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Angiogenesis, Metastasis, and the Cellular Microenvironment

Transfection of Keratin 18 Gene in Human Breast Cancer Cells Causes Induction of Adhesion Proteins and Dramatic Regression of Malignancy In vitro and In vivo

¹ Department of Gynecology and Obstetrics, Medical Center Marienhospital Herne, Ruhr-University Bochum, Bochum, Germany and ² Breast Care Institute, Berlin, Germany

Requests for reprints: Gerhard Schaller, Offenbacher Str. 8 14197, Berlin, Germany. Phone: 49-30-822-1838. E-mail: gerhard{at}schaller-berlin.de

	Abstract

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
This study shows that high keratin 18 (K18) expression in tumor cells is associated with reduced invasiveness in vitro and lack of tumorigenicity in nude mice. We previously showed that high K18 expression correlated with a good prognosis and that reducing K18 expression increased the aggressiveness of established breast cancer cell lines. To confirm these observations, we transfected the human K18 gene into the human breast cancer cell line MDA-MB-231 and isolated a stable overexpressing clone. The forced K18 expression was associated with a complete loss of the previously strong vimentin expression in the parent cell line, induction of the K18 dimerization partner K8, and up-regulation of adhesion proteins. These changes were accompanied by a dramatic reduction in the aggressiveness of the K18 transfectants in vitro and in vivo. We conclude that forced reexpression of K18 causes at least partial redifferentiation of the tumor cell, followed by a corresponding regression of malignant phenotype.

	Introduction

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
A characteristic feature of malignant transformation is the loss of cellular differentiation. This causes a whole series of structural changes which together enable the tumor cell to detach itself from its epithelial layer and metastasize. Cytoskeletal and cellular adhesion proteins play an important role in this process (1-3). The intermediate filaments of the cytoskeleton, which in epithelial cells are composed of keratins, seem to be closely involved in these changes (3, 4). The 20 different keratins discovered thus far (5, 6) show function- and tissue-specific expression (7). In the bilaminar breast epithelium, keratins 8 and 18 (K8 and K18) characterize the differentiation compartment (the luminal cells), whereas keratins 5 and 14 are expressed in the proliferation compartment. Expression of keratins 7, 17, and 19 is variable but generally low (5, 7). We have previously shown that 80% of all breast carcinomas exhibited a loss of the differentiation-associated keratins 8 and 18, and that loss of these keratins was associated with a significantly worse prognosis (8, 9). In breast cancer cell lines, invasiveness in vitro and metastatic potential in athymic mice also correlated inversely with the degree of K18 expression (8). Similar results were obtained by Sommer and Thompson, who showed that breast cancer cell lines became more aggressive as keratin filaments were replaced by vimentin, the intermediate filament-protein of mesenchymal cells (2-4). The importance of K18 in tissue integrity is seen in K18 knock-out mice, which die at an early embryonic phase due to hepatic hemorrhage (10).

The functionality of an epithelium, however, depends not only on the structural stability of individual cells but also on their overall cohesion, which is mediated by adhesion proteins. The two most important adhesive compartments in the epithelium are desmosomes and adhesion junctions (11). The structure-stabilizing parts of the cytoskeleton are anchored in these structures, which thus connect the skeletons of the cells involved and generate the high mechanical-loading capacity of epithelia (12, 13). Direct cell-cell contact is established by cadherins, i.e., E-cadherin for the adhesion junctions, and desmoglein and desmocollin for the desmosomes (14, 15). Both types of adhesive plaque contain numerous other proteins, which interact to organize the overall structure and bind the cytoskeleton. Desmosomal plakoglobin, which is identical to -catenin in the adhesion junctions, is the only adhesion protein that features in both desmosomes and adhesion junctions (16, 17). During malignant transformation, the tight network of reciprocal interactions in cellular adhesion is altered dramatically, such that the cell can leave the surrounding tissue and migrate. The finding that loss of K18 has a role in this dedifferentiation process suggests that reexpression of K18 in deficient tumor cells might reverse this process and reduce the malignant phenotype. To examine this hypothesis, we have transfected the complete human K18 gene into the MDA-MB-231 breast cancer cells, which normally exhibit very low K18 expression. Here we report the effects of K18 overexpression in these breast cancer cells in relation to invasiveness, metastatic potential, and adhesion protein expression. In a complementary series of experiments, we analyzed the effect of up-regulation of cell adhesion proteins (by cell selection for adhesiveness through numerous passages) on K18 expression, aggressiveness and metastatic potential.

	Results

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Isolation of Clones
A K18-transfected clone (MDA231-K18) and a control clone transfected with the neomycin resistance gene only (MDA231-neo) were isolated by limiting dilution. An "epithelial" clone was isolated by repeated passaging and selection for high adhesiveness, followed by limiting dilution. Comparison of the clones with wild-type MDA-MB-231 cells showed wild-type cells and MDA231-neo cells to have a characteristic "malignant" phenotype not seen in MDA231-K18 and MDA231-E cells. Thus, wild-type and MDA231-neo cells separated readily and grew in suspension. Their structure was spindle-shaped and fibroblastic. At low cell density in the culture flask, the cells were asymmetrical, suggesting high motility. An epithelial cell layer with close cell-cell contacts was not observed (Fig. 1A and C). MDA231-K18 and MDA231-E cells, on the other hand, exhibited a completely different morphology. Both failed to grow in suspension, but formed a dense confluent layer of highly prismatic cells with close contacts (Fig. 1B and D). Culturing these two clones for 2 weeks without passage caused the cell layer to become increasingly dense and the individual cells increasingly prismatic. Domes subsequently formed through cystic vaulting of the flat cell layer (Fig. 1E and F). This type of dome was never observed in the wild-type or in MDA231-neo cells.

View larger version (182K): [in this window] [in a new window] [Download PPT slide]   FIGURE 1. Morphology of MDA231 clones in culture. A. MDA231-wild-type. B. MDA231-epithelial type. C. MDA231-neo is the control clone transfected with the neomycin resistance gene only. D. MDA231-K18-transfected. Formation of domes by K18-transfected MDA231 cells (E) and by MDA231-epithelial type (F).

Intermediate Filament Pattern
Using immunoblotting, wild-type and sham-transfected cells expressed vimentin but not K18, although MDA231-K18 and MDA231-E showed strong K18 expression but only rudimentary vimentin expression (Fig. 2). The K18 level was even higher than in MCF7 cells, where we previously found the strongest expression comparing several established human breast cancer cell lines (9). The expression of K8, the keratin with which K18 is normally paired within cells, followed a similar pattern to K18 expression (Fig. 2), suggesting that forced expression of the K18 gene is accompanied by up-regulation of its partner molecule K8.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   FIGURE 2. Western blotting of the intermediate filaments K18, vimentin, and K8 expressed by MDA231 clones. wt, MDA231-wild-type; K18, keratin 18–transfected MDA231 cells; neo, control clone transfected with the neomycin resistance gene only; E, epithelial type selected clone of MDA231 parental cells.

View larger version (45K): [in this window] [in a new window] [Download PPT slide]   FIGURE 3. Western blotting of the adhesion proteins desmoglein, plakoglobin, and E-cadherin expressed by MDA231 clones: wt, MDA231-wild-type; K18, keratin 18–transfected MDA231 cells; neo, control clone transfected with the neomycin resistance gene only; E, epithelial type selected clone of MDA231 parental cells.

View larger version (13K): [in this window] [in a new window] [Download PPT slide]   FIGURE 4. Invasiveness of the MDA231 clones (Boyden chamber assay). MDA231-epithelial type selected subclone (E); MDA231-wild-type (wt); MDA231-K18-transfected (K18); MDA231-neomycin resistance gene only as control (neo). Invading cells were quantified via a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. Columns, means; bars, ± SE.

View larger version (33K): [in this window] [in a new window] [Download PPT slide]   FIGURE 5. Tumor growth in athymic mice following inoculation of MDA231 clones into the mammary fat pad. A. Tumors formed by MDA231 control cells, transfected with the neomycin resistance gene only (left), and MDA231 cells transfected with the K18 gene (right). B. Tumor growth versus time. The tumor volume was calculated with a caliper-like instrument as described in Materials and Methods. Points, means; bars, ± SD; wt, MDA231-wild-type; K18, keratin 18–transfected MDA231 cells; neo, control clone transfected with the neomycin resistance gene only; E, epithelial type selected clone of MDA231 parental cells.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   FIGURE 6. Proliferation of MDA231 clones in vitro [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay]. wt, MDA231-wild-type; K18, keratin 18–transfected MDA231 cells; neo, control clone transfected with the neomycin resistance gene only; E, epithelial type selected clone of MDA231 parental cells. A. Growth in standard culture medium over 5 days. Points, means; bars, ± SE. B. Growth in estrogen-free, charcoal-stripped culture medium versus standard culture over 5 days. Ratio of stripped/standard culture medium for each clone (n = 6).

	Discussion

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We previously showed in a retrospective study that high K18 expression in breast tumors was associated with a markedly improved prognosis for the patient, with the majority of patients with high K18 remaining recurrence-free throughout an observation period of >8 years (8). A similar correlation between K8/K18 expression and reduced tumorigenicity was also reported by Pankov et al. (18) for murine pancreatic carcinoma cells in a murine syngeneic transplant model. In the current study, we have obtained K18-overexpressing clones of the human breast cancer cell line MDA-MB-231 by transfection or cell selection, in order to further investigate and confirm the importance of K18 expression in aggressiveness, tumorigenicity, and metastatic potential.

MDA-MB-231 cells, which express virtually no K18, are characteristically aggressively invasive and metastatic (2, 19, 20). However, K18 gene transfer was found to change the expression pattern of many structural and adhesion proteins in the cell. The intermediate filaments of the cytoskeleton underwent a basic structural change. Vimentin, virtually the sole component of intermediate filaments in the wild-type and the sham-transfected controls, was suppressed and largely replaced by keratins. K8, the dimerization partner of K18 and barely expressed in wild-type cells, was induced following transfection. The regression of the vimentin filaments may be highly significant, because numerous clinical studies and in vitro investigations have shown that replacement of epithelial keratins by mesenchymal vimentin in malignant transformation is invariably associated with strong dedifferentiation and increased aggressiveness of the tumor cells (2, 18, 21–24).

K18 transfection also induced a strong increase in the expression of cellular adhesion proteins, including the cadherins of the desmosomes and zonula adherens, desmoglein, and E-cadherin, as well as plakoglobin (-catenin). This last molecule is particularly important for adhesion because it is the only one incorporated into both desmosomes and adhesion junctions.

There is clearly an interaction between the intermediate filaments of the cytoskeleton and the desmosomes, as well as the adherent junctions. The molecular basis for this "concerted action" is not yet known, but might involve transcription factors or signal transduction. Members of the E-cadherin system are known to be involved in several signaling pathways (14, 15, 25), and keratin filaments have also been implicated in these processes (26–28). More is known about the physical connections between structure and adhesion. Interactions of keratin filaments in desmosomal plaques have been extensively described (11, 12, 16, 28, 29), and it is suggested that keratin filaments are necessary for proper structure and function of desmosomes (13). Thus, it is possible that K18 transfection might not only regenerate the keratin filaments but that these could also reorganize the desmosomes by recruiting the requisite proteins and promoting their resynthesis. Conditions are less clear in the E-cadherin system. Here, there is no direct contact with the keratin filaments; however, the desmosomes may be influenced via plakoglobin, which is a component of both systems (30). Then, mediated by plakoglobin, the reorganizing force of the keratin filaments could extend via the desmosomes into the zonula adherens.

The changes in the cytoskeleton and adhesion apparatus were reflected in the altered morphology of the transfected cells. The wild-type and sham-transfected controls were spindle-shaped, typical of dedifferentiated carcinoma cells, and showed only weak adherence. The cells did not form coherent cell layers, became detached in high numbers, and remained viable in suspension. In contrast, the K18-transfected clone formed a dense epithelium from which no viable cells became detached. After some time, the confluent epithelium began to form "domes" through incipient epithelial transport. Such cell-layer protrusions can only develop in the presence of very tight contact surfaces between the cells. It may, therefore, be surmised that reorganization takes place not only in desmosomes and adherent junctions but also in epithelial-tight junctions.

The changes in the protein pattern and morphology of cells after transfer and expression of the K18 gene show a redifferentiation of the malignant tumor cell extending to the functional "original state" of the breast epithelium. This is accompanied by a marked decrease in aggressiveness and tumorigenicity, and emergence of a benign phenotype. Thus, the MDA-MB-231 line, which is characterized by very high invasiveness in the Boyden chamber model, became barely able to penetrate the matrix following K18 transfection. Moreover, the ability of the MDA-MB-231 cells to form tumors and metastasize in athymic mice was abrogated by K18 transfection. The redifferentiation of carcinoma cells triggered by K18 transfection thus markedly reduced the malignant phenotype.

The first step in metastatic spread is detachment of the cell from its coherent epithelial layer. This only succeeds if adhesion structures are already loosened. Once detached, a cell must reach and invade the vascular system to successfully metastasize. This can only be achieved if the cell architecture is flexible and plastically deformable. The intermediate filaments contribute decisively to the mechanical rigidity of the cell. If they are composed of vimentin, the cell is flexible, a property required, for example, by fibroblasts. Keratins, on the other hand, make the cell rigid in keeping with their function in the epithelium. Replacement of vimentin by keratin through K18 transfection thus impedes the motility of the cell and its ability to penetrate endothelial gaps.

We conclude from this study that forced expression of K18 leads to reorganization of the cellular adhesion structures with an expression pattern closely resembling that in healthy breast epithelium. These findings not only confirm the importance of K18 as a prognostic indicator in breast cancer, but may suggest that up-regulation of K18 could be used as a treatment strategy, either by biological modulation or through gene therapy approaches. Targeted delivery of liposome-encapsulated K18 expression vectors (for example by conjugation to anti-HER2/neu antibodies) seems a promising approach in the treatment of breast cancer (31). Such studies are currently under consideration.

	Materials and Methods

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Cell culture
MDA-MB-231 breast cancer cells were obtained from the American Type Culture Collection (HTB-26, Manassas, VA). For these cells, as well as for all subclasses described below, DMEM was used as the culture medium with the following additives: 10% FCS, 200 mmol/L glutamine, 10 IU/mL penicillin, and 10 µg/mL streptomycin (all from Biochrom/Seromed, Berlin, Germany). The cells were kept in a moist CO₂ incubator (5% CO₂ in air) and passaged weekly. The medium was changed twice between passages. Geneticin (G418, Sigma, Deisenhofen, Germany) 500 µg/mL was added to the culture medium for transfected cells.

Transfections
The expression vector pGC 1853, which codes the complete human K18 gene, was a generous gift from Dr. R.G. Oshima. The plasmid was grown in E. coli (MAX Efficiency DH5-Competent Cells, Life Technologies, Karlsruhe, Germany) and isolated with an endotoxin-free preparation kit (Endo-Free Plasmid Maxikit, Qiagen, Hilden, Germany). The transfection into MDA-MB-231 cells was carried out with PerFect Lipid pFx-6 (Invitrogen, Groningen, the Netherlands) at a DNA/lipid ratio of 3:1 in serum-free medium (OptiMem 1, Life Technologies) for 12 hours. A total of 1 µg DNA was used per 5 cm culture dish (Nunc, Roskilde, Denmark). Because the K18 vector carries no selection gene for eukaryote cells, a neomycin-resistance plasmid (pGEM bluescript, Stratagene, La Jolla, CA) was cotransfected at a ratio of 1:10. As a control, cells were also transfected with the resistance plasmid only. After 2 days, the cells were transferred into selection medium (500 µg/mL of G418). Stably transfected clones were isolated by limiting dilution. For selected clones, this step was repeated to ensure monoclonality. The K18-transfected clone was designated MDA231-K18, the mock-transfected control clone MDA231-neo.

Adhesive Subclone
A subclone of epithelial morphology was selected from the MDA-MB-231 wild-type cells by selective passaging. Weakly adherent cells were mechanically detached by tapping and shaking and were then discarded prior to the weekly passages. Thus, MDA-MB-231 cells with strong adhesive properties accumulated in the course of about 30 passages. The procedure described above was used to establish a uniform clone designated MDA231-E.

Western Blotting
Expression of K18, K8, vimentin, E-cadherin, desmoglein, and plakoglobin in the individual cell clones was determined by SDS-PAGE and Western blot. The specified proteins were solubilized with Empigen BB (Calbiochem, Bad Soden, Germany) as described by Lowthert et al. for K18 (32). The solubilization buffer consisted of 2% Empigen in PBS with inhibition of the proteolytic activity by addition of 600 µg/mL of Pefabloc SC and one tablet of Complete Mini per 8 mL of solution (both from Roche Diagnostics, Mannheim, Germany). This procedure releases >95% of the total K18 content from the cell pellet (31). Samples corresponding to 2.5 x 10⁴ cells were separated on a 10% SDS-PAGE gel in reducing conditions in a Mini Subcell system (Bio-Rad, Munich, Germany).

E-cadherin was an exception in that it could not be satisfactorily solubilized according to the specified protocol. Here, the cells were extracted directly in the culture flask with SDS loading buffer. To reduce the high viscosity of the solution, the DNA was sheared with an ultrasound probe.

Separated proteins were transferred to nitrocellulose membranes (Protran 85, Schleicher & Schuell, Dassel, Germany) at 50 V for 16 hours, nonspecific binding was blocked with 4% skimmed milk powder, and specific proteins detected by indirect chemiluminescence with monoclonal antibodies and the Lumi-Light Plus detection kit (Roche Diagnostics).

Antibodies used for detection of protein bands were as follows: K18, CK2 (Chemicon, Hofheim, Germany); K8, clone M20 (ICN/Cappel, Frankfurt, Germany); vimentin, clone 3B4 (DAKO, Hamburg, Germany); E-cadherin, clone 6F9 (Serotec via Biozol, Garching, Germany); desmoglein, CBL 174 (Cymbus Technologies clone DG 3.10, via Dianova, Hamburg, Germany); plakoglobin, CBL 175 (Cymbus Technologies clone PG 5.1).

Following chemiluminescent detection, membranes were exposed to X-ray film (X-OMAT AR, Kodak, Stuttgart, Germany) and scanned with a GS-700 imaging densitometer. The bands then analyzed with the quantification software Quantity One (Bio-Rad).

Invasion Assay
A total of 10⁵ cells/element were plated into Matrigel-coated filter elements with a pore size of 8 µm (Biocoat Matrigel Invasion Chambers, Becton Dickinson, Bedford, MA) in standard culture medium, with 20% FCS in the medium below the filter element. After 12 hours at 37°C, all cells remaining on the upper filter surface were removed with moistened cotton swabs, with care taken to prevent drying of the cells that had migrated to the underside of the membrane. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability test was used for quantification of these cells (CellTiter96 AQ, Promega, Mannheim, Germany). The filter elements were incubated in MTS solution for 4 hours, and absorbance was measured at 490 nm in an ELISA reader (Dynatech, Ulm, Germany). The baseline absorbance was obtained from a filter element incubated in the same medium and conditions but without cells.

Measurement of Tumorigenic and Metastatic Potential in Athymic Mice
A total of 10⁶ cells of the clones -wt, -neo, -K18 or -E and 100 µg of Matrigel (Tebu, Offenbach, Germany) per animal were inoculated into the fat pad of the breast of female athymic NCr-nu/nu mice (Charles River, Fredericksburg, VA; body weight, 21-24 g). Tumor growth was recorded twice daily with a caliper-like instrument. Tumor volumes were calculated by (widths² x length) / 2. The experiment was finished at day 83 after tumor cell inoculation. The animals were then killed and the tumor area, liver, lung, brain, and bone marrow (smears) were removed. The organs were trisected: one part was formalin-fixed; another was cryopreserved in liquid nitrogen; and the third was subjected to PCR (human -satellite DNA from chromosome 17) for detection of micrometastases (33). The formalin-fixed tissue was sectioned and stained for histologic identification of metastases. All animal experiments were done according to the German animal protection law and with permit from the local authorities.

Proliferation Assay
Cells were seeded at 2,000 cells/mL in 300 µL medium in microtiter plates and cultured for up to 5 days. Cell growth was then measured every 24 hours by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay as above; MTS solution was added to the cells, which were then incubated for 4 hours, followed by measurement of absorbance in the supernatant.

To test whether the proliferation is estrogen-dependent the clones were grown parallel in standard culture medium and in medium without phenol red, supplemented with charcoal-stripped FCS (Biochrom/Seromed). After 5 days, the cell growth was determined as mentioned above.

	Acknowledgements

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
We thank Professor R.G. Oshima, Director of the Burnham Institute in La Jolla, CA, for providing the human K18 expression vector. The excellent technical assistance of Blanka Duvnjak and Susanne Thamm is gratefully acknowledged. We are also greatly indebted to the German Research Foundation, without whose generous financial support the investigations would not have been possible.

	Notes

Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 
Grant support: Deutsche Forschungsgemeinschaft DFG Scha 833/1-1 and 1-2.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 7/ 6/04; revised 5/17/05; accepted 5/24/05.

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Top Notes Abstract Introduction Results Discussion Materials and Methods Acknowledgements References

 

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