The present invention relates to a gene encoding a new member of
the metalloproteinase family which has been found to be specifically
associated with invasive breast cancer, and to methods of diagnosis
for such cancer comprising detection of the marker or its nucleotide
sequence, and to treatment or prophylaxis by inhibiting, altering
the activity of or binding the marker, or by interfering with its
1. A cDNA encoding either mouse (SEQ. ID No. 3) or human (SEQ.
ID No. 1) stromelysin-3, or fragment thereof.
2. The vector of claim 1 wherein said vector directs the expression
of said cDNA.
3. The cDNA of claim 1 wherein said cDNA is contained in a vector.
4. The vector of claims 3 or 2 wherein said vector is selected
from the group consisting of plasmid, phage, cosmid, retrovirus
5. The cDNA of claim 1 wherein said cDNA encodes human stromelysin-3.
6. The cDNA of claim 5 wherein said cDNA is depicted in Sequence
ID No. 1.
7. The cDNA of claim 1 wherein said cDNA encodes mouse stromelysin-3.
8. The cDNA of claim 7 wherein said cDNA is depicted in Sequence
ID No. 3.
FIELD OF THE INVENTION
The present invention relates to tumor-associated enzyme markers.
Utilizing DNA sequences encoding stromelysin-3, and antibodies
capable of binding to stromelysin-3, the present invention provides
methods for diagnosing cancer, specifically malignant breast cancer.
BACKGROUND OF THE INVENTION
The number of deaths around the world from cancer each year continues
to be of major concern, with only a few treatments being available
for specific types of cancer, and these having no absolute guarantee
of success. Most treatments rely on a general "shotgun"
approach, killing off rapidly growing cells in the hope that rapidly
growing cancerous cells will succumb, either to the treatment, or
at least be reduced in numbers to allow the body's system to eliminate
The search for cures has been hampered by the discovery that different
forms of cancer require different treatments. Given that virtually
any part of the body can be affected by cancer, the task becomes
Nevertheless, despite their differences, cancers also share a number
of similarities. Prime amongst these is the growth of undifferentiated
tissue. However, even this is not 100% accurate, in that certain
cancerous cells do exhibit a degree of differentiation, and this
is shown in the sex cancers, such as those of breast and testicle,
where tumors may be positive or negative for hormone receptors.
Treatment of these tumors depends on the hormone state, and may
be as simple as administration of the relevant hormone antagonist,
such as tamoxifen.
Another factor which most cancers share is that, in order to be
fatal, they must metastasize. Until such time as metastasis occurs,
a tumor, although it may be malignant, is confined to one area of
the body. This may cause discomfort and/or pain, or even lead to
more serious symptoms, but if it can be located, it may be surgically
removed and, if done with adequate care, cause no further problems.
However, once metastasis sets in, surgical resection may remove
the parent tumor, but cancerous cells have invaded the body, and
only chemotherapy, or some particular form of targeting therapy,
stands any chance of success.
Thus, the ability to invade locally and to metastasize in organs
distant from the primary tumor (tumor progression) is the lethal
event in the course of most cancers. Alteration/degradation of the
extracellular matrix (ECM) surrounding the primary tumor, and modifications
of the tumor cell adhesive properties, are known to be crucial for
dissociation of the metastatic cells from the primary tumor cells
(Liotta, Cancer Res. 46:1-7 (1986); Hart et al., Biochim. Biophys.
Acta 989:65-84 (1989)).
Tumor angiogenesis is essential for both primary tumor expansion
and metastatic tumor spread, and angiogenesis itself requires ECM
degradation (Blood et al., Biochim. Biophys. Acta 1032:89-118 (1990)).
Thus, malignancy is a systemic disease in which interactions between
the neoplastic cells and their environment play a crucial role during
evolution of the pathological process (Fidler, I. J., Cancer Metastasis
Rev. 5:29-49 (1986)).
Identifying the alterations in gene expression which are associated
with malignant tumors, including those involved in tumor progression,
is clearly a prerequisite not only for a full understanding of cancer,
but also to develop new rational therapies against cancer. Mutations
and/or abnormal control of expression of two groups of cellular
genes (the proto-oncogenes and the tumor suppressor genes) have
been shown to lead in a multistep process to the loss of normal
growth control and to the acquisition of the transformed cell phenotype
(Weinberg, R. A., Cancer Res. 49:3713-3721 (1989)). However, the
molecular mechanisms which lead to tumor progression are much less
clear (Nowell, P. C., Cancer Res. 46:2203-2207 (1986); Fidler, I.
J., Cytometry 10:673-680 (1989)).
Thus, a further problem arises, in that the genes characteristic
of cancerous cells are very often host genes being abnormally expressed.
It is quite often the case that a particular protein marker for
a given cancer is over-expressed in that cancer, but is also expressed
elsewhere throughout the body, albeit at reduced levels.
Some of the proteins associated with cancers are enzymes which
break down the extracellular matrix, which is important for maintaining
cells in their proper relationship to each other. One such class
is the metalloproteinases (MMPs) (Matrisian, L. M., Trends Genet.
6:121-125 (1990)), so called because they bind zinc. However, none
has been found to be diagnostic, of cancer, or any particular tumors,
although the presence of some may be indicative.
MMPs are involved in a number of physiological and pathological
processes in which ECM remodelling and cell migration are implicated,
e.g. morphogenesis and embryonic development, rheumatoid arthritis,
and tumor invasion and metastasis. MMP inhibitors are known to be
able to block tumor invasion and angiogenesis, which are crucial
for tumor progression, in experimental models.
All members of the matrix metalloproteinase family are proteinases
which degrade at least one component of ECM, are secreted in a latent
form and require activation, such as proteolysis (e.g. by plasmin)
to become active. Interstitial collagenases specifically attack
connective tissue collagens (I to III), whereas type IV collagenases
(72 kD and 92 kD) degrade collagens present in the basement membrane
and fibronectin. Stromelysins (transins)-1 and -2, and also pump-1,
have a much broader substrate specificity, degrading proteoglycans,
laminin, fibronectin, and collagens (III to V).
In man, most of the malignant tumors are carcinomas, and among
non-smokers, breast cancer is the leading cause of mortality by
cancer in woman (Willett, W., Nature 338:389-394 (1989)). The expression
of several oncogenes has been reported to be altered in malignant
breast cells and tumors, but no particular pattern of oncogene/suppressor
gene expression can be consistently associated with breast cancer
(Gullick, W. J., Prog. Growth Factor Res. 2:1-13 (1990)).
However, the neoplastic cells of breast tumors are often embedded
in an adipose and mesenchymal stroma, which may also be important
in control of their proliferation and in their ability to metastasize.
Indeed, it is known that stroma cells can modulate, both positively
and negatively, the growth of normal mammary epithelium (Salomon
et al., in Breast Cancer: Cellular and Molecular Biology (eds.,
Lippman, M. E. and Dickson, R. B.), pp. 363-389 (Kluwer, Boston,
(1988)), and that interactions between the epithelial and stromal
components can influence epithelial carcinogenesis in the mammary
gland (DeOme et al., Cancer Res. 38:2103-2111 (1978)).
The existence of "activated" (Tremblay, G. Exp. Mol.
Pathol. 31:248-260 (1979)) and/or abnormal (Grey et al., Proc. Nat.
Acad. Sci. USA 86:2438-2442 (1989)) fibroblasts in malignant breast
tumors has been postulated, and it has been proposed that breast
cancer could represent a partial escape from dependence on a stromal
requirement or an abnormally strong response to a stromal component.
Owing to the nature of cancerous tissue, it is usually relatively
easy to set up a continuous culture, or cell line, of a given cancer,
a process which makes it easy to study the effects of a given treatment
regimen. A significant drawback to such systems lies in their very
nature--while the test treatment will establish whether it can act
directly against the cells, it is by no means certain what effect
the treatment will have in vivo, and biochemical analysis of such
lines is inevitably in the absence of the tissue normally surrounding
the tumor in vivo.
SUMMARY OF THE INVENTION
It is an object of the invention to identify genes whose expression
is increased in breast carcinomas, whereby breast carcinomas are
considered as malignant epithelial cells interacting with their
We have now found that a previously uncharacterized protein is
diagnostic of certain invasive cancers, especially breast carcinomas,
head and neck squamous cell carcinomas and skin (squamous and basal
cell types) carcinomas. The protein apparently belongs to the group
of metalloproteinases, and is referred to as stromelysin-3 herein.
DESCRIPTION OF THE FIGURES
FIG. 1. Northern blot analysis of total RNA from C1 breast carcinoma
RNA isolated from C1 breast carcinoma and fibroadenoma cells was
probed using four independently isolated cDNA probes as described
in example 1.
FIG. 2. Nucleotide sequence of stromelysin-3 cDNA.
The nucleotide sequence of the cDNA and the deduced amino acid
sequence of stromelysin-3 is presented. Starting from the 5' end,
the underlying nucleotide sequences correspond to: the punitive
signal peptide; the PRCGBPD sequence characteristic of prometalloproteinase,
the conserved histidine residues of the zinc binding domain and
the poly(A.sup.+) signal sequence.
FIG. 3. Comparison of Metalloproteinase Sequences.
Amino acid sequences are aligned and compared for stromelysin-3,
stromelysin-2, stromelysin-1 and collagenase-1, all putative metalloproteinase,
as described in example 3.
FIG. 4. Northern Blot Analysis of Human Metalloproteinase
Total RNA was prepared from four oestrogen receptor negative breast
carcinomas (C1, grade II; C2, C3 and C4, grade III), six oestrogen
receptor positive breast carcinomas (C5, C8 and C9, grade II; C6
and C7, grade III; Cio, grade I) and four breast fibroadenomas (F2-F5).
The RNA was probed with (a) stromelysin-3 RNA, (b) type 1 collagenase
RNA (COI) (c) 92 kd type 4 collagenase RNA (COIV 92k) (d) 72 kd
type 4 collagenase RNA (COIV 72k) (e) stromelysin-1 and 2 RNA (ST1/2)
and pump-1 RNA (PUI), as described in example 4.
FIG. 5. Northern Blot Analysis of Stromelysin-3 RNA From Various
Cell Lines and Tissues.
(a) Three normal and five metastatic auxiliary lymph nodes from
patients with breast cancer; (b) F o u r oestrogen receptive negative
(BT-20, MDA-231, SK-BR-3, HBL-100) and four oestrogen receptor positive
(T-47D, BT-474, ZR-75-1, MCF-7) breast carcinoma lines; (c) Ten
normal human tissues; and (d) HFL-1 Human Foetal Deployed Fibroblasts
(ATCC CCL 153) cultured in serum free medium (1 and 2), in the absence
(1) or presence (2) of tPA are cultured in serum free media supplemented
with 20 mg/ml insulin (3-6), in the absent (3) or presence (4) of
PDGF, (5) of EGF, or (6) of bFGF, were probed with stromelysin-3
sequences, as described in example 5.
FIG. 6. Localization of Stromelysin-3 RNA Transcripts in Sections
of Breast Carcinoma and Embryolimbuds.
Bright field micrographics of tissue sections (X 100) stained with
hematoxylin (A,C,E,G,I and K); and dark field images of the same
sections (still stained with hematoxylin) after in situ hybridization
with anti-sense stromelysin-3 cRNA (B,D,F,H,J and L) as described
in example 6.
FIG. 7. cDNA Sequence of Mouse ST3 cDNA.
cDNA sequence of mouse ST3 gene and comparison with human ST3 cDNA
DETAILED DESCRIPTION OF THE INVENTION
In a first aspect, the present invention provides a process for
the diagnosis of invasive cancer, especially breast, head and neck,
and skin carcinomas, comprising the detection of either stromelysin-3,
or a nucleotide sequence encoding stromelysin-3.
In an alternative aspect, the present invention provides the use
of an agent to interfer with the synthesis or activity of stromelysin-3
in the treatment or prophylaxis of invasive cancer, especially breast,
head and neck, and skin carcinomas.
It will be appreciated that metastatic tumors are invasive, but
that invasive tumors are not necessarily metastatic (for example
basal cell skin carcinomas).
As expression of the stromelysin-3 gene is specific to regions
of ECM degradation and apparently encodes a metalloproteinase, it
is assumed that its ECM degrading activity is crucial to tumor progression
into metastasis. Expression of stromelysin-3 by the stromal cells
is likely to break down an important part of the ECM, thereby allowing
cancerous cells to migrate away from the parent tumor.
Accordingly, any agent which can affect the activity of stromelysin-3
will have an effect on metastasis. Such agents will suitably be
those which either prevent synthesis of the protein, prevent maturation
of the protein, or alter the activity of the enzyme, either by blocking
or by altering its activity.
Expression of the stromelysin-3 gene was found to be, in the first
instance, diagnostic of breast cancer in the metastatic phase. In
fact, this result was achieved by the detection of mRNA in a variety
of resected tumors. Breast cancer was chosen, as this is responsible
for the highest death rate, by cancer, in the non-smoking female
Stromelysin-3 is a novel protein almost certainly belonging to
the MMP family, and is associated with invasive breast carcinomas,
irrespective of their hormonal status.
The members of the MMP family require an activation step, which
may be associated with removal of the pre- and pro-sequences, to
become active. The amino acid sequence of pro- and mature stromelysin-3
is notably different from those of the previously characterized
MMPs, and may exhibit distinct properties regarding maturation,
activation and specificity for ECM components.
The stromelysin-3 gene is expressed by all primary invasive breast
carcinomas, by some of their metastases, and in tissues in which
extensive ECM remodelling is known to occur (uterus, placenta and
limb bud) analyzed for such expression, but not in breast fibroadenomas
and normal adult tissues, suggesting that the stromelysin-3 gene
product plays an important role in breast cancer progression. Also
in agreement with this concept, the stromelysin-3 gene is not expressed
in most in situ breast carcinomas, with the exception of in situ
carcinomas of the comedo type, which are usually considered as preinvasive
lesions and are often associated with microinvasion. Thus the presence
of stromelysin-3 RNA transcripts in other than the low concentrations
found elsewhere in the body, other than uterus or placenta, is diagnostic
of a metastatic cancer or of a cancer with a high risk of becoming
Stromelysin-3 may be involved in the lytic processes which are
likely to be associated with invasive tumor growth. Alternatively,
it is possible that stromelysin-3 could also play a role in the
formation of desmoplasia, which is associated with most invasive
breast cancer lesions, and may represent a host reaction to prevent
further malignant cell spread (Ahmed, A., Pathol. Annu. 25(Pt2):237-286
(1990)). In such an instance, enhancement of stromelysin-3 activity
would be advantageous.
Further, the restricted expression of the stromelysin-3 gene in
stromal fibroblasts immediately surrounding the neoplastic cell
islands is strikingly in contrast to collagenase IV, another metalloproteinase
known to be associated with the malignant conversion of some tumorigenic
cells, and cathepsin D, a lysosomal aspartyl protease whose expression
is increased in breast carcinomas, both of which are expressed,
not in the fibroblasts, but in the neoplastic epithelial cells of
breast cancers (Monteagudo et al., Am. J. Pathol. 136:585-592 (1990);
Garcia et al., Steroid Biochem. 27:439-445 (1987)).
To identify the novel breast cancer marker, a cDNA library was
constructed, and substracted with poly (A+) RNA from a fibroadenoma
source. By this process, the cDNA library was enriched for sequences
characteristic of metastatic cancers.
A number of clones was grown up and screened using probes derived
from poly(A.sup.+) RNA from metastatic tumors and from fibroadenomas.
Those clones which bound more greatly to the probes derived from
metastatic cancer poly(A.sup.+) RNA were then grown up further.
Of the clones generated in this manner, one was found to be differentially
expressed to the extent that high rates of expression were only
found in malignant breast and pharyngeal cancers, head, neck, and
skin (squamous and basal cell type) carcinomas, as well as in the
uterus and placenta, in all of which there is a breaking down of
the ECM, which, when associated with cancer, allows cancerous cells
to spread around the body (metastasis).
In the case of the uterus and the placenta, breakdown of the ECM
occurs naturally, whilst the same event elsewhere is likely to be
characteristic of tumor growth.
It is also interesting to note that expression of the stromelysin-3
gene was found in interdigital differentiation during limb budding
in the foetus, which is associated with breakdown of the ECM.
Characterization of the cDNA sequence illustrated that there was
an open reading frame. Comparison of the encoded protein sequence
with a known library established that the protein belonged to a
family known to break down the ECM. Although the sequence of stromelysin-3
bears less similarity to the other members of its family than any
of the other members bear to each other, it does, nevertheless,
present a number of characteristic regions which serve to identify
the nature of the enzyme. Accordingly, the protein has been named
stromelysin-3, although it may be a collagenase, or may break down
a different constituent of the ECM altogether.
Construction of nucleotide probes to establish the occurrence of
stromelysin-3 mRNA revealed a tissue distribution as described above,
and also enabled photomicrographs to exactly locate the areas of
expression of the stromelysin-3 gene by labelling.
An analysis of the photomicrographs generated by this method showed,
somewhat suprisingly, that the stromelysin-3 gene was not expressed
in the cancerous cells itself, but in the surrounding stroma. In
addition, the stroma did not exhibit any evidence of stromelysin-3
mRNA when the basement membrane of the tumor was still intact (see
FIG. 6). The stromelysin-3 gene is expressed by all primary invasive
breast carcinomas, by some of their metastases nodes, and in tissues
in which extensive ECM remodelling is known to occur (uterus, placenta
and limb bud) analyzed for such expression, but not in breast fibroadenomas
and normal adult tissues, suggesting that the stromelysin-3 gene
product plays an important role in breast cancer progression. Also
in agreement with this concept, the stromelysin-3 gene is not expressed
in most in situ breast carcinomas, with the exception of in situ
carcinomas of the comedo type, which are usually considered as preinvasive
lesions and are often associated with microinvasion. Stromelysin-3
always occurs in the stroma of metastatic cancers, and does not
occur in the stroma of in situ primary tumors (tumors still having
a basement membrane and which are non-invasive). Thus the presence
of stromelysin-3 RNA transcripts in other than the low concentrations
found elsewhere in the body, other than uterus or placenta, is diagnostic
of a metastatic cancer or of a cancer with a high risk of becoming
Furthermore, expression of the stromelysin-3 gene was not detected
in any ER-positive or negative breast cancer cell lines, even though
some of them are known to secrete and possess receptors for EGF/TGF-.alpha.
and FGF (factors which are implicated in expression of the stromelysin-3
Accordingly, standard detection techniques applied to stromelysin-3,
its precursors or its coding nucleotide sequences may be used to
diagnose a metastatic cancer, or to confirm that a primary tumor
has not yet reached the fatal metastatic phase.
Such techniques may include detection with nucleotide probes, such
as in the manner described above, or may comprise detection of the
stromelysin-3 protein by, for example, antibodies or their equivalent.
The nucleotide probes may be any that will hybridize more strongly
to the sequence shown in the accompanying FIG. 2 than to other naturally
occurring sequences. Types of probe include cDNA, riboprobes, synthetic
oligonucleotides and genomic probes. The type of probe used will
generally be dictated by the particular situation, such as riboprobes
for in situ hybridization, and cDNA for Northern blotting, for example.
The most preferred probes are those which correspond to the negative
strand of the cDNA of FIG. 2. It is also possible to provide probes
which recognize introns located within the stromelysin-3 gene, but
this is not necessarily as reliable as detecting RNA transcripts.
Detection of the stromelysin-3 encoding gene, per se, will generally
serve no purpose in diagnosis, but other forms of assay to detect
transcripts and other expression products will generally be useful.
The probes may be as short as is required to differentially recognize
stromelysin-3 mRNA transcripts, and may be as short as, for example,
The form of labelling of the probes may be any that is appropriate,
such as the use of radioisotopes, for example, .sup.32 P and .sup.35
S. Labelling with radioisotopes may be achieved, whether the probe
is synthesized chemically or biologically, by the use of suitably
labelled bases. Other forms of labelling may include enzyme or antibody
labelling such as is characteristic of ELISA, but detection of mRNA
transcripts by labelled probes will generally be by way of X-radiography.
Detection of RNA transcripts may be achieved by Northern blotting,
for example, wherein a preparation of RNA is run on a denaturing
agarose gel, and transferred to a suitable support, such as activated
cellulose, nitrocellulose or glass or nylon membranes. Radiolabelled
cDNA or RNA is then hybridized to the preparation, washed and analyzed
In situ hybridization visualization may also be employed (Example
6), wherein a [.sup.35 S]-labelled antisense cRNA probe is hybridized
with a thin section of a biopsy sample, washed, cleaved with RNase
and exposed to a sensitive emulsion for autoradiography. The samples
may be stained with haematoxylin to demonstrate the histological
composition of the sample, and dark field imaging with a suitable
light filter shows up the developed emulsion.
Immunohistochemistry may be used to detect expression of stromelysin-3
in a biopsy sample. A suitable antibody is brought into contact
with, for example, a thin layer of cells, washed, and then contacted
with a second, labelled antibody. Labelling may be by enzyme, such
as peroxidase, avidin or by radiolabelling. Chromogenic labels are
generally preferable, as they can be detected under a microscope.
More generally preferred is to detect the protein by immunoassay,
for example by ELISA or RIA, which can be extremely rapid. Thus,
it is generally preferred to use antibodies, or antibody equivalents,
to detect stromelysin-3, but use of a suitably labelled stromelysin-3
substrate may also be advantageous.
It may not be necessary to label the substrate, provided that the
product of the enzymatic process is detectable and characteristic
in its own right (such as hydrogen peroxide for example). However,
if it is necessary to label the substrate, then this may also comprise
enzyme labelling, labelling with radioisotopes, antibody labelling,
fluorescent marker labelling or any other suitable form which will
be readily apparent to those skilled in the art.
Most preferred for detecting stromelysin-3 expression is the use
of antibodies. Antibodies may be prepared as described below, and
used in any suitable manner to detect expression of stromelysin-3.
Antibody-based techniques include ELISA (enzyme linked immunosorbent
assay) and RIA (radioimmunoassay). Any conventional procedures may
be employed for such immunoassays. The procedures may suitably be
conducted such that: a stromelysin-3 standard is labelled with a
radioisotope such as .sup.125 I or .sup.35 S, or an assayable enzyme,
such as horseradish peroxidase or alkaline phosphatase and, together
with the unlabelled sample, is brought into contact with the corresponding
antibody, whereon a second antibody is used to bind the first and
radioactivity or the immobilized enzyme assayed (competitive assay);
alternatively, stromelysin-3 in the sample is allowed to react with
the corresponding immobilized antibody, radioisotope- or enzyme-labelled
anti-stromelysin-3 antibody is allowed to react with the system
and radioactivity or the enzyme assayed (ELISA-sandwich assay).
Other conventional methods may also be employed as suitable.
The above techniques may be conducted essentially as a "one-step"
or "two-step" assay. The "one-step" assay involves
contacting antigen with immobilized antibody and, without washing,
contacting the mixture with labeled antibody. The "two-step"
assay involves washing before contacting the mixture with labeled
antibody. Other conventional methods may also be employed as suitable.
Enzymatic and radio-labelling of stromelysin-3 and/or the antibodies
may be effected by conventional means. Such means will generally
include covalent linking of the enzyme to the antigen or the antibody
in question, such as by glutaraldehyde, specifically so as not to
adversely affect the activity of the enzyme, by which is meant that
the enzyme must still be capable of interacting with its substrate,
although it is not necessary for all of the enzyme to be active,
provided that enough remains active to permit the assay to be effected.
Indeed, some techniques for binding enzyme are non-specific (such
as using formaldehyde), and will only yield a proportion of active
It is usually desirable to immobilize one component of the assay
system on a support, thereby allowing other components of the system
to be brought into contact with the component and readily removed
without laborious and time-consuming labor. It is possible for a
second phase to be immobilized away from the first, but one phase
is usually sufficient.
It is possible to immobilize the enzyme itself on a support, but
if solid-phase enzyme is required, then this is generally best achieved
by binding to antibody and affixing the antibody to a support, models
and systems for which are well-known in the art. Simple polyethylene
may provide a suitable support.
Enzymes employable for labelling are not particularly limited,
but may be selected from the members of the oxidase group, for example.
These catalyze the production of hydrogen peroxide by reaction with
their substrates, and glucose oxidase is often used for its good
stability, ease of availability and cheapness, as well as the ready
availability of its substrate (glucose). Activity of the oxidase
may be assayed by measuring the concentration of hydrogen peroxide
formed after reaction of the enzyme-labelled antibody with the substrate
under controlled conditions well-known in the art.
Other techniques may be used to detect stromelysin-3 according
to preference. One such is Western blotting (Towbin et al., Proc.
Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample
is run on an SDS PAGE gel before being transferred to a solid support,
such as a nitrocellulose filter. Anti-stromelysin-3 antibodies (unlabelled)
are then brought into contact with the support and assayed by a
secondary immunological reagent, such as labelled protein A or anti-immunoglobulin
(suitable labels including .sup.125 I, horseradish peroxidase and
Samples for diagnostic purposes may be obtained from any relevant
site. A sample obtained direct from the tumor, such as the stroma
or cytosol, may be ideal, but it may also be appropriate to obtain
the sample from blood, for example. However, if the sample is derived
from blood, highly sensitive assays may be required, as the amount
of stromelysin-3 would then be diluted through the bloodstream.
Such diagnosis may be of particular importance in monitoring progress
of a patient, such as after surgery to remove a tumor. If a reference
reading is taken after the operation, then another taken at regular
intervals, any rise could be indicative of a relapse, or possibly
a metastasis. The taking of such readings may need to take into
account activity in the uterus, for example.
Anti-stromelysin-3 antibodies may also be used for imaging purposes.
Besides enzymes, other suitable labels include radioisotopes, iodine
(.sup.125 I, .sup.121 I), carbon (.sup.14 C), salphee (.sup.35 S),
tritium (.sup.3 H), indium (.sup.112 In), and technetium (.sup.99m
Tc), fluorescent labels, such as fluorescein and rhodamine, and
However, for in vivo imaging purposes, the position becomes more
restrictive, as antibodies are not detectable, as such, from outside
the body, and so must be labelled, or otherwise modified, to permit
Markers for this purpose may be any that do not substantially interfere
with the antibody binding, but which allow external detection. Suitable
markers may include those that may be detected by X-radiography,
NMR or ESR. For X-radiographic techniques, suitable markers include
any radioisotope that emits detectable radiation but that is not
overtly harmful to the patient, such as barium or caesium, for example.
Suitable markers for NMR and ESR generally include those with a
detectable characteristic spin, such as deuterium, which may be
incorporated into the antibody by suitable labelling of nutrients
for the relevant hybridoma, for example.
In the case of in vivo imaging methods, an antibody or antibody
fragment which has been labelled with an appropriate detectable
imaging moiety, such as a radioisotope (for example, .sup.131 I,
.sup.112 In, .sup.99m Tc), a radio-opaque substance, or a material
detectable by nuclear magnetic resonance, is introduced (for example,
parenterally, subcutaneously or intraperitoneally) into the subject
(such as a human) to be examined.
The size of the subject, and the imaging system used, will determine
the quantity of imaging moiety needed to produce diagnostic images.
In the case of a radioisotope moiety, for a human subject, the quantity
of radioactivity injected will normally range from about 5 to 20
millicuries of technetium-99m. the labelled antibody or antibody
fragment will then preferentially accumulate at the location of
cells which contain stromelysin-3. The labelled antibody or antibody
fragment can then be detected using known techniques.
For a general discussion of this technological area, see S. W.
Burchiel et al., "Immunopharmacokinetics of Radiolabelled Antibodies
and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, eds., S. W. Burchiel and B. A. Rhodes, Masson
Publishing Inc. (1982)).
The antibodies may be raised against either a peptide of stromelysin-3
or the whole molecule. Such a peptide may be presented together
with a carrier protein, such as an albumin, to an animal system
or, if it is long enough, say 25 amino acid residues, without a
carrier. Human antibodies are unlikely to be able to recognize stromelysin-3,
as this protein will represent a self protein.
As used herein, the term "peptide" means any molecule
comprising 2 or more amino acids linked via a peptide bond. As such,
the term includes oligopeptides, polypeptides and proteins.
Polyclonal antibodies generated by the above technique may be used
direct, or suitable antibody producing cells may be isolated from
the animal and used to form a hybridoma by known means (Kohler and
Milstein, Nature 256:795 et seq. (1975)). Selection of an appropriate
hybridoma will also be apparent to those skilled in the art, and
the resulting antibody may be used in a suitable assay to identify
Antibodies, or their equivalents, may also be used in accordance
with the present invention for the treatment or prophylaxis of metastatic
cancers. Administration of a suitable dose of the antibody may serve
to block production, or to block the effective activity of stromelysin-3,
and this may provide a crucial time window in which to treat the
Prophylaxis may be appropriate even at very early stages of the
disease, as it is not known what actually leads to metastasis in
any given case. Thus, administration of the antibodies, their equivalents,
or factors, such as TIMPs (naturally occurring compounds which regulate
the MMPs-tissue inhibitors of metalloproteinases), which interfere
with stromelysin-3 activity, may be effected as soon as cancer is
diagnosed, and treatment continued for as long as is necessary,
preferably until the threat of the disease has been removed.
A preferred form of treatment is to employ the so-called magic
bullet technique, where a suitable toxin is attached to the antibodies
which then target the area of the tumor. Such toxins are well known
in the art, and may comprise toxic radioisotopes, heavy metals,
enzymes and complement activators, as well as such natural toxins
as ricin which are capable of acting at the level of only one or
two molecules per cell. It may also be possible to use such a technique
to deliver localized doses of hormone antagonists or any other suitable
physiologically active compounds, which may be used, for example,
to treat cancers.
It will be appreciated that antibodies for use in accordance with
the present invention, whether for diagnostic or therapeutic applications,
may be monoclonal or polyclonal as appropriate. Antibody equivalents
of these may comprise: the Fab' fragments of the antibodies, such
as Fab, Fab', F(ab').sub.2 and Fv; idiotopes; or the results of
allotope grafting (where the recognition region of an animal antibody
is grafted into the appropriate region of a human antibody to avoid
an immune response in the patient), for example. Other suitable
modifications and/or agents will be apparent to those skilled in
In addition to using antibodies to inhibit or remove stromelysin-3,
it may also be possible to use other forms of inhibitor. Such inhibitors
may be general (for ECM degrading enzymes, for example), or specific
for stromelysin-3. Tissue inhibitors of metalloproteinases (TIMPs)
are known to exist, and it is extremely likely that there is a specific
TIMP for stromelysin-3. Such a TIMP is easily identifiable by standard
Synthetic inhibitors of stromelysin-3 may also be manufactured,
and these will generally correspond to the area of the substrate
affected by the enzymatic activity. It is generally preferred that
such inhibitors correspond to a frozen intermediate between the
substrate and the cleavage products, but it is also possible to
provide a sterically hindered version of the binding site, or a
version of the binding site which will, itself, irreversibly bind
to stromelysin-3. Other suitable inhibitors will be apparent to
the skilled person.
Other methods for blocking stromelysin-3 activity may also be employed.
These may constitute denaturing agents, for example, although these
tend to be non-specific and could only be adequately employed if
they could be targeted, such as by the use of specific antibodies.
Other forms of stromelysin-3 blocking activity could be effected
by blocking the progress from pre-proprotein through to protein.
This process provides several target stages, and it is only necessary
to identify a stage which can be independently blocked so as not
to affect other vital enzymes, or which can again be targeted.
It may also be possible to use peptides or other small molecules
to selectively recognize a tertiary structure on stromelysin-3,
thereby blocking its enzymic activity. Such an activity blocker
need not necessarily bind the active site, but may serve to alter
or freeze the tertiary structure of stromelysin-3, destroying, suspending
or altering its activity. The blocker also need not necessarily
act by itself, but may be linked to another molecule for this purpose,
or may serve as a recognition site for a suitable inactivating agent.
Our studies have demonstrated that the occurrence of type I collagenase
and 92 kD type IV collagenase mRNAs is exclusively associated with
malignant tumors, although the reverse does not always hold (i.e.
tumors are not always associated with these proteins).
There is apparently a parallel between the expression of the stromelysin-3
gene and that of the tenascin gene, in invasive breast carcinomas.
The ECM glycoprotein tenascin (Chiquet-Ehrismann et al., Cell 47:131-139
(1986)) appears to play an essential role in epithelial mesenchyme
cell interactions and cell migration during normal development,
including that of the mammary gland during organogenesis.
Tenascin has consistently been found to be over-expressed in the
fibrous stroma of malignant breast tumors, and appears to be induced
in a similar manner to stromelysin-3. When compared with fibronectin,
tenascin is a poor substrate for attachment of mammary tumor epithelial
cells, suggesting that it may allow them to become invasive.
Thus, stromelysin-3 may act in concert with tenascin during the
invasive phase of breast cancer. Stromelysin-3 and tenascin may
also be co-expressed during embryogenesis in the regions where epithelium-mesenchyme
interactions are known to play an important role, and where cell
migration is taking place.
Accordingly, the present invention also provides a process for
the diagnosis of metastatic cancer as defined above, further comprising
the detection of any of the foregoing proteins, or a nucleotide
sequence encoding them.
The invention also provides a use in the treatment or prophylaxis
of metastic cancer, further comprising the use of an agent to bind
any of the foregoing proteins.
The present invention further provides a nucleotide sequence encoding
all or part of stromelysin-3. The sequence of stromelysin-3 is preferably
that shown in FIG. 2 of the accompanying drawings, whilst the nucleotide
sequence is also preferably that shown in FIG. 2. However, it will
be appreciated that the nucleotide sequence may be substantially
different from that shown in the Figure, due to degeneracy in the
genetic code, provided that it still encodes at least a part of
The necessary sequence may vary even further, according to the
use to which it is to be put. If it is intended for use to detect
RNA transcripts in biological samples, then it will usually be preferable
that it more nearly corresponds to the sequence given in FIG. 2.
However, the sequence may still vary, provided that hydbridization
is possible under the selected conditions of stringency.
A probe may be reverse-engineered by one skilled in the art from
the peptide sequence of FIG. 2. However use of such probes may be
limited, as it will be appreciated that any one given reverse-engineered
sequence will not necessarily hybridize well, or at all with any
given complementary sequence reverse-engineered from the same peptide,
owing to the degeneracy of the genetic code. This is a factor common
in the calculations of those skilled in the art, and the degeneracy
of any given sequence is frequently so broad as to yield a large
number of probes for any one sequence.
If the nucleotide sequence is required for expression of a stromelysin-3
peptide or entire enzyme, then there may be a considerably greater
leeway, both as described above with respect to the genetic code,
and also to the fact that some amino acid sequence of stromelysin-3
may be varied without significant effect on the structure or function
of the enzyme.
If such differences in sequence are contemplated, then it should
be borne in mind that there will be critical areas on the molecule
which determine activity. Such areas will usually comprise residues
which make up the binding site, or which form tertiary structures
which affect the binding site. In general, it is possible to replace
residues which form the tertiary structure, provided that residues
performing a similar function are used. In other instances, the
type of residue may be completely unimportant.
Accordingly, the present invention also includes any variants and
mutants on the sequence which still show substantial stromelysin-3
activity, or which exhibit characteristic regions of stromelysin-3
for use in generating antibodies, for example. Such variants and
mutants include deletions, additions, insertions, inversions, repeats
and type-substitutions (for example, substituting one hydrophilic
residue for another, but not strongly hydrophilic for strongly hydrophobic
as a rule). Small changes will generally have little effect on activity,
unless they are an essential part of the molecule, and may be a
side-product of genetic manipulation, for example, when generating
extra restriction sites, should such be desired. Modification may
also include replacement of one or more of the residues with any
other suitable residue, and such replacement may either be 1:1 or
any other suitable ratio, greater or less than unity.
Spot mutations and other changes in the coding sequence may be
effected to add or delete restriction sites, for example, to otherwise
assist in genetic manipulation/expression, or to enhance or otherwise
conveniently to modify the stromelysin-3 molecule.
It will also be appreciated that a stromelysin-3 equivalent will
be found in other animals, especially mammals, and sequence information
from such sources can be of particular importance to elucidate the
conserved regions of the stromelysin-3 molecule. For example, the
corresponding sequence in the mouse is.perspectiveto.80% conserved,
including such as the 10 amino acid sequence in the prodomain characteristic
of stromelysin-3. It will also be appreciated that animal sequences
corresponding to human stromelysin-3 sequences will be readily detectable
by methods known in the art and described above, and such sequences
and their peptides, as well as mutants and variants thereof, form
a part of the invention.
The sequences of the invention may also be engineered to provide
restriction sites, if desired. This can be done so as not to interfere
with the peptide sequence of the encoded stromelysin-3, or may interfere
to any extent desired or necessary, provided that the final product
has the properties desired.
As stated above, although hybridization can be an unreliable indication
of sequence homology, preferred sequences will generally be those
showing in excess of 50%, preferably 70% and more preferably 80%
homology with the sequence of FIG. 2.
As with the other metalloproteinases, stromelysin-3 is originally
expressed as a pre-proenzyme. Thus, two stages of cleavage are observed
in vivo. Cleavage is not necessarily a requirement for in vitro
expression, and it may be possible for E. coli, for example, to
express the mature protein.
Where it is desired to express stromelysin-3 or a characteristic
peptide thereof, any suitable system can be used. The general nature
of suitable vectors, expression vectors and constructions therefor
will be apparent to those skilled in the art.
By "characteristic" is meant any peptide which has a
sequence unique to stromelysin-3. Such a sequence may be important
to stromelysin-3 activity, or may just be a sequence not found in
other peptides. However, sequences important to stromelysin-3 activity
are generally preferred, as these are more likely to be conserved
within a population.
Suitable expression vectors may be based on phages or plasmids,
both of which are generally host-specific, although these can often
be engineered for other hosts. Other suitable vectors include cosmids
and retroviruses, and any other vehicles, which may or may not be
specific for a given system. Again, control sequences, such as recognition,
promoter, operator, inducer, terminator and other sequences essential
and/or useful in the regulation of expression, will be readily apparent
to those skilled in the art, and may be associated with the natural
stromelysin-3 sequence or with the vector used, or may be derived
from any other source as suitable. The vectors may be modified or
engineered in any suitable manner.
Correct preparation of nucleotide sequences may be confirmed, for
example, by the method of Sanger et al. (Proc. Natl. Acad. Sci.
USA 74:5463-7 (1977)).
A cDNA fragment encoding the stromelysin-3 of the invention may
easily be inserted into a suitable vector. Ideally, the receiving
vector has suitable restriction sites for ease of insertion, but
blunt-end ligation, for example, may also be used, although this
may lead to uncertainty over reading frame and direction of insertion.
In such an instance, it is a matter of course to test transformants
for expression, 1 in 6 of which should have the correct reading
frame. Suitable vectors may be selected as a matter of course by
those skilled in the art according to the expression system desired.
By transforming a suitable organism or, preferably, eukaryotic
cell line, such as HeLa, with the plasmid obtained, selecting the
transformant with ampicillin or by other suitable means if required,
and adding tryptophan or other suitable promoter-inducer (such as
indoleacrylic acid) if necessary, the desired stromelysin-3 may
be expressed. The extent of expression may be analyzed by SDS polyacrylamide
gel electrophoresis--SDS-PAGE (Lemelli, Nature 227:680-685 (1970)).
Suitable methods for growing and transforming cultures etc. are
usefully illustrated in, for example, Maniatis (Molecular Cloning,
A Laboratory Notebook, Maniatis et al. (eds.), Cold Spring Harbor
Labs, NY (1989)).
Cultures useful for production of stromelysin-3, or a peptide thereof,
may suitably be cultures of any living cells, and may vary from
prokaryotic expression systems up to eukaryotic expression systems.
One preferred prokaryotic system is that of E. coli, owing to its
ease of manipulation. However, it is also possible to use a higher
system, such as a mammalian cell line, for expression of a eukaryotic
protein. Currently preferred cell lines for transient expression
are the HeLa and Cos cell lines. Other expression systems include
the Chinese Hamster Ovary (CHO) cell line.
One valuable system is the baculovirus system, wherein butterfly
cells are cotransfected with a DNA vector encoding stromelysin-3,
or a suitable peptide, and baculovirus DNA. Recombination occurs
within the cell, and suitable baculovirus recombinants may be selected
by standard techniques. Thereafter, the recombinant may be used
to infect the cell line as desired, stromelysin-3 or peptide being
expressed on infection. A particular advantage of this system is
the amount of protein produced, which can be in the range of about
1 to about 500 mg/liter.
Although such systems tend not to be as easy to use as the E. coli
system, the advantage lies in the processing of the protein after
primary synthesis. E. coli, for example, does not employ the same
system for processing pre-proproteins as mammalian cells.
Other expression systems which may be employed include streptomycetes,
for example, and yeasts, such as Saccharomyces spp., especially
S. cerevisiae. Any system may be used as desired, generally depending
on what is required by the operator. Suitable systems may also be
used to amplify the genetic material, but it is generally convenient
to use E. coli for this purpose where only proliferation of the
DNA is required.
It may be advantageous to produce only the mature enzyme, for the
purposes of raising antibodies, as the sequence of the mature enzyme
is common to the pro- and prepro sequences also. However, it will
be appreciated that cleavage of the pro and prepro portions may
alter the tertiary configuration of the molecule, and so it is possible
that an antibody raised against the mature enzyme will not detect
the proenzyme, for example. Antibodies raised to the enzyme in either
of its earlier states and/or to the pre- or pro-peptides which are
cleaved may also prove useful.
The peptide or nucleotide sequence may be any that is characteristic
of stromelysin-3, having consideration to the purpose to which it
is to be put. Ideally, the sequences would be completely characteristic
of stromelysin-3, but the length of such sequences may vary according
to the region of the stromelysin-3 molecule. The most preferred
regions are those which are highly conserved, and which are not
shared with other proteins, although it may be advantageous if the
sequence is characteristic of the MMPs or, more particularly, those
MMPs associated with invasive tumors.
The invention includes and relates to equivalents of the above
peptide and nucleotide sequences, the term "equivalent"
being used in the sense of the preceding description, that is to
say, equivalents in the sense of sequences having substitutions
at the C- or N-terminals, or anywhere else.
The invention also includes mutants of the sequences, the term
"mutants" being used with reference to deletions, additions,
insertions, inversions and replacement of amino acid residues or
bases in the sequence subject to the restrictions described above.
The present invention further includes variants of the sequences,
which term is used in relation to other naturally occurring stromelysin-3
which may be discovered from time to time and which shares essentially
the same sequence as shown in FIG. 2, but which vary therefrom in
a manner to be expected within a large population. Within this definition
lie allelic variation and those peptides from other species showing
a similar type of activity and having a related sequence. Also included,
although less preferred, are animal sequences.
We have also discovered that stromelysin-3 expression can be stimulated
by, for example, growth factors and tumor promoters. Typical examples
of such factors include EGF FGF and PDGF and TPA. Thus, in conjunction
with the foregoing processes, detection of any of these factors
in a tumor sample may also help to diagnose the metastatic condition
of a cancer.
Thus, the invention also provides the treatment of a metastatic
cancer by altering the expression of the stromelysin-3 gene. This
may be effected by interfering with the factor required to stimulate
stromelysin-3 production, such as by directing specific antibodies
against the factor, which antibodies may be further modified to
achieve the desired result. It may also be possible to block the
receptor for the factor, something which may be more easily achieved
by localization of the necessary binding agent, which may be an
antibody or synthetic peptide, for example.
Affecting stromelysin-3 gene expression may also be achieved more
directly, such as by blocking of a site, such as the promoter, on
the genomic DNA.
Where the present invention provides for the administration of,
for example, antibodies to a patient, then this may be by any suitable
route. If the tumor is still thought to be, or diagnosed as, localized,
then an appropriate method of administration may be by injection
direct to the site. If the target is breast cancer, then an injection
to the breast may suffice, or an implant may be used. If TIMPs are
to be administered, for example, then it may also be possible to
employ a dermal patch for prolonged administration.
If the cancer is pharyngeal, then a further option may be oral
administration, for example, by means of gargling.
In either instance, administration may instead, or additionally,
be by injection, including subcutaneous, intramuscular, intravenous
and intradermal injections.
Formulations may be any that are appropriate to the route of administration,
and will be apparent to those skilled in the art. The formulations
may contain a suitable carrier, such as saline, and may also comprise
bulking agents, other medicinal preparations, adjuvants and any
other suitable pharmaceutical ingredients.
Suitable preparations may also include vaccines comprising stromelysin-3
or a characteristic peptide thereof. Such vaccines may be active
or passive, but passive is generally preferred as stromelysin-3
expression occurs in the uterus, and indefinite exposure to antistromelysin-3
antibodies may have undesirable effects. However, active vaccination
may be advantageous, especially where a patient has had a hysterectomy,
as no tissues will then normally express stromelysin-3. Other suitable
vaccines include recombinant viruses containing a nucleotide sequence
encoding a stromelysin-3 or a characteristic peptide thereof. One
suitable such virus is the vaccinia virus.
The following Examples serve to illustrate the present invention,
and are not intended to limit the invention in any manner.
Cloning of a breast cancer specific cDNA
A breast cancer cDNA library was constructed in the .lambda.gt10
vector using poly(A.sup.+) RNA from a surgical resection-sample
(referred to as tumor C1) of a primary breast cancer. 50,000 plaques
were differentially screened using (+) and (-) probes corresponding
to cDNAs reverse-transcribed from C1-poly(A.sup.+) RNA and poly(A.sup.+)
RNA from a breast fibroadenoma (referred to as F1), respectively.
FIG. 1 shows a Northern blot analysis of total RNA from C1-breast
carcinoma and F1-fibroadenoma using cDNA probes of four genes (A-D)
exhibiting higher levels of expression in the carcinoma than in
the fibroadenoma. Each lane contained 8 .mu.g of total RNA. The
filters were reprobed using the 36B4 probe which corresponds to
an ubiquitously expressed gene (Rio et al., Proc. Nat. Acad. Sci.
USA 84:9243-9247 (1987)).
Specifically, total RNA was prepared (Chirgwin et al., Biochemistry
18:5294-5299 (1979)) from surgical specimens stored in liquid nitrogen,
and poly(A.sup.+) RNA was selected by oligo(dT)-cellulose chromatography.
A breast cancer-enriched cDNA library was constructed using cDNA
prepared from an oestrogen receptor-negative, grade II, ductal carcinoma
(referred to as C1), in which stromal cells represented approximately
50% of the total cell population.
Prior to cloning, the single-stranded cDNA was substracted with
an excess of poly(A.sup.+) RNA from a breast fibroadenoma (referred
to as F1), and the single-stranded enriched material was purified
by hydroxyapatite chromatography (Davis et al., Proc. Nat. Acad.
Sci. USA 81:2194-2198 (1984); Rhyner et al., Neuroscience Res. 16:167-181
The breast cancer-enriched cDNA was made double-stranded and cloned
into the EcoRI site of the .lambda.gt10 vector. Three million recombinant
phages were obtained, and .perspectiveto.50,000 were differentially
screened using replica nylon filters (Biodyne A, Pall Corporation)
from plates containing .perspectiveto.5,000 cDNA clones.
(+) and (-) probes were made using C1-breast cancer cDNA and Fl-breast
fibroadenoma cDNA, respectively. Both probes were substracted (Davis
et al., Proc. Nat. Acad. Sci. USA 81:2194-2198 (1984); Rhyner et
al., Neuroscience Res. 16:167-181 (1986)) with an excess of total
human liver RNA before [.sup.32 P]-labeling using random priming
Hybridizations were for two days under stringent conditions (50%
formamide, 42.degree. C.) and washing was in 2.times.SSC, 0.1% SDS,
at 22.degree. C., followed by 0.1.times.SSC, 0.1% SDS at 55.degree.
C. 130 differentially labelled plaques were selected for a second
The cDNA inserts of five differential plaques taken at random were
purified by PCR amplification, [.sup.32 P]-labelled, and hybridized
to all of the differential plaques to identify related clones. This
procedure was repeated several times with differential plaques taken
at random, finally yielding four genes referred to as A to D, which
exhibited higher levels of expression in C1-carcinoma than in F1-fibroadenoma.
The Northern blots for C1-breast cancer and F1-breast fibroadenoma
were prepared using total RNA (8 .mu.g) separated by electrophoresis
in 1% agarose gels containing formaldehyde and transferred to Hybond-N
The blots were stained with methylene blue before prehybridization
to check for the integrity and amounts of transferred RNA. Hybridization
(18 h) and washing were performed under standard conditions, as
described above, using [.sup.32 P]-labeled cDNA inserts corresponding
to A-D genes.
The genes A and B, which were also expressed in normal colon (not
shown), were not examined further.
Although expressed in colon (not shown), the C gene was partially
characterized because of its high level of differential expression
(FIG. 1). It was also expressed in a variety of transformed epithelial
cell lines and in normal human skin (not shown). Sequencing of the
cDNA of one C clone indicated that the corresponding gene belongs
to the keratin gene superfamily (data not shown).
Finally, the D gene (also referred to herein as the stromelysin-3
gene) was further studied, because of its marked differential expression
between C1-carcinoma and F1-fibroadenoma (FIG. 1), and also because
it was not expressed in normal human colon and in a number of other
human tissues (infra).
Sequencing of Stromelysin-3 Gene and Encoded Protein
Several independent clones were isolated from a non-substracted
C1-breast cancer .lambda.gt10 cDNA library using a D cDNA insert
as probe, and sequenced. FIG. 2 shows the nucleotide sequence of
the full length D cDNA and the corresponding protein sequence.
The cDNA open reading frame, encoding a 488 amino acid-long protein,
is followed by a 714 base 3'-untranslated region containing a poly(A)
addition signal located 14 bases upstream from the 3'-end of the
RNA. A presumptive initiation methionine is located at nucleotide
position 10-12. Although the corresponding AUG is not associated
with and located in a sequence which conforms to the Kozak consensus
motif, translation is probably initiated at this AUG, since the
sequence immediately downstream corresponds to that for a hydrophobic
leader peptide, an expected feature (infra).
In FIG. 2, which shows the nucleotide sequence of stromelysin-3
cDNA and deduced amino acid sequence, the nucleotide residues are
numbered in the 5' to 3' direction and deduced amino acids in the
open reading frame are designated by their one-letter codes. Starting
from the 5'-end, the underlined nucleotide sequences correspond
to: the putative signal peptide (two potential cleavage sites are
marked by arrows); the PRCGVPD sequence characteristic of prometalloproteinases;
the conserved histidine residues of the zinc-binding domain (Matrisian,
L. M., Trends Genet. 6:21-125 (1990)); and the poly(A) addition
Specifically, a cDNA insert corresponding to the 3'-part of D cDNA
[250 bp including a 19 bp poly(AT) region] was [.sup.32 P]-labeled
by random priming synthesis and used to screen a non-substrated
.lambda.gt10 cDNA library generated from C1-breast tumor poly(A.sup.+)
RNA by the method of Gubler and Hoffmann (Gene 25:262-269 (1983)].
Several independent clones were identified and subcloned in M13
sequencing vector. DNA sequence was determined by the dideoxy method
using sequenase and the deaza-dGTP reagent kit from US Biochemical.
The sequence was analyzed using the PC/GENE software package.
Stomelysin-3, a putative metalloproteinase
FIG. 3 shows a comparison of the predicted amino-acid sequences
of human stromelysins and human type I collagenase.
(a) Amino-acid sequences were aligned using a multialignment program
(Higgins et al., Gene 73:237-244 (1988)). Amino-acid residues identical
in all of the four sequences are marked by stars. The arrows denote
putative signal peptide eleavage sites of stromelysin-3. The arrowhead
points to the cleavage which occurs on activation of type I procollagenase
and prostromelysins. The 10 amino-acid residues specific to stromelysin-3
at the level of this cleavage site are boxed. The PRCGVPD sequence
and the conserved residues of the putative zincbinding domain are
(b) Left, regions of similarity (in percent amino-acid identity)
between stromelysin-3, stromelysin-1 (ST1, Whitham et al., Biochem.
J. 240:913-916 (1986)), stromelysin-2 (ST2, Muller et al., Biochem.
J. 253:187-192 (1988)) and type I collagenase (COI, Whitham et al.,
Biochem. J. 240:913-916 (1986));
(b) Right, regions of similarity between ST1, ST2 and COI; P indicates
the signal peptide and the pro-domain; ENZ indicates the domain
corresponding to the mature active enzymes.
Thus, comparison of the derived protein sequence with the Swissprot
data library (release 14) showed that the new protein belongs to
the family of secreted matrix metalloproteinases (MMPs) (FIG. 3a).
Accordingly, the new protein possesses an hydrophobic N-terminal
leader sequence candidate (underlined in FIG. 2), and exhibits the
highly conserved sequence PRCGVPD (amino-acid residues 78-84), which
is characteristic of the prodomain of the MMPs, as well as having
the zinc binding site of MMPs (amino-acid residues 212-225-FIG.
3a) (Matrisian, L. M., Trends Genet. 6:121-125 (1990)).
By analogy with the other members of the family, the N-terminal
amino acid of the mature protein is likely to correspond to phenylalanine
98 of the pre-proprotein (Whitham et al., Biochem. J. 240:913-916
(1986)) (FIG. 3a). After optimal alignments, the similarity between
the putative mature protein is 40% with stromelysin-1 (Whitham et
al., Biochem. J. 240:913-916 (1986)), 38% with stromelysin-2 (Muller
et al., Biochem. J. 253:187-192 (1988)) and 36% with type I collagenase
(Goldberg et al., J. Biol. Chem. 261:6600-6605 (1986)) (FIG. 3b).
The substrate specificity of the new protein is not known. Herein,
it is referred to as stromelysin-3, although its similarity with
stromelysin-1 (40%) is clearly much below that existing between
stromelysin-1 and stromelysin-2 (79%), and even lower than the similarity
existing between type I collagenase and stromelysin-1 (53%) (FIG.
3b). Thus, while the protein is an MMP, the cognomen "stromelysin"
is not necessarily strictly accurate, but is convenient.
In addition, upstream of the PRCGVPD sequence, there is no significant
similarity between stromelysin-3 and the other MMPs with which it
is has been compared (FIG. 3). However, stromelysin-3 has a unique
short sequence (amino-acid residues 88-97) at a position corresponding
substantially precisely with the proprotein cleavage site of type
I collagenase and the stromelysins (Whitham et al., supra). Further,
stromelysin-3, as with type I collagenase and the other stromelysins,
does not exhibit the fibronectin-like domain characteristic of type
IV collagenases (Wilhelm et al., J. Biol. Chem. 264:17213-17221
Over-expression in Breast Carcinomas
The occurrence of stromelysin-3 RNA transcripts was studied in
resected samples of 30 breast carcinomas and five breast fibroadenomas.
FIG. 4 shows Northern blot analyses of human metalloproteinase
RNAs in breast tumors:
(a) stromelysin-3 RNA;
(b) type I collagenase RNA (COI);
(c) 92-kD type IV collagenase RNA (COIV 92K);
(d) 72-kD type IV collagenase RNA (COIV 72K);
(e) stromelysin-1 and -2 RNA's (ST1/2); and
(f) pump-1 RNA (PUI).
Total RNA was prepared from four oestrogen receptor-negative breast
carcinomas (C1, grade II; C2, C3 and C4, grade III), six oestrogen
receptor-positive breast carcinomas (C5, C8 and C9, grade II; C6
and C7 grade III; CIO, grade I) and four breast fibroadenomas (F2-F5).
Each lane contained 8 .mu.g of RNA. The 36B4 signal corresponds
to the RNA of a control gene (FIG. 1).
Specifically, several Northern blots were prepared in parallel
with identical RNA samples, as for FIG. 1, and hybridized with either
of the following cDNA probes: (a) 1.6 kb insert covering the 3'
-part of stromelysin-3 cDNA, (b) COI cDNA, (e) ST2 cDNA (which cross-hybridizes
with ST1 RNA), (f) PUI cDNA (COI, ST2 and PU1 probes kindly provided
by R. Breathnach, Muller et al., Biochem. J. 253:187-192 (1988)),
or 80 mer antisense oligonucleotide probes corresponding to (c)
COIV 92K (nucleotides 2144-2223, Wilhelm et al., J. Biol. Chem.
2641:7213-17221 (1989)) and (d) COIV 72K (nucleotides 1937-2016,
Collier et al., J. Biol. Chem. 263:6579-6587 (1988)).
The cDNA probes were (.sup.32 P)-labeled using random priming synthesis
(.perspectiveto.5.times.10.sup.8 cpm/.mu.g) and the oligonucleotides
were labeled using 5'-end kination .perspectiveto.10.sub.8 cpm/.mu.g).
Hybridizations were carried out under stringent conditions (42.degree.
C., 50% formamide) with .perspectiveto.10.sup.6 cpm/ml. The filters
were then washed in 2.times.SSC, 0.1% SDS, at 22.degree. C., followed
by 0.1.times.SSC, 0.1% SDS at 55.degree. C. Autoradiography was
for (a), 18 h, (b), 20 h, (c), (d) and (e), 4 days, (f), 2 days,
at -80.degree. C. with an intensifying screen.
Stromelysin-3 mRNA was found in all of the breast carcinomas, regardless
of whether they were oestradiol receptor (ER) positive (C5-C10)
or negative (C1-C4) (FIG. 4a), but not in the fibroadenoma samples,
with one exception (F2) where the level of expression was similar
to the lowest level observed in breast carcinomas.
The occurrence of RNA transcripts of the other members of the MMP
gene family was also investigated in the same samples (FIGS. 4b-f).
These other members of the MMP family can clearly be separated into
two classes, according to their pattern of expression in human breast
tumors. The first class includes the 72 kD type IV collagenase (COIV
72K, FIG. 4d), stromelysin-1 and -2 (ST1/2, FIG. 4e) and pump-1
(PU1, FIG. 4f), all of which genes were expressed in both malignant
and benign tumors. By contrast, the second class, which includes
stromelysin-3 (FIG. 4a), type I collagenase (COI, FIG. 4b) and the
92 kD type IV collagenase (COIV 92K, FIG. 4c) genes, shows over-expression
only in breast carcinomas, although only stromelysin-3 was consistently
The patterns of expression were not identical for the three genes
of the second class. Type I collagenase RNA transcripts were not
detected in the C5, C6, C7 and CIO carcinomas, and the 92 kD type
IV collagenase RNA transcripts were not seen in the C7 and CIO samples,
but the stromelysin-3 RNA transcripts were clearly detected in all
Thus, stromelysin-3 appears to be diagnostic of invasive breast
carcinomas, while type I collagenase and the 92 kD type IV collagenase
may also be specifically involved in breast cancer progression in
Expression in Cells from Various Sources
FIG. 5 shows Northern blot analyses of stromelysin-3 RNA in various
cell lines and tissues.
(a) Three normal and five metastatic auxillary lymph nodes from
patients with breast cancers;
(b) four oestrogen receptor-negative (BT-20, MDA-231, SK-BR3, HBL-100)
and four oestrogen receptor-positive (T-47D, BT-474, ZR75-1, MCF-7)
breast cancer cell lines;
(c) 10 normal human tissues,
(d) HFL-1 human foetal diploid fibroblasts (ATCC CCL 153) cultured
in serum-free medium (1 and 2), in the absence (1) or presence (2)
of TPA (10 ng/ml) or cultured in serum-free medium supplemented
with 20 .mu.g/ml insulin (3 to 6), in the absence (3) or presence
(4) of PDGF (20 ng/ml, British Biotechnology), (5) of EGF (20 ng/ml,
Collaborative Research) or (6) of bFGF (10 ng/ml, kindly provided
by Pettmann (FEBS Lett. 189:102-108 (1985))).
In (a), each lane contained 10 .mu.g of total RNA with the exception
of lane 5 (2 .mu.g) and lane 6 (20 .mu.g). In (b) and (c), each
lane contained 8 .mu.g of total RNA, and in (d), each line contained
5 .mu.g of cytoplasmic RNA.
Specifically, in (a), (b) and (c) the blots were made and processed
as indicated in FIG. 4 for stromelysin-3. In (d), confluent HFL-1
fibroblasts were kept in serum-free DMEM culture medium. After 24
hrs, fresh medium was added and supplemented or not with TPA or
growth factors, as indicated above. After 24 hrs of culture, the
cells were harvested and cytoplasmic RNA prepared (Gough, N.M.,
Analyt. Biochem. 173:93-95 (1988).
The blots were then prepared and processed as indicated in FIG.
4 for stromelysin-3, but the autoradiography was for three days.
92 kD collagenase IV RNA transcripts were found in 3 normal and
5 breast cancer metastatic lymph nodes, whereas stromelysin-3 RNA
transcripts were detected only in the metastatic nodes (FIG. 5a,
and data not shown).
In contrast to the results obtained with primary malignant breast
tumors and metastatic lymph nodes, no stromelysin-3 RNA transcripts
could be detected under similar conditions in eight human breast
cancer cell lines, irrespective of their ER status (FIG. 5b). Similarly,
stromelysin-3 RNA transcripts could not be detected in a number
of normal human adult tissues (FIG. 5c), with two notable exceptions,
uterus and placenta.
Stromelysin-3 is not apparently associated with all cancers, and
only low levels of stromelysin-3 RNA transcripts were found in RNA
samples from colon, ovary, kidney and lung cancers. However, high
levels of expression, comparable to those found in breast cancers,
were observed in larynx cancer RNA samples (data not shown).
Specific Expression in Stromal Cells of Invasive Tumors
The expression of the stromelysin-3 gene in primitive breast carcinomas,
but not in a number of established breast cancer cell lines, suggested
that the gene was expressed in the stromal cells surrounding the
tumor, rather than in the neoplastic cells themselves.
In situ hybridization experiments using a [.sup.35 S]-labelled
stromelysin-3 antisense riboprobe were performed using sections
from six carcinomas (tumors C1, C3, C5, C9, C10, referred to as
for FIG. 4, and tumor C11, an ER-positive carcinoma not shown in
Specifically, in situ hybridization was carried out as described
by Cox et al. (Dev. Biol. 101:485-502 (1984)). Deparaffinised and
acid-treated sections (6 .mu.m thick) were proteinase K-treated
and hybridized overnight with [.sup.35 S]-labelled antisense transcripts
from a stromelysin-3 cDNA insert (467 bp extending from nucleotides
1128 to 1594) subcloned in Bluescript II (Stratagene). Hybridization
was followed by RNase treatment (20 .mu.g/ml, 30 min, 37.degree.
C.) and stringent washing (2.times.SSC, 50% formamide, 60.degree.
C., 2 h), prior to autoradiography using NTB2 emulsion (Kodak).
Autoradiography was for 15 days. No significant labeling above background
was observed under similar conditions using a sense riboprobe (not
FIG. 6 shows the presence of stromelysin-3 RNA transcripts in sections
of breast carcinomas and embryo limb bud. a, c, e, g, i and k: bright
fields of tissue sections (x100) stained with haematoxylin; b, d,
f, h, j and l: the same sections (still stained with haematoxylin)
after in situ hybridization with an antisense stromelysin-3 cRNA
probe and dark field imaging.
a and b, grade II ductal breast carcinoma (tumor C1, see FIG. 4):
infiltrating cancer cells (C) are surrounded by a stroma rich in
fusiform cells (S); stromelysin-3 RNA transcripts are most abundant
in the stromal cells immediately surrounding the neoplastic epithelial
cells. c and d, grade III ductal breast carcinoma (tumor C3, see
FIG. 4): multiple islands of infiltrating breast cancer cells (C)
are surrounded by stromal cells; the expression of the stromelysin-3
gene is weaker in the central part of most of the stromal trabeculae
(S) i.e. in the region which is the farthest away from the neoplastic
cells. e and f: ductal carcinoma, (tumor C3, see FIG. 4) together
with two normal lobules (N); stromelysin-3 RNA transcripts were
detected exclusively in the stroma apposing the infiltrating cancer
cells (C), with the exception of a small area rich in lymphocytes
(arrow). g and h, ductal carcinoma (tumor C10, see FIG. 4): stromelysin-3
RNA transcripts can be detected above background in the stromal
cells surrounding the infiltrating (upper corner, right) but not
the in situ (star) breast cancer cells. i and j, ductal carcinoma
(tumor C11, ER-positive, grade II, carcinoma); left: carcinoma in
situ (stars), no stromelysin-3 RNA transcripts can be detected in
the stromal cells; right: infiltrating neoplastic cells surrounded
by stromal cells expressing the stromelysin-3 gene. k and l, interdigital
region of an 8-week-old human embryo limb bud: stromelysin-3 RNA
transcripts are detected in the mesoderm underlying the primitive
epiderm, most notably in the interdigital area (M); note that the
primitive epiderm (arrows), the cartilage in formation (PC), and
the surrounding mesoderm are not labelled.
In all cases, stromelysin-3 RNA transcripts were detected only
in the stromal cells surrounding the islands of malignant epithelial
cells which formed the invasive component of the tumors (FIG. 6:
panels a and b, for tumor C1; panels c, d, e and f for tumor C3;
panels g and h for tumor C10; panels i and j, right hand side, for
tumor C11; and data not shown for tumors C5 and C9).
In metastatic lymph nodes (same source as C5) the expression of
the stromelysin-3 gene was also restricted to the stromal cells
surrounding the metastatic epithelial cells (data not shown).
It is particularly notable that, in all cases, the malignant epithelial
cells themselves were not labelled, and that the highest levels
of expression were observed in the stromal cells in apposition to
the malignant cells. In marked contrast, no significant expression
could be detected in the stromal cells surrounding in situ carcinoma
lesions still surrounded by a basement membrane (panels g and h
for tumor C10; panels i and j, left hand side, for tumor G11), while
the labelling of stromal cells could be clearly observed in the
invasive component of the same tumors (panels g and h for tumor
CIO; panels i and j, right hand side, for tumor C11).
Also, no significant expression could be detected in stromal cells
located at a distance from the cancerous cells nor in the stromal
cells surrounding normal ducts and ductules (e.g. panels e and f).
No discrete foci of stromelysin-3 transcripts were detected in sections
of the F2 fibroadenoma weakly positive for stromelysin-3 RNA on
Northern blots (FIG. 4a and not shown).
Both fibroblasts and myofibroblasts are known to be present in
the stroma of invasive breast carcinomas (Ahmed, A., Pathol. Annu.
25(Pt2):237-286 (1990)). Using our in situ hybridization technique,
it was not possible to determine whether only one or both of these
cell types expressed stromelysin-3 gene.