation of the maspin promoter. Interestingly, the maspin promoter was completely methylated in maspin-negative normal peripheral blood lymphocytes. This indicates that the maspin promoter is not a functional CpG island and that cytosine methylation of this region may contribute to normal tissue-restricted gene expression. Chromatin accessibility studies with MCF-7 cells, which lack maspin expression and have a methylated maspin promoter, showed a closed chromatin structure compared with HMECs. Moreover, maspin gene expression could be re-activated in MCF-7 cells by treatment with 5-aza-2;-deoxycytidine, a DNA demethylating agent. Thus, aberrant cytosine methylation and heterochromatinization of the maspin promoter may silence maspin gene expression, thereby contributing to the progression of human mammary cancer (Domann F.E. et al., 2000).

Tumors:
- Analysis of breast cancer specimens demonstrated that loss of maspin expression occurred most frequently in advanced cancers. This supports the hypothesis that maspin functions as a tumor suppressor (Zou Z. et al., 1994).

- Maspin expression was reported as being a specific marker for breast cancer (Luppi M. et al., 1996). However, in another study, maspin was found non-suitable for RT-PCR detection of submicroscopic lymph node metastases in breast cancer, as its transcript was also detected in the great majority of control (from non-cancer patients) lymph nodes tested (Merrie AEH et al., 1999).

- Peripheral-blood samples were collected from 30 patients with histologically proven breast cancer before and 4 and 8 days after conventional chemotherapy for three consecutive courses. A total of 216 samples were screened for the presence of maspin mRNA by RT-PCR. Before therapy, all samples but one were negative. After chemotherapy, 11 patients (38%) had positive samples. No difference in the rate of positivity was observed between groups defined according to initial stage, type of chemotherapy, Ki-67-related proliferative activity, or CA 15.3 expression (Sabbatini R. et al., 2000).

Domann F.E. et al. (2000) Epigenetic silencing of maspin gene expression in human breast cancers. Int. J. Cancer 85, 805-810.
Luppi M. et al. (1996) Sensitive detection of circulating breast cancer cells by reverse-transcriptase polymerase chain reaction of maspin gene. Annal Oncol. 7, 619-624.
Merrie A.E.H. et al. (1999) Analysis of potential markers for detection of submicroscopic lymph node metastases in breast cancer. Br. J. Cancer 80, 2019-2024.
Sabbatini R. et al. (2000) Detection of circulating tumor cells by reverse transcriptase polymerase chain reaction of maspin in patients with breast cancer undergoing conventional-dose chemotherapy. J. Clin. Oncol. 18, 1914-1920
Schneider S.S. et al. (1995) A serine proteinase inhibitor locus at 18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc. Natl. Acad. Sci. USA 92, 3147-3151.
Seftor R.E.B. et al. (1998) Maspin suppresses the invasive phenotype of human breast carcinoma. Cancer Res. 58, 5681-5685.
Sheng S. et al. (1998) Tissue-type plasminogen activator is a target of the tumor suppressor gene maspin. Proc. Natl. Acad. Sci. USA 95, 499-504.
Zhang M. et al. (1997a) Expression of maspin in prostate cells is regulated by a positive ets element and a negative hormonal responsive element site recognized by androgen receptor. Proc. Natl. Acad. Sci. USA 94, 5673-5678.
Zhang M. et al. (1997b) Transactivation through Ets and Ap1 transcription sites determines the expression of the tumor-suppressing gene maspin. Cell Growth Differ. 8, 179-186.
Zou Z. et al. (1994) Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 263, 526-529.