Nuclear location of tumor suppressor protein maspin inhibits proliferation of breast cancer cells without affecting proliferation of normal epithelial cells
© Machowska et al.; licensee BioMed Central Ltd. 2014
Received: 22 July 2013
Accepted: 11 February 2014
Published: 28 February 2014
Maspin, which is classified as a tumor suppressor protein, is downregulated in many types of cancer. Several studies have suggested potential anti-proliferative activity of maspin as well as sensitizing activity of maspin for therapeutic cytotoxic agents in breast cancer tissue culture and animal models. All of the experimental data gathered so far have been based on studies with maspin localized cytoplasmically, while maspin in breast cancer tumor cells may be located in the cytoplasm, nucleus or both. In this study, the effect of maspin cytoplasmic and nuclear location and expression level on breast cancer proliferation and patient survival was studied.
Tissue sections from 166 patients with invasive ductal breast cancer were stained by immunohistochemistry for maspin and Ki-67 protein. The localization and expression level of maspin were correlated with estimated patient overall survival and percent of Ki-67-positive cells. In further studies, we created constructs for transient transfection of maspin into breast cancer cells with targeted cytoplasmic and nuclear location. We analyzed the effect of maspin location in normal epithelial cell line MCF10A and three breast cancer cell lines - MCF-7, MDA-MB-231 and SKBR-3 - by immunofluorescence and proliferation assay.
We observed a strong positive correlation between moderate and high nuclear maspin level and survival of patients. Moreover, a statistically significant negative relationship was observed between nuclear maspin and Ki-67 expression in patients with invasive ductal breast cancer. Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells. No such differences were observed in cells with cytoplasmic maspin. We found a strong correlation between nuclear maspin and loss of Ki-67 protein in breast cancer cell lines, while there was no effect in normal epithelial cells from breast. The anti-proliferative effect of nuclear maspin on breast cancer cells was statistically significant in comparison to cytoplasmic maspin.
Our results suggest that nuclear maspin localization may be a prognostic factor in breast cancer and may have a strong therapeutic potential in gene therapy. Moreover, these data provide a new insight into the role of cytoplasmic and nuclear fractions of maspin in breast cancer.
KeywordsMaspin Breast cancer Nuclear maspin Cell proliferation Ki-67
Maspin (Mammary Serine Protease Inhibitor) was first identified in normal mammary glands and breast cancer cells. Based on structural homology, maspin belongs to the serpin superfamily (serpin b5) . Since serpin b5 has no inhibitory activity against serine proteases, its role in cells is not fully understood .
In normal mammary glands, maspin is expressed, at a high level, in myoepithelial cells, while it is not found in luminal cells [1, 3]. Maspin may be located in cytoplasm, nucleus, at the cell surface and in extracellular matrix of myoepithelial cells in normal mammary glands . However, there have been no specific domains or sequences identified that may ensure maspin nuclear localization or secretion. Maspin is responsible for cell adhesion and mobility during embryogenesis and mammary gland development . Maspin is also important in the development of the mammary gland. At the early stage of lactation, maspin causes a lower level of milk proteins, including casein and WAP (whey acidic protein) .
Maspin is classified as a class II tumor suppressor. Maspin demonstrates proapoptotic, antimetastatic and antiangiogenic properties, exerting an inhibitory effect on tumor cell survival, mobility, invasiveness and metastasis ability, and also reduces the tumor tissue vascularization [7–10]. In many studies, it has been found that a decreased level of maspin causes cancer progression and transition from non-invasive to invasive cancer [11, 12]. In vitro studies showed that in primary cell lines derived from tumors maspin is expressed, while after several passages the maspin level decreases until complete loss [7, 13]. In secondary breast cancer cell lines maspin is absent . Clinical data indicate a positive correlation between higher maspin expression level and lower degree of differentiation, lower grade of tumor and improved survival of patients [10, 15].
Despite these data, there are some controversial and contradictory data about maspin prognostic significance and importance of its expression. In many cancer studies, including those related to breast cancer, a negative and positive correlation are described with reference to high or low maspin expression level as a prognostic factor of tumor development [16–19].
Many reports have suggested that biological significance, activity and clinical implications of maspin in various types of cancer depend on its subcellular localization [19–22]. In many types of cancers, including breast, ovarian, lung, larynx, renal and colon cancer, there has been indicated a positive correlation between nuclear maspin location and molecular markers of good prognosis, benign instead of malignant form of cancer, better patient survival and long-term remission [19, 20, 23–26]. However, the significance of nuclear maspin localization in cancer is still not clear enough to use maspin localization pattern as an unquestioned diagnostic or prognostic factor. Maspin’s mechanism of action, especially its nuclear fraction, is not very well understood and requires further examination for better understanding. Recently, a few attempts have been made to clarify this controversy of anticancer activity and molecular mechanism of action of maspin using different models [22, 27–29] but they have not clarified fully the essential question of the potential different activities of cytoplasmic and nuclear fraction of maspin, because in studies performed so far maspin was mainly localized in cytoplasm or ubiquitously in cytoplasm and cell nucleus [22, 30, 31].
That is why we made an attempt to develop a breast cancer tissue culture model system for studies of function of cytoplasmic and nuclear maspin independently. This breast cancer cell line model system together with clinical data from the patients allowed us to resolve the effect of nuclear and cytoplasmic maspin in breast cancer on proliferation and its potential as a genetic drug in breast cancer gene therapy.
Patient samples and ethical issues
Patients’ characteristics N = 166
< = 50
Lymph nodes (N)
Paraffin sections were placed on Superfrost + slides, deparaffinized in xylene, rehydrated in a series of decreasing concentrations of ethanol and washed in phosphate buffered saline (PBS). After antigen retrieval by boiling in a microwave in citrate buffer (pH 6.0), the endogenous peroxidase activity was blocked by incubating sections with 3% H2O2 solution for 30 minutes.
Slices were incubated for one hour at room temperature with primary antibodies diluted in background reducing buffer (DakoCytomation): mouse anti-human maspin (clone G167-7, BD Pharmingen), dilution 1:400 or mouse anti-Ki-67 (clone MIB-1, DakoCytomation), dilution 1:10. After washing three times with PBS for 5 minutes, slices were incubated for 15 min with secondary, biotinylated antibody, followed by 30 min incubation with streptavidin-horseradish peroxidase (DakoCytomation). Color was developed with 3,′-diaminobenzidine (DAB) for 3-7 minutes. Nuclei were stained with Mayer’s hematoxylin.
To evaluate maspin expression, a semiquantitative method  was used to determine reaction intensity and percentage of positive cells from three representative fields of the specimen at 40× magnification. Specimens were scored according to percentage: 1-5% - 1 point, 6-50% - 2 points, >50% - 3 points and reaction intensity: weak - 1 point, moderate - 2 points, strong - 3 points. Points for both percentage and intensity were added and specimens were categorized into four groups: negative reaction - 0 points (0 points for percentage and intensity), weak reaction - 1 point (1-2 points), moderate reaction - 2 points (3-4 points), strong reaction - 3 points (5-6 points). Evaluation of maspin expression was conducted independently for the cytoplasm and the nucleus using the same procedure.
To evaluate the proliferative status, three representative fields with the typical signal from Ki-67 antigen was chosen . At 40× magnification, nuclei showing expression of Ki-67 antigen in relation to all nuclei were counted. Proliferative status of tumors was evaluated as the percentage of positive cells and classified into two groups: less than 20% and more than 20% (according to the limit value of predictive and prognostic assessment of Ki-67 expression in breast cancer).
Human breast cancer cell line MCF-7 was cultured in Eagle’s medium (EMEM, Lonza) and MDA-MB-231 and SKBR-3 cell lines were cultured on Dulbecco’s modified Eagle’s medium (DMEM with low glucose, Lonza) supplemented with 10% fetal bovine serum (Sigma), 1% Glutamax (Gibco) and 1% antibiotic/antimycotic (Gibco). MCF10A cell line was cultured on DMEM/F12 medium (medium mixture, Lonza) supplemented with 5% horse serum (Gibco), 1% Glutamax (Gibco), 1% antibiotic/antimycotic (Gibco), 20 ng/ml epidermal growth factor (Sigma), 0.5 µg/ml hydrocortisone (Sigma), 100 ng/ml cholera toxin (Sigma) and 10 µg/ml insulin (Sigma). MCF-7 (ATTC HTB-22), SKBR-3 (ATTC HTB-30), MDA-MB-231 (ATTC HTB-26) and MCF10A (ATTC CRL-10317) cell lines were kindly provided by Dr Paweł Surowiak (Wrocław Medical University) and Prof. Hermann Lage (Institute of Pathology, Charite Campus Mitte, Humboldt University, Berlin). MCF-7 stable cell lines expressing enhanced green fluorescent protein (EGFP) and maspin-EGFP were cultured in supplemented Eagle’s medium with G418 antibiotic in a final concentration of 150 µg/ml. All cell lines were maintained at 37°C in 5% CO2.
Control plasmid pEGFP-C1 ensuring expression of EGFP was purchased from Clontech. Expression vector with maspin cDNA sequence (NM_002639) was constructed based on pReceiver M03 (GeneCopoeia). In order to obtain nuclear localization of maspin with fusion protein EGFP, the nuclear localization signal (NLS) was inserted between the sequences encoding maspin and EGFP. NLS was inserted by site-directed mutagenesis using QuikChange XL kit (Stratagene). Sequences of primers are the following: (forward/reverse) 5′-CTCCTTACGGTCAT CCAAAAAAGAAGAGAAAGGTC ATGGCTAGCGTGAGC-3′; 5′-GCTCACGCTAGCCAT GACCTTTCTCTTCTTTTTTGG ATGACCGTAAGGAG-3′. The amino acid sequence of NLS is PKKKRKV.
Stable and transient cell transfection
For generation of stable cell lines expressing EGFP, maspin-EGFP and maspin-NLS-EGFP, MCF-7 cells were plated on a 10 cm cell culture dish and after 24 hours transfected with complexes of plasmid DNA and MetafectenePro (Biontex). Six hours later cell culture medium was removed and replaced with complete medium and after 48 hours the post-transfection medium was replaced with complete selection medium containing antibiotic G418 to a final concentration of 400 µg/ml. The selection was monitored by EGFP expression and negative cell death and G418 concentration was gradually reduced.
For transient transfection to immunofluorescence analysis breast cancer cells were plated on a 24-well plate 24 hours before transfection. Six hours after transfection with complexes of MetafectenePro and plasmid DNA medium was replaced with fresh complete medium. After appropriate time cells were fixed and stained as described below.
For immunofluorescence experiments, cells were plated on glass coverslips, fixed in 4% paraformaldehyde for 20 minutes and permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were incubated in primary antibody solution overnight at 4°C, washed with PBS, and incubated for 1 hour with secondary antibody solution at room temperature and washed again in PBS. Coverslips were mounted on glass slides using DABCO mounting medium (Fluka) with DAPI. Staining was visualized on a Zeiss 510 Meta confocal microscope using the 63X objective. Primary antibodies used for staining were mouse anti-human maspin (BD Pharmingen, 1:25), mouse anti-human Ki-67 (DakoCytomation, 1:50), rabbit anti-human lamin C (a kind gift from prof. C.J Hutchison, 1:20). Secondary antibodies were donkey anti-mouse conjugated with TRITC and donkey anti-rabbit conjugated with Cy5 (both from Jackson ImmunoResearch). After 24 hours to 120 hours following transfection, EGFP-expressing cells were counted for expression of Ki-67 antigen in five to ten fields of view and the Ki-67-positive subpopulation was calculated as the percentage of all transfected cells.
Cell proliferation assay
For cell proliferation assay cells were plated on a 10 cm cell culture dish and after 24 hours transfected with complexes of plasmid DNA and MetafectenePro (Biontex). Medium was replaced every three days with complete medium (without G418). After 24 hours to 9 days following transfection EGFP-expressing cells and all cells were counted in four fields of view and the EGFP-positive subpopulation was calculated as the percentage of all cells.
SPSS software (SPSS version 17.0., Chicago, Illinois, USA) was used for statistical analysis of histopathological results. In order to analyze the correlation between the maspin protein level in cytoplasm and/or nucleus and the presence of Ki-67 antigen, contingency tables were used. Statistical significance of differences was assessed using the Pearson’s chi-square test or, if the assumptions were not met, Fisher’s exact test estimated with simulation methods. The correlation was evaluated by Spearman’s test. Significance was defined at the level of P-value ≤0.05 by the two-tailed test. Overall survival was estimated by the Kaplan-Meier method. The logrank test (the Mantel-Cox test) was used to evaluate statistical differences between two curves (for cytoplasmic pool of maspin and nuclear pool of maspin).
Microsoft Office Excel 2003 was used for statistical analysis of data obtained from experiments on cell lines. To evaluate the significance of differences between control and experimental groups the two-tailed Student’s t test was used.
Maspin expression and localization in normal and cancer tissue from breast
In normal breast tissue, maspin is expressed in myoepithelial cells (Figure 1a). During cancer development, there is a decline in maspin level in myoepithelial cells and maspin protein is expressed in other cells (Figure 1b-f). Breast cancer cells showed nuclear, cytoplasmic or mixed maspin location (Figure 1g-i).In order to analyze the subcellular location and assess the level of expression of maspin in normal and tumor tissues of breast, a collection of tissue sections from 166 patients with invasive ductal breast cancer was studied using immunohistochemistry. The maspin-specific staining intensity in ductal breast cancer specimens was compared with staining of myoepithelial cells from regions of normal mammary tissue (Figure 1a) and cancer in situ specimens (Figure 1b,c). The staining intensity in myoepithelial cells was generally stronger than in breast cancer tissue.
Maspin localization in patients’ specimens
Number of cases
Maspin intensity of staining
Maspin staining intensity
Correlation between maspin expression level, location and proliferation status and prognosis for patients
There was a statistically significant relationship observed between nuclear maspin and Ki-67 expression (p < 0.001). Spearman’s correlation analysis showed a negative correlation between level of maspin localized in nucleus and percentage of Ki-67 positive cells (r = 0.771; p < 0.001). In specimens in which less than 20% of cells expressed Ki-67, most cells showed moderate or strong staining intensity of nuclear maspin. Most cases of lack or weak staining intensity of nuclear maspin were observed in the second group - more than 20% of Ki-67 positive cells.
Cell culture model for studies of maspin’s subcellular fraction anticancer activity
Maspin location and proliferation of transfected MCF-7 cells
Nuclear maspin shows its anti-proliferative activity in other breast cancer cell lines
Nuclear maspin does not affect proliferation of normal epithelial cells
The lack of inhibition of proliferation of MCF10A cells by maspin indicates that antiproliferative activity of nuclear maspin is restricted solely to/against breast cancer cells. This indicates that nuclear maspin may be used as a genetic drug in breast cancer treatment.
In many publications the various maspin localization has been confirmed, including the nuclear localization, which initially was ignored as an artifact [18, 19, 37]. Nuclear maspin localization was observed in breast, prostate, lung, colorectal, pancreas and larynx cancers [19, 20, 23–26, 37].
Even if the anticancer effect of the nuclear fraction of maspin has been described in some reports, the precise significance of the maspin expression level and subcellular localization is still not completely understood. Moreover, recent reports on maspin function have focused on cytoplasmic maspin or undirected maspin introduction into the cells, which resulted in its cytoplasmic location [30, 31, 37]. Also the biological function of maspin and its influence on degree of differentiation, stage and invasiveness of cancer are not clearly established. Therefore we decided to investigate the effect of maspin localization on breast cancer with reference to clinical data from breast cancer specimens from patients (Figure 1).
Many reports have suggested that biological of maspin in various types of cancer depend on its subcellular localization [19–22]. Therefore, maspin protein level was evaluated independently in cytoplasm and nucleus in each specimen from patients.
In our study, a positive reaction to maspin was demonstrated in most specimens - 89.2%. A mixed reaction, localized both in nucleus and cytoplasm, was observed in most cases; only cytoplasmic localization was observed in 21.1% of cases and only nuclear in 13.3%. Similar data were obtained in studies in ovarian cancer, where most cases of low grading tumor exhibited maspin expression and most of them showed nuclear reaction . In other studies on breast cancer specimens, about 96% of samples showed nuclear maspin expression and a cytoplasmic signal was present in 35% of the cases .
To evaluate the effect of maspin localization on cell proliferation, the proliferation status of cells in samples from patients was determined by immunohistochemical staining for Ki-67 protein (Figure 1j,k). The expression of proliferation marker Ki-67 is one of the main indicators of tumor cell proliferation and tumor grading. Moreover, it is used as a prognostic factor that helps to predict an outcome of cancer treatment [33, 38]. Results obtained in our study indicated a statistically significant correlation between higher level of maspin protein in nuclei and decreased number of cells expressing Ki-67 (Figure 2c,d). These data suggest that the nuclear fraction of maspin has a strong influence on proliferation status of breast cancer cells in breast tissue, inhibiting their growth and division. The cytoplasmic fraction of maspin seems to have either an opposite effect than the nuclear pool or have no significant effect on proliferation status of breast cancer cells. Analyses of the Kaplan-Meier data indicated that in a time span of 2-5 years, lack of cytoplasmic maspin is beneficial over cytoplasmic maspin (Figure 2a,b). Also total lack of maspin is beneficial over low level of nuclear maspin (cumulative survival index 0.7 versus 0.2). The demonstrated correlation indicates the functional significance of each pool of maspin and suggests that maspin level and location may be considered for use as a prognostic marker as well.
To validate and compare results obtained in clinical studies, we decided to establish an in vitro model for assessing the functional significance of nuclear and cytoplasmic localization of maspin in breast cancer cell lines: MCF-7, MDA-MB-231 and SKBR-3. We also used normal epithelial cell line MCF10A as a control for normal breast tissue. These breast cancer cell lines do not express endogenous maspin because of its silencing mainly by epigenetic processes: CpG island methylation and histone deacetylation [39, 40]. Control, normal cell line MCF10A does express maspin but at a relative moderate level and the protein is rather uniformly distributed in the cytoplasm.
All of the previously used methods of maspin re-expression or overexpression [32, 41, 42] in tumors and breast cancer cell lines had some disadvantages. Classical cDNA transfection, transduction by adenoviruses, protein administration and re-expression by artificial transcription factors resulted in cytoplasmic or extracellular localization of maspin. Such in vitro experimental models did not address properly the problem of dual maspin location observed in breast cancer tissues. That is why our major aim was to develop an easy and appropriate model for studies of the effect of differently located maspin fractions on breast cancer cell lines.
In order to devise a simple model system allowing for controlled placement of maspin in cytoplasm and cell nucleus, we used EGFP protein as a fusion tag. EGFP protein (27 kDa, no NLS and NES signals) alone is capable of two-way diffusion through nuclear pore complexes . Our model allows for active transport of maspin with fusion protein but prevents free diffusion. For driving maspin-EGFP fusion protein into the cell nucleus we added a NLS signal sequence in a short linker protein fragment between maspin and EGFP (Figure 3b) to allow for proper folding of maspin and the EGFP part of the protein.Analyses of location of maspin fusion proteins in MCF-7 cells using confocal microscopy fully supported our idea of driving maspin fusion proteins to the cytoplasm and nucleus using EGFP and NLS (Figure 3c).In other breast cancer cell lines (SKBR-3 and MDA-MB-231) the expression level of maspin fusion protein was similar but the ratio between nuclear and cytoplasmic fractions of maspin NLS-EGFP fusion proteins was a little lower (lower efficiency of maspin placement to targeted location) (Figure 6). This effect may be caused by maspin retention or active transport by binding/interacting proteins. MCF10A cells show a similar ratio between the nuclear and cytoplasmic maspin protein (Figure 7c). When both endogenous and exogenous maspin is analyzed using antibodies against maspin protein the lower efficiency of maspin placement to the targeted location is achieved since endogenous maspin locates in cytoplasm (Figure 7c). Thus in transfected MCF10A cells the level of maspin protein in nuclei is comparable with all other cancer cell lines tested while cytoplasmic maspin (endogenous and exogenous together) is roughly about 10-20% higher than in tested cancer cell lines.
Taking the observations together, we successfully constructed a simple, efficient and complete model to study the influence of maspin localization on breast cancer cell growth, which can be used for further studies using transient transfection.Usefulness of this model was confirmed during our attempts to select cancer cell lines expressing nuclear maspin (maspin-NLS-EGFP), which failed completely and always within 2-3 weeks of selection. Since it was possible to select many cell lines expressing cytoplasmic maspin (Figure 3d) this demonstrated from the beginning the different and anti-proliferative activity of nuclear maspin. However, obtaining clones expressing maspin-EGFP required a longer period of time and lower concentration of selection antibiotic (because of cell death) than EGFP clones. Moreover, after transfection and a couple of days of selection, there were more EGFP-positive proliferating clones than in maspin-EGFP, despite the transfection efficiency being similar. Therefore, overexpression of maspin localized in cytoplasm may lead to slowdown of cell proliferation.
Nevertheless, there was no evidence that it was caused directly by a negative effect of cytoplasmic maspin on cells. It may be a result of overexpression of a larger protein with some biological functions, whilst EGFP is smaller and should not interfere with any cellular processes. However, previous reports demonstrated that exogenous maspin protein addition or transfection with plasmid encoding maspin (cytoplasmic location) resulted in increased cell sensitivity to cytotoxic agents, increased cell adhesion and inhibition of proliferation [42, 44].
Proliferation assays and Ki-67 protein staining performed on MCF-7 breast cancer cell line indicated that the antiproliferative activity of maspin is the strongest in cells with nuclear maspin. For this reason, we have not been able to generate a stable breast cancer cell line expressing maspin localized in the nucleus and it does not allow us for precise studies of signaling pathways triggered by maspin. In the literature, there was no information about attempts of generation of a stable cell line with nuclear maspin, so it seems that this was the very first attempt to do so. Perhaps the generation of such a stable cell line will be possible only when maspin-NLS will be under a strictly controlled inducible promoter.Anti-proliferative activity of nuclear maspin on MCF-7 cells (Figures 4 and 5) was confirmed on other breast cancer cell lines: SKBR-3 cells and highly invasive MDA-MB-231 cell line (Figure 6). This indicates that nuclear maspin may be used as a prognostic marker and possibly as a therapeutic agent for cancer treatment using engineered maspin cDNA, for obtaining nuclear location of maspin, as a genetic drug in gene therapy.In order to confirm the potential usefulness of nuclear maspin as a genetic drug we tested its potential negative effect on normal breast tissue using normal epithelial cell line MCF10A. This normal breast tissue cell line expresses maspin which is located mostly in cytoplasm (Figure 7c). Transient expression of maspin did not have any significant effect on proliferation of MCF10A cells or the number of Ki-67 protein expressing cells (Figure 7a,b,d), thus confirming the high potential of nucleus-directed maspin as an anticancer agent in gene therapy. But still, the major question of what is the molecular mechanism of maspin action remains unanswered.
The major difficulty clouding the biological activity of maspin is lack of sufficient knowledge of molecular mechanisms regulating maspin subcellular location in normal and cancer tissues. In the maspin protein sequence, neither characteristic domains nor sequences which may target maspin to the nucleus were found. Passive transport to the nucleus is rather unlikely due to maspin’s weight (42 kDa) and structure. This is verified experimentally since simple expression of maspin cDNA in many cancer cells results in cytoplasmic location of the protein. EGFP (27 kDa) expression results in dual nucleo-cytoplasmic location.
Many proteins may be translocated to the nucleus as a result of phosphorylation, for example the transcription factors IRF (interferon regulatory factors) . Maspin, in its structure, has many potential phosphorylation sites. In vitro studies on mammary epithelial cells transfected with maspin indicate that maspin protein is phosphorylated by a kinase domain from the epidermal growth factor receptor (EGFR) . This may regulate interaction of maspin with its binding partners which in turn, depending on their function and modifications, may translocate to and from the cell nucleus, affecting maspin location. On the other hand, maspin presence in a particular fraction may modulate the activity of interacting protein.
Proteins that may directly interact with maspin are not well known, although some studies in recent years suggest direct interaction of maspin with proteins associated with oxidative stress (GST - glutathione S-transferase) , heat shock proteins (HSP), histone deacetylase (HDAC1-histone deacetylase I) , and IRF6 (interferon regulatory factor 6) , possibly indirectly affecting function of such transcription factors as Egr-1 and CGF2 . Moreover, maspin may indirectly modulate the pro-apoptotic protein BAX (Bcl-2-associated X protein) , increase expression of the antiangiogenic thrombospondin and affect the expression of E2F1 [49, 50]. Maspin is also implied in increase of expression of proteins from chromatin remodeling complex SMARCA2 and decreased expression of cytokines inducing inflammation and cell proliferation . There was also observed maspin association with chromatin at the promoter of colony-stimulating factor-1 (CSF-1), which caused inhibition of tumor growth . Some of these interactions may play an important role in maspin antiproliferation activity or maspin translocation to the cell nucleus.
The most interesting crosstalk between breast cancer cell proliferation and maspin offers two interactions of maspin: with IRF6 transcription factor and with HDAC1 in a GST-dependent manner. IRF6 promotes changes in cell adhesion, mobility and cell cycle probably (among others) through cadherin and vimentin. IRF6 locates both in cytoplasm and cell nucleus and interacts with maspin only when it is phosphorylated . Recent studies demonstrated that IRF phosphorylation and subsequent proteasomal degradation are associated with induction of proliferation in normal breast epithelial cells (MCF10A). Concomitantly with this in breast cancer cell lines (MCF-7 and MDA-MD-231) overexpression of IRF6 together with maspin (located cytoplasmically) induced a synergistic effect of inhibition of proliferation but not total inhibition of proliferation . This result resembles our data on proliferation slowdown by cytoplasmic maspin (Figure 5a,b).
The second directly interacting partner is HDAC1 complexed with GST. Cytoplasmic maspin may sequester complexes containing HDAC1 and GST and modulating its transport to nucleus and activity. Nuclear maspin may inhibit HDAC1 in a GST-dependent manner (oxidative stress sensitive) and prevent chromatin remodeling and change in transcription in an oxidative dependent manner .
The intriguing question is whether the loss of Ki-67 protein in cells with nuclear maspin is the cause or the result of the anti-proliferative effect of nuclear maspin on cells. The presence of Ki-67 protein is commonly used as a proliferation marker but its cellular functions are diverse and complex. During interphase it plays a crucial role in structural organization of nuclei, transcription and splicing of ribosomal RNA. The function of Ki-67 protein during mitosis is not fully understood but it belongs to the group of chromosomal passenger proteins and is located on mitotic chromosomes during mitosis [33, 38]. Moreover, Ki-67 is necessary for forming a proper connection between microtubules and chromosome and for correct mitosis . The time frame of cell death of nuclear maspin-containing cells fits exactly the 3-day interval between loss of Ki-67 protein (72 h) and cell death (144 h) (Figures 4, 5, and 6).
Exogenous and cytoplasmic maspin may increase cell adhesion by activation of ERK1/2 and MAP kinases and independently the PI3K pathway (through atypical PKCzeta) and inhibit cell mobility by inhibition of Rac1 and PAK1 pathways. Increased cell adhesion may cause inhibition of cell proliferation [31, 54]. Therefore, overexpression of maspin localized in cytoplasm may lead to slowdown of cell proliferation, which may explain why breast cancer cells transfected with maspin-EGFP (cytoplasmic maspin) slow down proliferation but do not stop proliferating. Unfortunately, current knowledge of maspin does not provide any sufficient explanations for the function of nuclear maspin in complete blocking of proliferation of breast cancer cell lines in our model system. Since we also demonstrated that nuclear maspin is crucial for better prognosis and inhibition of proliferation of breast tumors in vivo it suggests that nuclear maspin may be successfully used as a prognostic marker and potential genetic drug for gene therapy for breast cancer.
Our results indicate that nuclear maspin localization may be a prognostic factor in breast cancer and may have a strong therapeutic potential. High level of nuclear maspin is associated with better survival among breast cancer patients and lower proliferation status; thus nuclear maspin can be considered as a new marker of good prognosis of breast cancer. We also observed the inhibitory effect of nuclear maspin on cell proliferation in vitro in the three most frequently used breast cancer cell line models (MCF-7, SKBR-3 and MDA-MB-231), while there was no effect on proliferation of normal MCF10A breast cells. Maspin localization in the cell nucleus correlates with consecutive loss of Ki-67 protein, cell proliferation inhibition and cell death. Our data suggest a strong potential of nuclear maspin in breast cancer gene therapy treatment and provide a new insight into the role of maspin in breast cancer.
Bcl-2-associated X protein
Dulbecco’s modified Eagle’s medium
Enhanced Green Fluorescent Protein
Epidermal growth factor receptor
Early growth response protein 1
Eagle’s minimum essential medium
Extracellular signal-regulated kinase 1/2
GC-binding factor 2
Histone deacetylase 1
Human Epidermal Growth Factor Receptor 2
Heat shock proteins
Interferon regulatory factor 6
- Kip 1:
Cyclin-dependent kinase inhibitor p27
Nuclear localization signal
Nuclear pore complexes
p21 protein activated kinase 1
Protein kinase C isotype zeta
Ras-related C3 botulinum toxin substrate 1
Signal transducer and activator of transcription 3
Whey acidic protein.
We would like to thank Dr. Pawel Surowiak for providing breast cancer cell lines and the normal epithelial cell line from breast. We also would like to thank Pathology Department of Lower Silesia Cancer Center for histologically evaluaed, archived specimens from patients. This work was partially supported by the Polish Ministry of Science and Higher Education “Statutory Grant (1013/S/WB/2011-2013) and Wroclaw Research Centre EIT + under the project “Biotechnologies and advanced medical technologies” BioMed (POIG.01.01.02-02-003/08) (for MM and RR), financed by the European Regional Development Fund (Operational Programme Innovative Economy, 1.1.2). Funding bodies had no influence on design, analysis and interpretation of data as well as in writing the manuscript and decision for publication.
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