- Email: [email protected]
Dominant and recessive genes involved in tumor cell invasion
Dominant Walter
and recessive genes . cell invasion
Birchmeier,
involved
Jiirgen Behrens, K. Michael
in tumor
Weidner,
Uwe H. Frixen
and J&g Schipper University
of Essen Medical
School,
Essen, Germany
The past year has seen the discovery and further analysis of several genes and protein products that are critically involved in the generation of invasive and metastatic tumor cells. Like oncogenes and tumor suppressor genes, the genes responsible for invasive and metastatic phenotypes can function in a dominant or recessive fashion. In this review, particular emphasis will be given to the dominantly acting genes encoding the cell adhesion molecule CD44 and the motility factor scatter factor, and the recessively acting genes encoding the cell adhesion molecule E-cadherin and nm23.
Current
Opinion
in Cell
Biology
Introduction
are
the the
key first
molecules involved step in metastasis?
in tumor
3:832-B40
creases me&static potential (see below) ]2-1. Motility factors and growth factors which influence cell motility can also affect invasive behaviour. For instance, scatter factor and fibroblast growth factor promote the invasiveness of epithelial cells in z&v [ 3’,4]. This year, the cDNA for the receptor of autocrine motility factor which appears to be involved in the progression of bladder carcinomas, has been characterized; the encoded protein shows homology to p53 [ 51.
The generation of malignant tumors in humans and in experimental animals is a multistep process. An accumulation of somatic mutations results in the loss of growth control of the cells involved, induces cell invasiveness and the vascularization of tumors, and finally leads to metas tasis of certain target organs. In the past decade, much progress has been made in the identification of genes responsible for the loss of growth control in tumors (oncogenes and tumor suppressor genes), whereas the specific genes involved in the later steps of tumor progression, invasion and metastasis are less well defined. Nevertheless, some progress has been made in the past year, which will be discussed in this review. Particular emphasis will be given to tumor invasiveness. Which invasion,
1991,
On the other hand, invasiveness of cells is also promoted by downregulation of expression of a different class of molecules that are the products of potentitiy recessive genes. The antagonists of proteases, protease inhibitors (e.g. plasminogen activator inhibitors, tissue inhibitor of metalloproteinase, etc.), are prime candidates here. Cell adhesion molecules are involved as well, because the invasive cells break off contacts to the tissue of origin. For instance, downregulation of expression of the epitheliumspecific cell adhesion molecule E-cadherin leads to increased motility and invasiveness of carcinoma cells in culture, and transfection with E-cadherin cDNA can reverse this (see below) [6-•,7**]. DCC (deleted in colon carcinomas), isolated as a tumor suppressor gene, encodes a neural cell adhesion molecule (NCAM)-like protein and might influence not only growth but also invasiveness and metastasis [w] Transfection of the fibronectin receptor cDNA can suppress tumor formation under certain conditions [!?I. This year has also seen the further characterization of the nm23 gene, which codes for a nucleoside diphosphate kinase. Transfection of the ~123 cDNA can suppress metastatic potential [lo-].
ceil
On the one hand, invasiveness is promoted by the upregulation of expression of critical components that can be viewed as products of dominant genes. Proteases (metaloproteinases, plasminogen activators etc.> are prime can didates in this group [la] because these molecules can generate the necessaty space for the invading tumor cells. Cell adhesion molecules and lectins are also thought to be involved because the invading cells continuously make new contacts on their migratoty pathway. For instance, a splice variant of the putative cell adhesion molecule CD44 has been discovered which, after transfection of the corresponding cDNA into low metastatic cells, inAbbreviations CAT-chloramphenicol NC-neural
832
cell
acetyltransferase; adhesion molecule;
@
HGF-hepatocyte KC-squamous
Current
Biology
growth factor; cell carcinoma;
Ltd
ISSN
0955-0674
LFM-lung SF-scatter
fibroblast-derived factor; S-simian
mitogen; virus.
Dominant
and
recessive
Judging from the rapid progress in the field, we can expect that new genes affecting invasive and metastatic phenotypes will be discovered in the next few years. The inverse
correlation
and invasiveness
between
differentiation
of carcinomas
The characteristics of invasiveness have been studied extensively in epithelial tumors, i.e. carcinomas, which represent over 90% of the human tumors. In the process of tumor progression, proliferating (transformed) epithelial cells break through the basement membrane and invade the underlying connective tissue. It seems that the loss of growth control and the phenomenon of invasion in carcinomas are distinct processes-growth can be affected in both benign and malignant tumors, but invasiveness is associated exclusively with malignancy. Carcinomas can be subdivided by both morphological and functional criteria: welI differentiated carcinomas mainly express epithelial tissue morphology, whereas poorly differentiated carcinomas are characterized by a more amorphous tissue structure, have fewer cell-cell junctions and are more highly invasive (see [6**] for a review). There are also heterogeneous carcinomas in which both differentiated and undifferentiated areas are found within one tumor (see also below). It has been shown that the differentiation and invasiveness of carcinomas can determine cancer prognosis. For instance, 80% of patients with well differentiated colorectal carcinomas survive 5 or more years, compared with only 25% of patients with diagnosis of poorly differentiated colorectal carcinomas (see [ 6**] >. The invasion potential of tumor cells has been studied in a variety of experimental systems in zjitro. For instance, the assay measuring invasion of ceUs into embtyonal chicken heart fragments (the so-called ‘Mareel as say’; see (1 I ] for a review) faithfully distinguishes between benign and malignant cells. Measuring the penetration of cells into a collagen matrix can be used eas ily to quantify invasive potential. Fig. 1 shows some of the features of these assays and illustrates the possibilities for experimental interference. For instance, motility factors or antibodies against cell adhesion molecules can be added directly (Fig. la) [3*,12], or the cells can be transfected with cDNA expression constructs prior to the use in the assay (Fig. lb) [6*=,7**]. The cell motility
factors
Cell motility factors have been described previously as a group of cytokines that selectively stimulate ceU migration with little or no effect on ceU proliferation. This group of proteins includes scatter factor (SF), which is secreted by mesenchymal cells and dissociates epithelial cells in a paracrine fashion [ 30,131, autocrine motility factor, which is derived from melanoma and breast carcinoma cells [ 141, and migration-stimulating factor, which affects Iibroblasts in an autocrine way [15]. We have recently reported [3*] that SF isolated from conditioned medium of human Iibroblasts promotes in-
genes
involved
in tumor
cell
invasion
Birchmeier et a/.
vasion of epithelial cells in vitro. The purified factor is a 9OkD glycoprotein that can be proteolytically cleaved into disulfide-linked 60 and 30kD subunits. Sequence analysis of seven ttyptic peptides from both subunits of SF revealed identity to sequences of human hepatocyte growth factor (HGF; Fig. 2a,b) [3*,16**,17*]. Subsequently, HGF was found to dissociate epithelial cells in tissue culture and to induce their invasion into collagen matrices. Conversely, SF was shown to promote the growth of hepatocytes in a similar fashion to HGF [ 16**]. The cDNA and putative amino acid sequence of SF from human fibroblasts (Fig. 2c) was found to be identical to both human HGF [ 16=*,18,19] and the newly described human lung fibroblast-derived mitogen (LFM) [20*]. It encodes a protein of 728 amino acid residues including a signal peptide at the amino-terminus (Fig. 2~). Heavy and light chains are produced from the common translation product by proteolytic processing between the Arg-Val residues indicated in Fig. 2b. The heavy chain consists of four repeated domains, so-called kringle modules, and the light chain shows homology to the serine protease domain of plasminogen. However, two amino acid residues essential for the catalytic properties of serine proteases, serine and histidine, are replaced by ty rosine and glutamic acid, respectively in SF/HGF/LFM [16,18,20*]. It is therefore unlikely that SF has protease activity. Thus, the domain structure of SF/HGF/LFM is unique and has not been observed previously in motility or growth factors. Southern blot analysis revealed a single gene encoding SF/HGF/LFM in the human genome, which is located on chromosome 7, bands q 11.2-21 [16**]. It is well known that several growth factors, besides promoting cell proliferation, can also influence differentiation and ceU motility. SF/HGF/LFM represents another factor which can exert such multimodal activities. It is remarkable, however, that SF/HGF/LFM mediates these Merent effects in different biological systems [3*,13,18]. Theoretically, this could be because of difFerent ceU surface receptors, different intracellular signal cascades, or the fact that identical signal cascades activate Merent target genes in the various cell types. A receptor for SF/HGF/LFM has been identified recently - the c-met protooncogene product, a transmembraneous tyrosinespecific protein kinase [ 21**,22*]. A number of genes involved in cell growth and differentiation as well as in tumor invasion and metastasis have been mapped to chromosome 7. The chromosome bands 7q11.2-q21, where the gene of SF/HGF/LMF is located, have been found to be affected by deletions, inversions and translocations in various types of leukemias. In malignant lymphomas and solid tumors, 7q11/21 aberrations were observed with lower frequencies than in leukemia [ 16**]. Further studies will show whether the gene for SF/HGF/LMF is in fact tiected in these malignancies. Because SF/HGF induces deditferentiation and invasiveness of epithelial (carcinoma) cells in vitro [13,16**], we hypothesized that this factor might also be involved in the progression of carcinomas toward a more malignant stage in vivo. Liver regeneration also requires extensive dedifferentiation of tissue, and it has been sug-
833
834
Cell-to-cell
contact
and
extracellular
matrix
Fig. 1. (a) Schematic representation of the collagen invasion assay with epithelial cells: possible experimental manipulations. Scatter factor and anti-E-cadherin antibodies produce dedifferentiated epithelial cells which enter the collagen gel. Conversely, transfection of poorly differentiated epithelial (carcinoma) cells with E-cadherin cDNA restores the epithelial phenotype and prevents invasiveness. fb) Invasion of chick heart tissue after treatment of Madin-Darby canine kidney (MDCK) epithelial cells with anti-E-cadherin antibody. Sections of d-day-old confrontation cultures between non-transformed MDCK cells and embryonic chick heart tissue are shown. The cultures were either incubated in the absence (i-iii) or in the presence (iv-ix) of 70 pg ml-l anti-E-cadherin monoclonal antibody Consecutive sections were stained with hematoxyline-eosine (left), anti-chick heart antiserum (middle) or anti-MDCK cell antiserum (right). M, MDCK cells; H, heart tissue. Arrowheads in (iv-ix) mark invasive MDCK cells after anti-E-cadherin treatment. Bars, 100 urn f121.
gested that HGF is involved in this process [ 18,191. Thus, the common denominator might be the involvement of SF/HGF in both types of dedifferentiation events, one being a highly uncontrolled process leading to an increased metastatic potential of epithelial cells, and the other being a tightly controlled process that is responsible for ordered regeneration of liver tissue. A contribution of SF/HGF/L.FM to other dedifferentiation processes-for example during epithelial-mesenchymal transitions in development-would be possible as well. Cell-cell
adhesion
molecules
The cell adhesion molecule E-cadherin (also called uvomorulin, LCAM, cell-CAM 128.80, Arc-l) is a 12OkD transmembrane glycoprotein, of which an 80 kD soluble tryptic fragment can be released extracellularly in the presence of Caz+. In early mouse development, E-cadherin functions as an adhesion component during compaction
of blastomers; at later stages, it is confined to epithelia originating from ecto-, meso- and endodermal tissue [ 23**,24]. In the epithelium of the small intestine, E-cadherin is enriched in the adherins junctions; in other epithelia, it is present at the lateral cell surfaces [25,26]. The E-cadherin cDNAs of mouse and chicken have been characterized; they code for a signal peptide at the ammo terminus, a large extracellular domain with five repeats, a single transmembrane sequence, and a 15 kD cytoplas mic domain [ 27-291. After transfection of the E-cadherin cDNA into fibroblasts, functional Ca2+-dependent contacts between the cells could be generated. E-cadherin belongs to the gene family of Ca2+ -dependent cell adhesion molecules; the closest relatives are N-cadherin (expressed in neural and muscle cells) and P-cadherin (originally identified in mouse placenta but also found in a restricted set of human epithelia [23”]). Further relatives include the desmosomal proteins desmoglein and desmocollin [30,31]. E-, N- and P-cadherin exhibit
Dominant
and
recessive
(a)
genes
involved
in tumor
cell
invasion
Birchmeier
et a/.
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MWVTKLLPALLLQIIVLLHI.LL.LPIAIPYAEG 32 QRKRRN’~IIIEFKKSAK’l”~LlKIUPAL.KIK’rKKVN’rAUQCANRCTRNKGI.Pt~CKAFVFDKARKQCLWFPFNSMSSGVKKEFGll~F~I.YSNKl~Y 128 C1IGKGRSYKG’I’VSITKSGIKCQPWSSHIPIIE~HGKDLQENYCRNPRGEEGGPWC~~SNPEVRY~VC~II’~SEVE
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Fig. 2. The amino acid sequence of human scatter factor (SF) and identity with human hepatocyte growth factor (HCF). (a) Peptides tiHvii) of SF (upper lines) were sequenced 13-l and are compared with the corresponding regions of HCF (lower lines). Note that all clearly identifiable residues of SF are identical to the corresponding sequences of HCF. lb) Location of these peptides within a model of SF/HCF (from 116**1). fc) Amino acid sequence of human SF as predicted from the cloned cDNA of fibroblasts (signal peptide, positions l-31; amino-terminal region, positions 322127; four kringle domains and connecting spacers, positions 12tr494; light chain exhibiting similarity to serine proteases, positions 495-728). The seven sequenced tryptic peptides are underlined. A five-amino-acid deletion in the first kringle domain found in a variant cDNA clone is boxed 116**1. a unique spatiotemporal expression pattern during embryogenesis, and cells expressing different cadherins sort out in rh-o, suggesting that these adhesion molecules play an important role during morphogenesis [ 23**].
breast, lung and pancreas carcinomas. Cell lines with an epithelioid phenotype were non-invasive and expressed E-cadherin, whereas cell lines with a libroblastoid phenotype were invasive and had lost E-cadhetin expression
Work in our laboratory pression with epithelial
(Fig. 3). Invasiveness of deditferentiated breast carcinoma cells (MDA-ME-435s) was prevented by transfectlon with E-cadherin cDNA and was again induced by treatment of
has correlated differentiation
the and
E-cadherin invasiveness
exof
carcinomas. Non-transformed Madin-Darby canine kidney epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition
the transfected tibodies (Fig. selective loss
of antibodies against assume a iibroblast-like
differentiation
E-cadherin; (i.e.
the separated dedilferentiated
cells then morphol-
ogy) and invade collagen gels and embryonal heart tissue (Fig. 1) [ 121. Epithelial cells transformed with Harvey and Moloney sarcoma viruses were found to be constitutively libroblast-like E-cadherin [ 121. examining various
C
and invasive, and they We also confirmed this human cell lines derived
do not express correlation by from bladder,
cells with 4) [6-,7*-l. of E-cadherin and
anti-E-cadherin These findings expression
invasiveness
of human
monoclonal indicate that can generate carcinoma
anthe decells
in zdtro. This correlation, a loss of E-cadherin expression coinciding with invasive properties, also holds for carcinoma cells in lliuo. In an extensive study, again from our laboratory, squamous cell carcinomas neck were analyzed for expression
(SCCs) of the of E-cadhetin
head and [ 32*]. A
835
836
Cell-to-ceil
contact
and extracellular
matrix
Bladder
RT112 RT4 EJ28
1
.. _-_-_-_-_-____-_-___-------------~---------------------------------
cx-1 WiDr COLO 205 SW948 SW620
Colon
1 I 1 I
MU-7 MDA-MB-361 ET-549 MDA-MB-231 MDA-MB-435S/l MDA-MB-436
Breast
1
I
..
I
( , I
I
I
1 ,
I 1
-______-_-__________-------------~--------------------------------Lung
LX-1 A-427 A-549 SK-MES-1
I 7 c3 I
1
------------_------_-------------.---------------------------------
Pancreas
Capan-l CapanDAN-G Hs 766T MIA PaCa-2
I 1 I ._
I
7
511,111,111,1,1,111,111,111
3.
I
3000
2500
1111,1111,1111,11~,,1111
2000
1500
1000
Invasion (cells cm-‘)
500
0
500
1000 E-cadherin (cpm/30000
1500
2000
2:
cells)
Fig. 3. E-cadherin expression and invasiveness in vitro of human carcinoma cell lines of various tissues. For the invasion experiments, human carcinoma cell lines were cultured on collagen gels for 3 days and invasive cells were quantified under the light microscope f121. The expression of E-cadherin was measured separately in an indirect cell-binding assay using monoclonal antibody 6F9 and l*sl-labeled goat anti-mouse IRC. Note the lack of invasion in the E-cadherin-expressing carcinoma cell lines and the invasive activity of the negative i&es 16**1. -
similar study on gastric adenocarcinomas has also been reported [33]. In SCCs, E-cadherin expression was found to be inversely correlated both with the loss of difterentiation of the tumor and with lymph node metastasis (Fig. 5) [32*]. Th e well differentiated SCCs expressed E-cadherin, often as strongly as the normal stratified epithelium, the moderately differentiated SCCs expressed intermediate amounts of E-cadherin or were heterogeneous, whereas the poorly differentiated SCCs were all E-cadherin-negative. In seven out of eight patients with infiltrated lymph nodes, we found that the carcinoma cells in the lymph nodes were E-cadherin-negative. These data indicate that the loss of the cell adhesion molecule E-cadherin in fact plays an important role in the progression of human SCCs; that is downregulation of expression is associated with dedifferentiation and metastasis of the tumor cells in vivo. The molecular mechanism responsible for E-cadherin downregulation in the dedifferentiated carcinoma cell lines and the poorly differentiated SCCs is not known. Downregulation could be caused by either mutations in the E-cadherin structural gene or by indirect suppression of E-cadherin gene expression. Interestingly, a new tumor suppressor gene in hepatocellular carcinomas has been localized on human chromosome 16; the common region of allele loss was narrowed down to the positions q22.1 and 23.2 [ 34**]. The human E-cadherin gene is located at position 16q22.1 [35]. Furthermore, loss
of heterozygosity on chromosome 16 was much more frequent in poorly differentiated (88%) than in well differentiated (18%) liver carcinomas. Thus, the E-cadherin gene is a good candidate for this tumor suppressor gene of chromosome 16. The tumor suppressor gene fat of Drosophila has also been identified as a cadherin-like molecule (C Goodman, personal communication). Loss of heterozygosity of chromosome 16 has also been observed in prostrate and breast carcinomas [ 36,371. Downregulation of E-cadherin expression in tumors by regulatory mechanisms can be studied only if normal regulation of E-cadherin gene expression is understood. We have therefore characterized the promoter of the E-cadherin gene [38] and begun to study regulatory proteins controlling it. We found that a promoter fragment ( - 178/ + 92 bp) mediates strong expression of a chloramphenicol acetyitransferase (CAT) reporter gene in epithelial cells (i.e. 60% of the level obtained with Simian virus (SV)40 promoter-enhancer constructs>, whereas in non-epithelial cells this promoter was either inactive or its activity was strongly reduced. By DNase I footprinting and gel retardation analysis, as weU as through functional dissection of the regulatory sequences, two regions that contribute to tissue-specific activity of the promoter were identified. First, a GCrich region at -25 to -58 generates basic epithelial promoter activity, most probably in combination with an ‘initiator’ element [39] present at the single transcription start site of the gene. Second,
Dominant
and
recessive
genes
involved
in tumor
cell
invasion
noma cells, but was silent in their poorly counterparts [ 381.
(i)
Invasive
Birchmeier
et al.
differentiated
cells cm.’
14001200lOOO800 600 400 20001
(ii)
Invasive
2
3
cells cm-’
1600 1400
1200 1000
r
800 600 400 200 0 ;en gels
Fig. 4. (a) lmmunofluorescence staining of mouse E-cadherin in transfected human breast carcinoma cells MDA-MB-435Sil. (i,ii) Human breast carcinoma clone cad-B1 transfected with mouse E-cadherin cDNA; fiii,iv) negative human clone neo-B4. The cells were stained with monoclonal antibody DECMA-1, which recognizes mouse E cadherin fii,iv). Note that the transfected clone cad-B1 expresses mouse E-cadherin at the cell-cell contacts. (b) Invasiveness of human breast carcinoma cells MDA-MB-435Sil transfected with the mouse E-cadherin cDNA. Cells were plated on collagen gels and Invasion was scored over 3 days. (9 E-cadherin-expressing human breast carcinoma clone cad-Bl; (ii) negative human clone neo-B4. 0, invasion in the absence of antibody; 0, invasion in the presence of the dissociating anti-mouse E-cadherin monoclonal antibody DECMA-1 I6”l.
a palindromic sequence at -86 (named E-pal) confers epithelial-specific activity to an SV40 promoter. The E-pal sequence is homologous to ®ulatory elements active in keratin promoters and it competes with these elements for nuclear factor binding [38,40]. Interestingly, the E-pal sequence stimulated transcription of a SV40 promoter-CAT construct in differentiated breast carci-
Fig. 5. E-cadherin expression in squamous cell carcinoma tissues of the head and neck. (a) Normal stratified squamous epithelium; (b) well differentiated carcinoma; fc) moderately differentiated carcinoma; fd) poorly differentiated carcinoma. The well differentiated carcinoma tissue shows a high expression of E-cadherin comparable with that of the normal epithelium, whereas the moderately differentiated carcinoma shows a weak signal. No E-cadherin expression is detectable in the poorly differentiated carcinoma. Bar, 40 pm.
CD44
and
nm23
Some years ago, Matzku et a[ t4I] generated monoclonal antibodies which reacted with a metastasizing cell line of the rat pancreatic adenocarcinoma BSp73, but did not recognize non-metastasizing variants. More recently, Gunthert et al. [2**] have screened cDNA expression libraries from the highly metastatisizing cell line with these monoclonal antibodies, and discovered the cDNA clone pMeta1. It codes for a splice variant of the presumed cell adhesion molecule CD44; the variant has a I62arninoacid residue insert in the extracellular domain, the epitope for the monoclonal antibody. Overexpression of pMeta1 in non-metastasizing pancreatic adenocarcinoma ceils establishes full metastatic behaviour in rats [2**]. This important work shows that cell surface differences between metastatic and non-metastatic states of cells can
837
838
Cell-to-cell
contact
and extracellular
matrix
.
indeed be described by molecular means. Furthermore, this work might give rise to new ways of therapeutic interference with metastatis, for example, by application of soluble parts of the CD44 extracellular domain. Steeg and colleagues performed differential cDNA cloning with related low and high metastatic murine K-1735 melanoma cells and discovered the recessive gene ~~m23 whose mRNA is more strongly expressed in low metastatic melanoma, breast carcinoma, and sarcoma cell lines than in the highly metastatic counterparts (42,431. The gene nm23 codes for a 17kD cytoplasmic protein with 75% amino acid identity to the predicted protein product of abnormal wing d&s (awd) of Drosophila (441. Mutations of this gene in Drcxc@ifa result in altered differentiation and necrosis during larval development. The nm23 gene product has nucleoside diphosphate kinase activity which seems to be important for microtubule polymerization [45]. Overexpression of the nm23 cDNA in highly metastatic melanoma ceU lines resulted in significantly reduced metastatic potential, independent of tumor cell growth [lo**]. Somatic allelic deletions of the nm.23 gene located on chromosome 17 were identified in human breast, renal, colorectal and lung carcinomas; in three out of nine cases, loss of heterozygosity was observed without any other detectable deletion of chromosome 17 [46]. It will now be important to examine the second allele for mutations in the nm23 gene, as weU as to understand the molecular function of the gene product and the mechanism by which it influences metastatic behaviour. Conclusions
Judging from the recent progress in the research of invasion and metastasis, it appears that the genes involved in these processes can be classified as either dominantly or recessively acting. The genes encoding CD44 [2-l and SF [16-l are thought to be dominant, and those encoding nm23 [lo**] and E-cadherin [6**] are thought to be recessive. In certain cases, we anticipate the boundaries between invasion/metastasis genes and oncogenes/tumor suppressor genes to become less distinct. SF, first identified as a motility and dediierentiation factor [ 131, was found recently to influence growth in other systems [16*-,18,20=]. A role for the SF receptor, the c-met-encoded tyrosine kinase [21**,22*], in both inva sion and growth control can be envisaged. On the other hand, the tumor suppressor gene DCC, which codes for a putative ceU adhesion-type component [8-l, might represent an invasion suppressor besides a growth-controlling gene and function in intercellular adhesion. It has also become obvious that invasion and metastasis genes, like oncogenes and tumor suppressor genes, can code for either extracellular, cell surface, cytoplasmic or nuclear components. A variety of molecules potentially important in invasion and metastasis have now been identified. In some cases, even the molecular mechanisms by which these proteins influence malignant behaviour are known. It will now be important to search for the actual mutations respon-
sible for invasive and metastatic behaviour of human tumors. For instance, we know that the SF gene is located at a fragile site on human chromosome 7 [lti*], that E-cadherin is a candidate for the tumor suppressor gene of chromosome 16 [6-*,34-l, and that cadherins can be tumor suppressor genes in other species. Definite proofs for mutations of these genes in human tumors are still lacking, however. To our knowledge, no mutations in the genes for CD44 or ~-123 have yet been discovered in human tumors. Such mutations could in principle either affect the structural genes of components directly involved in invasion and metastasis, or they could indirectly influence the regulatory systems of these genes. The names type I and type II tumor suppressor genes have been used to describe analogous differences in growth-related genes [ 471. The identified candidate genes responsible for invasiveness/metastasis have now to be investigated for both these types of mutations in human tumors. Acknowledgements We would like to thank Dr Carmen Birchmeier (Kdln) for critically reading the manuscript. and B Lelekakis for preparation of the type script. Our work was supported by the Deutsche Krebshilfe and the Deutsche Forschungsgemeinschaft
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F~UXEN LJ, BEHRENS J, SACHS M, EBERLE G, Voss 4 LOCHNER D, BIRCHMEI~R W: E-Cadherin-Mediated
Adhesion Prevents Invasiveness J Cell Biol 1991, 111:173-185.
B, WARDA
VLEMINCKX
..
Genetic Manipulation of E-cadherin Ual Tumor Cells Reveals an Invasion 1991, 66:107-l 19.
K, VAKAT
L JR, MAIUZL
M,
FIERS W.
VAN ROY
Expression Suppressor
8. ..
Gene that 247:4+56.
is Altered
in Colorectal
Cancers.
Scietlce
1990,
This work indicates, for the lirst time, that the gene encoding a putative cell adhesion moleculr (an NCAMlike component; a member of the immunoglobulin superfamily) c&n be a tumor suppressor gene [DCC (deleted in colon arcinoma) located on human chromosome lBq]. This work represents a milestone in the research on both cancer and cell adhesion.
9. .
G~~COTII
E: Elevated Levels of the a5Bl FiSuppresses the Transformed Phenotype 0vary Cells. Cell 1990, 60:84W59.
FG. R~lo\~~nii
bronectin Receptor of Chinese Hamster
Transformed chinese hamster ovary cells lose NmOdgeniCity when transfected with the cDNA for both chains of the ftbronectin receptor. The fibronectin rcyeptor can thus be viewed as a NmOr suppressor. 10. ..
LEONE A, FIATOW’ 11, KING CR, SANNDEEN M4 MARGLIUS IMK. IJO~TA LA, SIFT% KS: Reduced Tumor Incidence. Metastatic Potential, and Cytokine Responsiveness of MI 23-Trans-
fected
Melanoma
Cell.
Cefl 1991,
65:2%35.
Transfection of the cDNA for nnz?.5 converts into the non-metastatic state. Thus, nmZ.3 cessive metastasis gene. 11.
MARIEI.
MM:
Mefus&l.sis 12.
14.
15.
In Vitro 2:2.01-21x.
B~~HKENS J. MAKEEI. MM,
ing Tumor Propertics Adhesion. 13.
Invasion
Rc?l~ 19x3.
VAN ROY FM,
L1011-A IA, MANIXI:H CHING PK. SclIIFFhtmN
R, MIIR,!N~ E: Tumor
tor.
Sci I!W
GREY A-M.
of Analysis.
Null
Awd
SCI~OR AM.
16.
cells re-
Cancer
This
21. ..
22. .
19%.
RK.
Motility
Fac-
23. ..
by Fetal Acad Sci
Factor and Human Hepatocyte A& Sci l&l 1991. 88:700-7005.
Growth
Identity between SF, HGF and LFM was established by functional ysis, by immunological comparison. by partial protein sequencing,
analand
Growth
work
shows
Growth Factor Speciiic for Sci USA 1991, 86:802-806.
Factor.
that LFM is identical
to HGF
Factor Science
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RUDIKOFF
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KMIEC~K TE,
of the HepProto-onco-
c-Met
NAIDINI L, VIGNA E, NARSIMHAN R, GAUDINO MICHAL~PO~ILUS GK, COMOGLIO PM: Hepatocyte
TAKEICHI
M: Cadherin
phologic
Regulator.
An excellent review opment and tumor
24.
G. ZARNEGAR
R,
Growth Facthe Tyrosine Kinase Activity of the by Proto-oncogene c-MET. Oncogene
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Transformation Introduced
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K. TAKEICHI
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SL; Purifica-
Produced Proc N&l
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These two papers [21**,22*] show that HGF (SF) is the i&and for the c.mefprotooncogene. a transmembrane tyrosine kinase, and that l&and binding lea& to autophosphorylation of c-met on tyrosine residues. These two Studies have uncovered a new transmembrane signaling pathway.
83:3302-3306.
R~IsH’~-ON G, ELUS 1. .%I-OR
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G. KATZ DA, CeU Autocrine
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MN~AWA K, Tstr~oilc~~ H, NAKA D, TAKAHASHI K, OK~GAKI M, ARAKAKI N, NAKAY.Q.~A H, HIRONO S, SAKNAMA 0, T-HI K, GOHDA E. DAIKL~HARA Y, KITAMURA N: Molecular Cloning and
Identified Natl Acad
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of Human Scatter Factor. Prw Narl
20.
M, GRAY J: Scatter
of Epithelial
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et al.
1990, 346:228.
Expression of Human 1989, 342440-443.
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BIR01MEIER
tion of the Migration Stimulating Factor and Breast Cancer Patient Fibroblasts. 1l.U 1989, 86:2r3t+24r2. ..
melanoma a potentially
Cell Invasion: Epithellal Cells Acquire after the Loss of Uvomorulin-Mediated .I &ll Hiol 1989. 108:2+35-2+b7.
M. GHEHARIII
Proc
metastatic represene
Methods
E, PERRYhIm is a Fibroblast-Derived Modulator N&ire 1987. 327:23%2+2.
SIXXXR
19.
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FEAHON ER, CHO KR, NIGRO JM. KERN SE, SIMONS JW. TIII’ERT JM, HAMIL’I‘ON SR, PREISINCER AC, THOMAS G, KINZLER KW, VOGELSTXIN 8: Identification of a Chromosome 18q
SF/HGF is coded by one gene 7, bands ql1.2.21.
E, STOKER M: Scatter
GHERARDI
Birchmeier
invasion
Sequence Analysis of cDNA for Human Factor. Biocktn Biopbys Res Cotnmun
F:
A cDNA encoding the cell-cell adhesion molecule E-cadherin was transfected into invasive epithelial tumor cell lines, and highly expressing ctones were found to be non-invasive in two in ilifro assays, including the embtyonal heart invzion ~svdy.
cell
amino acids of the light L uence similarity between the amino-terminal chain of mouse SF and rat HGF (21 of 24 residues) was observed. 18.
by EpitheRole. Cell
in tumor
sequence comparison. on human chromosome
lure
DiRerentiated. non-invasive human carcinoma ceil lines of various tissues generally express the cell adhesion molecule E-cadherin, whereas dserenriated, invasive carcinoma cell lines are deficient in E-cadherin. Invasiveness of the latter cells could be prevented by transfection with the E-cadherin cDNA E-cadherin thus has the properties of an invasion suppressor.
7.
involved
by cDNA is located 17.
Cell-Cell Carcinoma Cells.
of Human
genes
Molecular Mechanisms of EMBO J 1987, 63647-3653.
KOCH PJ. WALSH MJ, SCHMELZ M, GOUXCHMIDT R, FAKE WW: Identification of Desmoglein,
Desmosomal Glycoprotein Family of CeU Adhesion 53:1-12.
as a Member Molecules. Eur
MD, ZIMBELMAN
a Constitutive of the Cadherin .J Cell Biol 1990,
839
840
Cell-to-cell 31.
contact
MECHANIC a Distinct Molecules.
and extracellular
matrix
S, RAYNOR K, HIU JE, COWIN P: Desmoco&ns Form Subset of the Cadherin Family of Cell Adhesion I+oc Nail Acad Sci USA 1991, 88:4476-rj80.
SCHIPPER JH, F~UXEN UH, BEHRENS J, UNGER 4 JAHNCU K. BIRCHMEIEH W: E-Cadherin Expression in Squamous Cell Carcinomas of Head and Neck: Inverse Correlation with Tumor Differentiation and Lymph Node Metastasis. Cunccr Xes 1991, in pres. All poorly differentiated squamous cell carcinomas of the upper aerodigestive tract were found to be E-cadherin.deficient. Lymph node metastases of head and neck carcinomas were also E-cadherin-nega tive, irrespective of the grade of the original tumor. These data indicate that downregulation of expression of E-cadherin is associated with de& iaerentiation and metastasis of carcinoma cells in t+to,
39
ShWI Control
40
LE~~I( A, Rosr;.h%rxc; M. VA~SI\R R. FIICHS E: Regulation of a Human Epidermal Keratin Gene: Sequences and Nuclear Factors Involved in Keratinocyte-Specihc Transcription. Genes Del3 199Q. 4:19Xi199H.
41.
MAVKII S. KOhII?‘OW’SKS D, MIIDENUERGER M. ZOLLER M: Charaterization of Bsp73. a Spontaneous Rat Tumour and its In Vioo Selected Viriants Showing DiITerent Metastasying Capacities. Imzbotr .Ilela.W& 1983 3:109-123.
42.
ST~XG PS, B~VI.Q~IA G. KOI’I’I:R L, THORGF.IK%ON UP. TA~MADCI: JE, LIOITA JE, SOI~EI. ME: Evidence for a Novel Gene Associated with Low Tumor Metastatic Potential. / Narl Cancer hsr 1988, 80:2m2oj.
43.
HENNESXY C. HENRY JA, bti~ FEB. WEST BR, ANGUS B, IENNAKD TWJ: Expression of the Anti-Metastatic Gene nm23 in Human Breast Cancer; Association with Good Prognosis. J Nafl Girlcer 1tt.Q 1991, 83:265--2X1.
44.
ROSENGARII AM, KR[I-rsctI HC. SI~F.AK~’ A. BICCS JR, BARKF.R E. MARCLUES IMK. KIX; CR. IJOTTA IA. STEEG PS: Reduced run 23/awd Protein in Tumor Metastasis and Aberrant Drosophila Development. Aallcre 19X9, 342:177-180.
45.
BICGS J, HERSPERGEK E. Snzc; PS, IJOITA L& SHEARN k A Drosophila Gene that is Homologous to a Mammalian Gene Associated with Tumor Metastasis Codes for a Nucleoside Diphosphate Kinase. Gull 1990. 63:933-940.
16.
&ONE 4 MCBRIDE ow. W~SU~N 4 WANG MG, ~GLARI) P, CROI’I’ CS, GOU’F.L JR, ~D~;RI!AI~ R. CAlJAHA,, R. LINEHAN WM. I&ES RC, HAKKls CC, L~TA IA. STEEG PS: Somatic Allelic Deletion of nm 23 in Human Cancer. Ccrncer Res 1991. 51:2-150-2193.
47.
LF.t! SW, TOMASEITO C, SACEK R: Positive Selection of Candidate Tumor-Suppressor Genes by Subtractive Hybridization. Proc Nafl Acad Sci l&t 1991, B&2825-2829.
32. .
33.
SHIMOYAMA Y. HIROHASHI in Gastric Carcinomas.
S: Expression of E- and P-Cadherin Cancer Res 1991. 51:21852192.
34. ..
TSUDA H, ZHANG W, SHIMOSATO Y, YOKOTA J, TERADA J, TERADA M, SUCIMUKA T, MNIUI~IKA T. HIROHASHI S: Allele Loss on Chromosome 16 Associated with Progression of Human Hepatocellular Carcinoma. Pra Nafl Acad Sci C&i 19$XI, 87:67914794. A new tumor suppressor gene associated with the loss of differentiation of carcinomas has been discovered on human chromosome 16. bands q22.1-23.2. 90% of poorly differentiated but only 20% of well differentiated hepatocellular carcinomas are a&ted by this mutation. 35.
NATT E, MAGENIS RE, ZIMMER J. MANsoiuu A, SCHERER G: Regional Assignment of the Human Loci for Uvomorulin (UVO) and Chymotrypsinogen B (CTRB) with the Help of Two Overlapping Deletions on the Long Arm of Chromomme 16. Qfogrmec CeN Gene/ 1983, 50:14+148.
36.
CAKTER BS, EWING CM, WARD WS, TREIGR BF, AALDEI& TW, SCHAIJEN JA, EPSTEIN Jl? Isucs WB: Allelic Loss of Chromosome 16q and 1Oq in Human Prostate Cancer. Proc Nat/ Acud Sci USA 1990, 87:8751+755.
37.
SATO T, TANIGAMI A, YAMAKAWA K. AI(Iy~hti F. WIIMI F, SMOTO G. N~LIIW Y: Allelotype of Breast Cancer: Cumulative Allele Losses Promote Tumor Progression in Primary Breast Cancer. Cancer Res 1990. 50:7184-7189.
38
BEHRENS J, L~WRICK 0, KLEIN-HITPA~S L, BIHCHMEIER w: The ECadherin Promoter: Functional Analysis of a GC-rich Region and an Epithelial Specilic Palindromic Regulator Element. PrM Nat1 Acad Sci I/.%4 1991, in press.
ST, BfinMoKI;. D: The ‘Initiator as a Transcription Element. CeN 1989, 57:103-l 13.
W Birchmeier. J Behrens. KM Weidner. IIH Frixen and J Schipper, In. StiNt hir Zellbiologie (Tumorforschung), llniversity of Essen Medical School, Virchowstnqse 173. D-q300 Essen 1, Germmy.
and recessive genes . cell invasion
Birchmeier,
involved
Jiirgen Behrens, K. Michael
in tumor
Weidner,
Uwe H. Frixen
and J&g Schipper University
of Essen Medical
School,
Essen, Germany
The past year has seen the discovery and further analysis of several genes and protein products that are critically involved in the generation of invasive and metastatic tumor cells. Like oncogenes and tumor suppressor genes, the genes responsible for invasive and metastatic phenotypes can function in a dominant or recessive fashion. In this review, particular emphasis will be given to the dominantly acting genes encoding the cell adhesion molecule CD44 and the motility factor scatter factor, and the recessively acting genes encoding the cell adhesion molecule E-cadherin and nm23.
Current
Opinion
in Cell
Biology
Introduction
are
the the
key first
molecules involved step in metastasis?
in tumor
3:832-B40
creases me&static potential (see below) ]2-1. Motility factors and growth factors which influence cell motility can also affect invasive behaviour. For instance, scatter factor and fibroblast growth factor promote the invasiveness of epithelial cells in z&v [ 3’,4]. This year, the cDNA for the receptor of autocrine motility factor which appears to be involved in the progression of bladder carcinomas, has been characterized; the encoded protein shows homology to p53 [ 51.
The generation of malignant tumors in humans and in experimental animals is a multistep process. An accumulation of somatic mutations results in the loss of growth control of the cells involved, induces cell invasiveness and the vascularization of tumors, and finally leads to metas tasis of certain target organs. In the past decade, much progress has been made in the identification of genes responsible for the loss of growth control in tumors (oncogenes and tumor suppressor genes), whereas the specific genes involved in the later steps of tumor progression, invasion and metastasis are less well defined. Nevertheless, some progress has been made in the past year, which will be discussed in this review. Particular emphasis will be given to tumor invasiveness. Which invasion,
1991,
On the other hand, invasiveness of cells is also promoted by downregulation of expression of a different class of molecules that are the products of potentitiy recessive genes. The antagonists of proteases, protease inhibitors (e.g. plasminogen activator inhibitors, tissue inhibitor of metalloproteinase, etc.), are prime candidates here. Cell adhesion molecules are involved as well, because the invasive cells break off contacts to the tissue of origin. For instance, downregulation of expression of the epitheliumspecific cell adhesion molecule E-cadherin leads to increased motility and invasiveness of carcinoma cells in culture, and transfection with E-cadherin cDNA can reverse this (see below) [6-•,7**]. DCC (deleted in colon carcinomas), isolated as a tumor suppressor gene, encodes a neural cell adhesion molecule (NCAM)-like protein and might influence not only growth but also invasiveness and metastasis [w] Transfection of the fibronectin receptor cDNA can suppress tumor formation under certain conditions [!?I. This year has also seen the further characterization of the nm23 gene, which codes for a nucleoside diphosphate kinase. Transfection of the ~123 cDNA can suppress metastatic potential [lo-].
ceil
On the one hand, invasiveness is promoted by the upregulation of expression of critical components that can be viewed as products of dominant genes. Proteases (metaloproteinases, plasminogen activators etc.> are prime can didates in this group [la] because these molecules can generate the necessaty space for the invading tumor cells. Cell adhesion molecules and lectins are also thought to be involved because the invading cells continuously make new contacts on their migratoty pathway. For instance, a splice variant of the putative cell adhesion molecule CD44 has been discovered which, after transfection of the corresponding cDNA into low metastatic cells, inAbbreviations CAT-chloramphenicol NC-neural
832
cell
acetyltransferase; adhesion molecule;
@
HGF-hepatocyte KC-squamous
Current
Biology
growth factor; cell carcinoma;
Ltd
ISSN
0955-0674
LFM-lung SF-scatter
fibroblast-derived factor; S-simian
mitogen; virus.
Dominant
and
recessive
Judging from the rapid progress in the field, we can expect that new genes affecting invasive and metastatic phenotypes will be discovered in the next few years. The inverse
correlation
and invasiveness
between
differentiation
of carcinomas
The characteristics of invasiveness have been studied extensively in epithelial tumors, i.e. carcinomas, which represent over 90% of the human tumors. In the process of tumor progression, proliferating (transformed) epithelial cells break through the basement membrane and invade the underlying connective tissue. It seems that the loss of growth control and the phenomenon of invasion in carcinomas are distinct processes-growth can be affected in both benign and malignant tumors, but invasiveness is associated exclusively with malignancy. Carcinomas can be subdivided by both morphological and functional criteria: welI differentiated carcinomas mainly express epithelial tissue morphology, whereas poorly differentiated carcinomas are characterized by a more amorphous tissue structure, have fewer cell-cell junctions and are more highly invasive (see [6**] for a review). There are also heterogeneous carcinomas in which both differentiated and undifferentiated areas are found within one tumor (see also below). It has been shown that the differentiation and invasiveness of carcinomas can determine cancer prognosis. For instance, 80% of patients with well differentiated colorectal carcinomas survive 5 or more years, compared with only 25% of patients with diagnosis of poorly differentiated colorectal carcinomas (see [ 6**] >. The invasion potential of tumor cells has been studied in a variety of experimental systems in zjitro. For instance, the assay measuring invasion of ceUs into embtyonal chicken heart fragments (the so-called ‘Mareel as say’; see (1 I ] for a review) faithfully distinguishes between benign and malignant cells. Measuring the penetration of cells into a collagen matrix can be used eas ily to quantify invasive potential. Fig. 1 shows some of the features of these assays and illustrates the possibilities for experimental interference. For instance, motility factors or antibodies against cell adhesion molecules can be added directly (Fig. la) [3*,12], or the cells can be transfected with cDNA expression constructs prior to the use in the assay (Fig. lb) [6*=,7**]. The cell motility
factors
Cell motility factors have been described previously as a group of cytokines that selectively stimulate ceU migration with little or no effect on ceU proliferation. This group of proteins includes scatter factor (SF), which is secreted by mesenchymal cells and dissociates epithelial cells in a paracrine fashion [ 30,131, autocrine motility factor, which is derived from melanoma and breast carcinoma cells [ 141, and migration-stimulating factor, which affects Iibroblasts in an autocrine way [15]. We have recently reported [3*] that SF isolated from conditioned medium of human Iibroblasts promotes in-
genes
involved
in tumor
cell
invasion
Birchmeier et a/.
vasion of epithelial cells in vitro. The purified factor is a 9OkD glycoprotein that can be proteolytically cleaved into disulfide-linked 60 and 30kD subunits. Sequence analysis of seven ttyptic peptides from both subunits of SF revealed identity to sequences of human hepatocyte growth factor (HGF; Fig. 2a,b) [3*,16**,17*]. Subsequently, HGF was found to dissociate epithelial cells in tissue culture and to induce their invasion into collagen matrices. Conversely, SF was shown to promote the growth of hepatocytes in a similar fashion to HGF [ 16**]. The cDNA and putative amino acid sequence of SF from human fibroblasts (Fig. 2c) was found to be identical to both human HGF [ 16=*,18,19] and the newly described human lung fibroblast-derived mitogen (LFM) [20*]. It encodes a protein of 728 amino acid residues including a signal peptide at the amino-terminus (Fig. 2~). Heavy and light chains are produced from the common translation product by proteolytic processing between the Arg-Val residues indicated in Fig. 2b. The heavy chain consists of four repeated domains, so-called kringle modules, and the light chain shows homology to the serine protease domain of plasminogen. However, two amino acid residues essential for the catalytic properties of serine proteases, serine and histidine, are replaced by ty rosine and glutamic acid, respectively in SF/HGF/LFM [16,18,20*]. It is therefore unlikely that SF has protease activity. Thus, the domain structure of SF/HGF/LFM is unique and has not been observed previously in motility or growth factors. Southern blot analysis revealed a single gene encoding SF/HGF/LFM in the human genome, which is located on chromosome 7, bands q 11.2-21 [16**]. It is well known that several growth factors, besides promoting cell proliferation, can also influence differentiation and ceU motility. SF/HGF/LFM represents another factor which can exert such multimodal activities. It is remarkable, however, that SF/HGF/LFM mediates these Merent effects in different biological systems [3*,13,18]. Theoretically, this could be because of difFerent ceU surface receptors, different intracellular signal cascades, or the fact that identical signal cascades activate Merent target genes in the various cell types. A receptor for SF/HGF/LFM has been identified recently - the c-met protooncogene product, a transmembraneous tyrosinespecific protein kinase [ 21**,22*]. A number of genes involved in cell growth and differentiation as well as in tumor invasion and metastasis have been mapped to chromosome 7. The chromosome bands 7q11.2-q21, where the gene of SF/HGF/LMF is located, have been found to be affected by deletions, inversions and translocations in various types of leukemias. In malignant lymphomas and solid tumors, 7q11/21 aberrations were observed with lower frequencies than in leukemia [ 16**]. Further studies will show whether the gene for SF/HGF/LMF is in fact tiected in these malignancies. Because SF/HGF induces deditferentiation and invasiveness of epithelial (carcinoma) cells in vitro [13,16**], we hypothesized that this factor might also be involved in the progression of carcinomas toward a more malignant stage in vivo. Liver regeneration also requires extensive dedifferentiation of tissue, and it has been sug-
833
834
Cell-to-cell
contact
and
extracellular
matrix
Fig. 1. (a) Schematic representation of the collagen invasion assay with epithelial cells: possible experimental manipulations. Scatter factor and anti-E-cadherin antibodies produce dedifferentiated epithelial cells which enter the collagen gel. Conversely, transfection of poorly differentiated epithelial (carcinoma) cells with E-cadherin cDNA restores the epithelial phenotype and prevents invasiveness. fb) Invasion of chick heart tissue after treatment of Madin-Darby canine kidney (MDCK) epithelial cells with anti-E-cadherin antibody. Sections of d-day-old confrontation cultures between non-transformed MDCK cells and embryonic chick heart tissue are shown. The cultures were either incubated in the absence (i-iii) or in the presence (iv-ix) of 70 pg ml-l anti-E-cadherin monoclonal antibody Consecutive sections were stained with hematoxyline-eosine (left), anti-chick heart antiserum (middle) or anti-MDCK cell antiserum (right). M, MDCK cells; H, heart tissue. Arrowheads in (iv-ix) mark invasive MDCK cells after anti-E-cadherin treatment. Bars, 100 urn f121.
gested that HGF is involved in this process [ 18,191. Thus, the common denominator might be the involvement of SF/HGF in both types of dedifferentiation events, one being a highly uncontrolled process leading to an increased metastatic potential of epithelial cells, and the other being a tightly controlled process that is responsible for ordered regeneration of liver tissue. A contribution of SF/HGF/L.FM to other dedifferentiation processes-for example during epithelial-mesenchymal transitions in development-would be possible as well. Cell-cell
adhesion
molecules
The cell adhesion molecule E-cadherin (also called uvomorulin, LCAM, cell-CAM 128.80, Arc-l) is a 12OkD transmembrane glycoprotein, of which an 80 kD soluble tryptic fragment can be released extracellularly in the presence of Caz+. In early mouse development, E-cadherin functions as an adhesion component during compaction
of blastomers; at later stages, it is confined to epithelia originating from ecto-, meso- and endodermal tissue [ 23**,24]. In the epithelium of the small intestine, E-cadherin is enriched in the adherins junctions; in other epithelia, it is present at the lateral cell surfaces [25,26]. The E-cadherin cDNAs of mouse and chicken have been characterized; they code for a signal peptide at the ammo terminus, a large extracellular domain with five repeats, a single transmembrane sequence, and a 15 kD cytoplas mic domain [ 27-291. After transfection of the E-cadherin cDNA into fibroblasts, functional Ca2+-dependent contacts between the cells could be generated. E-cadherin belongs to the gene family of Ca2+ -dependent cell adhesion molecules; the closest relatives are N-cadherin (expressed in neural and muscle cells) and P-cadherin (originally identified in mouse placenta but also found in a restricted set of human epithelia [23”]). Further relatives include the desmosomal proteins desmoglein and desmocollin [30,31]. E-, N- and P-cadherin exhibit
Dominant
and
recessive
(a)
genes
involved
in tumor
cell
invasion
Birchmeier
et a/.
fb) (i
1
A.F.V.F.D.K .K.A.F.V.F.D.K.A.
(ii)
86 W.D.S.O.?.P.H.E.HID.D.D/M.T.P.E.N.F .R.W.D.S.ll.Y.P.H.E. H .D. 329 N.P.D.G.S.E.S.P.X.X.F.T.T.D.P .R.N*P.D.G.S.E.S.P.W.C.F.T.T.D.P.N. 356 H.I.F.W.E.P.D.A.S.K. .R.H.I.F.W.E.P.D.A.S.K.L. 425 434 N.P.D.D.D.D.A.X.G.P
(iii)
(iv)
(VI (vi
(vii)
1
.R.;$.D.D.D.A.H.G.P.W. D.Y.E.A.X.L.G.I.H.D.V. .K.D.Y.E.A.W.L.G.I.H.D.V.H. 543 a.V.L.X.V.S.0.L.V.Y.G.P .K.D.V.L.N.V.S.O.L.V.Y.G.P.E. 563
M .T.P.E.N.F.K. 344
H-chain
370
450
L-chain
553 574
Cc) 1
31
MWVTKLLPALLLQIIVLLHI.LL.LPIAIPYAEG 32 QRKRRN’~IIIEFKKSAK’l”~LlKIUPAL.KIK’rKKVN’rAUQCANRCTRNKGI.Pt~CKAFVFDKARKQCLWFPFNSMSSGVKKEFGll~F~I.YSNKl~Y 128 C1IGKGRSYKG’I’VSITKSGIKCQPWSSHIPIIE~HGKDLQENYCRNPRGEEGGPWC~~SNPEVRY~VC~II’~SEVE
I27 IRN 210
21 I JO4 CH’I’CNGESYRGLHDllTESGKICQRWDHQTPHRHKFLPEHYPDKGFDDNYCRNPDGQPRPWCYTLDPll’rHWEYCAIK1’CADW’I’HNI)’I’I~VPI.~I”~E 305 390 CI~QGEGYRGTV~~IWNGIPCQRWDSQYPHEHDHTL’ENFKCKDLRENYCHNPDGSESPWCF7TDPNIRVGYCSQIPNCD~SHGQD 391 CYRCNGKNYHGNI.SQTRSGI.TCSHWDKNHEDLHRHIFWEPDASKI.NENYCHNPD~DAHGPWCYTGNPL~PWDYCPISRC~GD’~l’l’~~IVNl.l~lli’VlS~‘AK’I’K(VI.H
494
4YS VVNGIPTRTNIGWHVSI.RYHNKIIICGGSLIKESWVLTARQCFPSRDLKl~YEAW[.GIHDVHGRGD~KCKQVLNVSQI,VYGPEGS~l,Vl.MKl.Ai~l’AVl.l~l~~V 595
5 ‘J4
~*~I~LPNYG~*~IPEKTSCSVYGWGYTGI,INYDGLLRVAI~LY 695 720
VPG
6 94
IHGNEKCSQIIIIRGKVTLNESEICAGASKICSGPCE~~UYCGI~LVCUQIIKMI(NVI.CVI
HGCAIPNRPGIFVRVAYYAKWIllKIILTYKVPQS*
Fig. 2. The amino acid sequence of human scatter factor (SF) and identity with human hepatocyte growth factor (HCF). (a) Peptides tiHvii) of SF (upper lines) were sequenced 13-l and are compared with the corresponding regions of HCF (lower lines). Note that all clearly identifiable residues of SF are identical to the corresponding sequences of HCF. lb) Location of these peptides within a model of SF/HCF (from 116**1). fc) Amino acid sequence of human SF as predicted from the cloned cDNA of fibroblasts (signal peptide, positions l-31; amino-terminal region, positions 322127; four kringle domains and connecting spacers, positions 12tr494; light chain exhibiting similarity to serine proteases, positions 495-728). The seven sequenced tryptic peptides are underlined. A five-amino-acid deletion in the first kringle domain found in a variant cDNA clone is boxed 116**1. a unique spatiotemporal expression pattern during embryogenesis, and cells expressing different cadherins sort out in rh-o, suggesting that these adhesion molecules play an important role during morphogenesis [ 23**].
breast, lung and pancreas carcinomas. Cell lines with an epithelioid phenotype were non-invasive and expressed E-cadherin, whereas cell lines with a libroblastoid phenotype were invasive and had lost E-cadhetin expression
Work in our laboratory pression with epithelial
(Fig. 3). Invasiveness of deditferentiated breast carcinoma cells (MDA-ME-435s) was prevented by transfectlon with E-cadherin cDNA and was again induced by treatment of
has correlated differentiation
the and
E-cadherin invasiveness
exof
carcinomas. Non-transformed Madin-Darby canine kidney epithelial cells acquire invasive properties when intercellular adhesion is specifically inhibited by the addition
the transfected tibodies (Fig. selective loss
of antibodies against assume a iibroblast-like
differentiation
E-cadherin; (i.e.
the separated dedilferentiated
cells then morphol-
ogy) and invade collagen gels and embryonal heart tissue (Fig. 1) [ 121. Epithelial cells transformed with Harvey and Moloney sarcoma viruses were found to be constitutively libroblast-like E-cadherin [ 121. examining various
C
and invasive, and they We also confirmed this human cell lines derived
do not express correlation by from bladder,
cells with 4) [6-,7*-l. of E-cadherin and
anti-E-cadherin These findings expression
invasiveness
of human
monoclonal indicate that can generate carcinoma
anthe decells
in zdtro. This correlation, a loss of E-cadherin expression coinciding with invasive properties, also holds for carcinoma cells in lliuo. In an extensive study, again from our laboratory, squamous cell carcinomas neck were analyzed for expression
(SCCs) of the of E-cadhetin
head and [ 32*]. A
835
836
Cell-to-ceil
contact
and extracellular
matrix
Bladder
RT112 RT4 EJ28
1
.. _-_-_-_-_-____-_-___-------------~---------------------------------
cx-1 WiDr COLO 205 SW948 SW620
Colon
1 I 1 I
MU-7 MDA-MB-361 ET-549 MDA-MB-231 MDA-MB-435S/l MDA-MB-436
Breast
1
I
..
I
( , I
I
I
1 ,
I 1
-______-_-__________-------------~--------------------------------Lung
LX-1 A-427 A-549 SK-MES-1
I 7 c3 I
1
------------_------_-------------.---------------------------------
Pancreas
Capan-l CapanDAN-G Hs 766T MIA PaCa-2
I 1 I ._
I
7
511,111,111,1,1,111,111,111
3.
I
3000
2500
1111,1111,1111,11~,,1111
2000
1500
1000
Invasion (cells cm-‘)
500
0
500
1000 E-cadherin (cpm/30000
1500
2000
2:
cells)
Fig. 3. E-cadherin expression and invasiveness in vitro of human carcinoma cell lines of various tissues. For the invasion experiments, human carcinoma cell lines were cultured on collagen gels for 3 days and invasive cells were quantified under the light microscope f121. The expression of E-cadherin was measured separately in an indirect cell-binding assay using monoclonal antibody 6F9 and l*sl-labeled goat anti-mouse IRC. Note the lack of invasion in the E-cadherin-expressing carcinoma cell lines and the invasive activity of the negative i&es 16**1. -
similar study on gastric adenocarcinomas has also been reported [33]. In SCCs, E-cadherin expression was found to be inversely correlated both with the loss of difterentiation of the tumor and with lymph node metastasis (Fig. 5) [32*]. Th e well differentiated SCCs expressed E-cadherin, often as strongly as the normal stratified epithelium, the moderately differentiated SCCs expressed intermediate amounts of E-cadherin or were heterogeneous, whereas the poorly differentiated SCCs were all E-cadherin-negative. In seven out of eight patients with infiltrated lymph nodes, we found that the carcinoma cells in the lymph nodes were E-cadherin-negative. These data indicate that the loss of the cell adhesion molecule E-cadherin in fact plays an important role in the progression of human SCCs; that is downregulation of expression is associated with dedifferentiation and metastasis of the tumor cells in vivo. The molecular mechanism responsible for E-cadherin downregulation in the dedifferentiated carcinoma cell lines and the poorly differentiated SCCs is not known. Downregulation could be caused by either mutations in the E-cadherin structural gene or by indirect suppression of E-cadherin gene expression. Interestingly, a new tumor suppressor gene in hepatocellular carcinomas has been localized on human chromosome 16; the common region of allele loss was narrowed down to the positions q22.1 and 23.2 [ 34**]. The human E-cadherin gene is located at position 16q22.1 [35]. Furthermore, loss
of heterozygosity on chromosome 16 was much more frequent in poorly differentiated (88%) than in well differentiated (18%) liver carcinomas. Thus, the E-cadherin gene is a good candidate for this tumor suppressor gene of chromosome 16. The tumor suppressor gene fat of Drosophila has also been identified as a cadherin-like molecule (C Goodman, personal communication). Loss of heterozygosity of chromosome 16 has also been observed in prostrate and breast carcinomas [ 36,371. Downregulation of E-cadherin expression in tumors by regulatory mechanisms can be studied only if normal regulation of E-cadherin gene expression is understood. We have therefore characterized the promoter of the E-cadherin gene [38] and begun to study regulatory proteins controlling it. We found that a promoter fragment ( - 178/ + 92 bp) mediates strong expression of a chloramphenicol acetyitransferase (CAT) reporter gene in epithelial cells (i.e. 60% of the level obtained with Simian virus (SV)40 promoter-enhancer constructs>, whereas in non-epithelial cells this promoter was either inactive or its activity was strongly reduced. By DNase I footprinting and gel retardation analysis, as weU as through functional dissection of the regulatory sequences, two regions that contribute to tissue-specific activity of the promoter were identified. First, a GCrich region at -25 to -58 generates basic epithelial promoter activity, most probably in combination with an ‘initiator’ element [39] present at the single transcription start site of the gene. Second,
Dominant
and
recessive
genes
involved
in tumor
cell
invasion
noma cells, but was silent in their poorly counterparts [ 381.
(i)
Invasive
Birchmeier
et al.
differentiated
cells cm.’
14001200lOOO800 600 400 20001
(ii)
Invasive
2
3
cells cm-’
1600 1400
1200 1000
r
800 600 400 200 0 ;en gels
Fig. 4. (a) lmmunofluorescence staining of mouse E-cadherin in transfected human breast carcinoma cells MDA-MB-435Sil. (i,ii) Human breast carcinoma clone cad-B1 transfected with mouse E-cadherin cDNA; fiii,iv) negative human clone neo-B4. The cells were stained with monoclonal antibody DECMA-1, which recognizes mouse E cadherin fii,iv). Note that the transfected clone cad-B1 expresses mouse E-cadherin at the cell-cell contacts. (b) Invasiveness of human breast carcinoma cells MDA-MB-435Sil transfected with the mouse E-cadherin cDNA. Cells were plated on collagen gels and Invasion was scored over 3 days. (9 E-cadherin-expressing human breast carcinoma clone cad-Bl; (ii) negative human clone neo-B4. 0, invasion in the absence of antibody; 0, invasion in the presence of the dissociating anti-mouse E-cadherin monoclonal antibody DECMA-1 I6”l.
a palindromic sequence at -86 (named E-pal) confers epithelial-specific activity to an SV40 promoter. The E-pal sequence is homologous to ®ulatory elements active in keratin promoters and it competes with these elements for nuclear factor binding [38,40]. Interestingly, the E-pal sequence stimulated transcription of a SV40 promoter-CAT construct in differentiated breast carci-
Fig. 5. E-cadherin expression in squamous cell carcinoma tissues of the head and neck. (a) Normal stratified squamous epithelium; (b) well differentiated carcinoma; fc) moderately differentiated carcinoma; fd) poorly differentiated carcinoma. The well differentiated carcinoma tissue shows a high expression of E-cadherin comparable with that of the normal epithelium, whereas the moderately differentiated carcinoma shows a weak signal. No E-cadherin expression is detectable in the poorly differentiated carcinoma. Bar, 40 pm.
CD44
and
nm23
Some years ago, Matzku et a[ t4I] generated monoclonal antibodies which reacted with a metastasizing cell line of the rat pancreatic adenocarcinoma BSp73, but did not recognize non-metastasizing variants. More recently, Gunthert et al. [2**] have screened cDNA expression libraries from the highly metastatisizing cell line with these monoclonal antibodies, and discovered the cDNA clone pMeta1. It codes for a splice variant of the presumed cell adhesion molecule CD44; the variant has a I62arninoacid residue insert in the extracellular domain, the epitope for the monoclonal antibody. Overexpression of pMeta1 in non-metastasizing pancreatic adenocarcinoma ceils establishes full metastatic behaviour in rats [2**]. This important work shows that cell surface differences between metastatic and non-metastatic states of cells can
837
838
Cell-to-cell
contact
and extracellular
matrix
.
indeed be described by molecular means. Furthermore, this work might give rise to new ways of therapeutic interference with metastatis, for example, by application of soluble parts of the CD44 extracellular domain. Steeg and colleagues performed differential cDNA cloning with related low and high metastatic murine K-1735 melanoma cells and discovered the recessive gene ~~m23 whose mRNA is more strongly expressed in low metastatic melanoma, breast carcinoma, and sarcoma cell lines than in the highly metastatic counterparts (42,431. The gene nm23 codes for a 17kD cytoplasmic protein with 75% amino acid identity to the predicted protein product of abnormal wing d&s (awd) of Drosophila (441. Mutations of this gene in Drcxc@ifa result in altered differentiation and necrosis during larval development. The nm23 gene product has nucleoside diphosphate kinase activity which seems to be important for microtubule polymerization [45]. Overexpression of the nm23 cDNA in highly metastatic melanoma ceU lines resulted in significantly reduced metastatic potential, independent of tumor cell growth [lo**]. Somatic allelic deletions of the nm.23 gene located on chromosome 17 were identified in human breast, renal, colorectal and lung carcinomas; in three out of nine cases, loss of heterozygosity was observed without any other detectable deletion of chromosome 17 [46]. It will now be important to examine the second allele for mutations in the nm23 gene, as weU as to understand the molecular function of the gene product and the mechanism by which it influences metastatic behaviour. Conclusions
Judging from the recent progress in the research of invasion and metastasis, it appears that the genes involved in these processes can be classified as either dominantly or recessively acting. The genes encoding CD44 [2-l and SF [16-l are thought to be dominant, and those encoding nm23 [lo**] and E-cadherin [6**] are thought to be recessive. In certain cases, we anticipate the boundaries between invasion/metastasis genes and oncogenes/tumor suppressor genes to become less distinct. SF, first identified as a motility and dediierentiation factor [ 131, was found recently to influence growth in other systems [16*-,18,20=]. A role for the SF receptor, the c-met-encoded tyrosine kinase [21**,22*], in both inva sion and growth control can be envisaged. On the other hand, the tumor suppressor gene DCC, which codes for a putative ceU adhesion-type component [8-l, might represent an invasion suppressor besides a growth-controlling gene and function in intercellular adhesion. It has also become obvious that invasion and metastasis genes, like oncogenes and tumor suppressor genes, can code for either extracellular, cell surface, cytoplasmic or nuclear components. A variety of molecules potentially important in invasion and metastasis have now been identified. In some cases, even the molecular mechanisms by which these proteins influence malignant behaviour are known. It will now be important to search for the actual mutations respon-
sible for invasive and metastatic behaviour of human tumors. For instance, we know that the SF gene is located at a fragile site on human chromosome 7 [lti*], that E-cadherin is a candidate for the tumor suppressor gene of chromosome 16 [6-*,34-l, and that cadherins can be tumor suppressor genes in other species. Definite proofs for mutations of these genes in human tumors are still lacking, however. To our knowledge, no mutations in the genes for CD44 or ~-123 have yet been discovered in human tumors. Such mutations could in principle either affect the structural genes of components directly involved in invasion and metastasis, or they could indirectly influence the regulatory systems of these genes. The names type I and type II tumor suppressor genes have been used to describe analogous differences in growth-related genes [ 471. The identified candidate genes responsible for invasiveness/metastasis have now to be investigated for both these types of mutations in human tumors. Acknowledgements We would like to thank Dr Carmen Birchmeier (Kdln) for critically reading the manuscript. and B Lelekakis for preparation of the type script. Our work was supported by the Deutsche Krebshilfe and the Deutsche Forschungsgemeinschaft
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