Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR TREATING BREAST CANCER AND CHRONIC DISEASES
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application
No.
62/583,910, filed on November 9, 2017, the disclosure of which is hereby
incorporated by
reference in its entirety.
INCORPORATION BY REFERENCE OF TO MATERIALS SUBMITTED
ELECTRONICALLY
[0002] This application contains, as a separate part of the disclosure, a
Sequence Listing in
computer readable form (Filename: 52280A Seqlisting.txt; Size:842 bytes;
Created:
November 8, 2018), which is incorporated by reference in its entirety.
FIELD OF DISCLOSURE
[0003] The invention relates to materials and methods for treating breast
cancer, obesity,
nonalcoholic steatohepatitis (NASH), and chronic diseases.
BACKGROUND
[0004] Breast cancer is the most common non-skin cancer and the second leading
cause of
cancer mortality amongst women in the United States. Siegel et al. (2013). CA
Cancer J
Clin, 63, 11-30. Although mortality rates from breast cancer have decreased
due to
surveillance, early detection and use of adjuvant therapies, the five-year
survival of metastatic
disease is only 22%. Siegel, supra. A better understanding of the mechanisms
underlying
breast cancer metastasis is needed to develop new and more effective
therapies.
SUMMARY
[0005] Disclosed herein is a method of treating breast cancer, the method
comprising
administering to a mammalian subject in need thereof an inhibitor of Receptor
for Advanced
Glycation End-product (RAGE). The disclosure further provides a method of
inhibiting
breast cancer metastasis and/or inhibiting the onset of breast cancer,
comprising
administering to a mammalian subject in need thereof an inhibitor of RAGE. The
disclosure
further provides a method of treating obesity, nonalcoholic steatohepatitis
(NASH), or
chronic disease, the method comprising administering to a mammalian subject in
need
thereof an inhibitor of RAGE.
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BRIEF DESCRIPTION OF THE FIGURES
[0006] Figures 1A-1J. Increased RAGE expression promotes tumor metastasis.
Western
blot analysis of RAGE in (Fig.1A) human highly lung (4175) and bone (1833)
metastatic
variants of parental human 231 cells and (Fig.1B) in murine highly metastatic
4T-1 & E0771
compared to non-metastatic 67NR cells. Western blots were performed using anti-
RAGE
and anti-3 actin antibodies. (Fig.1C) Western blot analysis of 231 control &
RAGE
overexpressing cells (using anti-RAGE and anti-3 actin antibodies). (Fig.1D)
Scratch/wound
assay of 231 control & RAGE transfected cells at Oh / 16h post wounding. (E)
Relative cell
number (y-axis) invading through matrigel in transwell chamber after 24h to 1%
FBS stimuli.
(Fig. 1F) Proliferation quantified by crystal violet staining after 48h (y-
axis OD; white
bar=vector; black bar=RAGE). (Fig.1G) Soft agar colony formation assays of 231
control &
RAGE cells. Representative cell colonies in soft agar are shown. Colony number
per field
(y-axs) is shown in the bar graph (white bar=vector; black bar=RAGE). (Fig.1H)
231 vector
and 231-RAGE cells (1x106) were injected into mammary fat pad of NSG mice.
Tumor size
(y-axis) was measured over the course of 35 days (x-axis). (Fig.1I) Tumor
weight (y-axis)
was measured at time of sacrifice. Data shown are from 10 mice per group.
(Fig.1J)
Immunohistochemistry of lung tissue from 231-vector and 231-RAGE mice stained
with
human anti-CK7 to visualize tumor cell metastasis. Bar graph illustrates CK7
staining (y-
axis; pixel/field) for tested samples.
[0007] Figures 2A-2C. RAGE signaling through MAP kinase and EMT drives MDA-MB-
231 cell invasive gene expression and function. (Fig.2A) Western blot analysis
shows EMT
markers are increased with RAGE overexpression. (Fig.2B) Western blot of total
and
phospho-proteins in 231 & 231-RAGE expressing cells. (Fig.2C) Matrigel
invasion in
transwell chambers after 24h to 1% FBS. Assays performed with control (DMSO),
and
inhibitors for MEK (U0126, 10 [I,M), Akt (LY294002, 50 [I,M) and p38
(5B203580, 10 M).
Data are means SEM, n=3. *, P<0.05.
[0008] Figures 3A-3E. RAGE knockdown in human highly metastatic breast cancer
cells
downregulates cell invasion, anchorage-independent growth in soft agar, and
downstream
signaling. (Fig.3A) Western blot analysis of RAGE in shControl and RAGE sh66
231 and
4175 cells (using anti-RAGE and anti-f3 actin antibodies). (Fig.3B) 231 & 4175
cells with
RAGE shRNA (and shControl) were quantified for matrigel invasion in transwell
chambers
after 24h to 1% FBS stimuli, were quantified. (Fig.3C) Proliferation
quantified by crystal
violet staining after 48h. (Fig.3D) Soft agar colony formation assays of 231 &
4175 cells.
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Representative cell colonies in soft agar are shown. Colony number per field
is shown in the
lower panel. For all experiments, data are means SEM, n=3. *, P<0.05.
(Fig.3E) Western
blot of total and phospho-proteins in 4175 cells with RAGE shRNA and
shControl. For all
experiments, data are means SEM, n=3. *, P<0.05.
[0009] Figures 4A-4F. Knockdown of RAGE inhibitors tumor progression. (Fig.4A)
231
parental or (Fig.4B) 4175 cells (1x106) with RAGE sh66 or shControl were
injected into
mammary fat pad of NSG mice (top line=control; bottom line=treated). Tumor
size was
measured over the course of 35 days, and at time of sacrifice (35 days), tumor
weight was
measured. Data shown are from 10 mice per group. (Fig.4C-4F)
Immunohistochemical
analysis of tumors for proliferation (Fig.4C. Ki67), angiogenesis (Fig.4D.
CD34), and
leukocyte (Fig.4E. CD45) and macrophage (Fig.4F. F4/80) infiltration from 4175-
shControl
and 4175 RAGE sh66 tumors.
[0010] Figures 5A-5F. RAGE expression in tumor cells is required for breast
cancer
metastasis in vivo: xenograft models. Lung and liver tissue from 4175 or 231
tumor bearing
RAGE sh66 or shControl mice were analyzed for metastasis by
immunohistochemistry with
anti-human CK7 antibodies. Representative images are shown from tissue for 231
(A&B),
4175 time (Fig.5C and5D) and size (Fig.5E and 5F) -matched mice.
[0011] Figures 6A-6F. RAGE knockdown in mouse highly metastatic breast cancer
cells
downregulates cell invasion and breast cancer metastasis in vivo: syngeneic
models. (Fig.6A)
Western blot shows knockdown by different RAGE shRNAs (sh10 and sh12) compared
to
shControl in 4T-1 cells, and RAGE overexpression in 67NR cells compared to
vector control
(using anti-RAGE and anti-f3 actin antibodies). (Fig.6B) 4T-1 cells with RAGE
shRNA (and
shControl) were quantified for matrigel invasion in transwell chambers after
24h to 1% FBS
stimuli, were quantified. (Fig.6C and 6D) Soft agar colony formation assays of
(Fig.6C) 4T-
1 cells and (Fig.6D) 67NR cells. Representative cell colonies in soft agar are
shown. Colony
number per field is shown in the lower panel. For all experiments, data are
means SEM,
n=3. *, P<0.05. (Fig.6E) 4T-1 cells (1x106) with RAGE sh10, RAGE sh12 or
shControl were
injected into mammary fat pad of BALBc mice. Tumor size was measured over the
course of
35 days, and at time of sacrifice (35 days), tumor weight was measured. Data
shown are
from 8 mice per group. (Fig.6F) Lung tissue from 4T-1 tumor bearing RAGE sh10,
sh12 or
shControl mice were analyzed for metastasis by immunohistochemistry with H&E.
Representative images are shown.
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[0012] Figures 7A-7D. RAGE knockout in mice impairs tumor growth in vivo. AT-3
murine mammary tumor cells (0.5x106) were injected into mammary fat pad of
C57BL6
wild-type and RAGE knockout mice, and (Fig.7A) tumor progression monitored.
(Fig.7B
and 7C) Immunohistochemical analysis of tumors for angiogenesis (Fig.7B,
CD34), and
leukocyte (Fig.7C, CD45) infiltration from wild-type and RAGE -/- tumors.
(Fig.7D)
Western blot of total and phospho-ERK 1/2 in tumors from wild-type and RAGE -/-
mice.
[0013] Figures 8A-8H. The RAGE inhibitor FPS-ZM1 impairs cell invasion and
anchorage-independent growth in soft agar. (Fig.8A) 231 control & RAGE
transfected cells
were quantified for Matrigel invasion in transwell chambers after 24h to 1%
FBS stimuli in
the presence of FPS-ZM1 (1 t.M) or DMSO control. (Fig.8B) 4175 cells with RAGE
shRNA
(and shControl) were quantified for Matrigel invasion in transwell chambers
after 24h to 1%
FBS stimuli in the presence of FPS-ZM1 (1 t.M) or DMSO control, were
quantified.
(Fig.8C) 4T-1 cells were quantified for Matrigel invasion in transwell
chambers after 24h to
1% FBS stimuli in the presence of FPS-ZM1 (li.tM) or DMSO control. (Fig.8D)
Primary
human dissociated tumor (DT28) cells were quantified for Matrigel invasion in
transwell
chambers after 24h to 1% FBS stimuli in the presence of FPS-ZM1 (1 t.M) or
DMSO control.
(Fig.8E) Proliferation of 4175 cell treated with FPS-ZM1 (1, 10 and 25 t.M)
and DMSO
control quantified by crystal violet staining after 72h. (Fig.8F)
Proliferation of 4T-1 cell
treated with FPS-ZM1 (10 t.M) and DMSO control quantified by crystal violet
staining after
72h. (Fig.8G-8H) Soft agar colony formation assays of 4175 (Fig.8G.) and 4T-1
(Fig.8H.)
cells treated with FPS-ZM1 (1, 10 and 25 t.M) and DMSO control.
[0014] Figures 9A-9G. The RAGE inhibitor FPS-ZM1 reduces tumor progression and
metastasis of highly metastatic 4175 cells. 4175 cells were injected into
mammary fat pad of
NSG mice, and mice treated injected IP with lmg/kg FPS-ZM1 or vehicle control
twice per
week. Tumor size was measured over the course of 35 days, and shown as
(Fig.9A)
representative mice and (Fig.9B) for tumor size. Data shown are from 5 mice
per group.
(Fig.9C-9F) Immunohistochemical analysis of tumors for proliferation (Fig.9C,
Ki67),
angiogenesis (Fig.9D. CD34), and leukocyte (Fig.9E, CD45) and macrophage
(Fig.9F, F4/80)
infiltration from 4175-control and 4175 FPS-ZM1 treated tumors. (Fig.9G)
Representative
images of lung and liver tissues from control (DMSO) and FPS-ZM1 treated mice
stained
with anti-human CK7 antibodies to visualize metastasis. Non-tumor bearing
(NTB) controls
are shown.
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[0015] Figures 10A-10E. RAGE gene expression and clinical outcomes in human
breast
cancer. Relative expression of RAGE mRNA normalized to beta-actin examined
using breast
cancer datasets from OncomineTM. Breast cancer stromal datasets (Fig.10A-10C)
and from
metastatic datasets (Fig.10D and 10E) were compared.
[0016] Figure 11. RAGE inhibitors impair tumor growth of 231-4175 human breast
cancer
cells in NSG immunocompromised mice. 4175 cells were injected into mammary fat
pad of
NSG mice and mice injected IP with 1 mg/kg FPS-ZM1, TTP488, or vehicle control
(DMSO)
twice per week. Tumor size (volume, x-axis) was measured over the course of 35
days (y-
axis).
[0017] Figure 12. Tumor growth of 4T-1 breast cancer cells in BALBc mice. 4T-1
cells
were injected into mammary fat pad of BALBc mice, and mice injected IP with 1
mg/kg
FPS-ZM1, TTP488, or vehicle control (DMSO) twice per week. Tumor size (volume,
x-axis)
was measured over the course of 35 days (y-axis).
[0018] Figure 13. Mouse weight changes in type 2 diabetic mice on RAGE
inhibition.
Control (db/m) and diabetic (db/db) mice (5 mice per group) were treated by
intraperitoneal
(I.P.) injection with 1 mg/kg FPS-ZM1, TTP488, or vehicle control (DMSO) twice
per week
for 30 days. Mice were weighed weekly. Line graph illustrates weight (g, y-
axis) and day
(x-axis).
[0019] Figure 14. Combination therapy with FPS-ZM1 and doxorubicin impairs
tumor
growth in syngeneic breast cancer models. BALBc mice were injected with 4T-1
cells and
treated with control (DMSO), doxorubicin (5 mg/kg), FPS-ZM1 (1 mg/kg), or a
combination
of doxorubicin and FPS-ZMl. Doxorubicin was administered I.P. on days 3 and 7
(post
tumor implantation). FPS-ZM1 was administered I.P. on days 2, 6, 10, and 13.
N=6 mice
per group for all experiments.
[0020] Figure 15. RAGE inhibition with FPS-ZM1 reduces liver inflammation in
obese
mice. Dbdb mice were treated with FPS-ZM1 (1 mg/kg, twice per week I.P.) or
vehicle
(control). RNA was extracted from liver tissue and QPCR performed to determine
gene
expression levels. Four sets of bars are provided in the bar graph denoting
RNA of four
targets (from left to right, x-axis), CDE3, f480, IL6, and TNF, with relative
normalized
expression denoted on the y-axis. Within each set of bars, the left bar
corresponds to
treatment with vehicle; the right bar corresponds to treatment with FPS-ZMl.
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[0021] Figure 16. RAGE inhibition with FPS-ZM1 reduces liver expression of
RAGE and
RAGE ligands in obese mice. Dbdb mice were treated with FPS-ZM1 (1 mg/kg,
twice per
week I.P.) or vehicle (control). RNA was extracted from liver tissue and QPCR
performed to
determine gene expression levels. Three sets of bars are provided in the bar
graph denoting
RNA of three targets (from left to right, x-axis), RAGE, S10048, and S10049,
with relative
normalized expression denoted on the y-axis. Within each set of bars, the left
bar
corresponds to treatment with vehicle; the right bar corresponds to treatment
with FPS-ZML
[0022] Figures 17A-17B. The RAGE inhibitor FPS-ZM1 displays a dose dependent
effect
on tumor metastasis. Tumor growth (Fig.17A; y-axis=tumor size, x-axis=day) and
metastasis
(Fig.17B; y-axis=luminoscore, x-axis=DMSO, FPS1, FPS2) was measured in tumor
bearing
mice treated with either DMSO or FPS-ZM1 (1 or 2 mg/kg, twice per week).
[0023] Figures 18A-18B. Frequency and dose effects of the RAGE inhibitor FPS-
ZM1 on
tumor metastasis. Tumor growth (Fig.18A; y-axis=tumor size, x-axis=day) and
metastasis
(Fig.18B; y-axis=luminoscore, x-axis=DMSO, FPS dosing) was measured in tumor
bearing
mice treated with either DMSO or FPS-ZML FPS-ZM1 was given to mice either
twice per
week (lmg/kg), or every day (lmg/kg or 2mg/kg).
[0024] Figures 19A-19B. The RAGE inhibitor TTP488 displays a dose dependent
effect on
tumor metastasis. Tumor growth (Fig.19A; y-axis=tumor size, x-axis=day) and
metastasis
(Fig.19B; y-axis=luminoscore, x-axis=DMSO, TTP dosing) was measured in tumor
bearing
mice treated with either DMSO or TTP488 (1, 2.5, 5 mg/kg, twice per week).
[0025] Figure 20. FPS-ZM1 impairs tumor cell invasion. Cell invasion assays
were
performed using a Matrigel transwell invasion system. X-axis=cell line and
treatment; y-
axis= relative invasion (%).
[0026] Figure 21. RAGE inhibitors (FPS-ZM1 and TTP488) impair tumor cell
invasion.
Cell invasion assays were performed as in Figure 20. Cells (4T-1) were treated
with either
FPS-ZM1 (5 t.M), TTP488 (5 t.M) or DMSO control.
[0027] Figure 22. RAGE inhibitors (FPS-ZM1 and TTP488) impair tumor cell
inflammation. Cytokine and chemokine release from cells was assessed using
Proteome
Profiler Mouse XL Cytokine arrays with condition media from either control
(DMSO), FPS-
ZM1 (2 t.M) or TTP488 (2 t.M). Cytokine levels were quantified by western blot
and
densitometry. Data shows heat maps where protein levels are decreased. Data is
expressed
as a percentage of decrease for each cytokine.
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[0028] Figure 23. RAGE knockout in mice impairs tumor progression. The tumor
size of
wild-type (WT) and RAGE knockout mice (RKO) Py8119 breast cancer cells were
compared
over a period of 33 days.
[0029] Figures 24A-24B. RAGE knockout in MMTV-PyMT mice impairs tumor
initiation
and metastasis. Tumor latency (Fig. 24A) and tumor metastasis (Fig. 24B) were
measured for
MMTV-WT and MMTV-PyMT RAGE KO (RKO) mice.
[0030] Figure 25. RAGE inhibitors FPS-ZM1 impairs NASH in db/db mice. Db/db
mice
were treated with FPS-ZM1 (lmg/kg, twice per week) or DMSO control. NASH was
assessed by histology of liver and serum analysis of the liver enzyme ALT.
DETAILED DESCRIPTION
[0031] The Receptor for Advanced Glycation End-product (RAGE) is a multiligand
cell
surface molecule of the immunoglobulin superfamily. RAGE binds multiple
ligands
including, e.g., the non-enzymatic protein-adducts (AGEs) that form in the
hyperglycemic
state of diabetes (Siegel et al. (2013). CA Cancer J Clin, 63, 11-30; Taguchi
et al. (2000).
Nature, 405, 354-360)), various members of the S100/calgranulins (5100A4, A6-
9, SlOOB
and SlOOP) (Kalea et al. (2010). Cancer Res, 70, 5628-5638; Kang et al.
(2010). Cell Death
Differ, 17, 666-676; Liao et al. (2011). Asian Pac J Cancer Prey, 12, 1061-
1065)), amyloid
beta (AP), LPA, collagen I/IV, and the high mobility group box-1 (HMGB1)
protein (Larsson
et al. (2007). Int J Cancer, 121, 856-862). RAGE is able to bind broad ligand
classes due to
its highly positively charged ligand binding domain, which forms an
electrostatic trap for
these largely negatively charged ligands. Chavakis et al. (2003). J Exp Med,
198, 1507-1515;
Kock et al. (2010). Structure, 18, 1342-1352.
[0032] As described herein, RAGE-signaling in both the tumor and tumor
microenvironment is required for invasion and metastasis of highly metastatic
murine and
human breast cancer cells. Targeting RAGE with novel small molecule inhibitors
significantly impairs metastasis in vivo. The data described herein
demonstrate that
therapeutic blockade of RAGE-ligand signaling is a powerful approach to treat
invasive and
metastatic breast cancer. The disclosure provides a method of treating breast
cancer, the
method comprising administering to mammalian subject in need thereof an
inhibitor of
Receptor for Advanced Glycation End-product (RAGE). The disclosure also
provides a
method of inhibiting breast cancer metastasis, the method comprising
administering to
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mammalian subject in need thereof an inhibitor of RAGE. In various aspects,
the subject is a
human.
[0033] In various aspects, the RAGE inhibitor is a small molecule. For
example, a RAGE
inhibitor suitable for use in the context of the disclosure is TTP-488
(chemical name 344-12-
butyl-1- [4-(4-chlorophenoxy)-phenyl] -1H-imidazole-4-y1} -phenoxy)-propy1]-
diethylamine),
also known as azeliragon or PF-04494700). The structure of TTP-488 is provided
below.
HiC
14,C,\...4
:.
*
4
- 0 ..1 TTP-488
[0034] TTP-488 is an orally active, antagonist of RAGE-RAGE ligand
interaction, which
has shown reduction of amyloid accumulation in the brains of mice. In human
clinical trials,
TTP-488 did not display any adverse effects in both in Phase I and II trials.
Low dose
treatment with TTP-488 in Alzheimer patients demonstrated slower decline in
cognitive
function compared to controls.
[0035] Alternatively, the RAGE inhibitor is FPS-ZM1, the structure of which is
provided
below.
0
1
t
ISIFPS -ZM1
[0036] The disclosure provides a method of treating breast cancer or
inhibiting breast
cancer metastasis in a subject in need thereof. "Treating" breast cancer does
not require a
100% abolition of cancer in the subject. Any decrease in tumor load, tumor
burden, or tumor
volume; inhibition of tumor cell proliferation; eradication of tumor cells;
and the like
constitutes a beneficial biological effect in a subject. The progress of the
method in treating
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breast cancer (e.g., reducing tumor size or eradicating cancerous cells) can
be ascertained
using any suitable method, such as those methods currently used in the clinic
to track tumor
size and cancer progress. Tumor size can be figured using any suitable
technique, such as
measurement of dimensions or estimation of tumor volume. Tumor size can be
determined
by tumor visualization using, for example, CT, ultrasound, SPECT, spiral CT,
MRI,
photographs, and the like. Measurement of tumor size, detection of new tumors,
biopsy,
surgical downstaging, PET scans, and the like can point to the overall
progression (or
regression) of cancer in a human. Similarly, "inhibiting metastasis" does not
require a
complete blockage of metastasis; any degree of preventing, suppressing,
delaying the onset,
or slowing metastasis in the subject is contemplated.
[0037] The disclosure further provides a method of inhibiting the onset of
breast cancer.
The method comprises administering to a mammalian subject in need thereof an
inhibitor of
Receptor for Advanced Glycation End-product (RAGE). "Inhibiting the onset of
breast
cancer" does not require 100% prevention of the disease, although complete
prevention is
contemplated. "Inhibiting the onset" when used in the context of a disease or
disorder (for
example, breast cancer) also includes lessening the likelihood of the disease
or disorder onset
or slowing the onset of the disease or disorder.
[0038] The disclosure also provides a method of treating chronic disease,
optionally
associated with inflammation, comprising administration to a subject in need
thereof a RAGE
inhibitor, such as a RAGE inhibitor described herein. In various aspects, the
chronic disease
is obesity, non-alcoholic fatty liver disease (NAFLD), or nonalcoholic
steatohepatitis
(NASH). In various aspects, the subject has a body mass index of 30 or
greater. NAFLD is
characterized by the presence of steatosis, whereas NASH is characterized by
the histologic
presence of steatosis, cytological ballooning, inflammation, and fibrosis.
"Treating" the
chronic disease, such as obesity or NASH does not require a 100% abolition of
the disorder
in the subject. Any reduction in, e.g., weight, body mass index, liver
inflammation, or
accumulation of fatty deposits in the liver, is contemplated. For example, in
various aspects,
the method reduces the risk of developing the chronic disease (e.g., NAFLD or
NASH),
arrests or slows the development of the disease or clinical symptoms thereof,
or ameliorates
the chronic disease (e.g., promotes regression or reversal of the disease
state or symptoms
thereof). In this regard, the disclosure contemplates a method of reducing
liver inflammation
comprising administering to a subject in need thereof a RAGE inhibitor, such
as a RAGE
inhibitor described herein. Liver inflammation and steatosis are detected
using any of a
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number of techniques including, but not limited to, blood tests (to detect,
e.g., elevated liver
enzymes), ultrasound, computerized tomography (CT) scans, Magnetic resonance
imaging
(MRI), and biopsy.
[0039] In various aspects, the RAGE inhibitor is provided in a composition
(e.g., a
pharmaceutical composition) comprising a physiologically-acceptable (i.e.,
pharmacologically-acceptable) carrier, buffer, excipient, or diluent. Any
suitable
physiologically-acceptable (e.g., pharmaceutically acceptable) carrier can be
used within the
context of the disclosure, and such carriers are well known in the art. The
choice of carrier
will be determined, in part, by the particular site to which the composition
is to be
administered and the particular method used to administer the composition.
Suitable
composition formulations include aqueous and non-aqueous solutions, isotonic
sterile
solutions, which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the
formulation isotonic with the blood, and aqueous and non-aqueous sterile
suspensions that
can include suspending agents, solubilizers, thickening agents, stabilizers,
and preservatives.
The composition can be presented in unit-dose or multi-dose sealed containers,
such as
ampules and vials.
[0040] A particular administration regimen for a particular subject will
depend, in part,
upon the amount of RAGE inhibitor administered, the route of administration,
and the cause
and extent of any side effects. The amount administered to a subject (e.g., a
mammal, such as
a human) in accordance with the disclosure should be sufficient to affect the
desired response
over a reasonable time frame. For example, the administration regimen for
TTP488 may, in
various aspects, comprise daily administration of about 10 mg to about 60 mg
to a subject in
need thereof.
[0041] The disclosure further contemplates administering the RAGE inhibitor in
combination with one or more additional therapeutics. For instance, in the
context of cancer,
the therapeutic regimen for the subject may include administration of one or
more cytotoxic
agents or chemotherapeutic agents. Representative additional therapeutic
agents include, but
not limited to, 5-azacytidine, actinomycin D, amanitin, aminopterin,
anguidine, anthracycline,
anthramycin (AMC), auristatin, bevacizumab, bleomycin, busulfan, butyric acid,
camptothecin, carboplatin, carmustine, cemadotin, cisplatin, colchicin, a
combretastatin,
cyclophosphamide, cytarabine, cytochalasin B, dactinomycin, daunorubicin,
decarbazine,
diacetoxypentyldoxorubicin, dibromomannitol, dihydroxy anthracin dione, a
disorazole,
docetaxel, dolastatin (e.g., dolastatin 10), doxorubicin, daunorubicin,
duocarmycin,
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echinomycin, emetine, epothilones, esperamicin, ethidium bromide, etoposide,
fluorouracil,
gemcitabine, geldanamycin, glucocorticoid, irinotecan, lapatinib, melphalan,
mercatopurine,
methopterin, methotrexate, mithramycin, mitomycin, mitoxantrone, paclitaxel,
pertuzumab,
propranolol, pteridine, puromycin, taxol, tamoxifen, tenoposide, tetracaine,
teniposide,
topotecan, trastuzumab, vinblastine, vincristine, vindesine, vinorelbine, or a
derivative of any
of the foregoing. In various aspects, the RAGE inhibitor is administered in
combination with
doxorubicin. It will be appreciated that "in combination" does not restrict
the timing or order
in which the RAGE inhibitor and one or more additional therapies are
administered to the
subject. The administration of different therapeutic agents can occur
simultaneously or
sequentially, and by the same or different routes of administration.
[0042] The invention, thus generally described, will be understood more
readily by
reference to the following example, which is provided by way of illustration
and is not
intended to limit the invention.
EXAMPLES
Example]
[0043] This example demonstrates inhibition of breast cancer cell
proliferation and
metastasis in vivo using RAGE inhibitors.
Materials and Methods
[0044] Cell lines: The MDA-MB-231 breast cancer line and its highly metastatic
derivatives (4175 and 1833) are described in Minn et al. (2005). Nature, 436,
518-524;
Vernon et al. (2013). J Immunol, 190, 1372-1379. Cells were cultured in
Dulbecco's
Modified Eagle Medium (Lifetech, Carlsbad, CA, USA) and 10% fetal calf serum
(FCS,
Atlanta Biologicals, USA). Murine breast cancer cells 4T1 and 67NR were
obtained from
ATCC and Karmanos Cancer Institute respectively. AT-3 cells were previously
isolated
from a spontaneous mammary tumor from the MMTV-PyMT/B6 mouse model. The
dissociated primary human tumor cell line (DT28) established from an ER-
negative primary
breast tumors was cultured as previously described. Drews-Elger et al. (2014).
Breast Cancer
Res Treat, 144, 503-517. All cell lines were routinely tested for mycoplasma
using the
MycoAlertTM Mycoplasma Detection Kit (Lonza) and with PCR prior to injection
into
animals.
[0045] Generation of RAGE overexpression and gene knockdown cells: Lentiviral
vectors
encoding human RAGE cDNA (Precision LentiORF; Thermo Scientific) and human /
mouse
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RAGE shRNA (pGIPZ; Thermo Scientific) were used for RAGE overexpression and
silencing. Human RAGE cDNA (PLOHS 100006205) and empty vector pLOC control
were
used. Indicated shRNAs are as follows: shControl (RH54346), human RAGE sh65
(V3LHS 316665; TGGACTTGGTCTCCTTTCC (SEQ ID NO: 1)), human RAGE sh66
(V3LHS 316666; TACACTTCAGCACCAGTGG (SEQ ID NO: 2)), mouse RAGE sh10
(V3LMM 430610; TGACCTCCTTCCCTCGCCT (SEQ ID NO: 3)) and mouse RAGE sh12
(V3LMM 430612; TATTAGGGACACTGGCTGT (SEQ ID NO: 4)). Human RAGE cDNA
(PLOHS 100006205) and empty vector control. To generate lentivirus, lentiviral
vectors
were co-transfected with psPAX2 and pMD2.G (Addgene) into HEK-293T cells
(ATCC)
with Lipofectamine 2000 (Lifetech). Supernatants were collected at 48 hours
and cell debris
pelleted. MDA-MB-231, 4175. 4T-1, and 67NR were infected with viral
supernatant with 4
(ig/m1polybrene and stable expression selected with blasticidin (pLOC) or
puromycin
(pGIPZ). Stably transduced cells were tested for RAGE overexpression or
knockdown by
Western blotting (see below).
[0046] Western Blots: Cells were lysed with RIPA buffer with protease (Sigma)
and
phosphatase inhibitors (Sigma) according to manufacturer's instructions.
Western blotting
was performed using Invitrogen NuPAGE system as previously described. Jules et
al.
(2013). PLoS ONE, 8, e78267. Antibodies used were as follows; human RAGE
monoclonal
antibody (Millipore; MAB5328), RAGE polyclonal (Santa Cruz; H300), 13-actin
(Millipore;
MAB1501), E-cadherin (BD Biosciences; 610181), ZO-1 (Cell Signaling; 8193),
Vimentin
(Cell Signaling; 5741), MMP2 (Millipore), MMP9 (Millipore), Slug (Cell
Signaling; 9585),
Snail (Cell Signaling; 3879), Twist (Santa Cruz Biotechnology), and Zebl (Cell
Signaling;
3396). All phospho-status and total antibodies (MEK 1/2, ERK 1/2, p38,
SAPK/JNK, AKT)
were obtained from Cell Signaling. All antibody conditions are listed in Table
1.
TABLE 1
Antibody Supplier Catalog no. Application
Dilution
RAGE R&D Systems AF1145 WB 100
Beta-actin Cell Signaling 3700 WB 5000
E-cadherin BD Biosciences 610181 WB 500
ZO-1 Zymed 61-7300 WB 500
Vimentin Cell Signaling 3390 WB 1000
MMP2 Cell Signaling 4022 WB 1000
MMP9 Cell Signaling 3852 WB 1000
Slug Cell Signaling 9585 WB 1000
Twist Santa Cruz 81417 WB
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Zeb1 Cell Signaling 3396 WB 1000
Phospho-Akt (Thr308) Cell Signaling 2965 WB 1000
Phospho-Akt (Ser473) Cell Signaling 4060 WB 1000
Akt Cell Signaling 4691 WB 1000
Phospho-MEK1/2 Cell Signaling 2338 WB 1000
MEK1/2 Cell Signaling 9126 WB 1000
Phospho-p44/42 MAPK Cell Signaling 4370 WB 1000
(Erk1/2)
p44/42 MAPK (Erk1/2) Cell Signaling 9102 WB 1000
Phospho-p38 MAPK Cell Signaling 4511 WB 1000
p38 MAPK Cell Signaling 8690 WB 1000
Phospho-SAPKANK Cell Signaling 4668 WB 1000
SAPKANK Cell Signaling 9258 WB 1000
mDia1 BD Biosciences 610848 WB 500
Ki67 Abcam ab15580 IHC 100
CD34 eBioscience 14-0341-82 IHC 100
CD45 BD Biosciences 550539 IHC 100
F4/80 Abcam ab16911 IHC 100
CK7 Dako M701801-2 IHC
[0047] Wound healing assays: To assess cell migration, wound-healing (scratch)
assays
were used. Cells were plated in 6 well plates at 2.5 x 105 cells / well and
grown to
confluence to form a monolayer and serum starved overnight. A single scratch
was made per
well with a 200 ill pipette tip, and the cell media changed to 1% FBS. Cells
were fixed at 0
and 16 h after wounding and images acquired with a light microscope at 10x
magnification.
[0048] Cell invasion assay: Cell invasion assays were performed using
transwell
migration chambers as previously described; 17. 5 x 103 cells were seeded in
the upper
chamber of 8-i.tm porous transwell inserts (ThinCerts, Greiner) coated with
12.5 i.t.g of
Growth Factor Reduced Matrigel (BD Biosciences) in serum-free DMEM, and
incubated in
24 well plates with 1% FBS as a chemoattractant for 24 hours (48 hours for
DT28 cells).
Following incubation cells were fixed with methanol for 10 minutes and stained
with 2%
crystal violet in 2% ethanol solution. Non-migrated cells were removed from
transwell
chambers with a cotton swab. To quantify the cells, the cell stain was
extracted with 10%
acetic acid, transferred to a 96 well plate and measured at 595 nm using an
iMark Microplate
Reader (Biorad). For invasion assays with RAGE inhibitors, cells were pre-
treated for lh
prior to assays and re-added during invasion assays (both upper and lower
chambers) with
either FPS-ZM1 (1 t.M; Millipore) or equal volume DMSO control.
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[0049] Proliferation assays: Cells were counted and plated in triplicate at
35,000 cells per
well of a 12 well plate and grown for 48-72 hours. Cells were fixed with 4%
paraformaldehyde and stained with 0.1% crystal violet for 20 min. Cell number
was
quantified by extracting crystal violet stain with 10% acetic acid and
transferred to a 96 well
plate and measured at 595 nm using an iMark Microplate Reader (Biorad).
[0050] Soft agar assay: Cells (5 x 103 cells/well) were resuspended in 0.4%
agarose
(Sigma) in Iscove's Modified Dulbecco's Medium (IMDM) with 10% FBS and seeded
on top
of a 0.8% agarose layer (IMDM with 10% FBS) in 6 well plates and cultured for
14 days
(4T-1, 67NR cells) or 21 days (231, 4175 cells). Tumor colonies were stained
with 0.05%
TNT (Iodonitrotetrazolium chloride) in PBS overnight at 37 C and
representative images (5
per well) were acquired using a Nikon Eclipse TS100 microscope. The assay was
performed
in triplicate and repeated independently three times.
[0051] Animal studies: All animal studies were approved by the Institutional
Animal Care
and Use Committee of the University of Miami. NOD scid gamma (NOD.Cg-
Prkelcscid
Il2renlw-111SzJ; NSG), BALBc and C57BL6 wild-type mice were purchased from
Jackson
Laboratory. C57BL6 RAGE knockout mice (RAGE -/-) were previously described.
Myint et
al. (2006). Diabetes, 55, 2510-2522. As < 95% of the animals inoculated with
tumor cells
develop tumors and ¨90% develop lung metastasis (for NSG and BALBc mice); 10
mice per
group were used for all studies. Further, mice with tumor growth in exceeding
5% of body
weight or exhibiting 20% weight loss were terminated early and excluded from
the study.
Animals were randomized to each experimental group (RAGE shRNA vs. scramble
shRNA;
FPS-ZM1 vs. vehicle). Injections and measurements were performed by different
investigators, and mice injected/measured in a random manner.
[0052] 231 (vector/RAGE, shControl/RAGE sh66) and 4175 (shControl and RAGE
shRNA66) were injected in 100 [11 of Matrigel (1x106 cells) into the third
inguinal mammary
fat pad of 8-week old female NSG mice (10 mice per group) as previously
described. Drews-
Elger et al. (2014). Breast Cancer Res Treat, 148, 41-59. 4T-1 (shControl,
RAGE shRNA10
and RAGE shRNA12) cells were injected into the third inguinal mammary fat pad
of 8-week
old female BALBc mice. For AT3 cells, cells were injected into the third
inguinal mammary
fat pad of 8-week old female C57BL6 wild-type or RAGE knockout mice (10 mice
per
group). For experiments involving the RAGE inhibitor, NSG mice were injected
ip with
either DMSO (vehicle control) or with FPS-ZM1 (RAGE inhibitor) at a dose of
lmg/kg twice
per week. Tumor growth was monitored using calipers every 5 days and animals
were
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sacrificed when tumors reached 10% of body weight. Primary tumors and organs
were
harvested and fixed in 10% formalin and paraffin embedded for pathological
analysis.
Primary tumor and organs were stained with hematoxylin and eosin. Primary
tumors were
stained with antibodies against CD34 (EBioscience, 14-0341), Ki67 (Abcam,
ab15580),
CD45 (BD Biosciences, 550566) and F4/80 (Abcam). To quantitate metastasis of
tumor cells
in tissue, organs were stained with antibodies against human cytokeratin 7
(Leica
Biosystems). Slides were analyzed by a pathologist to confirm the presence of
metastases.
For both tumor and tissue sections, five view fields were selected randomly
from each section
(with 2 sections per tumor / tissue used) for each animal. To analyze and
quantify antibody
staining, percentage of area that stained positive was calculated with the use
of ImageJ1.34n
software (National Institutes of Health [NIH], Bethesda, MD) as previously
described.
Kurozumi et al. (2007). J Natl Cancer Inst, 99, 1768-1781.
[0053] Syngeneic drug models: 100,000 luciferase labelled 4T1 cells, were
injected into
the 4th mammary fat pad (anatomical right) of BALBc mice. Drug treatments (FPS-
ZM1 or
TTP488) during the study were given I.P. and varied on dosage schedule and
amount of drug
administered. Tumor size after initial palpability was measured twice weekly
by calipers. In
vivo imaging was performed using the Xenogen IVIS-200, which allowed for
quantitative
primary tumor and metastasis measurements. A luminoscore based on average
radiance and
total photon flux (photons/sec) was generated by drawing regions of interest
(ROIs) around
the lungs of each mouse.
[0054] Spontaneous models: MMTV-PyMT male mice were bred with either C57BL6
wild-type or RAGE knockout mice. Mouse genotype was confirmed by PCR for both
MMTV
transgene and knockout of the RAGE gene. Tumor palpability was monitored in
mice and at
time of sacrifice, lungs were isolated to identify metastasis.
[0055] Experimental metastasis assays: 25,000 luciferase labelled 4T-1 cells
were injected
into the tail vein of BALBc mice. RAGE inhibitor FPS-ZM1 (2mg/kg) or vehicle
control
(DMSO) were given IP every day. IVIS was used as above to quantify tumor
metastasis.
[0056] Chemotherapy and RAGE inhibitor experiments: 4T-1 cells / BALBc mice
were
used as described for syngeneic studies above. Mice were treated with either
vehicle control
(DMSO), doxorubicin (5mg/kg on days 3, 7 and 14 post-tumor injection), FPS-ZM1
(lmg/kg, 2x / week) or doxorubicin and FPS-ZM1 (same as single dose). Tumor
size was
measured as above. Metastasis was assessed by H&E ex vivo.
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[0057] In vitro assays: Invasion assays: cellular invasion was tested using
transwell
Matrigel invasion assays. Cells (50,000) were seeded in the upper chamber of a
transwell
insert coated with Matrigel. Cells were allowed to invade toward a lower
chamber with 1%
FBS as stimulant for 24 hours. For inhibitor studies, cells were incubated
with either FPS-
ZM1, TTP488 or DMSO control. Cytokine arrays: cell were grown on 100mm dishes
and
treated with either FPS-ZM1, TTP488 or DMSO control for 24 hours. Conditioned
media
was collected and cytokine release assessed using the Proteome Profiler Mouse
XL Cytokine
array.
[0058] OncomineTM analysis of RAGE mRNA expression: The OncomineTm database
was
used for initial analysis and extraction of RAGE expression from three breast
cancer stromal
datasets (Finak, Ma-4, and Karnoub) and two invasive breast cancer datasets
(Sorlie-2 and
TCGA). Finak et al. (2008). Nat Med, 14, 518-527; Karnoub et al. (2007).
Nature, 449, 557-
563; Ma et al. (2009). Breast Cancer Res, 11, R7; Sorlie et al. (2003). Proc
Natl Acad Sci U S
A, 100, 8418-8423; (2012). Nature, 490, 61-70. Log2 median-centered intensity
values for
RAGE gene expression were compared between normal breast stroma and stroma
from
primary breast lesions: invasive breast cancer (Finak), invasive ductal
carcinoma (Karnoub),
and DCIS (Ma-4). Log2 median-centered intensity values for RAGE gene
expression were
also compared between primary breast lesions and metastatic breast lesions
(Sorlie-2 and
TCGA). Comparisons of RAGE expression between groups were performed using one-
sided
Student's t-test.
[0059] Statistical analysis: All statistical analyses and visualization were
performed using
GraphPad Prism version 6.00 for Windows, (GraphPad Software, San Diego
California USA)
or R statistical software for Mac (R 3Ø3, R Foundation for Statistical
Computing, Vienna,
Austria). Power analysis was performed using Statmate 2.0 (GraphPad Software,
San Diego
California USA). Data are expressed as means ( SEM), with significance
considered as
P<0.05. In all experiments data was normally distributed and variance similar
between
groups.
Results
[0060] Metastatic human and mouse breast cancer cells show increased RAGE
expression:
To investigate the role of RAGE in driving breast cancer metastasis, RAGE
protein levels
were compared by immunoblotting in breast cancer lines with low and high
metastatic
ability; MDA-MB-231 (hereafter 231) and in highly metastatic variants of 231,
MDA-MB-
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231-4175 and MDA-MB-231-1833 (hereafter 4175 and 1833), previously selected
for
increased metastasis to lung and bone in xenograft models, respectively. Minn
et al. (2005).
Nature, 436, 518-524; Kang et al. (2003). Cancer Cell, 3, 537-549. The highly
metastatic
4175 and 1833 cells displayed greater RAGE protein than parental 231 cells
(Figure 1A).
The murine mammary metastatic 4T1 model, which spontaneously metastasizes from
the
primary site in vivo in syngeneic hosts, and an isogenic variant thereof,
67NR, with reduced
metastatic potential was studied. Aslakson et al. (1992). Cancer Res, 52, 1399-
1405;
Sirotnak et al. (1984). Cancer Chemother Pharmacol, 12, 26-30; Stewart et al.
(2007). J
Immunol, 179, 2851-2859. RAGE levels were higher in the highly metastatic 4T1
compared
to their isogenic non-metastatic variants 67NR (Figure 1B). Thus in both human
and murine
breast cancer models, RAGE levels were increased in highly metastatic lines
compared to
those with reduced metastatic ability.
[0061] RAGE increases breast cancer cell malignancy in vitro and metastasis in
vivo: To
further investigate whether RAGE mediates human breast cancer metastasis, the
effects of
RAGE on migration, invasion and soft agar colony formation in 231 cells were
determined.
1% FBS (1%) is a rich source of numerous RAGE ligands and was used to activate
RAGE
dependent effects. RAGE overexpression in 231 cells (Figure 1C) increased cell
migration
(Figure 1D) and invasion through Matrigel (Figure 1E). The increased cell
migration and
invasion did not result from an increase in cell number due to increased
proliferation, as no
change in cell number was seen between 231 versus 231-RAGE cells after 48
hours of cell
growth (Figure 1F). To test further RAGE effects on the tumorigenic properties
of 231 cells,
soft agar assays were performed. RAGE overexpression increased both number and
size of
231 cell colonies in soft agar, compared to controls (Figure 1G). Thus, RAGE
over-
expression increases migration and invasion, and promotes soft agar colony
formation of 231
cells in vitro.
[0062] To extend these data in vivo, 231 cells, with or without RAGE
overexpression, were
implanted into the mammary fat pad in immunocompromised NOD scid gamma (NSG)
mice.
RAGE overexpression in 231 cells did not affect tumor growth (Figure 1H) or
final tumor
weight (Figure 1I). In marked contrast, RAGE overexpression in 231 cells
significantly
increased the number and size of metastases in lung (Figure 1J).
[0063] RAGE signaling drives cellular invasion through a MEK-dependent
expression of
EMT regulators: Since RAGE expression is linked to poor breast cancer
survival, and since
RAGE is overexpressed in metastatic lines and mediates increased tumor cell
invasion and
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soft agar colony formation, the effects of RAGE on signaling pathways and
epithelial to
mesenchymal transition (EMT) mediators implicated in metastasis was studied.
RAGE
overexpression in 231 cells increased expression of EMT markers including
matrix
metalloproteins, MMP-2, MMP-9, and vimentin, whilst concurrently reducing
expression of
epithelial markers, E-cadherin and ZO-1 (Figure 2A). Furthermore, RAGE
overexpression
increased expression of EMT transcription factors Slug and Twistl and
increased 13-catenin,
while Snail and Zebl levels were unchanged (Figure 2B).
[0064] RAGE signals through diverse pathways, including various mitogen
activated
protein kinase (MAP) kinases (ERK 1/2, p38, SAPK/JNK), PI3K/Akt and JAK/STAT.
Taguchi et al. (2000). Nature, 405, 354-360; Hudson et al. (2008). J Biol
Chem, 283, 34457-
34468; Kislinger et al.. (1999). J Biol Chem, 274, 31740-31749; Huang et al.
(2001). J Cell
Biochem, 81, 102-113; Yeh et al. (2001). Diabetes, 50, 1495-1504; Lander et
al. (1997). J
Biol Chem, 272, 17810-17814; Huttunen et al. (1999). J Biol Chem, 274, 19919-
19924. To
test which pathway(s) mediate RAGE dependent migration and invasion in breast
cancer
cells, the activated phosphorylation status of signaling proteins was compared
by Western
blot in parental 231-controls and 231-RAGE cells. RAGE overexpression
increased
activation of Akt, p38 and MEK/ERK pathways, but not of SAPK/JNK. To test
which
RAGE activated pathways mediate the increased invasive phenotype, invasion
assays were
carried out with and without inhibitors of MEK (U0126), p38 (5B203580) or
PI3K/Akt
(LY294002). Inhibition of MEK, but not Akt decreased Matrigel invasion in 231-
RAGE
(Figure 2C). These data demonstrate that the increase in cell invasion occurs
through a
RAGE-MAP kinase signaling cascade.
[0065] RAGE knockdown in highly metastatic breast cancer cells impairs
Matrigel
invasion and anchorage-independent growth: Since RAGE overexpression increased
migration and invasion of 231 cells, the ability of RAGE gene knockdown to
impair these
effects in highly metastatic lines was tested. RAGE shRNA decreased RAGE
protein levels
in 231 and 4175 to >75% of shControl (Figure 3A). Cell invasion assays showed
shControl
highly metastatic 4175 cells showed greater invasion than shControl parental
231 transwell
Matrigel invasion assays as expected (Figure 3B). RAGE knockdown dramatically
reduced
Matrigel invasion by 4175 cells, and impaired that of the less metastatic
parental 231 line
(Figure 3B). To further confirm these findings, the effect RAGE knockdown by
different
shRNAs was observed (RAGE shRNA 65 & 66). As shown in Figure 3B, both RAGE
shRNAs reduced cell invasion. Whilst the increase in 4175 cells over time was
more rapid
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than 231 cells, RAGE knockdown had little effect on population increase in
4175 cells and
only a modest effect (p = 0.057) in 231 cells (Figure 3C). RAGE is a mediator
of soft agar
colony formation. Control 4175 cells formed more and larger colonies in soft
agar than
parental 231 cells (Figure 3D). shRNA-mediated RAGE knockdown in both 4175 and
231
cells significantly reduced both the number and size of soft agar colonies
formed compared to
shControls. These data further support a role for RAGE as a key mediator of
invasion in
these breast cancer models.
[0066] To test whether RAGE knockdown would impair downstream signaling
activated
by RAGE overexpression shown in Figure 2, the activated phosphorylation status
of
downstream signaling proteins was tested. RAGE shRNA in 4175 cells led to
decreased
activation of Akt, p38 and MEK/ERK pathways, compared to shControl 4175 cells
(Figure
3E). To further extend these mechanistic data, how RAGE knockdown affects
levels of its
signaling partner mDia-1 was explored. Hudson et al. (2008). J Biol Chem, 283,
34457-
34468. RAGE directly interacts with mDia-1 to induce intracellular signaling.
Western blot
analysis revealed RAGE shRNA 4175 cells had strikingly lower levels of mDia-1
(Figure
3E). Together these data suggest RAGE signaling regulates tumor cell
properties linked to
metastasis in breast cancer cells.
[0067] Loss of RAGE in metastatic BC cells impairs tumor growth and metastasis
in vivo-
xenograft models: To test directly whether RAGE mediates metastasis in vivo,
231 and 4175
cells, with or without RAGE knockdown (RAGE sh66 and shControl), were
implanted into
the mammary fat pad in NSG mice. Primary tumor growth and the emergence and
growth of
metastases were assayed over time. RAGE knockdown in 231 cell did not
significantly affect
tumor growth (Figure 4A); however, in 4175 cells RAGE knockdown led to a
decrease in
final orthotopic mean tumor volume and tumor weight compared to control 4175
cells
(Figure 4B).
[0068] To investigate the mechanisms by which RAGE knockdown affects tumor
growth
in 4175 cells, tumor sections were stained for Ki-67 to assess proliferation
(Figure 4C).
Tumors formed from RAGE knockdown 4175 cells displayed a 28% reduction in
proliferation by Ki-67 staining compared to shControl 4175 cells (Figure 4C).
Since RAGE
can affect constituents of the tumor microenvironment, the effects of RAGE
knockdown on
tumor angiogenesis (endothelial cell marker, CD34) and inflammatory cell
recruitment
(leukocyte cell maker (CD45), macrophage marker (F4/80)) were assessed by IHC.
Tumors
arising from RAGE knockdown 4175 cells displayed decreased vessel formation
(Figure 4D)
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and a significant decrease in leukocyte and macrophage recruitment (Figure
5E&F),
compared to shControl 4175 cells.
[0069] Since the effects of RAGE on breast cancer metastasis are not fully
known, it was
determined whether RAGE knockdown would affect the ability of 231 and 4175
cells to
metastasize from the primary mammary tumor site. All NSG mice implanted with
231 and
4175 shControl cells developed extensive metastasis to lung and liver (Figure
5A-F). In
marked contrast, RAGE knockdown in 231 and 4175 cells significantly reduced
the number
and size of metastases in both lung and liver (Figure 5A-D).
[0070] To account for differences in tumor growth resulting from RAGE
knockdown in
4175 cells, metastasis arising from tumors of similar size was quantitated.
Even from size
matched 4175 RAGE shRNA and shControl tumors (5 mice per group), RAGE
knockdown
cells showed fewer distant metastasis (Figure 5E&F). Whilst 4175 shControl
cells resulted in
extensive metastasis to both lung and liver (day 35), RAGE shRNA in 4175 cells
(day 43) did
not display lung or liver metastasis (Figure 5E&F).
[0071] Loss of RAGE in metastatic BC cells impairs tumor growth and metastasis
in vivo:
syngeneic studies: To validate the data in other metastatic models of breast
cancer, the
effects of RAGE gene knockdown in murine 4T-1 cells and consequences of RAGE
overexpression in the non-metastatic 67NR cells, were assessed. RAGE shRNA in
4T-1
using different murine specific shRNAs (RAGE sh10 & 12), decreased RAGE
protein levels
compared to shControl, whereas RAGE expression was increased in 67NR cells by
lentiviral
transduction of RAGE (Figure 6A). As in 4175 cells, RAGE knockdown by both
RAGE
sh10 and sh12, impaired 4T-1 cell invasion compared to shControl (Figure 6B).
RAGE
knockdown in 4T-1 cells also decreased the number and size of colonies formed
in soft agar
(Figure 6C). Furthermore, RAGE overexpression in non-metastatic 67NR increased
the
number and size of colonies formed in soft agar (Figure 6D).
[0072] Next, 4T-1 cells, with or without RAGE knockdown (RAGE sh10, RAGE sh12
and
shControl), were implanted into the mammary fat pad in BALBc mice. Primary
tumor growth
and the emergence and growth of metastases were assayed over time. RAGE
knockdown in
4T-1 with either RAGE sh10 or sh12 led to a decrease in final orthotopic mean
tumor volume
compared to control 4T-1 cells (Figure 6E). Whilst 4T-1 cells expressing
shControl resulted
in extensive metastasis in lung tissue (Figure 6F), RAGE knockdown in 4T-1
cells with both
RAGE sh10 and RAGE sh12, resulted in little or no lung metastases (Figure 6F).
Thus, these
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data validate that RAGE expression in tumor cells is important for regulating
their migration
and invasion, and is required to increase their metastatic potential.
[0073] RAGE knockout in mice impairs tumor growth and progression: The role of
RAGE
expression in non-tumor cells of the tumor microenvironment was tested.
Syngeneic studies
with AT-3 cells (MMTV-PyMT spontaneous BC cell model) injected into the
mammary fat
pad of wild-type and RAGE knockout C57BL6 immunocompetent mice were performed.
RAGE knockout mice (RAGE -/-) displayed striking impairment of tumor cell
growth with
AT3 cells compared to wild-type (RAGE +/+) mice (Figure 7A). Furthermore, IHC
analysis
of tumor tissue revealed that RAGE -/- mice had decreased vessel formation
(Figure 7B) and
a significant decrease in leukocyte recruitment (Figure 7C) to the tumor.
Western blot
analysis of total tumor lysate revealed that RAGE -/- mice to have decreased
activation of
ERK1/2 (Figure 7D) compared to wild-type mice. These data identify RAGE
expression in
non-tumor cells as an important regulator of tumor progression.
[0074] The RAGE antagonist, FPS-ZM1, impairs breast cancer cell invasion and
anchorage-independent growth: FPS-ZM1 is a RAGE antagonist that interacts with
the
ligand binding domain of the receptor to block RAGE signaling. Deane et al.
(2012). J Clin
Invest, 122, 1377-1392. This drug has been tested for potential effects in
Alzheimer's
disease, but has not been explored as an anticancer agent.
[0075] The effects of FPS-ZM1 on breast cancer growth and invasion in vitro
and
metastasis in vivo was studied. As shown in Figure 8A, FPS-ZM1 (1 t.M)
abrogated the
excess invasion caused by RAGE overexpression in 231-RAGE cells, and decreased
Matrigel
invasion by highly metastatic 4175 (Figure 8B). Importantly, as a test of drug
specificity,
FPS-ZM1 (1 t.M) did not affect cell invasion in 4175 RAGE knockdown cells
(Figure 8B).
To extend these data to other breast cancer models, FPS-ZM1 effects on
invasion of murine
4T-1 cells and of the dissociated primary human tumor cell line, DT28, were
tested. FPS-
ZM1 (1 t.M) treatment impaired invasion of both 4T-1 and DT28 cells compared
to DMSO
control (Figure 8C and 8D).
[0076] FPS-ZM1 did not affect cell proliferation or viability in any of the
cells tested even
at high doses (data shown for 4175 at 1, 10 and 25 i.t.M and for 4T-1 at 10
t.M, Figure 8E&F).
RAGE inhibitor effects on colony formation in soft agar assays also were
tested. In both
4175 and 4T-1 cells, FPS-ZM1 significantly impaired colony number and size in
a dose-
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dependent manner (Figure 8G&H). These data support a role for RAGE as a
therapeutic
target for breast cancer.
[0077] The RAGE inhibitor FPS-ZM1 displays a dose dependent effect on tumor
metastasis. Treatment of FPS-ZM1 led to a significant reduction in tumor
growth (Figure
17A). Assessment of metastasis showed a more striking effect of RAGE
inhibitors and a
dose-dependent effect of FPS-ZM1 on lung metastasis (Figure 17B).
[0078] The RAGE antagonist FPS-ZM1 impairs in vivo tumor progression and
metastasis
of highly metastatic 4175 cells: The data in this study show both in vitro and
in vivo that
targeting RAGE in both tumor cells and non-tumor cells of the tumor
microenvironment are
critical for tumor progression and metastasis. The effects of FPS-ZM1 on 4175
tumor
formation and metastasis from orthotopic primary tumors in NSG also were
tested.
Treatment of mice with FPS-ZM1 (1 mg/kg injection twice per week,
intraperitoneally
started 1 day after tumor cell implantation) impaired 4175 tumor growth
compared to vehicle
controls (Figure 9A&B). Next the effects of FPS-ZM1 were assessed by tumor
immunohistochemistry. Whilst a modest reduction was seen in proliferation by
Ki-67
staining compared to control treated 4175 cells (Figure 9C), FPS-ZM1 treatment
resulted in
tumor with markedly decreased vessel formation (Figure 9D) and a significant
decrease in
leukocyte and macrophage recruitment (Figure 9E and 9F). All 4175-implanted
vehicle
control mice developed extensive metastasis to lung and liver. In contrast,
FPS-ZM1 treated
mice displayed significantly fewer or no metastasis in either lung or liver
(Figure 9G). These
data suggest that RAGE inhibitor therapy impairs both primary and metastatic
breast cancer
growth. Together, the data suggest that RAGE is a critical regulator of tumor
progression
and metastasis through tumor cell intrinsic and extrinsic effects. Therefore,
therapeutic
targeting of RAGE presents a unique approach to inhibit metastasis through a
combined
effect on the tumor cell and other cells of the tumor microenvironment.
[0079] Frequency and dose effects of the RAGE inhibitor FPS-ZM1 on tumor
metastasis.
While FPS-ZM1 impaired tumor growth (Figure 18A and 18B), no dose dependent
effect on
tumor growth was observed. FPS-ZM1 given to mice every day at 2mg/kg, had the
greatest
effect on reducing tumor metastasis.
[0080] The RAGE inhibitor TTP488 displays a dose dependent effect on tumor
metastasis.
Mice treated with TTP488 showed impaired tumor growth compared to DMSO treated
mice.
Metastasis in mice was impaired with TTP488 treatment (Figure 19A and 19B).
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[0081] FPS-ZM1 impair tumor cell invasion. Treatment of cells with FPS-ZM1 (2
t.M)
impaired invasion of 4T-1, E0771 and Py8119 mouse breast cancer cell lines,
compared to
DMSO control (Figure 20). In addition, both FPS-ZM1 and TTP488 impaired tumor
cell
invasion, with TTP488 displaying a greater degree of inhibition than FPS-ZM1
(Figure 21).
[0082] RAGE inhibitors (FPS-ZM1 and TTP488) impair tumor cell inflammation.
Treatment of cytokine arrays with condition media from either control (DMSO),
FPS-ZM1 (2
i.t.M) or TTP488 (2 t.M) showed that FPS-ZM1 and TTP488) impair tumor cell
inflammation
(Figure 22).
[0083] RAGE knockout in mice impairs tumor progression. To confirm data in
wild type
and RAGE knockout C57BL6 mice with AT-3 cells and to test the role of RAGE in
non-
tumor cells of the tumor microenvironment, studies were performed using Py8119
breast
cancer cells. RAGE knockout mice (RKO) displayed striking impairment of tumor
cell
growth with Py8119 cells compared to wild-type (WT) mice (Figure 23).
[0084] RAGE knockout in MMTV-PyMT mice impairs tumor initiation and
metastasis. To
determine the role of RAGE in the initiation and progression of breast cancer,
wild-type and
RAGE knockout (RAGE KO) mice were crossed with the MMTV-PyMT spontaneous BC
model. MMTV-PyMT mice are a well-established mouse model that displays
widespread
transformation of the mammary epithelium resulting in rapidly forming mammary
tumors
and metastatic lesions primarily in lymph nodes and lung, closely mimicking
the human
clinical state. MMTV-PyMT RAGE KO displayed impaired tumor initiation and
growth
compared to MMTV-PyMT wild-type mice (Figure 24A). Further, MMTV-PyMT RAGE
KO mice had little or no metastatic lesions on the surface of the lungs,
whereas MMTV-
PyMT wild-type mice displayed extensive metastatic lesions (Figure 24B).
[0085] RAGE inhibitors FPS-ZM1 impair experimental metastasis. To dissect the
effects
of RAGE inhibition on metastasis from primary tumor growth, experimental
metastasis
assays were performed. Tumor cells were tail vein injected and metastasis
followed using
IVIS. Treatment of mice with FPS-ZM1 (2mg/kg, everyday) strongly impaired
tumor
metastasis compared to DMSO (control) treated mice. 7 mice per group imaged at
the same
exposure by IVIS (Data not shown).
[0086] RAGE is overexpressed in human breast cancer and is associated with
increased
metastasis: To determine whether RAGE is differentially expressed in breast
cancer tissues
from human subjects, microarray data from breast cancer patient samples in the
OncomineTm
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database was examined. Datasets were assessed that include breast stroma from
human
subjects. In the Finak, Karnoub and Ma-4 datasets, RAGE overexpression in
breast stroma
was associated with breast cancer compared to normal breast stroma (Figure 10A-
C). To
correlate these findings with outcomes in breast cancer, datasets were
examined that
compared the expression of genes in primary tumors versus distant metastases.
In both the
Sorlie-2 and TCGA datasets, increased RAGE expression was associated with the
metastatic
site versus primary tumor (Figures 10D and 10E). These data together
demonstrate that
increased RAGE expression is associated with invasive breast cancer and at the
metastatic
site.
[0087] Comparison of FPS-ZM1 and TTP-448: The activity of FPS-ZM1 and TTP-488
was compared in various animal models including xenograft and syngeneic breast
cancer. For
these studies, TTP488 and FPS-ZM1 were reconstituted in DMSO, and mice were
injected
twice per week with lmg/kg of FPS-ZM1, TTP-488 or vehicle control (DMSO). TTP-
488
impaired tumor growth to a similar degree compared to FPS-ZM1 (Figure 11). FPS-
ZM1
injection into mice strongly impaired tumor cell metastasis to both lung and
liver. TTP-488
also impaired metastasis (especially to liver). The activity of TTP-488
compared to FPS-
ZM1 also was tested in the syngeneic immunocompetent 4T-1/BALBc model. Similar
to
NSG mice, treatment of tumor bearing mice with either FPS-ZM1 or TTP-488
impaired
tumor growth (Figure 12).
[0088] Combination treatment of RAGE inhibitor and doxorubicin (DOX): A major
issue
with DOX is its cytotoxicity and the ability of breast cancer cells to become
chemoresistant.
Combination therapy with multiple cytotoxic agents have greater anti-tumor
effects than
DOX alone, but are associated with greater side-effects and reduced quality of
life for women
with breast cancer. The effect of combination therapy with DOX and FPS-ZM1 was
examined. Using the BALBc/4T-1 syngeneic model, mice were treated with either
DMSO
(control), doxorubicin (5mg/kg on days 3 and 7), FPS-ZM1 (lmg/kg on days 2, 6,
10 and 13)
or combination DOX & FPS-ZMl. Compared to control, DOX or FPS-ZM1 monotherapy
impaired tumor growth (Figure 14). Remarkably, combination therapy with DOX &
FPS-
ZM1 markedly impaired tumor growth to such a degree, that noticeable tumors
were only
detected on day 14. An additional experiment was performed using the BALBc/4T-
1
syngeneic model, wherein mice were treated with either DMSO (control), DOX
(5mg/kg on
days 3, 7 and 14), FPS-ZM1 (lmg/kg twice per week) or combination DOX & FPS-
ZMl.
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Analysis of lung metastasis demonstrated FPS-ZM1/DOX combination to have the
greatest
effect on metastasis compared to other groups.
Discussion
[0089] The data provided herein demonstrate that RAGE drives tumor cell
invasiveness
and metastasis in human and mouse breast cancer models through tumor intrinsic
and non-
tumor cell effects, and that the RAGE antagonist, FPS-ZM1, can impair these
processes.
RAGE protein expression levels were higher in highly metastatic breast cancer
cells lines of
both human (231) and mouse (4T-1) origin, and RAGE knockdown impaired both
Matrigel
invasion and soft agar colony formation. Further, RAGE knockdown in both 231
and highly
metastatic 4175 breast cancer cells decreased metastasis from the primary
orthotopic tumor
site to both lung and liver. Finally, the data show for the first time that a
novel RAGE
antagonist can powerfully suppress breast cancer cell invasiveness in vitro
and metastasis in
vivo. Taken together, these data provide evidence that RAGE contributes to
breast cancer
progression and metastasis, through effects on the tumor and its
microenvironment, and that
targeting of RAGE presents a novel therapeutic approach in breast cancer.
[0090] No studies to date have evaluated the role of RAGE on local progression
and
metastasis from the orthotopic site and the effects of RAGE antagonists. The
impact of
RAGE on tumor progression and metastasis using the metastatic MDA-MB-231 cells
and
their highly metastatic derived subline 4175 is described above. The highly
metastatic 4175
cells had higher RAGE expression compared to parental 231 cells. Consistent
with the
increased RAGE expression linked to malignancy, gene knockdown of RAGE by
shRNA
reduced Matrigel invasion and soft agar colony formation of both 231 and 4175
cells. These
data were validated in murine 4T-1 metastatic breast cancer cells and their
derived non-
metastatic 67NR variants. RAGE knockdown in 4T-1 cells impaired invasion and
soft agar
colony formation, whereas overexpression of RAGE in 67NR increased colony
formation in
soft agar. The in vivo studies described herein revealed that RAGE knockdown
in the 4175
cells decreased tumor growth at the orthotopic site, but did not affect tumor
growth of 231
cells. Further, RAGE knockdown prevented the emergence of distant metastasis
of 231 and
4175 cells in both lung and liver, in both a time and tumor size matched
manner.
[0091] The data provided herein further show for the first time that the use
of small
molecule RAGE antagonists is an effective treatment in multiple breast cancer
models. As
described above, RAGE plays a role in both the tumor cell (shRNA knockdown)
and non-
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tumor cells of the tumor microenvironment (RAGE host knockout). FPS-ZM1 is a
highly
attractive therapeutic approach to target multiple mechanisms that promote
progression and
metastasis. The data show FPS-ZM1 inhibits cell invasiveness and soft agar
colony
formation, but does not appear to inhibit tumor cell viability or
proliferation. These effects
were demonstrated not only in the 4175, 231 parental, and 4T-1 cells, but also
primary DT28
breast cancer cells dissociated from a patient with triple negative breast
cancer. Further, the
data show for the first time in vivo that RAGE inhibition (with FPS-ZM1)
affects tumor
progression and inhibits metastasis of tumor cells to lung and liver. FPS-ZM1
was originally
developed and tested in murine models of Alzheimer's disease, and was shown to
bind and
inhibit RAGE with high affinity. Deane et al. (2012). J Clin Invest, 122, 1377-
1392. These
studies also established that FPS-ZM1 had few toxic side effects, even at high
doses in
toxicity studies (500 mg/kg) in C56BL6j mice.
[0092] The data set forth herein provide strong evidence that RAGE is a key
mediator of
breast cancer metastasis in the models evaluated and strongly implicate it as
a mediator of
metastasis in vivo. The Example demonstrates a clear tumor cell intrinsic role
of RAGE in
affecting breast cancer cell invasion and metastasis. Furthermore, these data
from RAGE
knockout mice (and with FPS-ZM1) demonstrate a role of RAGE in breast cancer
through
cells of the breast tumor microenvironment. Further, these data show RAGE
knockdown in
4175 cells and treatment with the RAGE inhibitor reduces angiogenesis and
recruitment of
inflammatory cells to the tumor. The data implicate RAGE as a mediator of
breast
malignancy through effects on both the tumor cell itself and the associated
tumor
microenvironment.
Example 2
[0093] This example describes the effect of RAGE inhibition on obesity and
NASH.
Materials and Methods
[0094] Mouse studies: Db/db mice were obtained from jaxlabs and diabetic
status
confirmed by blood glucose monitoring. High-fat diet mice: C57BL6 wild-type
mice were
maintained on a high-fat diet for 32 weeks.
[0095] Ex vivo analysis: Histology: Liver from mice was used for histology.
H&E was
performed to visualize gross changes in liver. Trichrome staining was
performed to visualize
changes in collagen / liver fibrosis. Oil-red 0 staining was performed to
assess fat content /
accumulation in liver. F4/80 staining by IHC was performed to assess
macrophage
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accumulation / inflammation in liver. Serum: mouse serum was analyzed for the
liver enzyme
ALT.
Results
[0096] The impact of RAGE inhibitors on diabetes and obesity was examined in
db/db
mice. Db/db mice develop diabetes in a similar manner to type 2 diabetes in
humans.
Obesity develops at 3-4 weeks and hyperglycemia and frank insulin-resistant
diabetes is
present by 8 weeks in C57BL6 mice. Mice were treated with lmg/kg (twice per
week) of
FPS-ZM1 (compared to db/m control mice), compared to DMSO vehicle control.
Significant
weight loss was observed in obese dbdb mice, but no change in dbm mice (Figure
13).
[0097] A major consequence of the obesity epidemic is Nonalcoholic
Steatohepatitis
(NASH). Currently there is no effective standard treatment for NASH once
manifested.
Mice treated with RAGE inhibitor (FPS-ZM1, lmg/kg, twice per week) were
examined to
determine if NASH was altered. In db/db control mice, the liver displayed
extensive fat
accumulation indicative of NASH as detected by H&E staining of liver samples.
In contrast,
FPS-ZM1 treated db/db mice displayed far less fat accumulation in liver. As
NASH is
characterized by chronic liver inflammation, the effects of RAGE inhibitor FPS-
ZM1 on
inflammatory mediators of NASH was determined. Using RNA isolated from db/db
mice
described above, QPCR was performed for a range of major inflammatory
mediators of
NASH. RAGE inhibition led to lower levels of inflammatory macrophages (F4/80),
but did
not change total T-cell levels (CD3E). RAGE inhibition also led to a reduction
in the pro-
inflammatory mediators of NASH, IL-6 and TNF-alpha (Figure 15). The effects of
FPS-
ZM1 on accumulation of RAGE ligands and RAGE expression levels in db/db mouse
liver
also was examined. As shown in Figure 16, FPS-ZM1 decreased liver expression
of RAGE
and inhibited expression/accumulation of the RAGE ligands S100A8 and S100A9.
In
addition, mice treated with FPS-ZM1 demonstrate less fat accumulation in the
liver, and less
collagen (fibrosis). Analysis of ALT similarly shows a decrease in the serum
of db/db mice
compared to controls (Figure 25).
[0098] RAGE inhibitor FPS-ZM1 impairs NASH in mice on a high-fat diet. Mice
were fed
a HFD for 32 weeks to induce fat accumulation in the liver and NASH. Mice were
treated
throughout the study with either FPS-ZM1, TTP488 (lmg/kg / twice per week), or
DMSO
control. Liver histology demonstrated less fat accumulation in both TTP488 and
FPS-ZM1
treated mice compared to controls. Trichrome staining revealed less fibrosis
in RAGE
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inhibitor treated mice. F4/80 staining also demonstrated less inflammation in
RAGE inhibitor
treated mice. Data also showed that TTP488 displayed a greater inhibitory
effect than FPS-
Zml (data not shown).
[0099] In summary, the results described herein demonstrate that multiple RAGE
inhibitors (FPS-ZM1 and TTP-488) inhibit the development of a number of
chronic disease
states, including obesity and NASH.
[00100] The entire document is intended to be related as a unified disclosure,
and it should
be understood that all combinations of features described herein are
contemplated, even if the
combination of features are not found together in the same sentence, or
paragraph, or section
of this document. In addition, the invention includes, as an additional
aspect, all
embodiments of the invention narrower in scope in any way than the variations
specifically
mentioned above. With respect to aspects of the invention described or claimed
with "a" or
"an," it should be understood that these terms mean "one or more" unless
context
unambiguously requires a more restricted meaning. With respect to elements
described as
one or more within a set, it should be understood that all combinations within
the set are
contemplated. If aspects of the invention are described as "comprising" a
feature,
embodiments also are contemplated "consisting of" or "consisting essentially
of" the feature.
[00101] Although the applicant(s) invented the full scope of the claims
appended hereto,
the claims appended hereto are not intended to encompass within their scope
the prior art
work of others. Therefore, in the event that statutory prior art within the
scope of a claim is
brought to the attention of the applicants by a Patent Office or other entity
or individual, the
applicant(s) reserve the right to exercise amendment rights under applicable
patent laws to
redefine the subject matter of such a claim to specifically exclude such
statutory prior art or
obvious variations of statutory prior art from the scope of such a claim.
Variations of the
invention defined by such amended claims also are intended as aspects of the
invention.
Additional features and variations of the invention will be apparent to those
skilled in the art
from the entirety of this application, and all such features are intended as
aspects of the
invention.
[00102] All publications, patents and patent applications cited in this
specification are
herein incorporated by reference as if each individual publication or patent
application were
specifically and individually indicated to be incorporated by reference.
Although the
foregoing invention has been described in some detail by way of illustration
and example for
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purposes of clarity of understanding, it will be readily apparent to those of
ordinary skill in
the art in light of the teachings of this invention that certain changes and
modifications may
be made thereto without departing from the spirit or scope of the appended
claims.
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