Proc. Natl. Acad. Sci. USA Vol. 90, pp. 4261-4265, May 1993 Cell Biology Expression of complete keratin filaments in mouse L cells augments cell migration and invasion YI-WEN CHU*, RAYMOND B. RUNYANt, ROBERT G. OSHIMAt, AND MARY J. C. HENDRIX§¶ *Cancer Biology Program and §Department of Anatomy, College of Medicine, University of Arizona, Tucson, AZ 85724; tDepartment of Anatomy, University of Iowa, Iowa City, IA 52242; and tCancer Research Center, La Jolla Cancer Research Foundation, La Jolla, CA 92037 Communicated by Herbert E. Carter, February 1, 1993 (receivedfor review September 24, 1992) ABSTRACT Intermediate fiament proteins have been Keratins are usually expressed in epithelia; vimentin is used to diagnose the origin of specific cells. Classically, vimen- found in fibroblasts and endothelial cells whereas desmin and tin is found in mesenchymal cells, and keratins are present in neurofilaments are markers for muscle and neuronal tissue, epithelial cells. However, recent evidence suggests that the respectively (1). Although this cell lineage- and differentia- coexpression of these phenotype-specific proteins augments tion state-specific expression of IFs may be used in the tumor cell motility, and hence, metastasis. In the present study, diagnosis of tumors (11), noteworthy exceptions still exist in we used the mouse L-cell model to determine if a direct some tumor species and disease states. Work by Trask et al. correlation exists between the expression of additional keratins (12) showed that there are different sets ofkeratins expressed in these cells, which normally express only vimentin, and their in normal vs. tumor-derived mammary epithelial cells. The migratory ability. Mouse L cells were transfected with human peculiar switch of keratin expression during neoplastic trans- keratins 8, 18, and both 8 and 18. The results indicate that the formation may be a significant phenomenon associated with cells expressing complete keratin riaments have a higher tumor progression. Another interesting observation is the migratory and invasive ability (through extracellular matrix- unusual coexpression of vimentin and keratin in various coated filters) compared with the parental and control- tumor cells, which in a few documented cases correlates with transfected clones. Furthermore, there is an enrichment of metastatic potential. Some examples are as follows: a non- keratin-positive cells from a heterogeneous population of L metastatic rat pancreas adenocarcinoma expresses only vi- clones selected over serial migrations. This migratory activity mentin, whereas a metastatic variant contains a high amount was directly correlated with the spreading ability ofthe cells on ofkeratins in addition to vimentin (13). In a study with murine Matrigel matrix, in which the keratin-positive transfectants sarcoma cells, the ascites from the sarcoma were found to maintain a round morphology for a longer duration, compared coexpress keratin and vimentin, whereas the solid tumor with the other L-cell populations. Collectively, these data from the same sarcoma expresses only vimentin (14). In suggest that keratins may play an important role(s) in migra- several human breast cancer cell lines, vimentin, in addition tion, through a special interaction with the extracellular envi- to keratins, is expressed only in highly invasive cell lines but ronment, thereby influencing cell shape. not in cell lines of low metastatic potential (15). Last, human melanoma, which expresses vimentin as its classical IF Keratins make up the largest group in the intermediate marker, coexpresses keratins in recurrent and metastatic filament (IF) multigene family. There are at least 19 different states (16-18). Collectively, these data suggest that there is keratin proteins, which can be further subdivided into two an additional type of IF expression during the process of groups: type I (acidic) and type II (neutral-basic) keratins tumor metastasis that contributes to migratory and aggres- based on their sizes and isoelectric point (1). One major sive behavior in some manner. difference associated with keratins that distinguishes them To further elucidate if there is a direct correlation between from other IFs, such as vimentin and desmin, is that keratins the expression of additional keratins in cells and their migra- are expressed as heteropolymer pairs consisting of specific tory ability, we have used the mouse L-cell model. Mouse L type I and type II proteins. Their expression is highly cells are fibroblasts that express vimentin. For this study, regulated during embryonic development and cellular differ- these cells have been transfected with human keratin 8 and 18 entiation (2, 3). DNAs (19), and the formation of keratin filaments was The functional role(s) of IFs is still unclear (for review, see visualized by using indirect immunofluorescence staining. refs. 4-6). In epithelium, the keratin filaments are found Invasion analysis showed that the cells with complete keratin attached to the cell-cell adherence junctions, known as filaments have higher invasive ability and deformability ca- desmosomes (7), as well as to the cell-extracellular matrix pability to penetrate polycarbonate filters (containing 10-,um The pore size) than control cells, in addition to differential spread- (ECM) adhesion sites, known as hemidesmosomes (8). ing ability on an ECM. This study suggests that keratins may function ofthis interaction is thought to provide a mechanical a role in influencing cell scaffold for the epithelial sheet to maintain its integrity and play migration, by shape. structure. This hypothesis has been strengthened by recent MATERIALS AND METHODS investigations showing that transgenic mice expressing a Cells and Cell Culture. Mouse L cells transfected with mutant keratin 14 gene have a phenotype resembling a human different keratin DNAs have been described (19, 20) and for genetic skin disease known as epidermolysis bullosa simplex, our studies were cultured in the appropriate selection media. which is characterized by blistering of the skin due to the Immunofluorescence Staining. Cells were seeded onto glass cytolysis of the basal epidermal cells (9, 10). To our knowl- coverslips to 70% confluence, washed three times with edge, this is the first example demonstrating a relationship phosphate-buffered saline, fixed in cold methanol for 7 min, between abnormal keratin proteins and a human disease. Abbreviations: ECM, extracellular matrix; IF, intermediate fila- The publication costs of this article were defrayed in part by page charge ment; MICS, membrane invasion culture system; DMS, dynamic payment. This article must therefore be hereby marked "advertisement" morphology system. in accordance with 18 U.S.C. §1734 solely to indicate this fact. STo whom reprint requests should be addressed. 4261 Downloaded by guest on September 29, 2021 4262 Cell Biology: Chu et al. Proc. Natl. Acad. Sci. USA 90 (1993) and air dried. Indirect immunofluorescence staining was used RESULTS for staining keratin IFs, using mouse anti-human keratin 18 Keratin Filament Formation in Mouse L Cells. Although (CK5 antibody; ICN) followed by a rhodamine-conjugated keratins are the type of IFs present in epithelial cells, they secondary antibody (Organon Teknika-Cappel). All fluores- have been expressed in several nonepithelial cell lines by cence staining was observed with a Zeiss standard 18 fluo- using DNA transfection techniques (19, 26). Previously, we rescence microscope equipped with automatic rhodamine have transfected mouse L-cell fibroblasts with human simple and fluorescein filter sets. epithelial keratin 8, 18, or both 8 and 18 DNAs and obtained Invasion and Transfilter Migration Assays. The membrane several different stably transfected lines (19). They are invasion culture system (MICS; refs. 21 and 22) was used to termed LK8 cells, which are L fibroblasts that received the measure cell invasion. A polycarbonate membrane contain- plasmid (LK442-K8) containing keratin 8 cDNA driven by a ing 10-,um size pores (Poretics) was coated with a reconsti- human f3-actin promoter; LK18, which are L cells that were tuted basement membrane gel (Matrigel; Collaborative Re- cotransfected with vectors containing a neomycin-resistance search) and placed between the upper and lower well plates marker and a keratin 18 gene; and LK18+K8, which repre- of the MICS chamber. Subsequently, 5 x 104 cells were sent LK18 transfected again with the LK442-K8 plasmid. The resuspended in 10% NuSerum (Collaborative Research) and fate and expression of these transfected keratin DNAs have seeded into the upper wells. After incubation for 72 h at 37°C, been analyzed in detail (19), including RNA and protein cells that had invaded the Matrigel-coated membranes were analysis (19). Fig. 1 shows the results of the immunofluores- harvested from the lower wells, stained, and counted. Inva- cence staining of these cell lines with an anti-human keratin sion potential was calculated as the percentage of cells that antibody that recognizes simple epithelial keratins: the pa- invaded the Matrigel compared to the total number of cells rental L cells show negative staining as expected; LK8 and seeded (after correction for proliferation). LK18 also display no staining for keratin filaments, indicating The method for measuring transfilter migratory ability was a single type of keratin alone cannot form filaments in the modified from the procedure of McCarthy et al. (23). Instead cells. The LK18+K8 (clone 7) cells have a normal