Breast Cancer Gene Therapy Research Articles

A convenient plasmid system for construction of helper‐dependent adenoviral vectors and its application for analysis of the breast‐cancer‐specific mammaglobin promoter

Helper-dependent (HD) adenovirus (Ad) vectors, deleted of all viral coding sequences, have a higher cloning capacity, improved performance of tissue-specific promoters, and reduced toxicity in animals relative to first-generation Ad vectors, making these vectors promising tools for gene transfer in vitro and in vivo. However, the large size of HDAd precursor plasmids renders them relatively difficult to manipulate due to the paucity of unique restriction enzyme sites suitable for transgene insertion and to the size constraints imposed by the viral packaging machinery. We have constructed a series of HDAd precursor plasmids that allows cassette insertion at a unique site in the vector backbone. We have tested whether these vector backbones will support the tissue-specificity of inserted expression cassettes in a study of the activity of the potentially breast-cancer-specific mammaglobin promoter and enhancer. We report here the generation of a series of HDAd precursor plasmids, both with and without an additional reporter expression cassette, that were designed to accommodate a wide range in size of inserted DNA. The system was validated for transcriptional targeting studies by demonstrating the tissue-specificity and activity of the mammaglobin promoter rescued using this precursor system. In addition, we have extended our previous studies on the mammaglobin promoter by demonstrating that two copies of the mammaglobin enhancer fused to the minimal promoter surpassed the activity of the single enhancer/promoter by at least 10-fold in breast cancer cells while maintaining only minimal expression in normal cells both in vitro and in a mouse tumor model. This versatile plasmid system simplifies the construction of HDAd vectors and was valuable in demonstrating the targeting potential of the mammaglobin promoter for breast cancer gene therapy.

Gene therapy for breast cancer. — review of clinical gene therapy trials for breast cancer and mdr1 gene therapy trial in cancer institute hospital

Gene therapy for advanced breast cancer is anticipated to be a useful therapeutic approach. Strategies in ongoing clinical protocols can be divided into four groups: (1) suppression of oncogenes or transfer of tumor-suppressor genes; (2) enhancement of immunological response; (3) transfer of suicide genes; (4) protection of bone marrow using drug resistance genes. We have started a clinical study of multidrug resistance (MDR1) gene therapy. Advanced breast cancer patients received high dose chemotherapy and autologous peripheral blood stem cell transplantation(PBSCT)with MDR1-transduced hematopoietic cells, and then were treated with docetaxel. Two patients have been treated so far, and in vivo enrichment of MDR1-transduced cells with docetaxel treatment has been seen. Both patients are in complete remission and had no apparent adverse effects from the MDR1 gene transfer.

MUC1 gene overexpressed in breast cancer: structure and transcriptional activity of the MUC1 promoter and role of estrogen receptor alpha (ERα) in regulation of the MUC1 gene expression

BackgroundThe MUC1 gene encodes a mucin glycoprotein(s) which is basally expressed in most epithelial cells. In breast adenocarcinoma and a variety of epithelial tumors its transcription is dramatically upregulated. Of particular relevance to breast cancer, steroid hormones also stimulate the expression of the MUC1 gene. The MUC1 gene directs expression of several protein isoforms, which participate in many crucial cell processes. Although the MUC1 gene plays a critical role in cell physiology and pathology, little is known about its promoter organization and transcriptional regulation. The goal of this study was to provide insight into the structure and transcriptional activity of the MUC1 promoter.ResultsUsing TRANSFAC and TSSG soft-ware programs the transcription factor binding sites of the MUC1 promoter were analyzed and a map of transcription cis-elements was constructed. The effect of different MUC1 promoter regions on MUC1 gene expression was monitored. Different regions of the MUC1 promoter were analyzed for their ability to control expression of specific MUC1 isoforms. Differences in the expression of human MUC1 gene transfected into mouse cells (heterologous artificial system) compared to human cells (homologous natural system) were observed. The role of estrogen on MUC1 isoform expression in human breast cancer cells, MCF-7 and T47D, was also analyzed. It was shown for the first time that synthesis of MUC1/SEC is dependent on estrogen whereas expression of MUC1/TM did not demonstrate such dependence. Moreover, the estrogen receptor alpha, ERα, could bind in vitro estrogen responsive cis-elements, EREs, that are present in the MUC1 promoter. The potential roles of different regions of the MUC1 promoter and ER in regulation of MUC1 gene expression are discussed.ConclusionAnalysis of the structure and transcriptional activity of the MUC1 promoter performed in this study helps to better understand the mechanisms controlling transcription of the MUC1 gene. The role of different regions of the MUC1 promoter in expression of the MUC1 isoforms and possible function of ERα in this process has been established. The data obtained in this study may help in development of molecular modalities for controlled regulation of the MUC1 gene thus contributing to progress in breast cancer gene therapy.

654. Targeting Adenoviral Vectors for Use in Breast Cancer Gene Therapy

The results of many cancer gene therapy clinical trials to date suggest that improvements must be made to increase gene delivery, expression levels, and/or potency of the genes in order to induce tumour regression. However, increasing any of these parameters will require additional safeguards to protect surrounding normal cells. Our goal is to develop adenoviral vector systems that target tumour cells transcriptionally (using various tumour- and tissue- specific promoters) as well as transductionally (by modifying the viral fiber protein to enhance binding to receptors on the tumour cell surface).

AIM2 suppresses human breast cancer cell proliferation in vitro and mammary tumor growth in a mouse model

IFN-inducible proteins are known to mediate IFN-directed antitumor effects. The human IFN-inducible protein absent in melanoma 2 (AIM2) gene encodes a 39-kDa protein, which contains a 200-amino-acid repeat as a signature of HIN-200 family (hematopoietic IFN-inducible nuclear proteins). Although AIM2 is known to inhibit fibroblast cell growth in vitro, its antitumor activity has not been shown. Here, we showed that AIM2 expression suppressed the proliferation and tumorigenicity of human breast cancer cells, and that AIM2 gene therapy inhibited mammary tumor growth in an orthotopic tumor model. We further showed that AIM2 significantly increased sub-G(1) phase cell population, indicating that AIM2 could induce tumor cell apoptosis. Moreover, AIM2 expression greatly suppressed nuclear factor-kappaB transcriptional activity and desensitized tumor necrosis factor-alpha-mediated nuclear factor-kappaB activation. Together, these results suggest that AIM2 associates with tumor suppression activity and may serve as a potential therapeutic gene for future development of AIM2-based gene therapy for human breast cancer.

429. Adenoviral-Mediated Interleukin-15 Receptor-α Gene Therapy of Murine Breast Cancer

Background: Interleukin-15 (IL-15) is a critical cytokine involved in the activation, proliferation and survival of natural killer (NK) and CD8+ T cells. The high-affinity IL-15 receptor (IL-15Rα) expressed on monocytes and dendritric cells supplies IL-15 in trans to NK and T cells. Tumor cells engineered to express IL-15Rα may enhance the immune response to tumor. We studied the antitumor activity of adenoviral-mediated transfer of the murine IL-15Rα gene in a mouse breast cancer model.

Transcriptional targeting modalities in breast cancer gene therapy using adenovirus vectors controlled by α-lactalbumin promoter

The breast-specific antigen alpha-lactalbumin is expressed in >60% of breast cancer tissues. To evaluate the effect of gene therapy for breast cancer by controlling adenovirus replication with human alpha-lactalbumin promoter, we investigated the activity of a 762-bp human alpha-lactalbumin promoter. Alpha-lactalbumin promoter showed significantly higher activity in MDA-MB-435S and T47D breast cancer cells than in normal breast cell lines or other tumor cell lines. We then developed two novel breast cancer-restricted replicative adenoviruses, AdALAE1a and AdE1aALAE1b. In AdALAE1a, expression of adenoviral E1a gene is under the control of alpha-lactalbumin promoter, and in AdE1aALAE1b, expression of both E1a and E1b genes is under the control of a single alpha-lactalbumin promoter. Both breast cancer-restricted replicative adenoviruses showed viral replication efficiency and tumor cell-killing capability similar to wild-type adenovirus in MDA-MB-435S and T47D cells. The replication efficiency and tumor cell-killing capability of both viruses were attenuated significantly in cells that did not support alpha-lactalbumin promoter. AdE1aALAE1b showed better breast cancer-restricted replication than AdALAE1a, suggesting that a transcriptional targeting modality with alpha-lactalbumin promoter controlling both E1a and E1b gene expression is superior to alpha-lactalbumin promoter controlling only E1a gene expression. Importantly, we found that AdE1aALAE1b could be used to target hormone-independent breast tumors in vivo by inhibiting the growth of MDA-MB-435S s.c. tumors. These data showed that alpha-lactalbumin promoter could regulate the replication of adenovirus to target hormone-independent breast cancers, suggesting that alpha-lactalbumin promoter can be used to develop a novel therapeutic modality for hormone-independent breast cancer.

Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system

IntroductionIn view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Adenoviral vector mediated strategies for breast cancer gene therapy and virotherapy are a promising novel therapeutic platform for the treatment of breast cancer. However, the promiscuous tropism of adenoviruses (Ads) is a major concern. Employing tissue specific promoters (TSPs) to restrict transgene expression or viral replication is an effective way to increase specificity towards tumor tissues and to reduce adverse effects in non-target tissues such as the liver. In this regard, candidate breast cancer TSPs include promoters of the genes for the epithelial glycoprotein 2 (EGP-2), cyclooxygenase-2 (Cox-2), α-chemokine SDF-1 receptor (stromal-cell-derived factor, CXCR4), secretory leukoprotease inhibitor (SLPI) and survivin.MethodsWe employed E1-deleted Ads that express the reporter gene luciferase under the control of the promoters of interest. We evaluated this class of vectors in various established breast cancer cell lines, primary breast cancer cells and finally in the most stringent preclinical available substrate system, constituted by precision cut tissue slices of human breast cancer and liver.ResultsOverall, the CXCR4 promoter exhibited the highest luciferase activity in breast cancer cell lines, primary breast cancer cells and breast cancer tissue slices. Importantly, the CXCR4 promoter displayed a very low activity in human primary fibroblasts and human liver tissue slices. Interestingly, gene expression profiles correlated with the promoter activities both in breast cancer cell lines and primary breast cancer cells.ConclusionThese data suggest that the CXCR4 promoter has an ideal 'breast cancer-on/liver-off' profile, and could, therefore, be a powerful tool in Ad vector based gene therapy or virotherapy of the carcinoma of the breast.

A new targeting approach for breast cancer gene therapy using the Heparanase promoter

Gene therapy with adenoviral (Ad) vectors is a promising new approach in the treatment of cancer. Strategies to restrict adenoviral-mediated transgene expression are important to avoid gene transfer into normal cells. Heparanase (HPR) is overexpressed in breast cancer but downregulated in differentiated normal tissue. Expression of the HPR gene was evaluated in breast cancer cells. Biodistribution and liver tropism was evaluated in a mouse model. HPR is highly expressed in breast cancer tissue. The HPR promoter retained its fidelity in an adenovirus context and was activated in breast cancer cells but showed low activity in normal breast cells and the murine liver. We conclude that the HPR pathway is a promising target for the development of breast cancer directed gene therapy strategies

Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system

Successful adenoviral (Ad) vector-mediated strategies for breast cancer gene therapy and virotherapy have heretofore been hindered by low transduction efficiency. This has recently been understood to result from a relative paucity of expression of the primary adenovirus receptor, coxsackie-adenovirus-receptor (CAR), on primary tumor cells. To further investigate this issue, we evaluated the expression of CAR on breast cancer cell lines as well as primary breast cancer cells. With the exception of one patient sample, CAR expression was notably higher in the tumor cells from patients compared to CAR expression in the tumor cell lines. Furthermore, we explored CAR-independent targeting strategies to breast cancer tissue by exploring a panel of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs for their utility in breast cancer gene therapy and virotherapy. These targeting motifs included Ad 3 knob (Ad5/3), canine Ad serotype 2 knob (Ad5CAV-2), RGD (Ad5.RGD), polylysine (Ad5.pK7), or both RGD and polylysine (Ad5.RGD.pK7), and were tested using the breast cancer tissue slice model, which is the most stringent substrate system available. Of all the tested tropism modified Ad vectors, Ad5/3 exhibited the highest transductional efficiency in breast cancer. These preclinical results suggest that Ad5/3 is the most useful modification to achieve higher clinical efficacy of breast cancer gene therapy and virotherapy.

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector was further modified on a transductional level by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber, which allows the vector to use the alphavbeta3 and alphavbeta5 receptors as alternative receptors. The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines which have low expression level of CAR led to greater degree of tumor regression in vivo than did the intratumoral injection of control adenoviral vectors not so modified.

Targeted gene therapy for breast cancer with truncated Bid

We studied the efficiency of the proapoptotic factor tBid, targeted to tumor cells using the promoters of the hTERT, Survivin and Muc1 genes, in killing breast cancer cells. tBid is the active fragment of the proapoptotic protein Bid and is generated in response to death receptor activation. When placed under control of a strong CMV promoter, tBid was highly efficient in killing breast cancer cells. When expression of tBid was driven by tumor-specific promoters, the magnitude of killing was significant in cell lines with high levels of promoter activity. For successful gene therapy with targeted tBid, it is therefore crucial to be able to predict promoter activity prior to selection of the therapeutic construct. To test whether gene expression could serve as a predictor, we correlated expression of Survivin, hTERT and Muc1 genes with the activity of the corresponding promoters in a panel of breast cancer cell lines. Expression of the Muc1 gene correlated well with the activity of its promoter and the resultant tumor cell killing. For the hTERT and Survivin promoters, however, promoter activity did not correlate well with the expression of the corresponding genes. The implications and possible mechanism of these discrepancies are discussed.

Inducible expression of a degradation-resistant form of p27 Kip1 causes growth arrest and apoptosis in breast cancer cells

The cyclin-dependent kinase (CDK) inhibitor p27 Kip1 (p27) is an important regulator of cell cycle progression controlling the transition from G to S-phase. Low p27 levels or accelerated p27 degradation correlate with excessive cell proliferation and poor prognosis in several forms of cancer. Phosphorylation of p27 at Thr187 by cyclin E–CDK2 is required to initiate the ubiquitination-proteasomal degradation of p27. Protecting p27 from ubiquitin-mediated proteasomal degradation may increase its potential in cancer gene therapy. Here we constructed a non-phosphorylatable, proteolysis-resistant p27 mutant containing a Thr187-to-Ala substitution (T187A) which is not degraded by ubiquitin-mediated proteasome pathway, and compared its effects on cell growth, cell-cycle control, and apoptosis with those of wild-type p27. In muristerone A-inducible cell lines overexpressing wild-type or mutant p27, the p27 mutant was more resistant to proteolysis in vivo and more potent in inducing cell-cycle arrest and other growth-inhibitory effects such as apoptosis. Transduction of p27(T187A) in breast cancer cells with a doxycycline-regulated adenovirus led to greater inhibition of proliferation, more extensive apoptosis, with a markedly reduced protein levels of cyclin E and increased accumulation of cyclin D1, compared with wild-type p27. These findings support the potential effectiveness of a degradation-resistant form of p27 in breast cancer gene therapy.

312. Human Plasminogen Kringle 5-Engineered Murine Mammary Cancer Cells Arrest Tumor Growth and Promote Long-Term Survival

Objective: Neoangiogenesis has been strongly correlated with aggressive breast tumor growth and metastasis. However, since tumor-associated neovasculature is not of malignant origin, it retains the ability to respond physiologically to growth arrest signals and is the proposed basis for breast tumor regression in vivo. The fifth kringle (K5) domain of human plasminogen is distinct from angiostatin (K1-4), and has been shown -on its own- to act as a potent suppressor of angiogenesis. We propose that the K5 domain may serve as a potent angiostatic agent and that it may act as a useful therapeutic transgene within a breast cancer gene therapy strategy. To test this hypothesis, we have developed a K5-expressing retroviral vector, gene-modified murine DA3 mammary cells to produce soluble human K5 protein and characterized the anti-tumor potency of the de novo produced K5 peptide in vivo.

Transkriptionales Targeting zur zielgerichteten Krebsgentherapie des Mammakarzinoms

Purpose: Gene therapy with adenoviral (Ad) vectors is a promising new approach for different tumor types. Strategies to restrict adenoviral-mediated transgene expression are important to avoid side effects due to gene transfer into healthy cells. Heparanase (HPR) is highly overexpressed in human cancers including breast cancer but low or undetectable in differentiated, healthy tissue. Material and Methods: To evaluate the utility of HPR as a TSP for breast cancer gene therapy, RT-PCR was performed to evaluate the expression of the HPR gene in various established breast cancer cell lines, primary human breast cancer tissue samples and normal breast cell lines. We constructed an Ad vector, AdHPR.luc, encoding luciferase under the control of the HPR promoter to determine relative activity in a variety of breast cancers, normal human breast cell lines and purified breast cancer tissue samples. An Ad vector containing the ubiquitously expressed CMV promoter (AdCMV.luc) was used as control. Results: Quantitative RT-PCR revealed a 4.5-44.6fold,(p < 0.05) increased expression of the HPR gene in several breast cancer cell lines compared to a normal breast control cell line. When compared to the ubiquitous CMV promoter, the HPR promoter showed a high level of activity in four breast cancer cell lines (5.5 - 12.7% compared to CMV) and primary breast cancer patient samples (8.8-14.4%), whereas activity in normal breast cells was low. Conclusion: These findings show that the HPR pathway is a target for the development of breast cancer directed gene therapy strategies.

Exogenous PTEN gene induces apoptosis in breast cancer cells

To investigate the effects of exogenous PTEN gene on apoptosis of breast cancer cells. Human breast cancer cells of the line MDA468 were cultured. Recombinant plasmid pcDNA3.1-PTEN was constructed and transfected into the breast cancer cells with the lipofectAMINE 2000 transfection technique. Parental MDA468 cells and parental MDA468 cells transfected with blank vector pcDNA3.1(-) were used as control groups. RT-PCR was used to detect the expression of the PTEN mRNA and Western blotting was used to determine the expression of PTEN protein. Epithelial growth factor (EGF) was added into the cultures of MDA468 cells transfected with pcDNA3.1-PTEN or blank vector. Then Western blotting was used to detect the expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by EGF. The aapoptosis of the MDA468 cells was determined by flow cytometry with the double-staining method using FITC-conjugated annexin V and PI. Expressions of PTEN mRNA and protein were shown in the MDA468 cells transfected with pcDNA3.1-PTEN by RT-PCR and Western blotting. Both the expression of p-AKT and that of p-FAK were down-regulated in the MDA468 cells transfected with pcDNA3.1-PTEN in comparison with those in the control cells. The apoptotic rate of the MDA468 cells transfected with PCDNA3.1-PTEN was 21.68%, significantly higher than those of the blank control cells (1.17%) and pcDNA3.1(-)-transfected cells (3.55%, both P < 0.01). Exogenous PTEN suppress the growth of human breast cancer cell and induces apoptosis by phosphatase activity, which provides a new clue to gene therapy for breast cancer.

EpCAM is overexpressed in breast cancer and is a potential target for breast cancer gene therapy.

EpCAM (epithelial cell adhesion molecule) is a cell surface molecule that is known to be highly expressed in colon and other epithelial carcinomas. EpCAM is involved in cell-to-cell adhesion and has been the target of antibody therapy in several clinical trials. To assess the value of EpCAM as a novel target for breast cancer gene therapy, we performed real-time reverse transcription-PCR to quantify the level of EpCAM mRNA expression in normal breast tissue and primary and metastatic breast cancers. We found that EpCAM is overexpressed 100- to 1000-fold in primary and metastatic breast cancer. Silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a 35-80% decrease in the rate of cell proliferation in four different breast cancer cell lines. EpCAM siRNA treatment decreased cell migration by 91.8% and cell invasion by 96.4% in the breast cancer cell line MDA-MB-231 in vitro. EpCAM siRNA treatment was also associated with an increase in the detergent-insoluble protein fraction of E-cadherin, alpha-catenin, and beta-catenin, consistent with the known biology of EpCAM as a regulator of cell adhesion. Our hypothesis is that modulation of EpCAM expression can affect cell migration, invasion, and proliferation by enhancing E-cadherin-mediated cell-to-cell adhesion. These data provide compelling evidence that EpCAM is a potential novel target for breast cancer gene therapy and offer insights into the mechanisms associated with EpCAM gene silencing.

The Breast: Comprehensive Management of Benign and Malignant Diseases

The Breast: Comprehensive Management of Benign and Malignant Diseases

623. Transcriptional Targeting of Breast Cancer Employing the Heparanase Promoter

Breast cancer is a leading cause of death in the United States. Despite some advances in treatment of primary breast cancer, patients with metastatic disease still have no chance of cure and conventional treatment modalities remain palliative. Therefore, the identification of new agents with better antitumor activity merits a high priority in the treatment of advanced or metastatic breast cancer. In this regard, gene therapy with adenoviral (Ad) vectors is a promising new approach for different tumor types. A critical factor determining the utility of Ad vectors for cancer gene therapy is the selectivity of their transgene expression in cancer cells. Strategies to restrict adenoviral-mediated transgene expression are important to avoid side effects due to gene transfer into healthy cells, especially the liver. However, currently available TSPs used in the context of Ad-based breast cancer targeting, are generally not tumor-specific but tissue specific. In this context, Heparanase (HPR), a heparan sulfate-specific endo-β-D-glucuronidase, is highly overexpressed in human cancers including breast cancer and has been shown to play an important role in tumor metastasis. In contrast, HPR expression is low or undetectable in differentiated, healthy tissue. To evaluate the utility of HPR as a TSP for breast cancer gene therapy, RT-PCR was performed to evaluate the expression of the HPR gene in various established breast cancer cell lines, primary human breast cancer tissue samples and normal breast cell lines. We constructed an Ad vector, AdHPR.luc, encoding luciferase under the control of the HPR promoter to determine relative activity in a variety of breast cancer, normal human breast cell lines and purified breast cancer tissue samples. An Ad vector containing the ubiquitously expressed CMV promoter (AdCMV.luc) was used as control. Biodistribution and liver tropism was evaluated after i.v. virus injection into a mouse model. Quantitative RT-PCR reveals a 4.5 – 44.6 fold, (p<0.05) increased expression of the HPR gene in several breast cancer cell lines compared to a normal breast control cell line. In primary breast cancer patient samples the average HPR mRNA copy number was significantly increased (90 fold, p<0.05). When compared to the ubiquitous CMV promoter, the HPR promoter showed a high level of activity in four breast cancer cell lines (5.5% – 12.7% compared to CMV) and primary breast cancer patient samples (8.8%– 14.4%), whereas activity in normal breast cells (1% compared to CMV) was low. The mouse liver toxicity and biodistribution profile of AdHPR.luc is significantly (2.3 fold, p<0.05) repressed compared to AdCMV.luc. In conclusion, we have identified the HPR promoter as a candidate for transcriptional targeting of breast cancer. In an Ad backbone, this promoter appears to be highly active in human breast cancer cells. These findings place the HPR pathway as a target for the development of breast cancer directed gene therapy strategies.

Multiple functions of maspin in tumor progression and mouse development.

Maspin is a unique serpin with diverse biological functions. Initially identified from human normal mammary epithelial cells, maspin expression is either reduced or completely silenced in breast cancers. Numerous studies have implicated maspin function in cancer progression and angiogenesis. Maspin has also been targeted for breast cancer gene therapy. Recently, transgenic and gene knockout mouse models have been used by our laboratory to identify the biological functions of maspin in vivo. In this review, we summarize the multiple functions of maspin in tumor progression and mouse development. These data demonstrate that maspin not only plays a role in tumor progression and metastasis but also is a key regulatory molecule for normal mammary gland and embryonic development.