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Cancer Patent Abstract
This invention relates to the AIB1 protein as a coactivator that
potentiates the transcriptional activity of nuclear hormone receptors.
The gene is amplified in a subset of human breast cancers. One splice
variant of AIB1 transcribes a mRNA that lacks the exon 3 sequence.
.DELTA.3-AIB1 mRNA encodes a 130 kDa protein that lacks the N-terminal
basic helix-loop-helix and a portion of the PAS dimerization domain.
This 130 kDa protein was detected in MCF-7 breast cancer cells at
levels 5-10% of the full length protein, whereas in non transformed
mammary epithelium lines the .DELTA.3-AIB1 protein is present at
significantly lower levels compared to the full length AIB1. The
abundance of .DELTA.3-AIB1 mRNA is increased in human breast cancer
specimens relative to that in normal breast tissue. Functional reporter
gene assays revealed that the ability of .DELTA.3-AIB1 to promote
transcription mediated by the estrogen or progesterone receptors
was significantly greater than that of the full-length protein.
The .DELTA.3-AIB1 isoform was also more effective than AIB1 in promoting
transcription induced by epidermal growth factor. Thus, over expression
of .DELTA.3-AIB1 plays an important role in sensitizing breast tumor
cells to hormone or growth factor stimulation.
Cancer Patent Claims
The invention claimed is:
1. An isolated nucleic acid sequence that encodes a steroid receptor
coactivator Amplified In Breast (AIB1) protein isoform, wherein
the isoform is .DELTA.3-AIB1, and said nucleic acid comprises SEQ
ID NO:3.
2. A vector that contains the nucleic acid of claim 1.
3. An isolated recombinant cell that contains the nucleic acid
of claim 1.
Cancer Patent Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to coactivators of hormone and growth
factor activity in cancer cells and, specifically, to isoforms of
the coactivator amplified in breast. In particular, the invention
relates to compositions, diagnostic kits and methods for utilizing
coactivator isoforms for the diagnosis, treatment and prevention
of cancers such as breast cancer.
2. Description of the Background
Ligands such as estrogen and progesterone that interact with nuclear
receptors regulate gene expression predominantly at the transcriptional
level. The ligand-bound receptors interact specifically with DNA
and activate transcription by recruiting a preinitiation complex.
Although such gene activation was originally thought to be mediated
by interaction of the receptors with components of the basal transcriptional
machinery (1-6), a variety of screening techniques has identified
a family of receptor-interacting proteins known as nuclear receptor
coactivators (7-11). A common characteristic of this superfamily
of proteins is that, when overexpressed in the presence of nuclear
receptors, they potentiate ligand induction of transcription (12,
13). The related p160 group of coactivators, which include steroid
receptor coactivators (Src-1), Src-2, and Src-3 (which is also known
as AIB1, ACTR, RAC3, TRAM-1, and p/CIP) (14-20), possess several
similar structural features including a receptor interaction domain
(RID), a bHLH (basic helix-loop-helix)PAS (Per-Arnt-Sim homology)
dimerization domain, and a CBP interaction domain (CID) (13). Coactivators
are thought to function as bridges between nuclear receptors and
either other coactivators or the basal transcriptional machinery
(13). It was discovered that coactivators possess a histone acetylase
domain (15, 21-24), which suggests that these proteins also might
serve to regulate chromatin structure.
A portion of human chromosome 20 q that is frequently amplified
in breast cancer contains the gene for the nuclear coactivator AIB1
(amplified in breast cancer 1) (25). The AIB1 gene is amplified
in five to ten percent of breast cancers and the abundance of the
corresponding mRNA and protein is increased in 30-50% of breast
tumors and also breast cancer cell lines (14, 25-27). It has recently
been shown that AIB1 binds directly to ER (28) and that AIB1 is
rate-limiting for estrogen-induced growth of MCF-7 cells (29). However,
the overall role of AIB1 for breast tumorigenesis has not been clear
since AIB1 potentiates not only the action of estrogen (14, 16)
and progesterone (16) receptors, but also that of various other
nuclear receptors (9, 15, 17-20) and transcription factors (30,
31). In addition, several splice variants of SRC family members
have been described, although the functions of these variants remain
unknown (13).
SUMMARY OF THE INVENTION
The present invention overcomes the problems and disadvantages
associated with current strategies and designs and provides new
isoforms of the coactivator AIB1 including compositions, diagnostic
kits, and treatments utilizing and derived from these isoforms.
Further, the invention also provides novel siRNA molecules directed
to these isoforms and methods for the treatment and prevention of
cancer.
The invention relates to the identification and isolation of isoforms
of the transcriptional coactivator, AIB1 protein. Isoforms include,
but are not limited to, deletion mutants, addition mutants, and
splice variants of the wild-type protein. Isoforms can be identified,
for example, by an altered molecular weight or by an altered level
of expression, which may be over expression or under expression,
in breast cancer tissue and other tissues and cell lines. One splice
variant is .DELTA.3-AIB1, whose transcript encodes a N-terminal
truncated version of AIB1 that lacks the HLH and PAS A domain and
has a molecular weight of 130 KDa. In functional studies, it has
been determined that the .DELTA.3-AIB1 protein is a significantly
more effective coactivator of estrogen, progesterone and EGF signaling
as compared to the wild type ER, providing a role for this AIB1
isoform in hormone and paracrine signaling in breast cancer.
One embodiment of the invention is directed to isoforms of the
AIB1 transcriptional coactivator protein. Isoforms are potent transcriptional
coactivators of nuclear receptors (e.g. retinoid, steroid such as
estrogen receptor or progesterone receptor, thyroid), and/or potentiate
growth factor activities such as signaling pathways. Transcriptional
coactivators function by enhancing transcription induced by a transcription
factor. Enhancement is thought to be produced by binding to a transcription
factor which forms a complex that promotes transcription of a gene,
and not by direct binding of the coactivator to a site on the respective
genome. Preferably, coactivation by an isoform is significantly
greater than coactivation produced by wild-type AIB1 protein. Preferably,
the isoform contains a deletion of all or significant portions of
exon 3 in the amino terminus of the protein (exon 3 encompasses
NT positions 266-438 as counted from a transcription start site),
and is over expressed in cancer cells such as, for example, breast
tumor tissue and prostate tumor tissue. Although there may exist
alternate transcription start sites, exon 3 is the first exon 3'
of the exon containing a translation start site.
Another embodiment of the invention is directed to isolated nucleic
sequences that encode isoforms of the invention. Preferably the
nucleic acid encodes the isoform .DELTA.3-AIB1. The invention further
encompasses vectors that contains the nucleic acid of isoforms of
the invention, and also recombinant cells, which may be either eukaryotic
or prokaryotic, containing nucleic acids or vectors of the invention.
Another embodiment of the invention is directed to diagnostic kits
for the detection of cancer comprising chemical substances that
are specifically reactive and preferably bind to AIB1 and isoforms
of the invention. The diagnostic kits preferably contain antibodies
or antibody fragments directed against AIB1 and/or isoform proteins
of the invention, or identifiable fragments of these proteins that
are distinguishable. Antibodies may be monoclonal or polyclonal,
or antibody fragments which may comprise recombinant or humanized
proteins. The invention further relates to antibodies or antibody
fragments that are specifically reactive to the isoform of the invention.
Preferably such antibodies or antibody fragments are IgG isotypes.
Another embodiment of the invention is directed to methods for
the detection of cancer in a patient comprising contacting a biological
sample obtained from the patient to one or more chemical substances
that specifically bind to AIB1 and/or isoform of the invention (either
the protein or RNA product of transcription), and detecting binding
of one or more of the chemical substances. Chemical substances are
preferably antibodies specific to the coactivator proteins or nucleic
acids that are complementary to their genetic sequences. The method
may further comprise comparing the relative amount of isoform in
the sample with the amount of wild-type AIB1 protein in the sample,
for example, to determine a stage of the cancer such as a hormone-independent
phenotype.
Another embodiment of the invention is directed to pharmaceutical
compositions comprising an agent that specifically binds to the
isoform of the invention and prevents a coactivation function when
administered to a patient. Preferably compositions contain a pharmaceutically
acceptable carrier such as, for example, alcohols, buffers, fatty
acids, glycerol, oils, polysaccharides, saccharides, salts, sugars,
water, and combinations thereof.
Another embodiment of the invention is directed to small interfering
RNA molecules (siRNA) that inhibit expression of a transcriptional
coactivator protein such as, for example, AIB1, isoforms of AIB1
such as the p160 group of coactivators, Src-1, Src-2, Src-3, and
other isoforms, fragments and combinations thereof. Preferably,
the siRNA is specifically targeted to inhibiting the expression
of one protein isoform and does not target other isoforms. One preferred
siRNA targets the .DELTA.3-AIB1 mRNA and contains sequences homologous
and complementary to that mRNA. Preferably the siRNA contains a
sequence, a portion of which is derived from exon 2 of the mRNA
that encodes .DELTA.3-AIB1, and another portion of which is derived
from exon 4 of the mRNA that encodes .DELTA.3-AIB1. The portions
are each preferably from about 5 to about 16 nucleotides in length,
making the total length of the RNA molecule about 12 to about 32
nucleotides long. That sequence together with sequence complementary
thereto, forms the double-stranded siRNA molecule.
Another embodiment of the invention is directed to pharmaceutical
compositions comprising siRNA that inhibits expression of transcriptional
coactivator proteins and a pharmaceutically acceptable carrier.
Preferably pharmaceutically acceptable carriers include alcohols,
buffers, fatty acids, glycerol, oils, polysaccharides, saccharides,
salts, sugars, water, and combinations thereof. Pharmaceutical composition
of the invention may further include one or more anti-neoplastic
agents effective in the treatment of cancer such as, for example,
agents that inhibit cell growth, agents that inhibit cell proliferation,
agents that inhibit cellular differentiation, anti-angiogenic agents,
antibodies, antibody fragments, anti-sense agents, chemical agents,
cytokines, toxins, and combinations thereof.
Another embodiment of the invention is directed to methods for
treating or preventing a tumor comprising administering to a patient
a therapeutically effective dose of a pharmaceutical composition
of the invention. Administration is preferably by direct injection
to the tumor. These method may further comprise administering additional
tumorigenic therapy to the patient such as, for example, drug therapy,
radiation therapy, surgery, and combinations thereof.
Another embodiment of the invention is directed to transgenic animals
and methods for the creation of transgenic animals containing nucleic
acid sequences that express AIB1 and/or isoforms, or siRNA directed
against AIB1 or isoforms of the invention. Preferably the animal
is a mouse and the isoform is .DELTA.3-AIB1.
Other embodiments and advantages of the invention are set forth
in part in the description which follows, and in part, will be obvious
from this description, or may be learned from the practice of the
invention.
DESCRIPTION OF THE FIGURES
FIG. 1 Characterization of a splice variant of human AIB1 showing:
a the structure of human AIB1; b the positions of PCR products corresponding
to the full-length (AIB1) and truncated (.DELTA.3-AIB1) transcripts;
and c the positions of the splice junctions in .DELTA.3-AIB1 mRNA
and of the encoded protein domains.
FIG. 2 Immunoblot analysis of AIB1 isoforms in extracts of MCF-7
cells and transfected CHO cells.
FIG. 3 Comparison of the abundance of .DELTA.3-AIB1 mRNA between
malignant and nonmalignant human breast tissue and cell lines showing:
a total RNA isolated from MCF-7, MCF-10A, and A1N4 cells subjected
to Southern blot analysis with a probe specific for exon 4 of AIB1;
b total RNA isolated from six normal breast and eight breast cancer
tissue samples analyzed as in a.
FIG. 4 Effects of AIB1 and .DELTA.3-AIB1 on the activation of estrogen
receptor .alpha. and progesterone receptor .beta. in CHO cells showing:
a cells transfected with either the empty pcDNA3 vector (control),
pcDNA3-AIB1, or pcDNA3-AIB1-.DELTA.3, together with an expression
vector for human estrogen receptor .alpha., an ERE-luciferase reporter
plasmid, and pRL-CMV (inset shows immunoblot analysis of transfected
cell lysates probed with antibodies to AIB1); and b cells transfected
as in a with the exception that the estrogen receptor vector is
replaced with a vector for human progesterone receptor .beta., and
the ERE-luciferase plasmid is replaced with a luciferase reporter
construct containing the mouse mammary tumor virus (MMTV) promoter.
FIG. 5 Effects of AIB1 and .DELTA.3-AIB1 on the activation of estrogen
receptor .alpha. and progesterone receptor .beta. in COS-1 cells
showing: a cells transfected and analyzed as in FIG. 4a; b cells
transfected with either pcDNA3, pcDNA3-AIB1, or pcDNA-AIB1-.DELTA.3,
together with an expression vector for human progesterone receptor
.beta., luciferase reporter plasmid containing the MMTV promoter
and pRL-CMV.
FIG. 6 Effects of AIB1 and AIB1-.DELTA.3 on activation of the FGF-BP
gene promoter by EGF in ME-180 cells.
FIG. 7. AIB1 siRNA decreases endogenous AIB1 protein levels in
a dose dependent manner a Histogram of siRNA dose verses mRNA levels;
and b cell number.+-.growth factor was determined with AIB1 up or
down and is expressed relative to control.
FIG. 8 Growth factor dependent proliferation of MCF-7 cells is
reduced by a tetracycline-regulated AIB1 targeted ribozyme.
FIG. 9 a RT-PCR for AIB1 and .DELTA.3-AIB1 RNA from normal and
breast cancer tissue wherein the signal between breast and normal
tissue was compared using an arbitrary scale; and b sequence of
exon junctions for AIB1 and .DELTA.3-AIB1 isoform (SEQ ID NOS 1-3,
respectively in order of appearance.
DESCRIPTION OF THE INVENTION
As embodied and broadly described herein, the present invention
is directed to novel isoforms of the coactivator AIB1. In particular,
the invention relates to compositions containing these isoforms
and pharmaceutical compounds that relate to their method of action,
to diagnostic kits for detecting such isoforms in cells, to methods
for utilizing the compositions and kits, and to methods for the
prevention and treatment of cancer including, but not limited to,
breast cancer.
A portion of human chromosome 20 q that is frequently amplified
in breast cancer contains the gene for the nuclear receptor coactivator
AIB1 (amplified in breast cancer 1) (25). Nuclear receptors include,
but are not limited to, bile acid receptors, perxoidone proliferator
receptors, retinoid receptors, steroid receptors such as, for example,
estrogen receptors and progesterone receptors, thyroid receptors,
vitamin D receptors and others, which regulate gene expression predominantly
at the transcriptional level. Coactivators are proteins whose expression
enhances transcriptional activation of nuclear regulators, i.e.
molecules that regulate gene expression. These coactivators are
thought to function by binding to transcription factors and forming
a complex that enhances induced transcription, and not by binding
directly to the genome. The AIB1 gene is amplified in five to ten
percent of breast cancers and the abundance of the corresponding
mRNA and protein is increased in some breast tumors and breast cancer
cell lines (14, 25-27). It has recently been shown that AIB1 binds
directly to ER (28) and that AIB1 is rate-limiting for estrogen-induced
growth of MCF-7 cells (29). However, the overall role of AIB1 for
breast tumorigenesis is not clear since AIB1 potentiates not only
the action of estrogen (14, 16) and progesterone (16) receptors,
but also that of various other nuclear receptors (9, 15, 17-20)
and transcription factors (30, 31). Isoforms of AIB1 are closely-related
polypeptides derived from the same gene and functionally similar.
Isoforms include, but are not limited to, polypeptides translated
from different transcription and/or translation start sites, frame-shift
mutations, and splice variants of the mRNA that exist as functional
proteins. Several splice variants of Src family members have been
described in vitro, although their existence in vivo is not known
and the functions of these cDNA, if any, remain unknown, none were
translated into protein (13).
It was surprisingly discovered that an isomer of AIB1, a splice
variant of AIB1 that has exon 3 deleted, the .DELTA.3-AIB1 mRNA,
is translated in vivo in breast cancer cells into an NH.sub.2-terminal
truncated form of AIB1. This N-terminally truncated version of AIB1
has lost most of the predicted dimerization domains and thus is
more promiscuous with respect to potential interaction partners
(48). The predicted size of this truncated protein is approximately
130 kDA and a protein with this molecular weight was identified
in Western blot analysis of MCF-7 cells (48). In support of the
significance of the N-terminal region, it was found that over expression
of the .DELTA.3-AIB1 isoform potentiates nuclear hormone (ER, PR)
and growth factor (EGF) induction of transcription to a much greater
extent than full-length AIB1 (48). This indicates that over expression
of this isoform in cancer plays a role in sensitizing breast cancer
cells to hormone and/or growth factor induced changes in phenotype
during the malignant progression of breast cancer.
.DELTA.3-AIB1 has several unusual properties of interest. First,
on a per molecule basis, .DELTA.3-AIB1 is a potent transcriptional
coactivator of steroid receptors such as, for example, the estrogen
receptor and the progesterone receptor (see e.g. Example 4), and
also growth factor signaling (e.g. see Example 5). It is also a
more potent coactivator than full-length AIB1 protein as measured
by conventional transcriptional assays such as, for example, a transient
transfection assay (see e.g. Example 4). For a given amount of vector
transfected, the amount of .DELTA.3-AIB1 protein produced in transient
assays is only about 1% to 10% that of the full-length protein (48).
Despite this, the coactivating effect of .DELTA.3-AIB1 is several-fold
greater than full-length AIB1 making it highly effective on a molar
basis. This result is unexpected given that previous studies of
NH.sub.2-terminal deletion mutants of the AIB1-related protein Src-1
did not reveal an impact of this region on nuclear receptor signaling
(9, 30). One reason for the increased activity of .DELTA.3-AIB1
is that the conformation of this isoform is more favorable than
that of the full-length protein for interaction with nuclear receptors
or for recruitment of other coactivators such as CBP/p300. Another
reason is indicated by the observation that the bHLH-PAS domains
of Src-1 interacts with and potentiates the activity of members
of the TEF family of transcription factors (30). Thus, full-length
AIB1 is unavailable for interaction with nuclear receptors because
it is sequestered or squelched by other intracellular factors. In
contrast, AIB1-.DELTA.3, which lacks an intact bHLH-PAS domain,
would not bind to potential repressors such as TEF and would be
available for nuclear receptor coactivation. This may explain why
relatively small amounts of recombinant .DELTA.3-AIB1 are able to
induce significant potentiation of nuclear receptor activity in
transfected COS-1 cells with a high background of endogenous full-length
AIB1. This model also predicts that the relative coactivating effects
of AIB1 and .DELTA.3-AIB1 are likely cell-type specific, depending
on the endogenous expression of AIB1-sequestering molecules such
as TEF. A recent report described that the human MMS19 protein can
interact with the PAS-HLH domain of AIB1 and can regulate ER-mediated
transcriptional activity (43). The lack of interaction of .DELTA.3-AIB1
with this protein explains some of its increased effectiveness in
vivo. The data indicates that expression of the AIB1 isoform sensitizes
cells to the effects of estrogen and progesterone.
The second aspect of the function of the .DELTA.3-AIB1 isoform
is that it also increases EGF signaling in ME-180 squamous carcinoma
cells. This may be through direct interactions with a nuclear receptor.
However, analysis of the fragment of the FGF-BP gene promoter (nt
-118 to +62, relative to the transcription start site) used in this
study did not reveal obvious consensus recognition sites for known
nuclear receptors. In fact, EGF induction of this promoter is dependent
on the factors AP-1 and c/EBP.beta. (32), either of which may interact
directly or indirectly with AIB1. Alternatively it may be that a
common intermediary of both nuclear receptor and AP-1 signaling
such as CBP/p300 (44, 45) may be the target of the superactivating
effects of the .DELTA.3-AIB1 isoform. Whatever the mechanism of
the increased potentiation of growth factor signaling by the .DELTA.3-AIB1
isoform, the data suggest that an increase in the abundance of the
.DELTA.3-AIB1 isoform in mammary epithelial cells may be an important
step in tumor progression and to the development of a more aggressive,
hormone-independent phenotype.
Of major interest for breast cancer is that the .DELTA.3-AIB1 mRNA
is over expressed in breast cancer cell lines and in human breast
and ovarian tumors. While the increase in expression in tumors may
be due, in part, to dilution effects of surrounding stromal tissue,
this seems unlikely given the lower .DELTA.3-AIB1 mRNA expression
in non transformed versus malignant mammary epithelial cell lines.
To date, a number of laboratories, have reported overexpression
of AIB1 mRNA and protein in breast tumor tissue although the assessment
of the portion of breast cancers overexpressing AIB1 varies widely
between groups (14, 27, 46, 47). In addition, some groups have determined
that AIB1 overexpression is correlated with ER and PR status (26)
while others have found an inverse relationship with steroid receptor
expression, but a positive correlation with HER-2 and p53 expression
(47). However, all of these RT-PCR or immunohistochemical analyses
of expression levels have not distinguished the .DELTA.3-AIB1 isoform
signal from that of the wild type. The data indicates that overexpression
of relatively low levels of the .DELTA.3-AIB1 isoform can sensitize
cells to estrogen, progesterone and growth factors. Therefore, measurement
of increased levels of .DELTA.3-AIB1 levels is likely a sensitive
indicator of the progression of breast cancer to a more hormone-independent
phenotype.
Using ribozyme targeting that down regulates AIB1, it was determined
that down regulation leads to loss of estrogen reduction and proliferation
of breast cancer cells in vivo. To analyze over expression of .DELTA.3-AIB1
in transgenic animals, a transgenic animal was made that expressed
the .DELTA.3-AIB1 protein under CMV control. The phenotype observed
in these animals is that at about 7 weeks old, the male mice develop
large mammary glands. Upon whole mount examination of these mice,
it was determined that the mammary glands contain massive stromal
proliferation leading to large fat tissue in the breast. Thus, the
effect of the .DELTA.3-AIB1 isoform is to increase proliferation
of stromal cells and/or increase the differentiation of fibroblasts
to adipocytes. This same phenotype is seen in the female, although
not as pronounced, but the stromal tissue is already quite large
at seven weeks and also in other fat tissue and the stroma of the
female. This indicates that the .DELTA.3-AIB1 isoform can play a
role in aberrant expression or proliferation of stroma in breast
cancer and other endocrinological malignancies and pathologies.
Since a previously made AIB1 knockout mouse has been shown to be
a small animal with defects in IGF signaling, the defect from the
over expression of .DELTA.3-AIB1 is likely from over expression
of IGF1 in the liver as can be determined by measuring IGF1 in the
serum. These animals will likely develop a fat phenotype later in
life indicating that .DELTA.3-AIB1 and AIB1 have a wider role in
fat metabolism. Its also possible that the effect of .DELTA.3-AIB1
is not mediated through IGF1, but through the PPAR system (perxoidone
proliferator receptor), which is known to be central to fat metabolism.
Accordingly, one embodiment of the invention is directed to protein
isoforms of the AIB1 coactivator. Preferably these isoforms are
potent transcriptional coactivators of nuclear receptors including,
but not limited to, retinoid receptors, steroid receptors such as
estrogen and progesterone, thyroid receptors, and vitamin D receptors.
Further, it is also preferred that these isoforms potentiate growth
factor signaling pathways such as signaling through, for example,
EGF, FGF, and PTN, and preferably significantly or measurably more
so than the wild-type AIB1 protein. Isoforms of the invention include
proteins that contain deletions, additions or mutations (e.g. point,
frame shift). Isoform deletions include, but are not limited to,
deletions of a portion of the C terminus, a portion of the N-terminus,
the bHLH domain, the PAS A domain, the PAS B domain, RID, CID, exons
1, 2, 3, 4, 5, 6, 7, 8, 9, the intervening introns, and/or portions
or combinations thereof. Preferably, the isoform contains a deletion
of exon 3 in the amino terminus of the protein. Also preferably,
the isoform is over expressed or under expressed in cancer cells
including, but not limited to cancers of the breast, gastric cancers,
head and neck cancers, ovarian cancer, pancreatic cancer, prostate
cancer, squamous cell cancers, and tumors, as well as other epithelial
cell cancers, or combinations thereof, and thereby can be used to
detect and identify such cancerous conditions and metastasis related
thereto both in vivo and in biological samples in vitro.
Another embodiment of the invention is directed to isolated nucleic
acid sequences that encode one or more isoforms of the invention.
Preferably the nucleic acid, which may be DNA, RNA or PNA, encodes
the isoform .DELTA.3-AIB1 containing a deletion of exon 3 from about
positions 267 to 439. Please note, currently there is no accepted
exon numbering. The numbering for the exons herein is based on the
experiments set forth in the examples section and cited publications.
However, based on the information provided herein such as specific
sequence information of the AIB1 gene (FIG. 9), the locations of
the binding sites along AIB1 (FIG. 1 and FIG. 9), and the transcription
and translation start sites (FIG. 1 and FIG. 9), any different exon
numbering system can be readily and easily correlated to those disclosed
herein by those of ordinary skill in the art. The invention further
encompasses vectors (e.g. plasmids, cosmids, viral, or shuttle vectors)
that contains the nucleic acid of isoforms of the invention, and
also recombinant cells, which may be either eukaryotic or prokaryotic
(e.g. Enterobacter, Escherichia coli, or Bacillus subtilis), containing
nucleic acids or vectors of the invention.
Another embodiment of the invention is directed to diagnostic kits
for the detection of cancer comprising chemical substances that
are specifically reactive to one or more protein isoforms of the
invention, or nucleic sequences that encode these isoforms. Such
chemical substances include, for example, aptamers, antibodies,
ligands, nucleic acids (e.g. that are complementary to genes or
mRNA), protein binding partners (e.g. ligands), and fragments and
combinations thereof. The diagnostic kits preferably contain antibodies
or antibody fragments, such as, for example, monoclonal or polyclonal
antibodies, or antibody fragments which may be recombinant or humanized
proteins, directed against a particular isoform or portion of an
isoform. Antibodies may be covalently or non-covalently labeled
with one or more report molecules, such as, for example, a fluorescent
label, for detection and identification. By selecting only portions
of isoforms, those with specific regions (e.g. binding domains,
exons, etc.), can be identified and if desired selected. Further,
identification and selection can be used to determine the relative
amounts of protein or nucleic acid molecules in a sample in vitro,
or in vivo. Samples may be almost any biological sample obtained
from a patient, such as a human, including but not limited to blood,
biological fluids, cells, plasma, and most any other tissues. Preferably
such antibodies or antibody fragments are IgG isotypes, but may
be IgA, IgD, IdE or IgM, or fragments (e.g. Fv fragments) or combinations
thereof. The types of cancer that can be detected, identified and
possibly therapeutically and/or prophylactically treated include,
but are not limited to breast cancers, bone cancers, endothelial
cancers, epithelial cancers, gastrointestinal cancers, head and
neck cancers, ovarian cancers, metastatic cancers, neuroblastomas,
pancreatic cancers, prostate cancers, squamous cell cancers, stomach
cancers, and tissue-specific as well as non-tissue specific tumors.
This basically include most any disorder that demonstrates either
increased or decreased expression of AIB1 or an AIB1 isoform.
Another embodiment of the invention is directed to methods for
the detection of a cancer in a patient comprising contacting a biological
sample obtained from the patient to one or more chemical substances
that specifically bind to protein isoforms of the invention (or
their genetic sequences), and detecting binding of one or more of
the chemical substances thereto. The patient may be a human or other
primate, or another mammal, Samples may be liquid or solid, such
as blood, cells, plasma, tissues or other biological materials,
but are preferably tissue samples of the area suspected to contain
a cancerous region. The method may further comprise comparing the
relative amount of isoform in the sample (e.g. protein or mRNA)
with the amount of wild-type AIB1 (e.g. protein or mRNA), or the
amounts of one isoform or fragment in comparison to others. This
can help to determine a stage of the cancer as being more or less
aggressive, a phenotype such as a hormone-independent phenotype,
a relative resistance or sensitivity to conventional treatment,
or the amount of steroid or growth factor being sequestered by one
or more isoforms for detection or treatment purposes.
Another embodiment of the invention is directed to pharmaceutical
compositions comprising an agent that specifically binds to the
isoform of the invention and prevents a coactivation function when
administered to a patient. Preferably compositions contain one or
more pharmaceutically acceptable carriers such as, for example,
alcohols, buffers, fatty acids, glycerol, oils, polysaccharides,
saccharides, salts, sugars, water, and combinations thereof.
Another embodiment of the invention is directed to small interfering
RNA (siRNA) molecules directed against mRNA that encode transcriptional
coactivator proteins. These siRNA molecules are double-stranded
and typically between about 10 bp and 50 bp in length, preferably
between about 15 bp and 30 bp, and more preferably between about
18 bp and 25 bp. Methods for manufacturing such RNA molecules with
any desired sequence have been previously described (see U.S. Pat.
No. 5,795,715), as has their use in controlling gene expression
(see 51 and 55-65). These molecules are directed against transcriptional
coactivators and, preferably, the transcriptional coactivator proteins
AIB1, Src-1, Src-2, Src-3, and related isoforms such as, for example,
.DELTA.3-AIB1. The siRNA molecule directed against the .DELTA.3-AIB1
mRNA contains sequences that are homologous and complementary to
a mRNA that encodes .DELTA.3-AIB1, and that hybridize to each other
to form the double-stranded RNA molecule. Preferably, siRNA molecules
contains sequences derived from each side of the region that is
deleted such that only the particular isoform mRNA is targeted.
For example, a preferred siRNA contain a sequence from a portion
of exon 2 of the mRNA that encodes .DELTA.3-AIB1, and another portion
of which is derived from exon 4 of the mRNA that encodes .DELTA.3-AIB1.
Another preferred anti-AIB1 siRNA for identifying isoforms containing
a deletion of the PAS A binding site contains a sequence wherein
a portion is derived from the sequence on one side of the PAS A
binding site and another portion is derived from a sequence on the
other side of the PAS A binding site. Preferably the portions are
similar in length to provide good interaction across the deletion
and contiguous in the resulting mRNA, but may be non-contiguous
containing 1, 2, 3, 4, 5, 6, or more nucleotides between the portions.
Alternatively, siRNAs may contain sequences that represent additions
or new sequences of the isoform mRNA not found in the wild-type
mRNA. Portions, and typically the corresponding sequences of the
siRNA, are each preferably from about 4 to about 20 nucleotides
in length, more preferably from about 6 to 16 nucleotides in length,
and more preferably from about 8 to 12 nucleotides in length. Most
preferably, the siRNA is capable of targeting one or a small number
of isoforms of the particular coactivator, but not the wild-type
coactivator mRNA. The greater the ability to distinguish an isoform
from wild type, the greater the targeting ability to that isoform
and the therapeutic benefit against disease that over expression
or under expression of the isoform may cause. Further, the greater
the ability to not interfere with wild type function, the less likely
will be side effects and related complications of more general anti-coactivator
therapy.
Another embodiment of the invention is directed to pharmaceutical
compositions comprising siRNA that inhibits expression of one or
more transcriptional coactivator proteins and, optionally, a pharmaceutically
acceptable carrier. Preferably pharmaceutically acceptable carriers
include alcohols, buffers, fatty acids, glycerol, oils, polysaccharides,
saccharides, salts, sugars, water, and combinations thereof. Pharmaceutical
composition of the invention may further include one or more anti-neoplastic
agents effective in the treatment of cancer such as, for example,
agents that inhibit cell growth, agents that inhibit cell proliferation,
agents that inhibit cellular differentiation, anti-angiogenic agents,
antibodies, antibody fragments, anti-sense agents, chemical agents,
cytokines, toxins, and combinations thereof. Such combination treatments
may be administered simultaneously or sequentially as may be determined
from appropriate clinical trials.
Another embodiment of the invention is directed to methods for
treating or preventing cancer (e.g. hard or soft tumor, leukemia,
lymphoma) comprising administering to a patient a therapeutically
effective dose of a pharmaceutical composition containing one or
more types of siRNA molecules targeted to one or more form of AIB1
and/or AIB1 isoforms of the invention. Therapeutic and/or prophylactic
benefit may be to treat or prevent a disease or simply to reduce
side effects attributable to the activity of transcription factors
(e.g. transcriptional enhancers such as TEF {transcription enhancing
factor}, transcriptional repressors), ligand/receptor interactions
(e.g. PTN {pleiotrophin}, RTK {receptor tyrosine kinase}), growth
factors (e.g. EGF {epidermal}, FGF {fibroblast}, HGF {hepatocyte},
VEGF (vascular endothelial}, PDGF {platelet-derived}, IGF {insulin-like}),
or tumor suppressor gene products (e.g. p53). As certain isoforms
have a higher per molecule effectiveness, thus requiring lower amounts
for therapeutic benefit, the dose response curve is significantly
and positively shifted. This significantly lowers the risk of potentially
harmful side effects, and provides a higher maximum effectiveness
as compared to wild-type protein. Administration is preferably by
direct injection to the site of the cancer such as the tumor, but
may be systemic as determined empirically from clinical trials.
Therapeutically effective doses can be determined by those of ordinary
skill in the art and depend, in part, on the size of the patient
or size of the area being treated and route of administration. Typically,
systemic administration requires therapeutically effective doses
that attain blood levels of from 2 nM to 2 mM, preferably about
2 .mu.M. Localized administration may require that these same concentrations
be achieved, but only in the localized areas of treatment (e.g.
the tumor), and thus much lower overall amounts may be used. Treatments
can be repeated often because the composition is non-toxic and/or
non-carcinogenic, and safe for multiple and/or continued administration.
These method may further comprise administering additional anti-cancer,
anti-metastatic, or anti-tumor therapy to the patient such as, for
example, drug therapy, radiation therapy, surgery, and combinations
thereof.
Another embodiment of the invention is directed to transgenic animals
and methods for the creation of transgenic animals containing nucleic
acid sequences that express isoforms of the invention or siRNA molecules
directed against isoforms of the invention. Preferably the animal
is a mouse, a primate or another mammal, and the isoform contains
a deletion of one or more of the ligand binding domains such as
?3-AIB1. Transgenic animals can be used as models for drug testing
and to determine the effectiveness of siRNA therapy.
The following examples illustrate embodiments of the invention,
but should not be view as limiting the scope of the invention.
EXAMPLES
Example 1
Detection of the .DELTA.3-AIB1 Isoform
In this study, it was first determined if there were naturally
occurring splice variants of AIB1 present in breast cancer cells
that might encode proteins with altered function relevant to breast
cancer progression. All cell lines used were obtained from the tissue
culture core facility of the Lombardi Cancer Center. MCF-7, ME-180,
and COS-1 cells were cultured in Iscove's modified Eagle's medium
(IMEM) (Life Technologies) supplemented with 10% fetal bovine serum
(FBS). MCF-10A and A1N4 cells were grown in a 1:1 mixture of IMEM
and Ham's F-12 medium (Life Technologies) that was supplemented
with 5% horse serum, EGF (20 ng/ml), insulin (10 .mu.g/ml), and
hydrocortisone (500 ng/ml). CHO cells were maintained in F-12 Nutrient
Mixture (Life Technologies) supplemented with 10% FBS.
Shown in FIG. 1 is a characterization of the splice variants of
human AIB1. FIG. 1a shows the stricture of human AIB1 showing the
22 known exons (filled boxes) and the corresponding introns (open
boxes). The exon/intron regions which are spliced to form the various
functional domains of the AIB1 protein are indicated by horizontal
bars. Shown in FIG. 1b is detection of the .DELTA.3-AIB1 splice
variant in total RNA from MCF-7 cells. Total RNA was subjected to
RT-PCR with primers specific for exons 1 and 9 of AIB1. Reaction
products were resolved on a 1% agarose gel and transferred to a
PVDF membrane, which was then cut and the lanes were separately
subjected to hybridization with .sup.32P-labeled oligonucleotides
specific for exons 2, 3, or 8 of AIB1. The positions of PCR products
corresponding to the full-length (AIB1) and truncated (.DELTA.3-AIB
1) transcripts are indicated. Shown in FIG. 1c is a comparison of
the structures of AIB1 and .DELTA.3-AIB1 mRNAs. The alternative
splicing event that results in the loss of exon 3 causes the open
reading frame (ORF) to shift and terminate at a TAA codon in exon
4. A potential initiation site (AUG) for .DELTA.3-AIB1 mRNA is present
at nt 778; the use of this site would be consistent with the .DELTA.3-AIB1
protein lacking the NH.sub.2-terminal 26 kDa of full-length AIB1.
Shaded regions indicate the open reading frame, and exons in the
mRNAs are numbered. The positions of the splice junctions in .DELTA.3-AIB1
mRNA and of the encoded protein domains are indicated. UTR, untranslated
region.
The exon-intron structure of AIB1 was assembled as shown in FIG.
1a by comparing the published sequence of the cDNA (14) with the
contiguous genomic sequence available through the NCBI database.
The most 5' exon of AIB1 was arbitrarily designated as exon 1, with
the result that the first codon is located in exon 2. The initial
strategy was to determine if RNA from MCF-7 cells ,which overexpress
AIB1(14), contained any splice variant forms of AIB1 RNA. To achieve
this, reverse transcription and polymerase chain reaction (RT-PCR)
analysis was performed using total RNA from MCF-7 human breast cancer
cells with primers amplifying the region between exons 1 and 9.
Isolation of total RNA and synthesis of cDNA by RT were performed
as described previously (33). Amplification of AIB1 cDNA sequences
was achieved by PCR according to the following protocol: incubation
at 95.degree. C. for 5 min followed by 30 cycles of 95.degree. C.
for 1 min, 60.degree. C. for 1 min, and 72.degree. C. for 90 sec.
Oligonucleotides used as primers for PCR or as probes for hybridization
were for exon 1, exon 2, exon 3, exon 4, exon 5, exon 8, and exon
9. PCR products were separated by electrophoresis on a 1% agarose
gel, transferred to a polyvinylidene difluoride membrane, and hybridized
with a .sup.32P-labeled oligonucleotide probe. Quantification of
PCR products was performed with a Phosphorimager (Molecular Dynamics
445SI).
This revealed two PCR products that differed in size by .about.150
bp. These PCR products were then subjected to Southern Blot analysis
and individual lanes from the membrane were probed separately with
oligonucleotides specific for each exon from 2 to 8. Typical hybridizations
with exons 2, 3 and 8 are shown in FIG. 1b. This analysis revealed
that the smaller PCR product hybridized with all probes except that
specific for exon 3 (FIG. 1b), indicating that the lower band corresponds
to an RNA splice variant (designated .DELTA.3-AIB1) of AIB1 that
lacks the exon 3 sequence. The PCR product was subsequently subcloned
and sequenced, confirming that nucleotides (nt) 267 to 439 (exon
3) of the full-length AIB1 cDNA were missing (FIG. 1c).
The full-length AIB1 cDNA was subcloned from pCMX-ACTR into pcDNA3
(Invitrogen) with the use of the flanking KpnI and XhoI sites, thereby
creating the expression vector pcDNA3-AIB1. The smaller of the two
RT-PCR products generated from MCF-7 cell total RNA with exon 1-
and exon 9-specific primers (FIG. 1b) was subdloned into pCRII (Invitrogen).
The resulting plasmid was digested with BamHI and HpaI, recognition
sequences which flank the splice sites of AIB1-.DELTA.3 cDNA, and
the released fragment was purified and used to replace the corresponding
sequence of pcDNA3-AIB1, thereby creating pcDNA3-AIB1-.DELTA.3.
The pcDNA3-AIB1 and pcDNA3-AIB1-.DELTA.3 vectors contain identical
5' and 3' untranslated regions, differing only in the loss of exon
3 in the latter. Inserts were verified by sequencing.
Example 2
Translation of the .DELTA.3-AIB1 mRNA in Vitro and in Vivo
To determine if an AIB1 related protein was encoded by the .DELTA.3-AIB1
mRNA, in vitro transcription and translation of .DELTA.3-AIB1 cDNA
was performed with the TnT coupled reticulocytelysate system (Promega).
Plasmid DNA (1 .mu.g) was combined with 25 .mu.l of rabbit reticulocyte
lysate, 2 .mu.l of TnT reaction buffer, 1 .mu.l of T7 RNA polymerase,
1 .mu.l of amino acid mixture, 1 .mu.l of Rnasin ribonuclease inhibitor
(40 U), and 1 .mu.l of Transcend biotin-lysyl-tRNA, and the final
volume was adjusted to 50 .mu.l. The reaction was performed at 30.degree.
C. for 90 min, after which 5 .mu.l of the reaction mixture were
subjected to SDS-polyacrylamide gel electrophoresis and either to
immunoblot analysis with antibodies to AIB1 or to direct detection
with streptavidin-conjugated horseradish peroxidase (1:10,000 dilution
in phosphate-buffered saline containing 0.05% Tween-20) and enhanced
chemiluminescence.
Western blot analysis with an AIB1 specific antibody of the proteins
translated in vitro revealed the production of a 130-kDa protein
(FIG. 2). Interestingly, a similar 130-kDa protein, in addition
to the 156-kDa full-length AIB 1, was consistently detected by immunoblot
analysis of MCF-7 cell extracts with antibodies to AIB1 on 5-20%
polyacrylamide gels (27).
Whole cell extracts were prepared as described previously (32),
and equal portions (30 .mu.g of protein) were resolved either on
denaturing 4-20% polyacrylamide gradient gels or on 4% polyacrylamide
gels containing Tris-glycine. Separated proteins were transferred
to a nitrocellulose membrane and then subjected to immunoblot analysis
with a 1:500 dilution of a mouse monoclonal antibody specific for
amino acids 376 to 389 of human AIB1 (Transduction Laboratories),
horseradish peroxidase-conjugated goat antibodies to mouse immunoglobulin
(1:10,000 dilution; Amersham Pharmacia Biotech), and enhanced chemiluminescence
reagents (Amersham Pharmacia Biotech).
To determine if the MCF-7 130 kDa species and the in vitro transcription
translation product had identical electrophoretic properties, high-resolution
electrophoresis was performed on 4% polyacrylamide gels containing
Tris-glycine followed by immunoblot analysis. This analysis demonstrated
that the mobility of the 130-kDa protein detected in MCF-7 cell
extracts was identical to that of the 130-kDa protein produced by
in vitro transcription and translation of .DELTA.3-AIB1 cDNA (FIG.
2). This observation suggested that the 130-kDa MCF-7 cell protein
was translated from .DELTA.3-AIB1 mRNA present in these cells.
To verify that the .DELTA.3-AIB1 mRNA was translated in vivo, transient
transfection of CHO cells (FIG. 2; see FIG. 4a) or COS-1 cells (see
FIG. 5a) with the .DELTA.3-AIB1 cDNA was performed. COS-1 and CHO
cells were plated at densities of 2.times.10.sup.5 and 5.times.10.sup.5
cells per well, respectively, in six-well plates, and were cultured
for 24 h at 37.degree. C. under 5% CO.sub.2 in IMEM or Ham's F-12,
respectively, supplemented with 5% FBS that had been treated with
dextran-coated charcoal. The medium was then replaced with IMEM
containing Lipofectamine Plus (Gibco BRL) and expression and reporter
plasmids as indicated. After incubation for 3 h, the medium was
replaced with IMEM (COS-1 cells) or Ham's F-12 (CHO cells), each
containing 5% dextran-coated charcoal-treated FBS and nuclear receptor
ligands. Cells were incubated for 24 h and then disrupted in passive
lysis buffer (Promega). Portions (20 .mu.l) of the resulting cell
extract were assayed for both firefly and renilla luciferase activities
with the Dual-Luciferase reporter assay system (Promega).
ME-180 cells were plated at a density of 5.times.10.sup.5 cells
per well and cultured for 24 h in IMEM supplemented with 5% dextran-charcoal-treated
FBS. They were then incubated for 3 h in IMEM supplemented with
Lipofectamine Plus and expression and reporter plasmids. Cells were
washed and then incubated in IMEM for an additional 3 h before incubation
for 18 h with EGF (5 ng/ml) in serum-free IMEM and subsequent lysis.
Because of high background induction of pRL-CMV expression by EGF,
firefly luciferase activity was normalized by protein concentration
as described previously (32).
Analysis of cell extracts demonstrated that this indeed resulted
in the production of a 130-kDa protein, whereas transfection with
the full-length AIB1 cDNA yielded only the 156-kDa full-length protein.
This latter observation demonstrated that the 130-kDa protein was
clearly not the product of proteolytic processing of the full-length
protein. Electrophoretic mobility of the 130-kDa protein synthesized
in cells transfected with the .DELTA.3-AIB1 cDNA was identical to
that of both the 130-kDa AIB1 species present in MCF-7 cell extracts
and the product of in vitro transcription-translation of the .DELTA.3-AIB1
cDNA (FIG. 2). Together these data indicated that the endogenous
.DELTA.3-AIB1 mRNA present in MCF-7 cells encodes a 130-kDa protein.
Examination of the sequence of .DELTA.3-AIB1 mRNA indicated that
the open reading frame of AIB1, which initiates at nt 184 in the
full length mRNA would terminate after 90 amino acids in the splice
variant (FIG. 1c). This predicted low molecular weight product was
not detected in vivo or in vitro. The 130 kDA species was detected
by an AIB1 antibody raised against amino acids 376-389 in the amino
terminus of the protein. This suggests that the .DELTA.3-AIB1 isoform
most likely represents an NH.sub.2-terminally truncated form of
AIB1 whose synthesis is initiated at an internal translation start
site downstream of the splice junction, but prior to amino acid
376. Such internal translational initiation has been described for
various mRNAs with extended 5 untranslated regions (34-37). The
difference in size between the 156-kDa full-length AIB1 protein
and the 130-kDa species suggested that the latter lacks .about.210
amino acids of the former, including all of the bHLH region (residues
16 to 88) and most of the PAS A domain (residues 116 to 171) (FIG.
1c). This would place the initiation codon for the 130 kDa protein
most likely at the codon at 778 (FIG. 1c). Interestingly, for cells
transfected with equivalent amounts of cDNA, the intracellular concentration
of .DELTA.3-AIB1 protein was .about.10% of that of full-length AIB1
(FIG. 2; see FIG. 4a and FIG. 5a), suggesting that translation initiation
of the splice variant was inefficient, possibly because of the long
5' untranslated region of the .DELTA.3-AIB1 mRNA.
Example 3
.DELTA.3-AIB1 mRNA is Over Expressed in Human Breast Cancer
Given that the .DELTA.3-AIB1 splice variant was first detected
in a breast cancer cell line, it was next examined whether its expression
was restricted to tumor cells. MCF-7 cells are derived from a pleural
effusion of metastatic breast cancer, whereas MCF-10A and A1N4 cells
are not malignantly transformed and were derived from atypical human
breast epithelial hyperplasia (38) and from human mammary epithelial
cells treated with benzopyrene (39), respectively.
FIG. 3 shows a comparison of the abundance of .DELTA.3-AIB1 mRNA
between malignant and nonmalignant human breast tissue and cell
lines. FIG. 3(a) shows total RNA isolated from MCF-7, MCF-10A, and
A1N4 cells subjected to RT-PCR with primers specific for exons 2
and 5 of AIB1. Reaction products were resolved on a 1% agarose gel
and then subjected to Southern blot analysis with a .sup.32P-labeled
oligonucleotide probe specific for exon 4 of AIB1. FIG. 3(b) shows
total RNA, isolated from six normal breast and eight breast cancer
tissue samples, analyzed as in FIG. 3(a). The amounts of PCR products
corresponding to AIB1 and .DELTA.3-AIB1 mRNAs were quantitated by
densitometry, and the abundance of the latter was expressed as a
percentage of that of the former. The signal of the full-length
AIB1 transcript was compared between breast tumors and normal breast
tissue with the use of an arbitrary scale; the signals in tumor
and normal samples were 1.0.+-.0.46 and 0.7.+-.0.24 (means.+-.SEM),
respectively, and they did not differ significantly (P>0.05;
Student's t-test). The inset shows a typical blot of 8 of the 14
samples.
RT-PCR followed by Southern blot analysis revealed that the amounts
of .DELTA.3-AIB1 mRNA in MCF-10A and AIN4 cells were lower than
that of MCF-7 cells (FIG. 3a). By subsequent real-time PCR analysis,
using primers specific for AIB1 or its isoform, it was assessed
that the ratio of .DELTA.3-AIB1 mRNA/full length AIB1 is 5% in MCF-7
cells, whereas in MCF-10A and AIN4 cells the ratio is 0.5% (data
not shown). The abundance of the .DELTA.3-AIB1 mRNA was compared
in a series of eight human breast tumors with that in normal tissue
obtained from six women undergoing breast reduction mammoplasty.
Frozen tissue samples were obtained from the Lombardi Cancer Center
Histopathology and Tissue Shared Resource Core. Six normal samples
were obtained from individuals undergoing reduction mammoplasty
(mean age at time of surgery, 29 years; range, 19 to 54 years);
the eight primary breast carcinoma specimens were obtained from
women with a mean age at the time of surgery of 51 years (range,
29 to 64 years). The amounts of PCR products corresponding to AIB1
and .DELTA.3-AIB1 mRNAs were quantitated by densitometry, and the
abundance of the latter was expressed as a percentage of that of
the former. The signal of the full-length AIB1 transcript was compared
between breast tumors and normal breast tissue with the use of an
arbitrary scale; the signals in tumor and normal samples were 1.0.+-.0.46
and 0.7.+-.0.24 (means.+-.SEM), respectively, and they did not differ
significantly (P>0.05; Student's t-test). The inset shows a typical
blot of 8 of the 14 samples. The amount of the full-length AIB1
mRNA in tumor samples was slightly greater than that in the normal
tissue samples, but this difference was not significant (FIG. 3b).
In contrast, the abundance of the .DELTA.3-AIB1 mRNA in the tumor
specimens was significantly greater than that in the normal tissue
samples, with all but one of the tumors showing an increased amount
of this transcript compared with the normal range.
Example 4
Effect of the .DELTA.3-AIB1 Isoform on Nuclear Receptor Function
The effect of the deletion of the bHLH and PAS A domains was next
examined in .DELTA.3-AIB1 on protein function. AIB1 acts as a coactivator
for several nuclear receptors, including those for estrogen and
progesterone, which are important in breast carcinogenesis. FIG.
4 shows the effects of AIB1 and .DELTA.3-AIB1 on the activation
of estrogen receptor .alpha. and progesterone receptor .beta. in
CHO cells. In FIG. 4a, cells were transfected with either the empty
pcDNA3 vector (3 .mu.g) (control), pcDNA3-AIB 1(0.3 or 3 .mu.g),
or pcDNA3-.DELTA.3-AIB1 (3 .mu.g), together with an expression vector
for human estrogen receptor .alpha. (100 ng), an ERE-luciferase
reporter plasmid (1 .mu.g), and pRL-CMV (0.1 ng) (Promega). After
incubation for 24 h with either 10 nM estradiol-17.beta. or 100
nM of the estrogen receptor antagonist ICI 182,780, cells were lysed
and assayed for luciferase activity. The inset shows immunoblot
analysis of transfected cell lysates that were fractionated on 4
to 20% polyacrylamide gradient gels and probed with antibodies to
AIB1. FIG. 4b shows cells transfected as in FIG. 4a with the exception
that the estrogen receptor vector was replaced with a vector for
human progesterone receptor .beta. (20 ng), and the ERE-luciferase
plasmid was replaced by a luciferase reporter construct containing
the mouse mammary tumor virus (MMTV) promoter (2 .mu.g). Cells were
incubated for 24 h in the absence or presence of the progesterone
analog R5020 (1 nM) before preparation of lysates for luciferase
assay. The firefly luciferase activity of cell lysates was divided
by the renilla luciferase activity (internal control), and this
ratio (normalized reporter activity) for control cells incubated
in the absence of agonist was assigned a value of 1. Data are means.+-.SEM
of values from three independent experiments, each performed in
triplicate. *P<0.005 versus corresponding value for cells transfected
with 3 .mu.g of the AIB1 vector (Student's t test).
Transient Transfection Assays
CHO cells were transfected with expression vectors encoding full-length
AIB1 or .DELTA.3-AIB1, an expression vector for estrogen receptor
.alpha., and a luciferase reporter plasmid containing an estrogen
response element (ERE). Transfection of CHO cells with 3 .mu.g of
the AIB1 expression vector resulted in a 1.4-fold increase in estrogen-induced
luciferase activity, whereas transfection with 3 .mu.g of the vector
for .DELTA.3-AIB1 resulted in a 3.8-fold increase in the estrogen
response (FIG. 4a). Given that the abundance of recombinant AIB1
in the transfected cells was about 10 times that of .DELTA.3-AIB1,
CHO cells were also transfected with 0.3 .mu.g of the AIB1 vector,
which yielded about the same amount of intracellular recombinant
protein as did 3 .mu.g of the .DELTA.3-AIB 1 vector (FIG. 4a). Comparison
of transfected cells containing approximately equal amounts of recombinant
protein thus revealed that AIB1 and .DELTA.3-AIB1 potentiated or
enhanced the estrogen response by factors of 1.1 and 3.8, respectively.
Similar transfection experiments were also performed with COS-1
cells and the effects of AIB1 and .DELTA.3-AIB1 on the activation
of estrogen receptor a and progesterone receptor .beta. determined.
Briefly, cells were transfected and analyzed as in FIG. 4a (the
amount of pcDNA3-AIB1 was 3 .mu.g) (FIG. 5a). Cells were transfected
with 1 .mu.g of either pcDNA3, pcDNA3-AIB1, or pcDNA-.DELTA.3-AIB1,
together with an expression vector for human progesterone receptor
.beta. (10 ng), a luciferase reporter plasmid containing the MMTV
promoter (1 .mu.g), and pRL-CMV (0.1 ng). After incubation for 24
h in the absence or presence of 0.5 nM R5020, cells were lysed and
assayed for luciferase activity (FIG. 5b). Data are means.+-.SEM
of values from three independent experiments, each performed in
triplicate. *P<0.005 versus the corresponding value for cells
transfected with the AIB 1 vector.
Transfection experiments with COS-1 cells (which express endogenous
AIB1) also demonstrated a greater potentiation of the estrogen response
by .DELTA.3-AIB1 than by full-length AIB1 (FIG. 5a). The differences
between full-length and the .DELTA.3-AIB 1 isoform were seen at
different concentrations of estrogen (0.1 to 10 nM) and thus were
not due to a change in the affinity of the hormone for its receptor
but rather suggests enhanced efficacy of the signaling (data not
shown). Similar results were obtained in COS-1 cells with an expression
vector encoding progesterone receptor .beta.; the transcriptional
response to the progesterone analog R5020 was thus potentiated to
a greater extent by .DELTA.3-AIB1 than by AIB1 in both CHO and COS-1
cells (FIG. 4b, FIG. 5b). Of particular note was that small amounts
of transfected .DELTA.3-AIB1 protein had significant effects on
ER and PR induced transcription even against a relatively high background
of full-length AIB1 (FIG. 5).
Example 5
Effect of the .DELTA.3-AIB1 Isoform on EGF Signaling
The fact that members of the p160 SRC family act as coactivators
in intracellular signaling pathways that activate transcription
factors other than nuclear receptors (30,31) prompted an examination
of whether .DELTA.3-AIB1 might be able to sensitize breast cancer
cells to growth factor signaling. Overexpression of members of the
families of epidermal growth factor (EGF) ligands or EGF receptors
is important in the malignant progression of breast cancer (40).
Such growth factors also contribute to the hormone-independent phenotype
of breast tumors and the HER-2 receptor is a target of current therapies
(41). To determine whether AIB1 and .DELTA.3-AIB1 affect EGF signaling,
ME-180 human squamous cell carcinoma cells were transfected with
the respective expression vectors and with a luciferase reporter
plasmid containing the promoter of the fibroblast growth factor-binding
protein (FGF-BP) gene. FGF-BP functions as an angiogenic switch
molecule (42) that is overexpressed in breast cancer, and whose
gene is activated by EGF in squamous cell and breast cancer cell
lines (32).
Cells were transfected with 3 .mu.g of either pcDNA3, pcDNA3-AIB1,
or pcDNA3-AIB1-.DELTA.3, together with a luciferase reporter plasmid
containing the human FGF-BP gene promoter (1 .mu.g) (32) (FIG. 6).
After incubation of cells for 18 h in the absence or presence of
EGF (5 ng/ml) in serum-free medium, cell extracts were prepared
and assayed for luciferase activity. Activity was normalized by
protein concentration, and the normalized activity values were then
expressed relative to that of cells transfected with pcDNA3 and
not exposed to EGF. Data are means.+-.SEM of values from three independent
experiments, each performed in triplicate. *P<0.01 versus the
corresponding value for control cells.
As reported previously EGF induced a 2.5-fold increase in reporter
activity in control cells transfected with the empty expression
vector (FIG. 6). The basal EGF induction was increased slightly
by transfection of the full length AIB1 expression vector whereas
EGF induction was increased approximately 6-fold by expressing recombinant
AIB 1-.DELTA.3.
Example 6
In Vivo Targeting of Nuclear Receptor Coactivator AIB1 with siRNA
In this example, it was determined whether selective reduction
of the gene expression of a nuclear receptor coactivator AIB1 or
its more active isoform ?3-AIB1 leads to an inhibition of the growth
of human breast cancer cells in cell culture and of xenograft tumors
in mice.
Inhibition of Proliferation of Cells in Culture
MCF-7 cells were transiently transfected with varying concentrations
(log M is given) of either siRNA targeted against AIB1 mRNA or scrambled,
nonspecific siRNA (control) (FIG. 7). After 72 hrs, whole cell extracts
were harvested and probed with a monoclonal antibody to AIB1. The
membrane was stripped and reprobed for actin as a loading control.
Bands were quantitated by densitometry. Percent AIB1 levels were
calculated by taking the ratio of AIB1 levels in the control siRNA
versus AIB1 siRNA transfected cells. An IC50 of 0.2 .mu.M and an
effect size of 90% were estimated from the concentration-response
(FIG. 7, right panel).
In addition, growth factor dependent proliferation of MCF-7 cells
is reduced by a tetracycline-regulated AIB1 targeted ribozyme (FIG.
8). AIB1 siRNA decreases endogenous AIB1 protein levels in a dose-dependent
manner. siRNA dose verses mRNA levels and cell number.+-.growth
factor were determined with AIB1 up or down and is expressed relative
to control. Thus, using a regulatable AIB1-directed ribozyme, it
was found that down regulation of endogenous AIB1 levels in MCF-7
breast cancer cells results in the loss of estrogen-sensitive growth.
This is mainly through reduction in estrogen-dependent inhibition
of apoptosis.
Thus, by designing siRNA molecules to target AIB1 or .DELTA.3-AIB1,
cellular levels of AIB1 can be reduced. The IC50 for this effect
is 0.2 mM which indicates that it is possible to achieve tissue
levels of siRNA for a therapeutic effect in vivo with only a few
mg of siRNA per dose. The 5' region of the AIB1 mRNA was chosen
as the initial target because it bears no homology to other mRNA
sequences as determined by a Blast search of the entire human genome.
After a single liposome transfection into MCF-7 breast cancer cells
this siRNA produced an up to 90% reduction in AIB1 protein levels
as measured by Western blot analysis (FIG. 9). This effect was maintained
for up to 72 hrs after addition of siRNA to the cells for a time
period of 4 hours. Under this objective, alternative regions of
AIB1 mRNA are targeted and tested to produce reductions in AIB1
gene expression at a lower IC50. To narrow down the chosen regions,
areas common to Src-1 and Src-2 are excluded from this analysis
as are common domain sequences such as the histone acetyl transferase
domain and PAS/HLH sequence domains of known proteins. The remaining
areas are used as candidates for a detailed Blast search. The best
ten sequences are selected to make siRNA molecules. To specifically
target .DELTA.3-AIB1, the junction of exons 2 to 4 is targeted which
is a fusion that unique to this isoform (FIG. 9). A series of siRNA
molecules between -13 and +13 relative to the junction (-9/+13,
-10/+12, +11/+11, -12/+10, -13/+9 etc.) are synthesized and their
effect on target .DELTA.3-AIB1 relative to full-length AIB1 determined
(FIG. 9b). Residual levels of both AIB1 isoforms are measured by
Western blotting after electrophoretic separation of the isoforms
on 4% polyacrylamide gels as described previously. Each siRNA molecule
is tested at 5 concentrations of 1, 0.3, 0.1, 0.03 and 0.01 mM to
determine the maximum reduction and the IC50. MCF-7 cells are the
initial cell line used and confirmatory experiments are also run
in T47D and MDA MB231 human breast cancer cells. These cells lines
are chosen because they represent excellent model systems for human
breast cancer: MCF-7 cells are responsive to estrogens, harbor the
20 q AIB1 amplicon and express high levels of AIB1/.DELTA.3-AIB1
(14). T47D cells contain higher levels of AIB1 and .DELTA.3-AIB1
than normal mammary epithelium yet several fold lower levels than
MCF-7 cells (14). They do not harbor the 20 q amplicon, are estrogen
responsive and, at least with respect to AIB1 expression levels,
mimic a large portion of clinical breast cancers. MDA MB231 cells
are chosen because they respond to growth factors for proliferation,
are hormone-independent (ER-negative) and are a highly tumorigenic
and metastatic in animal models. MDA MB231 cells are relatively
invasive and have been well characterized in terms of both in vitro
and in vivo invasive and chemotactic characteristics. This cell
line responds to EGF and IGF with increased chemotaxis and to HGF
with increased invasive capacity (49, 50). This cell line expresses
intermediate levels of AIB1. Typically, the Prizm/Graphpad program
is used for curve fittings. For a 5-point siRNA dose response curve
plus a negative control in triplicate, the cellular levels of AIB1
isoforms can easily be quantitated using serial Western blots as
shown in FIG. 7. Once the concentration that achieves approximately
80% efficacy has been established for each siRNA molecule, the duration
of the effect of each molecule can be examined at that concentration.
Potency and thus, the drug concentration needed for the targeted
effect level, can be tested empirically and those siRNAs chosen
for use in animals that show the best dose/time/response (lowest
dose to get a 3 day effect >50%). In the experiment described
in FIG. 7, it was found that the effects of the siRNA directed at
AIB1 were preserved for at least 72 hours. Some reports have claimed
that siRNA effects can be prolonged to >100 hours in vitro after
a single administration (51). Therefore, time course experiments
are run for up to 120 hours with the optimal dose of each siRNA
to determine the duration of the RNA silencing and whether this
differs amongst the siRNAs chosen.
To determine if siRNA-induced reduction of cellular levels of AIB1
or .DELTA.3-AIB1 can change the phenotype of breast cancer, cell
lines are tested to examine if siRNA-mediated depletion of AIB1
or .DELTA.3-AIB1 result in estrogen or growth factor-induced changes
in cellular proliferation and soft agar colony formation. It is
first determined if there are changes in doubling time or in colony
formation under anchorage-independent growth conditions in soft
agar. In addition to spontaneous growth, cells are cultured in serum
only or EGF (10 to 100 ng/ml), IGF-1 (10 to 100 ng/ml), heregulin
(10 to 100 ng/ml), HGF (hepatocyte growth factor; 10 to 100 ng/ml).
The growth responses after 2, 4, 6 and 8 days are compared in the
presence or absence of siRNA (+/- growth factor) at the optimal
concentration of siRNA previously determined. For the growth assays,
it is only necessary to test the siRNA molecules that have had effective
and long lasting effects as well as those that are specific for
.DELTA.3-AIB1. A single addition of siRNA is used initially and
several dosings of siRNA (every other day) for those conditions
that showed effects only after the initial dosing. For the soft
agar assays, cells are cultured in the presence or absence of EGF,
IGF-1, heregulin, HGF or serum (concentrations as above) with or
without siRNA (only one dose is used initially). After at least
7 days of incubation, colonies are counted with an image analyzer.
In addition, experiments with the MCF-7 and T47D cells can include
estrogen to determine whether AIB1 contributes to synergism between
growth factor and estrogen-mediated signaling.
Cell cycle/apoptosis. Since growth factors contribute to cell cycle
progression as well as inhibition of apoptosis in human breast cancer
cells, whether any siRNA-induced effects on cell growth can be analyzed.
This might have resulted from a reduced ability of the cells to
progress through the cell cycle or whether this effect is based
on their altered susceptibility towards apoptosis. A similar analysis
with estrogen-induced cell growth was performed which found that
AIB1 was predominantly acting through inhibition of apoptosis rather
than through changes in cell cycle progression (29). However, AIB1
has been demonstrated to be important for the expression of cell
cycle genes (52) and AIB1 modulation of cell-cycle progression is
more important for growth factor signaling than for hormone signaling.
Cell cycle analysis is performed by FACS analysis and apoptosis
is measured with propidium iodide-annexinV-FITC dual staining and
FACS analysis. Both approaches have been described (29).
These experiments provide a comprehensive view of the impact of
AIB1 reductions induced by siRNA on gross phenotypic changes regulated
by growth factors and/or hormones. For specificity controls, it
can be determined if other related genes such as Src-1 are unaffected
by the treatment control. In addition, mutated siRNA which has several
bases changed can be used to determine specificity.
Inhibition of Growth of Xenograft Tumors in Mice
The deletion of the AIB1 (CIP) gene in mice resulted in a surprising
phenotype where mice had reduced overall growth due, in part, to
reduced serum IGF levels (53, 54), but mainly because IGF signaling
was reduced in AIB1 .sup.-/- cells (53). Serum induction of proliferation
in AIB1.sup.-/- cells was unaltered (53). In human breast cancer
cells it was found that the isoform .DELTA.3-AIB1 can strongly potentiate
EGF signaling (48) and similar to the AIB1 knock-out in mice. The
reduction of overall AIB1 levels in MCF-7 cells inhibits IGF-1 and
heregulin induced growth, but does not affect growth under control
conditions, i.e. in the presence of serum (FIG. 8). Thus, for the
targeting of AIB1, siRNA against isoforms of AIB1 is a viable therapy
for the treatment of human breast cancer.
These observations indicate that a central role of AIB1 in a defined
number of signaling pathways induced by growth factors that are
known to play pivotal roles in the malignant progression of breast
cancer. Over expression of growth factor receptors (HER-2/neu and
EGFR) are correlated with a more aggressive phenotype and with a
decreased responsiveness to antiestrogen therapy. Thus, over expressed
AIB1 and, in particular, the isoform .DELTA.3-AIB1 is a master regulator
that drives a more aggressive phenotype of breast cancer and AIB1
and .DELTA.3-AIB1 can be used as therapeutic targets in breast cancer.
The validity of AIB 1 as a therapeutic target is based on the facts
that: (i) AIB1 is a rate-limiting regulator for estrogen and growth
factor signaling in breast cancer; (ii) AIB1 is over expressed selectively
in breast cancer cells and not in non-transformed tissues; (iii)
the knock-out of the AIB1 gene in mice indicates that the side-effects
of AIB1 reduction are limited to the growth phase of the body before
adulthood and to the reproductive system indicating that that in
an adult, target-specific side effects are very likely small; (iv)
selective reduction of the .DELTA.3-AIB1 isoform is possible by
generating siRNAs that target the splice junction of exons 2/4 (see
FIG. 9b) and, thus, only deplete this isoform. Accordingly, targeting
.DELTA.3-AIB1 isoform is more effective against cancer than targeting
full-length AIB1. As shown in FIG. 1a, the .DELTA.3-AIB1 isoform
is selectively over expressed in breast cancer cells and cancer
tissues from patients and is barely detectable in normal breast
tissues. In addition, this isoform is significantly more potent
than full-length AIB1 on a molar basis and thus contributes to a
large percentage of AIB1 effects on growth and proliferation in
cancerous tissue; and (v) because AIB1 is selectively over expressed
in tumor tissues, the side effects from therapeutic targeting of
the .DELTA.3-AIB1 isoform are expected to be minimal.
The use of short, double-stranded RNA molecules that cause RNA
interference (RNAi) have proven to be an effective and selective
method of reducing cellular mRNA (51, 55, 56, 57, 58). A short,
double-stranded RNA (dsRNA) is synthesized to generate small interfering,
siRNA that matches a sequence in the target gene. Upon introduction
to cells, the endogenous cellular enzymatic degradation system triggers
degradation of the mRNA that matches its sequence via the siRNA/mRNA
complex. If the siRNA is short (21-23 mer), the double-stranded
RNA apparently evades cell defense mechanism to long double-stranded
RNA of viral origin, which normally provokes a total shut down in
cellular protein synthesis. The advantage of using siRNA molecules
is that they are short and stable, easily synthesized and are able
to degrade cellular RNA at very low concentrations, much lower than
that amount used with antisense oligonucleotides or ribozymes (51,
55, 56, 57, 58). In addition, siRNA can produce a prolonged down
regulation of mRNA in cells in culture. The respective data for
AIB1 targeting are shown in FIG. 3. A 90% reduction of AIB1 protein
in MCF-7 human breast cancer cells with an IC50 of approximately
0.2 mM was observed after a single addition of siRNA and this reduction
is sustained for at least 72 hrs. Thus, in hand is an siRNA species
that can target AIB1 effectively in cells in culture. It is next
determined if it is possible to selectively target AIB1 and its
isoform in vivo and, thus, if this prevents breast cancer development
and proliferation.
Therapeutic Administration of siRNA Directed Against AIB1
Utilizing the most potent of the AIB1 and .DELTA.3-AIB1 siRNAs
identified, one can determine a regimen of administration of siRNA
to animals to produce a sustained reduction of AIB1 in animal tumors
and ultimately an anti-tumorigenic effect in vivo. Distribution
and approximate tissue concentration is determined after siRNA administration.
From this, an optimal dose and dosing interval is assessed for the
animal. Fluorescently tagged (FITC) active siRNA molecules are prepared
and injected ip or iv into nude mice that carry at least one MCF-7
xenograft tumor each. The tissue levels of siRNA are estimated in
the tumor, liver, muscle, kidney and brain at several time points
after injection. From cell culture studies, it was shown that tissue
concentrations of 0.1 to 0.3 micromolar can reduce AIB1 protein
significantly (see FIG. 7). To achieve this concentration in vivo,
one would need to inject approximately 2 to 6 mg of siRNA i.p. per
mouse. To maintain these levels will depend on the half life of
the siRNA liposome complex in vivo and this is determined using
fluorescein conjugated molecules. Animals are sacrificed at 6, 12,
24, 48 and 72 hours of i.p. injection of the tagged siRNA and the
levels of fluorescein conjugated molecule determined by fluorescence
detection in the homogenates of liver, kidney, muscle, brain and
tumor. FITC labeled siRNA is imaged after treatment of MCF-7 cells
in vitro. In parallel with the measurement of siRNA levels by fluorescence,
AIB1 levels are measured in the tumors by Western blot. Thus, the
time course of drug concentrations is compared with the time-effect
relationship. After analysis of the first series with a single intraperitoneal
dose, the dose interval (bid, tid etc.) and/or dose level or even
the route of administration (intratumoral, i.v.) can be modulated
if that promised better efficacy based on AIB1 levels. To study
efficacy of siRNA not only on the target gene, but on tumor growth,
MBA-MB-231, MCF-7 or T47D cells are implanted into mammary glands
of athymic nude mice and their growth followed. In the first series
of experiments, systemic administration of siRNA, most likely twice
per week i.p., initiates after the formation of palpable tumors.
Five mice/group and two tumor inoculum sites per mouse comprise
one treatment or one control arm. One can start with two to three
i.p. doses of siRNA per week, but this would be modified to more
frequent (daily) or less frequent (weekly) dosing as time course
studies require. Dosing is continued for the whole duration of the
experiment. Tumor growth is monitored for up to two months following
implantation and tumor size estimated from the product of perpendicular
diameters of the tumor (twice weekly). Tumors are stained for proliferation
(by PCNA staining) and mitotic cells, apoptosis (TUNEL staining)
and the number of blood vessels. Statistical considerations and
evaluations are well known. Targeting AIB1 by regulatable ribozymes
in MCF-7 xenograft tumors will not only delay tumor growth, but
will induce complete regression of implanted tumors due to siRNA.
Treatment with siRNA would be stopped after tumor regression and
patients is followed to monitor for possible tumor re-growth. The
effect of siRNA is tested on large established tumors of approximately
1 cm in their largest diameter. The extent to which apoptosis may
be induced can be assayed by harvesting and staining of the tumor
tissues after 2 to 3 weeks of siRNA treatment.
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