Abstrict
The present invention provides a method for diagnosing breast cancer
in a subject. The present invention also provides a method for treating
breast cancer in a subject. Finally, the present invention provides
a method for assessing the efficacy of breast cancer therapy in
a subject who has undergone or is undergoing treatment for breast
cancer.
Claims
What is claimed is:
1. A method for detecting the presence or absence of breast cancer
in a non-lactating subject, comprising determining whether or not
mammary gland sodium/iodide symporter (mgNIS) is expressed in breast
tissue of the subject, wherein expression of mgNIS in the breast
tissue is detected using an antibody, or a fragment thereof, specific
for mgNIS, and expression of mgNIS in the breast tissue is indicative
of the presence of breast cancer in the subject, and no expression
of mgNIS in the breast tissue is indicative of the absence of breast
cancer in the subject.
2. The method of claim 1, wherein the expression of mgNIS is detected
in vitro or in vivo.
3. The method of claim 1, wherein the antibody is labeled with
a detectable marker.
4. A method for detecting the presence or absence of breast cancer
in a non-lactating subject, comprising determining whether or not
mammary gland sodium/iodide symporter (mgNIS) is expressed in breast
tissue of the subject, wherein expression of mgNIS in the breast
tissue is detected in vitro using at least one nucleic acid probe
that specifically hybridizes to nucleic acid encoding mgNIS, and
expression of mgNIS in the breast tissue is indicative of the presence
of breast cancer in the subject, and no expression of mgNIS in the
breast tissue is indicative of the absence of breast cancer in the
subject.
5. The method of claim 4 wherein the nucleic acid probe is DNA.
6. The method of claim 4, wherein the nucleic acid probe is labeled
with a detectable marker.
7. The method of claim 4, wherein the nucleic acid probe is RNA.
Description BACKGROUND OF THE INVENTION
Breast cancer is the most common malignancy among women, and has
one of the highest fatality rates of all cancers affecting females.
In fact, breast cancer remains the leading cause of cancer deaths
in women aged 20-59 (Greenlee et al., 2000).
Most breast cancers appear as a slowly growing, painless mass.
There are a number of physical signs which might suggest the presence
of breast cancer, and these may be discovered through a breast examination.
Mammography, xerography, and termography are other established methods
of detecting malignant breast masses (Cancroft and Goldsmith, 1973).
Breast cancer metastasizes by direct extension, and via the lymphatics
and the blood stream. Distant spread of the disease may be confirmed
by lymph node biopsy, by x-ray surveys of skeleton and chest, and,
when appropriate, by liver and bone scans using radioisotopes. Nevertheless,
while history, physical examination, and mammography may strongly
suggest breast cancer, a diagnosis can only be made by microscopic
examination of tissue removed by excisional biopsy or by aspiration
cytology or biopsy. At present, then, there is no means of diagnosing
breast cancer in a patient without necessitating the removal of
tissue.
Regarding treatment, breast cancer therapy depends mainly on the
extent of the disease and the patient's age. There are a number
of methods currently used to treat breast cancer, including surgery,
radiotherapy, hormone therapy, and chemotherapy. Successful cancer
therapy is directed to the primary tumor and to any metastases,
whether clinically apparent or microscopic. Because breast tumors
may be cured with combined modality therapy, each of the above methods
may be used alone, or in conjunction with one or more other therapies.
Thus, local and regional therapy, surgery, or radiotherapy is often
integrated with systemic therapy (e.g., chemotherapy). Adjunctive
chemotherapy, in particular, has a definite role in the treatment
of patients with breast cancer and axillary lymph node involvement.
When there is no evidence that cancer has spread peripherally from
the breast, the treatment most often recommended is surgery, namely,
a mastectomy. Many, if not most, primary operable Stage I and Stage
II breast carcinomas can be conservatively managed by partial mastectomy
(lumpectomy) plus a standard axillary node dissection, followed
by irradiation of the remaining breast tissue. Chemotherapy is sometimes
used as an adjuvant to surgery. Radiotherapy may also be used as
an adjuvant to surgery, particularly in conjunction with a partial
mastectomy and a standard axillary node dissection. For recurrent
cancer, palliative radiotherapy can be valuable in controlling local
chest wall or cervical lymph node recurrences, and in relieving
pain from skeletal metastases.
Hormone therapy, by addition or subtraction, is of greatest use
in the palliation of symptoms of breast cancer, or in delaying advance
of the disease. Hormone therapy is often combined with radiotherapy
when cancer recurs following mastectomy, and when the tumor is so
advanced that surgery is contra-indicated or only palliative. The
presence or absence of estrogen- and progesterone-receptor protein
in primary or metastatic tumor tissue is used to predict which patients
may be expected to respond to additive or ablative hormone therapy
(Thorpe, 1976).
Cytotoxic chemotherapy is an additional method currently used in
the treatment of breast cancer. Prophylactic chemotherapy may be
useful in patients at high risk of developing recurrent cancer (i.e.,
those with axillary lymph node metastases). Chemotherapy is also
used in patients with recurrent breast cancer, sometimes in conjunction
with hormonal manipulations and/or tamoxifen. The most commonly
used, and most effective, chemotherapeutic agent is 5-fluorouracil.
Chemotherapeutic agents have demonstrated value in halting or delaying
the appearance of metastases, especially in premenopausal patients,
and in treating recurrences.
Despite the various mechanisms for detecting, diagnosing, and treating
breast cancer, the disease remains the most common cancer in women,
and is one of the most fatal (Greenlee et al., 2000). Clearly, alternative
strategies for detection of micrometastatic disease, and for more
effective and targeted systemic therapies, are needed to improve
survival in breast cancer patients. Accordingly, new methods of
diagnosis and treatment of breast cancer are still needed, and would
be welcome additions to the arsenal of methods currently used in
the fight against breast cancer.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that mammary
gland sodium/iodide (Na.sup.+ /I.sup.-) symporter (mgNIS), a glycoprotein
that catalyzes the active transport of iodide, is found in mammary
tumoral cells. This discovery has broad implications in the diagnosis
and treatment of breast cancer, and in the monitoring of breast
cancer therapy.
Accordingly, it is an object of the present invention to provide
a method for diagnosing breast cancer in a subject, by detecting
expression of mgNIS in breast tissue of the subject.
It is also an object of the present invention to provide a method
for treating breast cancer in a subject, by diagnosing breast cancer
in the subject by detecting expression of mgNIS in breast tissue
of the subject, and treating the breast cancer diagnosed in the
subject.
Finally, it is an object of the present invention to provide a
method for assessing the efficacy of breast cancer therapy in a
subject who has undergone or is undergoing treatment for breast
cancer, by monitoring expression of mgNIS in breast tissue of the
subject.
Additional objects of the present invention will be apparent from
the description which follows.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Characterization of I.sup.- accumulation and NIS expression
in rat lactating MG and stomach. (A)-(C) In vivo scintigraphic imaging
of .sup.99m TcO.sub.4.sup.- -injected adult female rats. (A) Radiotracer
(1.5 mCi) was injected into the tail vein of nubile animals, and
pin-hole images of the body were obtained at 30 min post-injection.
(B) Lactating rats were imaged 5 min after .sup.99m TcO.sub.4.sup.-
injection. (C) To ascertain inhibition of .sup.99m TcO.sub.4.sup.-
uptake, .sup.99m TcO.sub.4.sup.- and 20 mg perchlorate were co-injected
into the tail vein, followed by static imaging at 5 min. (D) Immunoblot
analysis. Membrane fractions isolated from rat tissues were electrophoresed,
electrotransferred onto nitrocellulose, and immunoblotted with affinity-purified
anti-NIS antibody (Ab) and horseradish peroxidase (HRP) linked anti-rabbit
IgG (Amersham-Pharmacia, Piscataway, N.J.). Approximately 40 .mu.g
of membrane fractions were incubated (either with or without N-glycosidase
F) overnight at 37.degree. C., followed by SDS polyacrylamide gel
electrophoresis and Western blot analysis. Lanes 1 and 2: thyroid;
lanes 4 and 5: lactating MG; lanes 7 and 8: stomach; lane 10: non-lactating
MG; lane 11: muscle; and lane 12: lung. CNBr-treated membrane fractions
from thyroid (lane 3), lactating MG (lane 6), and stomach (lane
9) were electrophoresed, electroblotted onto nitrocellulose, and
immunoblotted with anti-NIS Ab, and HRP-linked secondary Ab as described
above. Immunohistochemical analysis of rat thyroid (E), lactating
MG (F), and stomach (G) tissues was performed with the same anti-NIS
Ab. Magnifications: .times.200.
FIG. 2. Analysis of mgNIS expression in murine MG at different
physiological stages. Fresh-fixed, snap-frozen tissue sections of
nubile (A), lactating (B), and previously lactating (48 h after
weaning of pups) (C) MGs, with hematoxylin and eosin staining. Magnifications:
.times.30. (D) Immunoblot analysis with anti-NIS Ab. MG membrane
fractions (40 .mu.g) from nubile animals (lane 1); lactating dams
(lane 2); and lactating dams after litter weaned for 24 h (lane
3), for 48 h (lane 4), and for 48 h followed by re-establishment
of nursing for 24 h (lane 5). (E) Immunoblot analysis of MG at various
stages of gestation with anti-NIS Ab. Membrane fractions (40 .mu.g)
of MG tissue, prepared from mice at the third (3d), eleventh (11d),
and eighteenth (18d) days of gestation, were analyzed by immunoblots
using anti-NIS Ab, as described above. (F) Radioiodide accumulation
in MG from mice at various stages of gestation. Animals at indicated
days of gestation (3.sup.rd, 11.sup.th, or 18.sup.th) received a
single dose of 1 .mu.Ci .sup.125 I.sup.- by subcutaneous injection
in 100 .mu.l of PBS. One hour later, MG were surgically removed,
and radioactivity was quantified in a .gamma.-counter (LKB 1282
Compugamma, Maryland). Obtained values were standardized according
to the weight of MG removed from each mouse, and were expressed
as the ratio of radioiodide in MG tissue versus blood.
FIG. 3. In vivo effect of oxytocin on functional expression of
mgNIS in nubile mice. (A) Radioiodide transport in MG of oxytocin-treated
nubile mice. Seven- to eight-week-old female mice were treated subcutaneously
with phosphate buffered saline solution (PBS), 10 I.U. of prolactin
(PRL), 1 I.U. of oxytocin (OXY), or prolactin and oxytocin (PRL+OXY)
for three days. Then, 1 .mu.Ci of .sup.125 I.sup.- was injected
intraperitoneally. One hour later, MG were surgically removed and
tracer accumulation was quantified in a .gamma.-counter (LKB 1282
Compugamma, Md.). Obtained values were standardized according to
the weight of removed tissue, and expressed as the ratio of radioiodide
in MG tissue versus blood. (B) Immunoblot analysis of MG from hormonally-treated
mice. Membrane fractions isolated from MG of nubile female mice
were either sham treated with PBS, or hormonally-treated (as described
in (A)) with prolactin (PRL), prolactin and oxytocin (PRL+OXY),
or oxytocin (OXY), as described in (A). Isolated membrane fractions
were electrophoresed, electrotransferred onto nitrocellulose, and
analyzed by immunoblots using anti-NIS Ab. (C) Ex vivo scintigraphic
imaging of .sup.99m TcO.sub.4.sup.- distribution in tissues from
oxytocin-treated nubile mice. Oxytocin-treated animals (n=4) were
divided into two groups. One group was intravenously injected with
.sup.99m TcO.sub.4.sup.- through the tail, while the other group
received .sup.99m TcO.sub.4.sup.- together with 2 mg of perchlorate.
Thirty minutes later, spleen, and skeletal muscle from the limb,
stomach, and MG, were removed from both groups of animals (upper
squares: .sup.99m TcO.sub.4.sup.- ; lower squares: .sup.99m mTcO.sub.4.sup.-
+perchlorate), and placed 4 cm apart from each other on a petri
dish. Images of organs were then obtained with a pin-hole gamma
camera for 5 min. Perchlorate-inhibited accumulation of .sup.99m
TcO.sub.4.sup.- was detected in stomach and MG of OXY-treated animals.
(D) Quantification of .sup.99m TcO.sub.4.sup.- accumulation in various
organs from OXY- or PBS-treated mice. Tracer accumulation was monitored
using image analysis software (ImageQuant, Macintosh). Regions of
interest were drawn around removed individual organs, and average
counts per pixel were obtained. Counts were divided by the weight
of the organs (cpm/mg tissue), and standardized by dividing them
by cpm detected in the blood (cpm/mg blood) of each animal. .sup.99m
TcO.sub.4.sup.- accumulation in the organs from: PBS-treated animals
(open bars); OXY-treated animals (dotted bars); and OXY-treated
animals after .sup.99m TcO.sub.4.sup.- +perchlorate injection (dark
bars).
FIG. 4. Immunoblot analysis of MG from ovariectomized nubile mice
treated with various hormones. Membrane fractions isolated from
MG of nubile mice were either sham treated with PBS (lane 1) or
treated for three consecutive days with: progesterone (1 I.U.) (lane
2); 17-.beta.-estradiol (1 .mu.g) (lane 3); 17-.beta.-estradiol
and progesterone (1 I.U.) (lane 4); oxytocin (1 I.U.) (lane 5);
oxytocin and 17-.beta.-estradiol (lane 6); prolactin (10 I.U.) (lane
7); oxytocin, prolactin, and 17-.beta.-estradiol (lane 8); and progesterone
together with oxytocin, prolactin, and 17-.beta.-estradiol (lane
9). Isolated membrane fractions were electrophoresed and electrotransferred
onto nitrocellulose, followed by immunoblot analysis with anti-NIS
Ab.
FIG. 5. Analysis of mammary adenocarcinomas in MMTV-ras and MMTV-neu
transgenic mice. (A) Photograph of MMTV-ras transgenic mouse with
a mammary adenocarcinoma. Transgenic mice were anaesthetized with
intramuscular sodium pentobarbital injections. Then, .sup.99m TcO.sub.4.sup.-
(B, G), .sup.99m Tc-labeled human serum albumin (.sup.99m Tc-HSA)
(C), or .sup.99m TcO.sub.4.sup.- +perchlorate (D, H) was administered
by tail vein injections. (E) Dynamic curves of percent injected
dose per pixel were calculated by drawing regions of interest around
tumoral tissue and the mediastinum, and dividing the counts per
pixel (average counts) within each region of interest by the initial
whole-body count value, thereby yielding a percent injected dose
per pixel. (F) Immunoblot analysis of membrane fractions isolated
from MMTV-ras tumor with anti-NIS Ab. (I) Immunoblot analysis of
membrane fractions isolated from MMTV-neu tumors with anti-NIS Ab.
In an MMTV-neu mouse with two adjacent mammary glands containing
tumors, each tumor is indicated with an arrow and numbered. Lanes
"tu1", "tu2", and "mg" correspond
to proteins extracted from tumor 1, tumor 2, and contralateral normal
mammary gland, respectively. (J) Tissue section exhibiting intense
immunoreactivity to anti-NIS Ab in this poorly-differentiated adenocarcinoma,
and (K) parallel section in the same area of "tu1" reacting
with anti-HER-2/neu Ab. (L) Contralateral normal gland ("mg")
exhibiting ductal structure surrounded by fatty stroma, demonstrating
no reactivity to HER-2/neu. Magnifications: .times.200.
FIG. 6. Immunohistochemical expression of NIS in human thyroid
and breast tissues. (A) Papillary carcinoma of the thyroid, revealing
distinct malignant cell NIS immunoreactivity with polyclonal anti-NIS
Ct-2 Ab. Magnification: .times.12. (B) Parallel section demonstrating
competitive inhibition of mgNIS expression in the presence of Ct-2
peptide. Magnification: .times.12. (C) Invasive ductal carcinoma
of the breast. mgNIS expression is detected with polyclonal anti-NIS
Ct-2 Ab. Magnification: .times.16. (D) Competitive inhibition of
immunoreactivity with corresponding peptide. Magnification: .times.16.
(E) Parallel section treated with monoclonal anti-NIS Ab, again
showing identical distribution of immunoreactivity as shown in (C).
Magnification: .times.16. (F) Normal ductal-lobular units in the
vicinity of the breast cancer shown in (C) and (E), as detected
with monoclonal anti-NIS Ab. Magnification: .times.40. (G) Higher
magnification of another invasive ductal carcinoma, showing focal
areas of immunoreactivity with a very distinct intracellular staining
pattern. Magnification: .times.160. (H) Ductal carcinoma in situ
featuring intraductal comedonecrosis and intense immunoreactivity
(>95%) of malignant cells, as detected with monoclonal anti-NIS
Ab. Magnification: .times.66. (I) Gestational breast tissue manifesting
characteristic adenomatous-lactational changes which occur in the
latter half of pregnancy, and demonstrating mgNIS expression in
epithelial cells, as probed with polyclonal anti-NIS Ct-2 Ab. Magnification:
.times.80.
FIG. 7. .sup.99m Tc-pertechnetate scintigraphy of a woman with
a locally-advanced cancer of the right breast and an enlarged thyroid.
The patient was scanned after injection of 15 mCi of .sup.99 mTc-labeled
pertechnetate (.sup.99m TcO.sub.4.sup.-). Tracer accumulates in
the thyroid and stomach. Tracer is present in the heart and liver
due to vascular pooling. The arrow indicates distinct accumulation
in the area of the breast cancer.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for diagnosing breast cancer
in a subject who has, or may have, breast cancer. As used herein,
"subject" is a mammal, including, without limitation,
a cow, dog, human, monkey, mouse, pig, or rat, but is preferably
a human. The method of the present invention comprises detecting
expression of mammary gland sodium/iodide (Na.sup.+ /I.sup.-) symporter
(mgNIS) in breast tissue of the subject. As used herein, "mgNIS"
includes mgNIS protein, cDNA, and mRNA. The appropriate form of
mgNIS will be apparent based on the particular techniques discussed
herein. According to the method of the present invention, the expression
of mgNIS in breast tissue may be detected in vitro or in vivo. In
accordance with the present invention, where expression of mgNIS
is detected in vitro, a sample of breast tissue or cells from the
subject may be removed using standard procedures, including biopsy
and aspiration. Preferably, the sample of breast tissue or cells
is removed using multidirectional fine-needle aspiration biopsy
(FNAB). This method of removal is preferred, as it is less invasive
than a standard biopsy. Cells which are removed from the subject
using FNAB may be analyzed using immunocytofluorometry (FACS analysis),
for example, as discussed below. Furthermore, the expression of
mgNIS in breast tissue may be detected by detection methods readily
determined from the known art, including, without limitation, immunological
techniques, hybridization analysis, fluorescence imaging techniques,
and/or radiation detection.
For example, according to the method of the present invention,
the expression of mgNIS may be detected using an agent reactive
with mgNIS. As used herein, "reactive" means the agent
has affinity for, binds to, or is directed against mgNIS. The agent
may be in the form of an antibody, a Fab fragment, an F(ab').sub.2
fragment, a peptide, a polypeptide, a protein, and any combinations
thereof. A Fab fragment is a univalent antigen-binding fragment
of an antibody, which is produced by papain digestion. An F(ab').sub.2
fragment is a divalent antigen-binding fragment of an antibody,
which is produced by pepsin digestion. Preferably, the agent is
a high-affinity antibody labeled with a detectable marker. Where
the agent is an antibody, the expression of mgNIS may be detected
from binding studies using one or more antibodies immunoreactive
with mgNIS, along with standard immunological detection techniques,
such as Western blotting.
As used herein, the antibody of the present invention may be polyclonal
or monoclonal, and may be produced by techniques well known to those
skilled in the art. Polyclonal antibody, for example, may be produced
by immunizing a mouse, rabbit, or rat with purified mgNIS. Monoclonal
antibody may then be produced by removing the spleen from the immunized
mouse, and fusing the spleen cells with myeloma cells to form a
hybridoma which, when grown in culture, will produce a monoclonal
antibody. In addition, the antibodies used herein may be labeled
with a detectable marker. Labeling of the antibody may be accomplished
using one of the variety of different chemiluminescent and radioactive
labels known in the art. The detectable marker of the present invention
may be, for example, a nonradioactive or fluorescent marker, such
as biotin, fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine,
which can be detected using fluorescence and other imaging techniques
readily known in the art. Alternatively, the detectable marker may
be a radioactive marker, including, for example, a radioisotope.
The radioisotope may be any isotope that emits detectable radiation,
and need not be a radioactive isotope that is selectively taken
up by mgNIS. For example, the radioisotope may include those which
are not selectively taken up by mgNIS, such as .sup.35 S, .sup.32
P, or .sup.3 H.
Alternatively, the expression of mgNIS in breast tissue of a subject
may be detected through hybridization analysis of nucleic acid extracted
from a sample of breast tissue or cells from the subject. According
to this method of the present invention, the hybridization analysis
may be conducted using one or more nucleic acid probes which hybridize
to nucleic acid encoding mgNIS. The probes may be prepared by a
variety of techniques known to those skilled in the art, including,
without limitation, restriction enzyme digestion of mgNIS nucleic
acid; and automated synthesis of oligonucleotides whose sequence
corresponds to selected portions of the nucleotide sequence of the
mgNIS nucleic acid, using commercially-available oligonucleotide
synthesizers, such as the Applied Biosystems Model 392 DNA/RNA synthesizer.
The nucleic acid probes used in the present invention may be DNA
or RNA, and may vary in length from about 8 nucleotides to the entire
length of the mgNIS nucleic acid. The mgNIS nucleic acid used in
the probes may be derived from mammalian mgNIS. The nucleotide sequences
for both rat and human NIS are known (Dai et al., 1996a; and Smanik
et al., 1996). Using these sequences as probes, the skilled artisan
could readily clone corresponding mgNIS cDNA from other species.
In addition, the nucleic acid probes of the present invention may
be labeled with one or more detectable markers. Labeling of the
nucleic acid probes may be accomplished using one of a number of
methods known in the art--e.g., nick translation, end labeling,
fill-in end labeling, polynucleotide kinase exchange reaction, random
priming, or SP6 polymerase (for riboprobe preparation)--along with
one of a variety of labels--e.g., radioactive labels, such as .sup.35
S, .sup.32 P, or .sup.3 H, or nonradioactive labels, such as biotin,
fluorescein (FITC), acridine, cholesterol, or carboxy-X-rhodamine
(ROX). Combinations of two or more nucleic acid probes (or primers),
corresponding to different or overlapping regions of the mgNIS nucleic
acid, may also be used to detect expression of mgNIS, using, for
example, PCR or RT-PCR.
In the further alternative, the expression of mgNIS in breast tissue
of a subject may be detected using a detectable agent that is selectively
taken up by mgNIS. The detectable agent may be a radioisotope that
is selectively taken up by mgNIS. The detectable agent may be, for
example, radioiodide (.sup.125 I.sup.- or .sup.131 I.sup.-) or .sup.99m
Tc-pertechnetate (.sup.99m TcO.sub.4.sup.-), but is, more preferably,
radioiodide. Radioactivity emitted by the radioisotope can be detected
by techniques well known in the art. For example, gamma emission
from the radioisotope may be detected using gamma imaging techniques,
particularly scintigraphic imaging.
The present invention also provides a method for treating breast
cancer in a subject or patient. The method of the present invention
comprises the steps of: a) diagnosing breast cancer in the subject
or patient by detecting expression of mgNIS in breast tissue of
the subject or patient; and b) treating the breast cancer diagnosed
in the subject or patient. The expression of mgNIS in breast tissue
of the subject or patient may be detected by any of the methods
described above. The breast cancer diagnosed in the subject or patient
may be treated by any method or combination of methods commonly
used to treat breast cancer, including, without limitation, surgery,
radiotherapy, hormone therapy, chemotherapy, immunotherapy, and
systemic therapy. Preferably, however, breast cancer which is diagnosed
by detecting expression of mgNIS is treated by an anti-cancer agent
that is selectively taken up by mgNIS or an anti-cancer agent that
is reactive with mgNIS.
According to the method of the present invention, the breast cancer
diagnosed in the subject or patient may be treated by administering
to the subject or patient an anti-cancer agent that is selectively
taken up by mgNIS. For example, the anti-cancer agent may be a radioisotope
which is selectively taken up by mgNIS, and which has an anti-cancer
effect. In one embodiment of the present invention, the anti-cancer
agent is radioiodide (.sup.125 I.sup.- or .sup.131 I.sup.-). Using
in vitro assays, it may also be possible to screen for other agents
which are selectively taken up by mgNIS. For example, to determine
if a particular agent is selectively taken up by mgNIS, the agent
may be brought into contact with a sample of tissue or cells known
to contain mgNIS, thereby permitting detection of uptake of the
agent by mgNIS.
In the alternative, the breast cancer diagnosed in the subject
or patient may be treated by administering to the subject or patient
an anti-cancer agent that is reactive with mgNIS. Preferably, the
anti-cancer agent is a high-affinity antibody bound to a chemotherapeutic
cytotoxin. Antibodies directed against mgNIS may be prepared according
to the method described above. As used herein, "chemotherapeutic
cytotoxin" refers to any chemical substance which kills or
destroys malignant breast cells, either independently or in conjunction
with an additional agent or treatment. The chemotherapeutic cytotoxin
may include any of the anti-cancer drugs or chemotherapeutic agents
known in the art, including, for example, chlorambucil, cyclophosphamide,
doxorubicin, 5-fluorouracil, melphalan, methotrexate, and vincristine.
In addition, the chemotherapeutic cytotoxin may be a photoreactive
compound which, when exposed to light of a particular wavelength,
undergoes a chemical reaction, and thereby destroys the malignant
breast cells.
It is also within the confines of the present invention to use
detected levels of mgNIS expression as a clinical or pathologic
staging tool, to determine which treatment options may be appropriate.
In particular, detected levels of mgNIS expression may be used to
determine whether any of the treatment methods of the present invention
is appropriate.
The present invention further provides a method for assessing the
efficacy of breast cancer therapy in a subject or patient who has
undergone or is undergoing treatment for breast cancer. The method
of the present invention comprises determining whether mgNIS is
expressed in breast tissue of the subject or patient, wherein an
absence of mgNIS expression is indicative of successful breast cancer
therapy. The expression of mgNIS may be detected by all of the various
methods described above. This method of the present invention provides
a means of monitoring the effectiveness of breast cancer therapy
by permitting the periodic assessment of levels of mgNIS expression
in breast tissue of the subject or patient.
According to the method of the present invention, levels of mgNIS
expression may be assessed in the subject or patient at any time
following the initiation of breast cancer therapy. For example,
levels of mgNIS expression may be assessed while the subject or
patient is still undergoing treatment for breast cancer. Where levels
of mgNIS expression continue to be detected in the breast tissue
of the subject or patient, a physician may choose to continue with
the breast cancer treatment. Where levels of mgNIS expression decrease
through successive assessments, it may be an indication that the
breast cancer treatment is working, and that treatment doses could
be decreased or even ceased. Where levels of mgNIS do not rapidly
decrease through successive assessments, it may be an indication
that the breast cancer treatment is not working, and that treatment
doses could be increased. Where mgNIS expression is no longer detected
in breast tissue of a subject or patient, a physician may conclude
that the breast cancer treatment has been successful, and that such
treatment may cease. It is also within the confines of the present
invention to assess levels of mgNIS expression following completion
of the subject's or patient's breast cancer treatment, in order
to determine whether breast cancer has recurred in the subject or
patient. Furthermore, it is within the confines of the present invention
to us assessed levels of mgNIS expression as a clinical or pathologic
staging tool, to determine the extent of breast cancer in the subject
or patient, to determine appropriate treatment options, and to provide
prognostic information.
The present invention is described in the following Experimental
Details section, which is set forth to aid in the understanding
of the invention, and should not be construed to limit in any way
the scope of the invention as defined in the claims which follow
thereafter.
EXPERIMENTAL DETAILS
1. Structure and Function of the Sodium/Iodide Symporter
The metabolism of iodide (I.sup.-) is commonly associated with
the thyroid gland more than with any other tissue or organ in mammals.
I.sup.- is an essential constituent of the thyroid hormones T.sub.3
and T.sub.4. Under physiological conditions, most of the ingested
dietary I.sup.- is accumulated in the thyroid by means of a highly
specialized active I.sup.- transport mechanism (Carrasco, 1993).
I.sup.- transport in the thyroid is catalyzed by the sodium/iodide
(Na.sup.+ /I.sup.-) symporter (NIS), a key glycoprotein located
in the basolateral plasma membrane of the thyroid follicular cells
(Dai et al., 1996a and 1996b). NIS-catalyzed I.sup.- accumulation
is a sodium-dependent active transport process, driven by the Na.sup.+
gradient maintained by the Na.sup.+ /K.sup.+ ATPase. The membrane
topology of NIS has been studied by biochemical, biophysical, and
immunological techniques. A secondary structure model, predicting
13 transmembrane segments--with an extracellular and an intracellular
C-terminus--has been proposed (Levy et al., 1997; and Levy et al.,
1998a). Detailed electrophysiological studies have established that
NIS activity is electrogenic. Studies have also shown that 2 Na.sup.+
ions are transported with one anion, thereby demonstrating unequivocally
a 2:1 Na.sup.+ :I.sup.- stoichiometry (Eskandari et al., 1997).
Several congenital cases of I.sup.- transport defects, arising from
mutations in NIS, have been identified (Fujiwara et al., 1997; Matsuda
and Kosugi, 1997; and Levy et al., 1998b. For reviews, see De la
Vieja et al., 2000; and Dohan et al., 2000). This is a rare condition,
most often diagnosed in patients who exhibit co-existence of goiter
with congenital hypothyroidism, low or no thyroidal uptake of radioiodide,
and little or no I.sup.- uptake by the salivary glands and gastric
mucosa.
Other than the thyroid, only a few tissues exhibit active I.sup.-
transport. These include lactating mammary gland (MG), salivary
glands, and gastric mucosa (Carrasco, 1993). The functional link
between I.sup.- transport in the lactating MG and in the thyroid
is particularly clear and noteworthy: I.sup.- accumulated in lactating
MG, and secreted into milk, is used by the nursing newborn for thyroid
hormone biosynthesis (Mountford et al., 1986; and Stubbe et al.,
1986). An adequate supply of I.sup.- for sufficient thyroid hormone
production is essential for proper development of a newborn's nervous
system, skeletal muscle, and lungs (Stubbe et al., 1986; DeGroot,
1989; and Werner and Ingbar, 1991). Severe iodine deficiency at
this early stage in life results in mental retardation, and in some
cases, dwarfism (Werner and Ingbar, 1991). Like thyroidal I.sup.-
transport, I.sup.- is actively translocated in the MG from the bloodstream
into the cytoplasm of the epithelial cells, from where it is secreted
into the milk.
The degree and pattern of I.sup.- accumulation in the thyroid,
as revealed by scintigraphic imaging, is used as an aid in the differential
diagnosis of thyroid nodules. Moreover, radioactive I.sup.- (radioiodide)
plays a major therapeutic role in the postoperative management of
differentiated thyroid carcinoma (DTC) because of its effectiveness
in ablating remnant thyroid tissue and metastases (Werner and Ingbar,
1991; Mazzaferri, 1999). In contrast, the ability of mammary tissue
to actively transport I.sup.- has not, thus far, been examined for
its possible utility in the diagnosis and/or treatment of breast
cancer.
2. Materials and Methods
A. Generation of site-directed antibodies
The following peptides, corresponding to C-terminal sequences of
human NIS protein, were synthesized by solid phase synthesis (Carrasco
et al., 1986) and used to generate polyclonal human anti-NIS antibodies
(Abs): peptide P-857, KELEGAGSWTPCVGHD (SEQ ID NO:1) corresponding
to residues 618-633, and peptide P-858, GHDGGRDQQETNL (SEQ ID NO:2),
corresponding to residues 631-643 of NIS, were used to generate
Abs Ct-1 and Ct-2, respectively. The protocol described in Levy
et al. (1997) was followed to generate and purify polyclonal antibodies.
Peptide NEDLLFFLGQKELE (SEQ ID NO:3) corresponding to residues 598-621,
was used to generate the site-directed monoclonal Ab, as described
in Harlow and Lane (1988).
B. Immunohistochemical analysis
Immunoreactivity was carried out using the immunoperoxidase method
(Amenta and Martinez-Hernandez, 1995). In brief, 5 .mu.m sections
were deparaffinated through 3 changes of xylene, followed by passage
through alcohols to distilled water. All slides were subjected to
antigen retrieval using 10% citrate buffer (DAKO Carpinteria, Calif.).
Thereafter, slides were cooled and rinsed twice in TBST solution
(0.3 M of NaCl, 0.1% Tween 20, and 0.05 M of Tris-HCl) (pH:7.6)
for 5 min. All incubations were carried out in a humid chamber at
room temperature, and all subsequent washes were done with TBST.
Endogenous biotin activity was blocked with sequential avidin biotin
incubation (DAKO Biotin Blocking System, Carpenteria, Calif.), followed
by serum-free protein block provided in the Catalyzed Signal Amplification
kit (DAKO, Carpinteria, Calif.), as indicated by the supplier. Slides
were incubated for 15 min with anti-rat NIS, Ct-1, Ct-2, or monoclonal
primary Abs diluted in the provided blocking solution to a concentration
of 1:500 (rat tissues), 1:600 (human breast, polyclonal Abs), 1:750
(human thyroid, polyclonal Abs), and 1:100 (human tissues, monoclonal
Ab), respectively. The initial concentration of polyclonal and monoclonal
Abs were 1 .mu.g/.mu.l and 0.5 .mu.g/.mu.l, respectively. Tissues
were sequentially incubated with biotinylated secondary antibodies,
then streptavidin, followed by an amplification step performed with
biotinyl tyramide, as described by the supplier (DAKO, Carpinteria,
Calif.). Lastly, slides were incubated with streptavidin-horseradish
peroxidase prior to chromogen reaction using diaminobenzidine (DAB)
tetrachloride Tris-HCl buffer (pH 7.6) containing 0.8% peroxide,
for 3 to 5 min. Sections were rinsed in water, dehydrated in graded
ethanols, counterstained or mounted with permount, and examined
by light microscopy. Immunoreactivity was competitively inhibited
in the presence of 0.7 .mu.M of corresponding synthetic peptides
used to generate Abs. Non-specific immunoreactivity was evaluated
with unrelated rabbit and mouse immunoglobulins (DAKO Carpenteria,
Calif.). CAM 5.2 Ab against low molecular weight keratins 8 and
18 (Becton Dickinson, San Jose, Calif.) was used to identify epithelial
cells. All counterstains for immunohistochemical studies were done
with Toluidine blue. Immunoreactivity was analyzed by light microscopy
and graded on a scale of 0 to 4+. Tissues were judged positive for
NIS expression when at least 20% or more of the cells exhibited
.gtoreq.2+.
C. Hormonal treatment of animals
Oxytocin (.alpha.-hypophamine), prolactin, progesterone (4-pregnene-3,20-dione),
and 17-.beta.-estradiol (1,3,5[10]-estratriene-3,17-.beta.-diol)
were purchased from Sigma, St. Louis, Mo. Either ovariectomized
or surgically unmodified 8- to 10-week-old CD1(ICR)IBR mice (Charles
River Laboratories, Wilmington, Mass.) were treated once a day with
subcutaneous injections of 1 I.U. of oxytocin, 10 I.U. of prolactin,
1 .mu.g 17-.beta.-estradiol, or 1 I.U. of progesterone (intraperitoneally)
for three consecutive days, either individually or in the indicated
combination. A final injection was done on the fourth day; two hours
later, mammary glands were removed for analysis. Oxytocin and prolactin
were dissolved in sterile distilled water, 17-.beta.-estradiol was
dissolved in 90% alcohol, and progesterone was dissolved in sesame
oil (Sigma, St. Louis, Mo.). Except for progesterone, indicated
doses of hormones were injected after being taken in 200 .mu.l sterile
PBS solution. Progesterone (final concentration 10 mg/ml) was injected
into 100 .mu.l of sesame oil. Control animals which were sham treated
with sesame oil were systematically included in experiments when
progesterone was administered. At least three identically-treated
animals were analyzed in each case.
D. In vivo transport studies
1 .mu.Ci of .sup.125 I.sup.- (100 mCi/ml, Amersham-Pharmacia, Piscataway,
N.J.) was added to 100 .mu.l of PBS and intraperitoneally administered
to hormonally-treated animals. One hour later, animals were sacrificed,
and various organs were surgically removed and placed in pre-weighted
eppendorf tubes. Approximately 500 .mu.l of blood was also taken
from the inferior vena cava of each animal during surgery. Tubes
were weighed and counted in a .gamma.-counter (LKB-Wallac, Gaithersburg,
Md.). Radioactivity accumulated in each organ was determined in
terms of cpm/mg of tissue, standardized with radioactivity detected
per mg of blood, and expressed as the ratio of cpm detected in the
organ of interest versus blood. Data were obtained from the analysis
of at least three animals in each experiment.
E. Tissue retrieval and immunoblot analysis
Sprague-Dawley female rats (over 8 weeks old) at different physiological
stages, and hormonally-treated CD1(ICR)BR female mice, were sacrificed
in a CO.sub.2 chamber before excision of thoracic, abdominal, and
inguinal MG. Organs, which were removed from mice or rats were blended
with a polytron homogenizer (Brinkmann Instruments, Westbury, N.Y.)
for 1 min, and homogenized with a stirrer-type glass-teflon homogenizer
(Caframo-Wiarton, Ontario, Canada) (Levy et al., 1997). Membrane
fractions were prepared in the presence of protease inhibitors,
as described above (Kaminsky et al., 1994). SDS-PAGE electrophoresis
and electroblotting to nitrocellulose were performed as described
in Levy et al. (1997). All samples were diluted 1:2 with loading
buffer (Harlow and Lane, 1988) and heated at 37.degree. C. for 30
min prior to electrophoresis. Immunoblot analyses were carried out
with 2 .mu.g of affinity-purified anti-NIS Ab (Levy et al., 1997)
and a 1:1500 dilution of a horseradish peroxidase-linked donkey
anti-rabbit IgG (Amersham-Pharmacia, Piscataway, N.J.). Both incubations
were performed for 1 h. Polypeptides were visualized by the enhanced
chemiluminescence (ECL) Western blot detection system (Amersham-Pharmacia,
Piscataway, N.J.).
F. Peptidyl N-glycosidase F treatment
Membranes (40 .mu.g) were resuspended in 10 .mu.l of 0.5 M Tris-HCl
(pH 8.0), and 18 .mu.l of water was added with either 3 .mu.l of
N-glycosidase F (600 milliunits, Boehringer Mannheim) or 2 .mu.l
of 50% glycerol. Membranes were then incubated overnight at 37.degree.
C. (18 h). After overnight incubation, samples were diluted 1:2
with loading buffer (15 .mu.l) and incubated at 37.degree. C. for
30 min prior to electrophoresis (Levy et al., 1997).
G. CNBr fragmentation of proteins
Pieces of nitrocellulose containing the corresponding immunoreactive
NIS species were excised and cut into smaller pieces of .about.1mm.times.2mm,
then incubated for 1 h in the dark, at room temperature, with 300
.mu.l of CNBr (.about.300 mg/ml) in 70% formic acid. Samples were
centrifuged to pellet the nitrocellulose pieces; the formic acid
containing the released digested peptides was lyophilized in a speed-vac
at medium heat (.about.1 h). Dried peptides were resuspended in
75 .mu.l water and lyophilized again, followed by resuspension into
sample buffer (30 .mu.l). Samples were neutralized with a small
volume (<5 .mu.l) of 100 mM of Tris (pH 9.1) prior to 15% SDS/PAGE
electrophoresis.
H. Cell aspiration and FACS analysis
A multidirectional fine-needle aspiration biopsy (FNAB) from the
tumor was performed with a 22-gauge needle connected to a 20 ml
plastic syringe that was attached to a metal holder. The aspirate
was resuspended and fixed in CytoLyt solution (Cyto Corporation,
Boxborough, Mass.), maintained at 4.degree. C., and analyzed within
24-48 hr. The cells which were resuspended in the CytoLyt solution
were then passed through a 35-mm nylon mesh, centrifuged at 500
g, and resuspended and permeabilized in PBS (phosphate buffered
saline) containing 0.1% BSA (bovine serum albumin) and 0.2% saponin
(PBSAP). 200,000 cells/tube were incubated for 1 hr at room temperature
in 100 ml PBSAP containing 10-80 nM of one of the two affinity-purified
antiNIS antibodies raised against the intracellular C-terminal end
of NIS (antiKELE *..antiETNL).
After washing in 1 ml PBSAP, the cells were incubated for 1 hr
on ice, in the dark, with fluorescein isothiocyanate (FITC)-conjugated
antirabbit IgG antibody in 100 .mu.l of BSA-SAP-PBS (1:1000 dilution).
Cells were washed once again with 1 ml of BSA-SAP-PBS, and resuspended
in 300 .mu.l of PBS. The fluorescence of 10,000 cells per tube was
assayed by flow immunocytofluorometry (FACScan; Becton Dickinson
& Co., Mountainview, Calif.); threshold and forward scatter
was used to gate the cells and eliminate debris and cell aggregates.
A histogram plot was generated, and mean fluorescence intensity
of each sample was determined using CELLQuest software.
2. Results
A. Physiology and regulation of I.sup.- transport in mammary tissue
1. In vivo analysis of I.sup.- accumulation in rat mammary tissue
To characterize the active accumulation of I.sup.- in milk, lactating
rats were administered a single .sup.125 I.sup.- intraperitoneal
injection, and the time course of .sup.125 I.sup.- transport was
assessed in milk samples at given intervals. .sup.125 I.sup.- was
concentrated more than .about.60 fold in milk with respect to blood,
with saturation occurring at .about.2 h (not shown). .sup.125 I.sup.-
was also concentrated in lactating MG; however, it was not concentrated
in skeletal muscle from the same rat, or in MG from a non-lactating
female rat. To assess the overall tissue distribution of I.sup.-
in vivo, .sup.131 I.sup.- or pertechnetate (.sup.99m TcO.sub.4.sup.-)
was administered to female rats that were then imaged by scintigraphy.
.sup.99m TcO.sub.4.sup.-, a gamma emitter that is actively concentrated
in the thyroid by thyroid NIS (tNIS), offers the practical advantage
of a much shorter half-life (t.sub.1/2 =6 h) than .sup.131 I.sup.-
(t.sub.1/2 =8 days) (Papadopoulos et al., 1967). Therefore, .sup.99m
TcO.sub.4.sup.- was used in most experiments.
When injected into non-lactating rats, the radioisotope was initially
(30 min) observed primarily in the stomach, whereas at later time
points it was concentrated predominantly in the thyroid (FIG. 1A).
In stark contrast, in lactating rats, .sup.99m TcO.sub.4.sup.- was
visualized in the stomach and was then rapidly concentrated (within
5 min) in all pairs of MGs (FIG. 1B), thereby demonstrating an avid
concentrating activity of the anion in lactating MG. In lactating
animals, .sup.99m TcO.sub.4.sup.- eventually accumulates in the
thyroid as well (not shown). Simultaneous injection of perchlorate--a
potent inhibitor of active I.sup.- transport (Carrasco, 1993)--and
.sup.99m TcO.sub.4.sup.- into a lactating rat effectively prevented
.sup.99m TcO.sub.4.sup.- concentration in thyroid, stomach, and
lactating MG (FIG. 1C). The pattern shown in FIG. 1C corresponds
to distribution of .sup.99m TcO.sub.4.sup.- in the vascular compartment
and the urinary tract, without active accumulation of .sup.99m TcO.sub.4.sup.-
in any tissue, as a result of the blocking effect of perchlorate.
These data show that .sup.131 I.sup.- /.sup.99m TcO.sub.4.sup.-
accumulation in the thyroid, lactating MG, and stomach, as observed
by scintigraphic imaging, is specific and inhibited by perchlorate.
II. Identification of mammary gland NIS (mgNIS)
The availability of anti-NIS antibody (Ab) raised against rat tNIS
made it possible for the first time to apply an immunological approach
to attempt the specific identification of mgNIS (Levy et al., 1997).
Anti-NIS Ab reacts with a rat tNIS polypeptide of .about.100 kDa
(Levy et al., 1997) (FIG. 1D, lane 1). The same Ab identified a
single broad polypeptide of .about.75 kDa in rat lactating MG membranes,
i.e., mgNIS (FIG. 1D, lane 4). In contrast, immunoreactivity was
absent in non-lactating MG (FIG. 1D, lane 10) and in membranes from
lung and muscle--tissues that do not actively transport I.sup.-
(FIG. 1D, lanes 11 and 12). Immunoreactivity against both the .about.75
and .about.100 kDa polypeptides was competitively blocked by addition
of the synthetic eliciting peptide that contains the last 16 amino
acids of NIS (not shown).
Given that tNIS is known to be a highly glycosylated protein (Levy
et al., 1997 and 1998a), the difference in electrophoretic mobilities
between tNIS (.about.100 kDa) and mgNIS (.about.75 kDa) may represent
differences in glycosylation. To investigate this possibility, alkaline-extracted
membrane proteins from thyroid and lactating MG were treated with
N-glycosidase F, an enzyme that removes N-linked carbohydrates.
Under these conditions, anti-NIS Ab recognized a .about.50 kDa polypeptide
in either thyroid (FIG. 1D, lane 2) or lactating MG (FIG. 1D, lane
5). Significantly, both non-glycosylated NIS in FRTL-5 cells (a
line of highly functional thyroid cells) and NIS expressed in E.
coli exhibited an identical electrophoretic mobility (Le., .about.50
kDa) (Levy et al., 1997). Therefore, the .about.75 kDa and .about.50
kDa immunoreactive polypeptides detected in lactating MG are glycosylated
and non-glycosylated mgNIS, respectively. These findings are consistent
with the recently reported full identity between cDNAs that encode
the human thyroid and mammary NIS proteins (Spitzweg et al., 1998).
Immunoreactivity against a gastric polypeptide of .about.110 kDa
was also observed with anti-NIS Ab (FIG. 1D, lane 7), and was blocked
by excess synthetic NIS carboxy terminus peptide (not shown), consistent
with the accumulation of I.sup.- in stomach (FIG. 1A). Upon deglycosylation
the gastric polypeptide also migrated at .about.50 kDa (FIG. 1D,
lane 8), strongly suggesting that these polypeptides correspond,
respectively, to the glycosylated and non-glycosylated species of
the gastric I.sup.- transporter (gNIS). Methionine-specific cleavage
of tNIS, mgNIS, and gNIS using CNBr indicates that NIS is the same
protein in each of the three tissues--thyroid, mammary gland, and
gastric (FIG. 1D, lanes 3, 6, and 9).
In addition, immunohistochemical analysis was carried out on formalin-fixed
paraffin-embedded tissue sections derived from rat thyroid, MG,
and stomach. Distinct basolateral plasma membrane reactivity was
evident in the thyroid follicular cells (FIG. 1E), lactating MG
epithelial cells (FIG. 1F), and the surface epithelial cells of
the gastric mucosa (FIG. 1G). Notably, basal chief cells exhibited
less NIS immunoreactivity, whereas parietal cells were entirely
devoid of immunoreactivity. As in the immunoblots, immunoreactivity
was absent in striated muscle, cartilage, and nubile MG (not shown).
Taken together, all the above observations indicate that active
I.sup.- accumulation in the thyroid, lactating MG, and stomach is
mediated by NIS.
III. Expression of mgNIS in various physiological stages
The expression of mgNIS was analyzed in nubile, lactating, and
previously lactating (PL) MG (i.e., MG of dams separated from their
litters). Considerable morphological changes are evident in the
MG during gestation and lactation. The fatty stroma, characteristic
of the nubile gland (FIG. 2A), is replaced by alveolar-ductal structures
containing luminal secretions and epithelial cells with intracellular
milk fat globules (Joshie et al., 1976; and Dulbecco et al., 1982)
(FIG. 2B). Weaning promotes rapid involutional changes, such as
alveolar dilatation and flattening of the epithelium (Helminen et
al., 1968; and Martinez-Hernandez et al., 1976) (FIG. 2C). Alkaline-extracted
membranes from MG tissue in each of the above-mentioned stages were
subjected to immunoblot analysis. mgNIS was absent in nubile MG
(FIG. 2D, lane 1), but clearly present in lactating MG (FIG. 2D,
lane 2). Twenty-four hours after weaning, mgNIS expression was significantly
decreased in PL MG (FIG. 2D, lane 3), and by 48 h mgNIS was not
detectable (FIG. 2D, lane 4). Remarkably, mgNIS expression was reversible
upon re-initiation of suckling (FIG. 2D, lane 5). Clearly, mgNIS
expression is upregulated during lactation, rapidly down-regulated
upon cessation of lactation, and exquisitely regulated in a reversible
manner by suckling.
To investigate whether mgNIS expression starts in response to suckling
or before (i.e., during gestation), immunoblot analysis was conducted
on membrane fractions from MGs during various stages of the mice
20-day gestation period (FIG. 2E). Expression of a .about.75 kDa
mgNIS protein was barely detectable at mid-gestation (lane 11d),
whereas higher levels of mgNIS expression, together with a significant
increase in .sup.125 I.sup.- transport in MG cells, were reached
towards the end of gestation (FIGS. 2E and 2F, lane 18d). These
data clearly indicate that the induction of mgNIS expression precedes
suckling (FIG. 2E); however, after delivery, suckling is essential
for continued milk production and increased expression of mgNIS
in the mammary epithelial cells (FIG. 2D).
B. Hormonal regulation of mgNIS expression
I. Effects of oxytocin and prolactin on mgNIS expression in intact
animals
In all mammals, mammary gland development, milk protein synthesis,
and lactation result from the combined effects of, among others,
estrogen, progesterone, prolactin, and oxytocin (Lyons et al., 1958;
and Topper et al., 1981). In mice, prolactin, a glycoprotein released
from the anterior pituitary, plays an essential role in lobuloalveolar
development of the mammary gland during gestation, and in the induction
of synthesis and secretion of milk proteins (Vonderhaar, 1987).
For its part, oxytocin, a nonapeptide hormone released from the
posterior pituitary, is essential for the milk ejection response
to suckling during lactation (Young et al., 1996). Intact and ovariectomized
(see following section) adult nubile mice were systematically treated
with various combinations of these hormones, then analyzed for mgNIS
expression using immunohistochemistry and immunoblot analysis. As
both prolactin and oxytocin are released simultaneously in response
to suckling (Wakerley et al., 1978; and Higushi et al., 1985), nubile
rats and mice were treated with these hormones to discern whether
the reversible suckling-dependent upregulation of mgNIS expression
is caused by oxytocin, prolactin, or both. It was observed that
oxytocin alone (but not in combination with prolactin) induced mgNIS
expression (FIG. 3B, lane OXY), leading to .sup.125 I.sup.- transport
in MG tissue (FIG. 3A). This demonstrates that the functional expression
of mgNIS is upregulated by oxytocin, even in nubile animals, and
that this regulatory effect is unexpectedly antagonized by prolactin.
The ability of oxytocin to induce mgNIS expression in the relatively
undeveloped and undifferentiated MG of nubile animals indicates
that mgNIS synthesis can occur independently of gestation and lactation-related
changes. The antagonist effect of prolactin on oxytocin action was
an unexpected result because mgNIS is upregulated in response to
suckling, which, as indicated above, stimulates the release of both
hormones (Wakerley et al., 1978; and Higushi et al., 1985). This
observation is further analyzed below.
Significant increases in mgNIS activity, due to oxytocin administration,
were also demonstrated in mice by scintigraphic imaging (FIGS. 3C
and 3D). In nubile mice, surgical removal of the MG for imaging
purposes was necessary to differentiate between the signals from
the stomach and those from the few epithelial cells present in the
MG. Oxytocin-treated and sham-treated nubile mice received a single
dose of .sup.99m TcO.sub.4.sup.-, with or without perchlorate. Thirty
minutes later, internal organs were surgically removed and .sup.99m
TcO.sub.4.sup.- accumulation in individual organs was monitored
with a pin-hole gamma-camera (FIG. 3C). The accumulation of tracer
in these organs was quantified (FIG. 3D). As expected, accumulation
of .sup.99m TcO.sub.4.sup.- in the stomach of both oxytocin-treated
and sham-treated animals was significantly higher than in the spleen
or skeletal muscle from the limb (FIGS. 3C and 3D). This accumulation
of .sup.99m TcO.sub.4.sup.- was inhibited by perchlorate, revealing
that the tracer was transported specifically via mgNIS. Thereafter,
accumulation of tracer in MGs from oxytocin-treated animals was
compared with accumulation in MGs from sham-treated animals. Perchlorate-inhibited
.sup.99m TcO.sub.4.sup.- accumulation in MGs was significantly higher
in oxytocin-treated than in sham-treated mice (FIGS. 3D). The levels
of mgNIS expression in imaged MGs of oxytocin-treated animals were
similar in all individual mice tested, as assessed by Ad immunoblot
analysis (not shown). Thus, the .sup.99m TcO.sub.4.sup.- accumulation
in MGs of these animals is due to perchlorate-inhibited mgNIS activity,
and not to individual variations in mgNIS expression. In conclusion,
results from transport assays (FIGS. 3A and 3D), immunoblot analysis
(FIG. 3B), and scintigraphic imaging (FIG. 3C) demonstrate that
functional mgNIS expression is upregulated by an increase in the
circulating concentration of oxytocin in nubile animals,
II. Effects of 17-.beta.-estradiol, progesterone, oxytocin, and
prolactin in ovariectomized mice
To further examine the hormonal regulation of mgNIS expression,
and particularly to assess the roles of gonadal steroid hormones
(estrogens and progesterone), nubile ovariectomized (OVX) adult
mice were used. Mice were administered 17-.beta.-estradiol, progesterone,
oxytocin, and prolactin, both individually and in different combinations,
and mgNIS expression was assessed in mammary tissue by immunoblot
analysis (FIG. 4). No mgNIS expression was detected in control PBS-treated
animals (FIG. 4, lane 1). Significantly, administration of oxytocin
alone to ovariectomized mice did not cause an increase in mgNIS
expression (FIG. 4, lane 5), starkly contrasting with the effect
of oxytocin in intact animals (FIG. 3B). Indeed, of all the hormones
tested individually, only 17-.beta.-estradiol led to a clearly discernible
increase in mgNIS expression (FIG. 4, lane 3). Combined administration
of 17-.beta.-estradiol and oxytocin resulted in mgNIS expression
(FIG. 4, lane 6) modestly higher than 17-.beta.-estradiol alone
(FIG. 4, lane 3), suggesting that the upregulating effect of oxytocin
on mgNIS expression may require estrogen. Although it has not been
studied in mammary tissue, estrogen has been reported to upregulate
accumulation of the oxytocin receptor gene mRNA in the uterine epithelium
and hypothalamus of the rat (Larcher et al., 1994; and Bale and
Dorsa, 1995 and 1997). A similar effect of estrogen in the breast
would be consistent with the inventors' observations.
By far the greatest increase in mgNIS expression was observed upon
administration of 17-.beta.-estradiol, oxytocin, and prolactin together
(FIG. 4, lane 8). This indicated that, not only does prolactin not
antagonize the upregulating effect of oxytocin, it actually enhances
it when estrogen levels are high. The antagonizing action of prolactin
on oxytocin upregulation of mgNIS expression in intact nubile mice
clearly occurs in the presence of comparatively lower (endogenous)
estrogen levels (see above section). Tellingly, when progesterone
was added to the 17-.beta.-estradiol/oxytocin/prolactin combination,
mgNIS expression was significantly decreased (FIG. 4, lane 9). Contrastingly,
a comparison of mgNIS expression in estrogen-treated versus estrogen-
and progesterone-treated animals revealed that progesterone did
not interfere with 17-.beta.-estradiol enhancement of mgNIS expression
(FIG. 4, lanes 3 and 4). These observations are consistent with
the notion that progesterone may act as an oxytocin inhibitor due
to competitive binding of progesterone to the oxytocin receptor
in the breast, much as it reportedly occurs in the uterus (Fuchs
et al., 1983; and Grazzini et al., 1998). In conclusion, the combination
of estrogen, prolactin, and oxytocin (in the absence of progesterone)
leads to the highest mgNIS expression in ovariectomized mice. This
combination of hormones closely resembles the relative hormonal
levels prevalent in mice and rats during lactation (McCormack and
Greenwald, 1974; and Rosenblatt et al., 1988), when the action of
mgNIS as an I.sup.- supplier to the nursing pups is most beneficial.
C. Active I.sup.- transport in experimental mammary tumors
I. Expression and activity of mgNIS in experimental mammary tumors
in transgenic mice
In the course of breast development, the menstrual cycle, gestation,
and lactation, mammary epithelial cells undergo extensive proliferation,
differentiation, and involution in response to hormonal regulation.
Physiologically, MG epithelial cells express mgNIS only during lactation,
following the culmination of intense glandular proliferation and
differentiation. Because neoplastic transformation represents an
abnormal proliferative process with altered cellular differentiation
(Fitzgibbons et al., 1998), it seems plausible that mgNIS could
be expressed in cancer. The inventors sought to assess whether mgNIS
is functionally expressed in experimental mammary tumors in female
transgenic mice carrying either an activated ras (a cytoplasmic
GTPase) oncogene or overexpressing the neu oncogene, each of them
under the transcriptional control of the MMTV promoter/enhancer
(Sinn et al., 1987; and Guy et al., 1992). The tyrosine kinase receptor
encoded by the neu (known as c-erbB2 in humans) proto-oncogene is
amplified and overexpressed in as many as 30% of human breast cancers
(Slamon et al., 1987; Paterson et al., 1991; DiGiovanna, 1999; and
Siegel et al., 1999).
To assess whether specific active I.sup.- uptake (a result of mgNIS
expression) occurred in the mammary tumoral cells, transgenic animals
were imaged in vivo with either .sup.131 I.sup.- or .sup.99m TcO.sub.4.sup.-.
The results were striking. Specific active .sup.99m TcO.sub.4.sup.-
transport in the tumors was indeed demonstrated on imaging. First,
injected .sup.99m TcO.sub.4.sup.- was accumulated in the stomach
and tumor (FIGS. 5B for an MMTV-ras mouse and 5G for an MMTV-neu
mouse). Second, .sup.99m TcO.sub.4.sup.- accumulation was prevented
when the isotope was co-injected with perchlorate (FIGS. 5C for
MMTV-ras and 5H for MMTV-neu), thereby showing that the observed
accumulation in the tumor and stomach was specifically mediated
by NIS. Injection of .sup.99m Tc-HSA, a vascular space marker that
is distributed non-specifically solely according to blood pool,
showed that the observed accumulation pattern in the MMTV-ras tumor
was not due to non-specific increase of the blood pool (FIG. 5D).
Quantification of the accumulation of the various tracers in the
tumor, as a function of time, is also shown for the ras mouse. The
quantification values underscore both the absence of tumoral accumulation
of .sup.99m TcO.sub.4.sup.- when co-injected with perchlorate, and
the lack of accumulation of the vascular space marker (FIG. 5E).
MMTV-ras and MMTV-neu mice were subsequently sacrificed, and tumoral
tissue was retrieved for histological, immunohistochemical, and
immunoblot analysis. Both kinds of transgenic animals developed
mammary adenocarcinomas that were high-grade, poorly differentiated,
estrogen- and progesterone-receptor negative (not shown), and positive
for mgNIS expression by immunohistochemistry. The mgNIS immunohistochemical
staining pattern of the tumors was intracellular, and not merely
evident exclusively at the plasma membrane (FIG. 5J). Furthermore,
mgNIS expression was demonstrated by immunoblot analysis in both
ras (FIG. 5F) and neu (tu1) (FIG. 5I) tumors, showing that I.sup.-
transport activity is concomitant with mgNIS protein expression.
Immunoblot analysis of non-tumoral MG tissue from the contralateral
side of the same MMTV-neu mouse revealed no mgNIS expression (FIG.
5I). Parallel tissue sections probed with anti-HER-2/neu Ab demonstrated
that neu expression was absent from normal glands, but was significant
in transformed MG, especially in the mgNIS-expressing tumor (FIGS.
5K and 5L). This indicates that any factors that led to the expression
and activation of mgNIS in tumoral mammary tissue were not operative
in non-tumoral tissue in the same neu mouse. No 99.sup.m TcO.sub.4.sup.-
transport activity was observed using imaging, and no mgNIS expression
was detected using immunoblots, either in a second carcinoma in
the same MMTV-neu mouse (tu2) (FIG. 5I) or in the tumor of a different
MMTV-neu mouse (not shown). These observations indicate that the
absence of tracer uptake in these tumors, based on imaging, correlates
with lack of mgNIS expression in the same tumors, based on immunological
analysis. In conclusion, functional expression of mgNIS has been
demonstrated in experimental mammary tumors caused in transgenic
mice by activation or overexpression of the ras and neu oncogenes.
Because increased amplification of the neu gene homologue in humans
(c-erbB2) correlates with highly aggressive tumors with negative
prognosis even for patients without lymph node involvement (Guy
et al., 1992; and Siegel et al., 1999), these findings in experimental
mouse models may be relevant to human breast cancer. The results
suggest that mgNIS might be expressed in human breast tumors, that
these tumors might be clinically detected by scintigraphic imaging,
and that mgNIS-mediated transport might provide an alternative modality
for the detection and/or treatment of breast tumors and metastatic
disease.
D. Expression of mgNIS in human breast cancer
Having shown that mgNIS is functionally expressed in experimental
transgenic mice adenocarcinomas, the inventors examined human breast
tissue specimens (29 malignant [23 invasive carcinomas and 6 ductal
carcinomas in situ], 13 non-tumoral from tissue in the vicinity
of the tumors, and three biopsies from pregnant women) for mgNIS
expression (Table 1). Specimens were studied by immunohistochemical
analysis using two site-directed polyclonal (Ct-1 and Ct-2) anti-NIS
Abs and one monoclonal anti-NIS Ab, each of the three directed against
different epitopes of the C-terminus of human NIS (FIG. 6) (see
Materials and Methods). In all experiments, competitive inhibition
of immunoreactivity with the C-terminal eliciting peptide verified
the specificity of the antibody reaction (FIG. 6D). As an additional
control, the use of rabbit and mouse immunoglobulins against unrelated
antigens confirmed that no staining resulted from non-specific immunoreactivity
with human antigens other than mgNIS in breast tissue. Breast epithelial
cells were identified on parallel sections with anti-cytokeratin
Abs (Zeng et al., 1999) to distinguish them from other stromal cell
populations (not shown). Sections were graded by the intensity of
the immunoperoxidase reaction on a scale of 0 to 4+, and by the
percentage of reacting epithelial cells. Tissues exhibiting 2+ to
4+ staining in 20% or more of the epithelial cells were considered
positive for mgNIS expression. Archival thyroid specimens displaying
clear mgNIS expression, both in differentiated thyroid cancerous
areas (either papillary or follicular) and in adjacent normal-appearing
follicles, were selected as positive experimental controls (FIG.
6A, thyroid papillary carcinoma; FIG. 6B, competitive inhibition
of immunoreactivity with C-terminal eliciting peptide).
The findings (summarized in Table 1) were compelling: 87% of the
23 invasive breast cancers (FIGS. 6C, E, and G) and 83% of the 6
ductal carcinomas in situ (FIG. 6H) expressed mgNIS, as compared
to only 23% of the 13 non-cancerous samples adjacent to, or in the
vicinity of, the tumors (FIG. 6F). Ductal carcinomas in situ (FIG.
6H), in which mgNIS expression was as marked as in invasive carcinomas
(FIGS. 6C, E, and G), are associated with an increased risk of developing
a subsequent invasive carcinoma (Fitzgibbons et al., 1998), and
represent a possible intermediate step between benign atypical ductal
hyperplasia and invasive carcinoma (Rosen, 1997). The pattern of
mgNIS expression in all malignant breast cells (FIGS. 6C, E, G,
and H) was identical to that noted in
TABLE 1 mgNIS expression in human breast cancer mgNIS Estrogen
Breast Histology Number Positive Receptor Positive Invasive carcinomas
23 20 (87%) 56% Ductal carcinoma in Situ 6 5 (83%) ND* Noncancerous
in vicinity of 13 3 (23%) ND* tumor Gestational tissues 3 3 (100%)
ND* ND*: not determined
control thyroid sections (FIG. 6A), consisting of a predominant
granular pattern of intracellular distribution that suggests organellar
localization of NIS. This contrasts with the distinct basolateral
plasma membrane staining observed in rat mammary gland tissues (FIG.
1F).
Staining in non-cancerous samples that were mgNIS-positive (FIG.
6F) was less intense than in malignant tissue (FIGS. 6C, E, G, and
H). All three gestational biopsies exhibited florid adenomatous-lactational
changes characteristic of this physiological stage, and were clearly
mgNIS positive (FIG. 6I). In two of these gestational samples, the
epithelial cells within the fibroadenomatous tissue displayed florid
hyperplasia, similar to the non-affected adjacent tissue (not shown).
This suggests that mgNIS expression in fibroadenomatous tissue is
responsive to hormonal changes that take place during gestation.
Interestingly, mgNIS expression was noted throughout the gestational
tissue sampled, including areas with distinct basolateral plasma
membrane immunoreactivity (FIG. 6I) similar to lactating rat mammary
gland (FIG. 1F). The observed expression of mgNIS in gestational
samples is consistent with the upregulation of mgNIS during pregnancy
and lactation (see previous sections).
In conclusion, over 80% of the breast cancers analyzed express
mgNIS, suggesting that mgNIS is frequently upregulated during malignant
transformation in human breast. The high prevalence of mgNIS expression
in human breast cancer, and the observation in transgenic mice that
mgNIS-positive tumors exhibit active I.sup.- transport, together
suggest that radioiodide is potentially a novel alternative therapeutic
modality in breast cancer.
3. Discussion
A. mgNIS catalyzes active I.sup.- transport in rat lactating mammary
glands
The ability of cancerous thyroid cells to actively accumulate I.sup.-
via thyroid NIS (tNIS) provides a unique and effective delivery
system to detect and target these cells for destruction with therapeutic
radioiodide, largely without harming other tissues. The effectiveness
of radioiodide therapy in treating thyroid cancer relies on the
capacity of malignant thyroid cells to retain sufficient I.sup.-
transport activity to accumulate the isotope, even though this activity
is decreased, relative to healthy thyroid cells, as a result of
malignant transformation. In fact, a major characteristic of the
healthy thyroid gland is that it exhibits tNIS activity for life,
within boundaries set by such thyroid regulatory factors as TSH
and I.sup.- itself (Levy et al., 1997; and Eng et al., 1999). In
contrast, the potential effectiveness of radioiodide therapy in
breast cancer depends on whether mgNIS becomes functionally expressed
in cancerous mammary cells as a result of malignant transformation,
given that mgNIS is normally not expressed in healthy epithelial
mammary cells, except during pregnancy and lactation. Thus, it is
notable that a single transport protein--NIS--catalyzes the same
fundamental process--active Na.sup.+ -dependent I.sup.- transport--in
both of these tissues, but is regulated differently in each. These
differences affect not only how NIS functions in health, but also
how it can play a role in cancer management in both tissues. Insofar
as NIS is functionally expressed to a sufficient degree in cancerous
cells, whether of thyroid, breast, or any other origin, radioiodide
emerges as a proven and effective therapeutic tool.
As shown in FIG. 1, I.sup.- transport in rat lactating mammary
glands is pronounced, active, specific, and inhibited by perchlorate,
as assessed in vivo both by tracking the accumulation of I.sup.-
in milk and by analyzing the tissue distribution of I.sup.- by scintigraphy.
This indicates that I.sup.- transport in rat lactating mammary glands
is a protein-mediated active transport process. mgNIS has been identified
as a single broad polypeptide of .about.75 kDa in rat lactating
mammary gland membranes, as observed by immunoblot analysis with
anti-NIS Abs raised against tNIS. It has also been demonstrated
that mgNIS is identical to both tNIS and gNIS, although it is differently
glycosylated than either of them in their respective tissues. Consistent
with this, the presence of identical NIS transcripts has been reported
in rat thyroid, mammary gland, and stomach (Spitzweg et al., 1998).
Thus, it is clearly established that mgNIS is the protein that catalyzes
active I.sup.- transport in rat lactating mammary glands.
B. Oxytocin, prolactin, estradiol, and progesterone play significant
roles in the regulation of mgNIS expression
To initiate the identification of factors involved in the regulation
of mgNIS, mgNIS expression was examined in nubile, pregnant, lactating,
and previously lactating rats and/or mice by immunohistochemistry
and immunoblot analysis, as well as by in vivo scintigraphic imaging
(FIGS. 1 and 2). Findings indicated that mgNIS is barely detectable
at any time other than lactation and the second half of gestation.
At approximately mid-gestation, mgNIS becomes detectable; thereafter,
its expression increases considerably as gestation progresses (FIG.
2E). By the 18th day of gestation in mice, the MG show a 15-fold
accumulation of subcutaneously-injected radioiodide, as compared
with the blood concentration of the tracer. Therefore, suckling
is not required for the functional expression of mgNIS. Still, both
suckling and its cessation clearly become the major stimuli that
regulate the functional expression of mgNIS after delivery (FIG.
2D). These findings suggest that there are suckling-dependent and
suckling-independent factors that regulate the functional expression
of mgNIS.
The inventors first considered the effect of suckling-dependent
hormones, oxytocin and prolactin, on mgNIS expression. The striking
ability of oxytocin alone to induce functional mgNIS expression
in nubile animals has already been mentioned above (FIG. 3), as
has the surprising antagonistic effect of prolactin on mgNIS upregulation
by oxytocin. Interestingly, the relative migration of oxytocin-induced
mgNIS from nubile animals is slightly slower (100 kDa) (FIG. 3B,
lane 4) than mgNIS from lactating animals (.about.75 kDa) (FIG.
2D, lane 2). This difference is due to glycosylation, as the relative
migration of mgNIS from either group of animals (oxytocin-treated
nubile as compared to lactating) becomes indistinguishable upon
deglycosylation (not shown). It is significant that the first enzyme
in the N-linked glycosylation pathway, UDP-GlcNAc:dolichol phosphate
N-acetylglucosamine-1-phosphate transferase (GPT), is constitutively
expressed in all tissues except mammary gland, where its expression
is modulated by lactogenic hormones (Rajput et al., 1994; and Ma
et al., 1996). This suggests that differences in the levels of lactogenic
hormones between oxytocin-treated nubile and intact lactating animals
are likely to account for the observed glycosylation differences
in mgNIS.
Subsequent studies of suckling-independent hormonal regulators
of mgNIS expression, such as estradiol and progesterone, also shed
light on the effects of the suckling-dependent hormones, oxytocin
and prolactin (FIG. 4). The inventors' findings may be summarized
as follows: 1. in ovariectomized mice, 17-.beta.-estradiol alone
caused a pronounced increase of mgNIS; 2. in contrast to its effect
in intact animals, oxytocin alone did not increase mgNIS expression
in ovariectomized animals; 3. in ovariectomized animals, combined
17-.beta.-estradiol and oxytocin led to a modestly higher expression
of NIS than did 17-.beta.-estradiol alone; 4. maximum mgNIS expression
resulted from a combination of 17-.beta.-estradiol, oxytocin, and
prolactin in ovariectomized animals, even though prolactin antagonized
mgNIS upregulation by oxytocin in intact animals; 5. progesterone
inhibits the stimulatory effect on mgNIS expression of combined
17-.beta.-estradiol, oxytocin, and prolactin.
The above results indicate that a threshold level of circulating
estrogens is necessary for optimal mgNIS expression overall, and,
in particular, for the upregulation of mgNIS by oxytocin. The co-operative
role played by estrogens in regard to oxytocin's effect on mgNIS
may be explained by the likelihood that the oxytocin receptor gene
mRNA is upregulated by estrogen in the breast, as it has been reported
in the hypothalamus and uterus (Zingg et al., 1995 and 1998). In
addition, a direct effect of estrogen on mgNIS transcription could
also occur. Consistent with this notion is the observation that
the NIS promoter contains several half-site estrogen responsive
elements (EREs) (Ohno et al., 1999). Hence, oxytocin alone increases
mgNIS expression in intact animals, in which endogenous estrogens
are present, but not in ovariectomized animals, in which estrogens
are absent.
In intact animals, prolactin's antagonistic effect on the increase
of mgNIS expression by oxytocin may be due to the reported inhibitory
effect of prolactin on steroidogenesis (Dorrington and Gore-Langdon,
1981 and 1982; Gitay-Goren et al., 1989; Krasnow et al., 1990; and
Villanueva et al., 1996). In intact (non-gestational, non-lactating)
animals, exogenous prolactin would cause endogenous estrogen levels,
which are lower than in gestational or lactating animals, to decrease
below the threshold, thereby preventing concomitantly-administered
oxytocin from stimulating mgNIS expression. On the other hand, no
antagonistic effect of prolactin administered simultaneously with
oxytocin is observed in ovariectomized animals that received a high
amount of exogenous estrogen. Under these conditions, steroidogenesis
is entirely bypassed. Moreover, the results show that prolactin
actually leads to higher mgNIS expression when given, together with
estrogen and oxytocin, to ovariectomized animals (FIG. 4, lane 8).
This suggests that, in the presence of high levels of estrogen,
prolactin may have a separate direct or indirect stimulatory effect
on mgNIS expression by a mechanism currently unknown. It appears
that endogenous oxytocin increases mgNIS expression in lactating
animals, despite the presence of endogenous prolactin, because,
under the hormonal conditions of lactation, steroidogenesis most
probably overcomes the steroidogenesis-inhibitory effect of prolactin.
The concentration of endogenous prolactin in intact animals, even
during lactation, is considerably lower (.about.1.2 .mu.g/ml) than
the one used experimentally (300 .mu.g/injection) (Mattheij et al.,
1982). Hence, during lactation, estrogens would remain above the
necessary threshold necessary for oxytocin to stimulate mgNIS expression,
and for prolactin to enhance, rather than antagonize, this effect.
Finally, given the central role played by oxytocin in the increase
of mgNIS expression, it is likely that the ability of progesterone
to inhibit the stimulatory effect on mgNIS expression of combined
17-.beta.-estradiol, oxytocin, and prolactin may involve a direct
inhibition of oxytocin-receptor signaling by competitive binding
of progesterone to the oxytocin receptor in the breast. This non-genomic
effect of progesterone has, thus far, been observed in the uterus
(Fuchs et al., 1983; Grazzini et al., 1998; and Zingg et al., 1998).
In addition, it is possible that progesterone also inhibits oxytocin
receptor gene expression in the breast, as proposed for the regulation
of this gene in the cervix of the uterus (Umscheid et al., 1998).
The fall of progesterone levels in mammals following delivery, therefore,
coexists with the onset of suckling and the release of oxytocin.
According to the inventors' results, this would, in turn, optimize
the ability of oxytocin to upregulate mgNIS expression during lactation.
C. mgNIS is functionally expressed in experimental mammary tumors
in transgenic mice, and is detectable by scintigraphic imaging in
vivo
The studies of experimental mammary tumors in transgenic mice,
as described above, seemed potentially relevant to human breast
cancer because the inventors were addressing a fundamental biological
question conceivably applicable to all mammals: Can malignant transformation
of mammary epithelial cells lead to functional expression of mgNIS?
The results (FIG. 5) provide compelling evidence that the answer
to this question is yes: scintigraphic imaging in vivo dramatically
demonstrated pronounced, active, specific, and perchlorate-inhibited
mgNIS activity in mammary tumors of non-gestational and non-lactating
female transgenic mice which either carried an activated ras oncogene
or overexpressed the neu oncogene. Immunoblot analysis (using anti-NIS
Ab) of mammary tissue from these mice demonstrated a correlation
between mgNIS activity observed by scintigraphy and mgNIS expression:
mgNIS was expressed only in tumoral mammary tissues that had displayed
mgNIS activity. No mgNIS expression was detected in non-tumoral
mammary tissue (which, without exceptions, exhibited no mgNIS activity),
or even in non-I.sup.- -transporting tumoral mammary tissue. Thus,
malignant mammary cells are detectable by in vivo scintigraphic
imaging in mice, and could, therefore, be specifically targeted
for destruction with higher doses of radioiodide.
D. mgNIS is specifically expressed in >80% of human breast cancer
tumors, as detected by immunohistochemistry with anti-NIS Abs
It remained to be revolved whether mgNIS is expressed in human
breast tumors, and, if so, whether the prevalence of mgNIS expression
in breast cancer is high. The finding that >80% of the analyzed
human breast cancer samples expressed mgNIS in at least 20% of their
cells (Table 1 and FIG. 6) resolves both of these issues in the
affirmative. In all cases, mgNIS expression detected by immunohistochemistry
was specific, as it was inhibited by excess C-terminus eliciting
peptide. However, a wide variety of immunohistochemical pattern
was observed from one tumor to another. Some tumors exhibited diffuse,
moderate to light immunoreactivity in nearly all of their cells
(FIGS. 6C and 6E); others showed distinct areas of complete negativity
interspersed with focal areas of intense positivity, or a single
confined positive region (FIG. 6G). In stark contrast, only 23%
of non-cancerous tissue samples that were either adjacent to or
in the vicinity of the tumors showed mgNIS staining; moreover, even
when present, staining was noticeably less pronounced (FIG. 6F)
than in tumoral samples (FIGS. 6C, 6E, and 6H). Therefore, as in
transgenic mice, mgNIS is expressed in humans primarily in neoplastic
mammary tissue, as a result of malignant transformation; it is not
expressed to any significant extent in apparently healthy tissue.
The expression of mgNIS during gestation appears to be an indicator
of the physiological, hormone-driven differentiation of breast epithelial
cells from non-lactating to lactating. Nevertheless, the degree
to which some mgNIS expression was observed in non-cancerous (and
non-lactating) tissue samples in the vicinity of the tumors may
reflect either the presence of morphologically normal cells in the
early stages of malignant transformation, or low-grade mgNIS expression
in actually healthy cells. In any case, the above results suggest
that therapeutic doses of radioiodide could be used in humans to
selectively destroy breast cancer cells, provided mgNIS is functional
in human breast cancer, as it is in transgenic mice mammary tumors.
The results obtained from the studies on transgenic mice, and the
similarity between the human breast cancer immunohistochemical patterns
and those observed in thyroid cancer, indicate the likelihood that
mgNIS expressed in human breast tumors is active. Clearly, the most
direct way to determine the activity of mgNIS in breast cancer is
to subject patients to scintigraphic evaluation with .sup.99m TcO.sub.4.sup.-.
One patient who was undergoing neoadjuvant hormonal therapy for
locally-advanced breast cancer presented initially with an enlarged
thyroid. Accordingly, she underwent whole-body .sup.99m TcO.sub.4.sup.-
scanning. Significantly, the tracer was concentrated in the area
of this patient's breast tumor (FIG. 7), which was a needle-biopsy-proven
invasive ductal carcinoma (not shown). This is an extremely meaningful
(if preliminary) finding that supports the notion that the use of
radioiodide therapy as an adjuvant to surgical treatment of primary
breast cancer, or as directed against systemic metastatic disease,
may be highly effective.
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All publications mentioned hereinabove are hereby incorporated
in their entirety. While the foregoing invention has been described
in some detail for purposes of clarity and understanding, it will
be appreciated by one skilled in the art, from a reading of the
disclosure, that various changes in form and detail can be made
without departing from the true scope of the invention in the appended
claims. |