U.S. patent application number 16/654326 was filed with the patent office on 2020-04-02 for inhibitors of proteins specific for the secretome of a chondrocyte for use in the treatment of breast cancer metastasis.
The applicant listed for this patent is ALBERT-LUDWIGS-UNIVERSITAT FREIBURG. Invention is credited to JON CHRISTENSEN, STEFAN GUNTHER, XAVIER LUCAS, PRASAD SHASTRI.
Application Number | 20200103409 16/654326 |
Document ID | / |
Family ID | 48143183 |
Filed Date | 2020-04-02 |
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United States Patent
Application |
20200103409 |
Kind Code |
A1 |
SHASTRI; PRASAD ; et
al. |
April 2, 2020 |
INHIBITORS OF PROTEINS SPECIFIC FOR THE SECRETOME OF A CHONDROCYTE
FOR USE IN THE TREATMENT OF BREAST CANCER METASTASIS
Abstract
The present invention relates to a method for identifying
inhibitors of breast cancer metastasis based on a screening with
proteins that are specific for the secretome of a chondrocyte,
preferably cytokines and/or chemokines. The ligands as identified
lead to a decrease of the migration and/or a re-differentiation of
a breast cancer cell and/or a reduction of the number and/or size
of breast cancer metastases. The present invention further relates
to a method for detecting breast cancer metastasis, comprising the
step of detecting at least one protein that is specific for the
secretome of a chondrocyte, and for methods for treating and/or
preventing breast cancer metastasis in a patient in need thereof,
comprising the step of administering an effective amount of at
least one ligand for one protein that is specific for the secretome
of a chondrocyte to said patient in need thereof.
Inventors: |
SHASTRI; PRASAD; (BREISACH,
DE) ; CHRISTENSEN; JON; (FREIBURG, DE) ;
LUCAS; XAVIER; (DUNDEE, GB) ; GUNTHER; STEFAN;
(FREIBURG, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALBERT-LUDWIGS-UNIVERSITAT FREIBURG |
FREIBURG |
|
DE |
|
|
Family ID: |
48143183 |
Appl. No.: |
16/654326 |
Filed: |
October 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14786099 |
Oct 21, 2015 |
10495642 |
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PCT/EP2014/058264 |
Apr 23, 2014 |
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16654326 |
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61815041 |
Apr 23, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/10 20130101;
G01N 2333/5418 20130101; G01N 2333/5412 20130101; G01N 2333/5421
20130101; G01N 2500/04 20130101; G01N 33/574 20130101; G01N
33/57415 20130101; G01N 2333/522 20130101; C07K 7/08 20130101; A61K
38/00 20130101; G01N 2333/5428 20130101; G01N 33/68 20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61K 38/10 20060101 A61K038/10; G01N 33/68 20060101
G01N033/68; C07K 7/08 20060101 C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 23, 2013 |
EP |
13164922.0 |
Claims
1. A method for identifying inhibitors of breast cancer metastasis,
comprising the steps of: a) providing at least one protein that is
specific for the secretome of a chondrocyte; b) contacting said at
least one protein with at least one putative ligand of said at
least one said protein of the secretome, and c) detecting a binding
between said at least one putative ligand and said at least one
said protein of the secretome.
2. The method according to claim 1, wherein said at least one
protein that is specific for the secretome of a chondrocyte is
selected from the group consisting of cytokines, chemokines, CXCL5,
GCP-2, MIP-3a, NAP-2, IL-6, IL-7, IL-8, IL-10, and GRO-a.
3. The method according to claim 1, further comprising the steps
of: d) in case of a binding of said ligand to said at least one
protein, detecting whether said binding between said ligand to said
at least one protein leads to a decrease of the migration and/or a
re-differentiation of a breast cancer cell and/or a reduction of
the number and/or size of breast cancer metastases.
4. The method according to claim 1, wherein said ligand is an
inhibitor of the expression, stability and/or biological function
of said at least one protein.
5. A ligand for at least one protein that is specific for the
secretome of a chondrocyte, wherein said ligand is an inhibitor of
the expression, stability and/or biological function of said at
least one protein, and is a peptide library molecule, an aptamer, a
combinatory library molecule, a cell extract derived molecule, a
small molecular drug, a bacterial metabolite, a phage display
molecule, an antibody or fragment thereof, a protein, a protein
fragment an aptamer, or an antibody or fragment thereof.
6. The ligand according to claim 5, wherein said at least one
protein is selected from the group consisting of cytokines,
chemokines, CXCL5, GCP-2, MIP-3a, NAP-2, IL-6, IL-7, IL-8, IL-10,
GRO, and GRO-a.
7. The ligand according to claim 5, wherein said ligand is part of
a fusion protein; is part of a carrier molecule that optionally
comprises at least one anticancer agent that is conjugated to said
ligand; is covalently bound to bisphosphonate or other bone-tissue
homing moieties; and/or is part of a diagnostic agent that
optionally comprises at least one detectable moiety.
8. A method for producing a pharmaceutical composition, comprising
formulating at least one ligand of claim 5 with at least one
pharmaceutically acceptable excipient.
9. A pharmaceutical composition, produced according to claim 8,
wherein said ligand is formulated with other ligands, and/or
chemotherapeutically active substances.
10. The pharmaceutical composition according to claim 9, wherein
said ligand is part of a fusion protein; is part of a carrier
molecule that optionally comprises at least one anticancer agent
that is conjugated to said ligand; and/or is part of a diagnostic
agent that optionally comprises at least one detectable moiety.
11. A method for detecting breast cancer metastasis, comprising the
step of detecting at least one protein that is specific for the
secretome of a chondrocyte selected from the group consisting of
chemokines, cytokines, CXCL5, GCP-2, MIP-3a, NAP-2, IL-6, IL-7,
IL-8, IL-10, GRO, and GRO-a in a biological sample obtained from a
subject having primary breast cancer; wherein the presence of said
at least one protein that is specific for the secretome of a
chondrocyte is indicative for breast cancer metastasis and/or an
increased risk for breast cancer metastasis.
12. The method according to claim 11, wherein said detecting
comprises detecting a binding of a ligand for at least one protein
that is specific for the secretome of a chondrocyte, wherein said
ligand is an inhibitor of the expression, stability and/or
biological function of said at least one protein, and is a peptide
library molecule, an aptamer, a combinatory library molecule, a
cell extract derived molecule, a small molecular drug, a bacterial
metabolite, a phage display molecule, an antibody or fragment
thereof, a protein, or a protein fragment.
13. The method according to claim 11, wherein said sample is
selected from blood, plasma, urine, a sample comprising
chondrocytes, and tissue samples.
14. The method, according to claim 3, wherein said breast cancer
metastases are bone and/or lung metastases.
15. The method, according to claim 4, wherein said ligand is a
peptide comprising an amino acid sequence selected from
ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID NO: 2), or is
a ligand that binds to CXCL5 in a similar or the same manner as the
peptide comprising ALWPPNLHAWVP (SEQ ID NO: 1) or AHSVSNSDVLGI (SEQ
ID NO: 2).
16. The ligand, according to claim 5, wherein said ligand is a
peptide comprising an amino acid sequence selected from
ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID NO: 2), or is
a ligand that binds to CXCL5 in a similar or the same manner as the
peptide comprising ALWPPNLHAWVP (SEQ ID NO: 1) or AHSVSNSDVLGI (SEQ
ID NO: 2).
17. A method for inhibiting breast cancer metastasis wherein said
method comprises administering, to a subject in need of such
inhibition, a ligand of claim 5.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation Application of U.S. Ser.
No. 14/786,099, filed Oct. 21, 2015; which is a National Stage
Application of International Application Number PCT/EP2014/058264,
filed Apr. 23, 2014; which claims the benefit of U.S. Provisional
Application No. 61/815,041, filed Apr. 23, 2013, and European
Application No. 13164922.0, filed Apr. 23, 2013; all of which are
incorporated herein by reference in their entirety.
[0002] The Sequence Listing for this application is labeled
"SEQ-9-23-15.TXT", which was created on Sep. 23, 2015, and is 2 KB.
The entire content is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The present invention relates to a method for identifying
inhibitors of breast cancer metastasis based on a screening with
proteins that are specific for the secretome of a chondrocyte,
preferably cytokines and/or chemokines. The ligands as identified
lead to a decrease of the migration and/or a re-differentiation of
a breast cancer cell and/or a reduction of the number and/or size
of breast cancer metastases. The present invention further relates
to a method for detecting breast cancer metastasis, comprising the
step of detecting at least one protein that is specific for the
secretome of a chondrocyte, and for methods for treating and/or
preventing breast cancer metastasis in a patient in need thereof,
comprising the step of administering an effective amount of at
least one ligand for one protein that is specific for the secretome
of a chondrocyte to said patient in need thereof.
BACKGROUND OF THE INVENTION
[0004] Secreted proteins are involved in a variety of physiological
processes, including cell signaling and matrix remodeling, but are
also integral to invasion and metastasis of malignant cells
(Pavlou, Maria P.; Diamandis, Eleftherios P. (2010). "The cancer
cell secretome: A good source for discovering biomarkers?". Journal
of Proteomics 73 (10): 1896-906).
[0005] The secretome is defined as the bulk of peptides, proteins,
growth factors, cytokines and other active substances produced and
secreted by cells. Secretomics has thus been especially important
in the discovery of biomarkers for cancer.
[0006] The two main biological sources for cancer secretomics are
cancer cell line supernatants and proximal biological fluids, the
fluids in contact with a tumor. Cancer cell line supernatant is an
attractive source of secreted proteins, but it is unclear whether a
cell line secretome is a good representation of an actual tumor in
its specific microenvironment and a standardized cell line is not
illustrative of the heterogeneity of a real tumor (Karagiannis,
George S.; Pavlou, Maria P.; Diamandis, Eleftherios P. (2010).
"Cancer secretomics reveal pathophysiological pathways in cancer
molecular oncology". Molecular Oncology 4 (6): 496-510). Using
secretomic analysis of prostate cell lines, one study was able to
discover multiple proteins found in higher levels in the serum of
cancer patients than in healthy controls (Makridakis, Manousos;
Vlahou, Antonia (2010). "Secretome proteomics for discovery of
cancer biomarkers". Journal of Proteomics 73 (12): 2291-305).
[0007] There is also a great need for biomarkers for the detection
of breast cancer--currently biomarkers only exist for monitoring
later stages of cancer. Secretomic analysis of breast cancer cell
lines led to the discovery of the protein ALCAM as a new biomarker
with promising diagnostic potential (Makridakis, Manousos; Vlahou,
Antonia (2010). "Secretome proteomics for discovery of cancer
biomarkers". Journal of Proteomics 73 (12): 2291-305).
[0008] Metastasis, the major cause of death for cancer patients, is
a complex and multistep process in which secondary cancers are
formed in other nonadjacent organs. The development of metastasis
includes several steps that consist of cellular transformation and
abnormal growth, new blood or lymphatic vessels formation,
dissemination of tumor cells into the circulation, attachment to
the target organs and growth in target sites. The migratory and
invasive ability of cancer cells is required in many of these
steps, and is therefore associated with metastasis.
[0009] In addition to cancerous cells, also "normal" cells, such
as, for example, chondrocytes secrete proteins, and thus have a
secretome. In cartilage, the majority of the substances secreted by
chondrocytes in vivo are extracellular matrix (ECM) components,
especially collagen type II and aggrecan. However, there are
numbers of other factors that are secreted as well, like
epithelial-derived neutrophil-activating peptide (ENA-78),
macrophage inflammatory proteins (MIP-1.beta.), epidermal growth
factor (EGF), transforming growth factor beta (TGF-.beta.) and
tissue inhibitors of metalloproteinases (TIMP1 and TIMP 2) (Polacek
M, Bruun J A, Johansen O, Martinez I. Comparative analyses of the
secretome from de-differentiated and re-differentiated adult
articular chondrocytes. Cartilage 2010; and De Ceuninck F,
Dassencourt L, Anract P. The inflammatory side of human
chondrocytes unveiled by antibody microarrays. Biochem Biophys Res
Commun 2004; 323(3):960-969).
[0010] CXCL5 is a small cytokine belonging to the CXC chemokine
family that is also known as epithelial-derived
neutrophil-activating peptide 78 (ENA-78). It is produced following
stimulation of cells with the inflammatory cytokines interleukin-1
or tumor necrosis factor-alpha. CXCL5 is usually associated with
bacterial induced inflammation. The gene for CXCL5 is encoded on
four exons and is located on human chromosome 4 amongst several
other CXC chemokine genes. CXCL5 has been implicated in connective
tissue remodeling.
[0011] Kuo et al. (in: Kuo P L, Chen Y H, Chen T C, Shen K H, Hsu Y
L CXCL5/ENA78 increased cell migration and
epithelial-to-mesenchymal transition of hormone-independent
prostate cancer by early growth response-1/snail signaling pathway.
J Cell Physiol. 2011 May; 226(5):1224-31) describe the analysis of
CXCL5/ENA78, which is highly expressed in androgen-independent
prostate cancers, and is responsible for cell migration and
epithelial-to-mesenchymal transition in two androgen-independent
prostate cancer cell lines. Inducement of PC-3 and suggest that
inhibition of CXCL5/ENA78-mediated ERK/Egr-1/Snail signaling would
an attractive therapeutic target for androgen-independent prostate
cancer.
[0012] US 2008-0206766 relates to compositions and methods for the
detecting, treating, and empirically investigating cellular
proliferation disorders and cellular motility disorders. In
particular, the present invention provides compositions and methods
for using CXCL chemokines (e.g., CXCL1, CXCL5, CXCL6, CXCL12), CXCL
receptors (e.g., CXCR1, CXCR2, CXCR4, CXCR7), and/or pathway
related compounds (e.g., NF-kappaB, ERK 1/2, ELK-1) in the
diagnosis, treatment, and empirical investigation of prostate
disorders (e.g., prostate cancer, benign prostatic hypertrophy,
prostatitis).
[0013] US 2010-004304 describes that chronic inflammation is an
important risk factor for the development of cancer. The
proinflammatory cytokine IL-6 is implicated in cancer because it is
important for the activation of STAT, a key regulator of cancer
growth, survival, metastasis, immune evasion and angiogenesis.
Increased IL-6 and Stat-3 exists in vitro in pancreatic cancer,
malignant melanoma, papillary thyroid cancer, breast cancer, colon
cancer, and prostate cancer cells with high basal expression of
Toll-like receptor 3 (TLR3) and Wnt5a. IL6/STAT3 activation,
mediated by overexpressed TLR3 signaling, appears important in the
tumor growth process; it may increase Wnt5a signaling, and be
associated with increased cellular growth and migration. Using a
novel inhibitor of pathologic TLR3 signaling (5-phenylmethimazole
[C10]) they demonstrated decreases in these markers plus
suppression of cell growth and migration in human pancreatic
cancer, malignant melanoma, papillary thyroid cancer, breast
cancer, colon cancer, and prostate cancer cells.
[0014] The metastasis of a tumor is the dominant contributor to
fatality. In breast and prostate cancer, bone and lung are the
primary sites of metastasis. Therefore, the discovery of molecules
for therapeutic targets that are unique to the bone or lung
environment are critical for the development of new and more
efficient treatment regimens.
BRIEF SUMMARY
[0015] It is therefore an object of the present invention, to
provide new and effective targets and therapeutics based on
secretomics for the diagnosis, prevention and/or treatment of
metastases of breast cancer ("secondary breast cancer"). Other
objects and advantages will become apparent to the person of skill
upon studying the following description and the examples of the
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0016] The following figures, sequences, and examples merely serve
to illustrate the invention and should not be construed to restrict
the scope of the invention to the particular embodiments of the
invention described in the examples. For the purposes of the
present invention, all references as cited in the text are hereby
incorporated in their entireties.
[0017] FIGS. 1A-1B: Analysis of the secretome of chondrocytes for
(1A) cytokines and (1B) chemokines (see also tables and 1 and
2).
[0018] FIGS. 2A-2B: The chondrocyte secretome induces morphological
changes in migrating MBA-MB-231 cells. 2A) cell shape factors of
three samples of chondrocytes (AC) after the addition of
chondrocyte-conditioned medium. 2B) Photograph of the morphological
changes of sample AC03.
[0019] FIGS. 3A-3B: The Inhibition of CXCL5/ENA-78 in chondrocyte
secretome reverse (rescues) morphology of MDA-MB-231 cells. 3A)
reduction of migration of chondrocytes (AC) after the addition of
an inhibitor of CXCL5/ENA-78. 3B) Photograph of the morphological
changes of sample AC03.
[0020] FIGS. 4A-4B: Inhibition of CXCL5/ENA-78 in chondrocyte
secretome reduces migration of MDA-MB-231 cells. 4A) Effect of
peptide 001417C; 4B) Effect of peptide 001418C.
[0021] FIG. 5: Chondrocyte-conditioned medium promotes mitogentic
signaling through PI3K/AKT pathway in MCF7A--Western blot showing
activation of AKT and ERK1/2 pathway in MCF7A upon treatment with
chondrocyte-conditioned medium (AC CM).
[0022] FIG. 6: Inhibition of AC CM induced proliferation in MCF7A
using PI3K inhibitors (e.g. LY294002).
[0023] FIG. 7: Primary amino acid sequence of CXCL5 (SEQ ID NO: 3).
Residues in contact with peptide #1 (SEQ ID NO: 1 at 4 Angstroms)
are underlined.
[0024] FIG. 8: Screenshot of the CXCL5 surface colored in white and
with the region in contact with the peptide #1 (SEQ ID NO: 1)
highlighted in gray. Peptide #1, in the proposed binding mode, is
shown in balls and sticks.
[0025] FIGS. 9A-9C: Inhibition data mAB: Inhibition of chondrocyte
secreted CXCL5 inhibits migration and invasion of MDA-MB-231 breast
cancer cells. 9A) Percent reduction of MDA-MB-231 cells migrating
towards chondrocyte conditioned media, with increasing amount of
CXCL5 inhibitory antibody (Ab), n=4. 9B) Inhibition of CXCL5 does
not reduce the migration of MDA-MB-231 cell when osteoblast
conditioned media is used as chemoattractant, n=3. 9C) Filters were
coated with Matrigel and MDA-MB-231 cells were allowed to invade
through the matrix. Chondrocyte conditioned media was used as
chemoattractant supplemented with CXCL5 inhibitory antibody.
[0026] FIGS. 10A-10D: Inhibition data InhPep: CXCL5 binding
peptides can inhibit migration and invasion of MDA-MB-231 cells
towards chondrocyte conditioned media. 10A) Migration of MDA-MB-231
cells towards chondrocyte conditioned media supplemented with CXCL5
binding peptides InhPEP-1 or InhPEP2, n=3. 10B) Migration of
MDA-MB-231 cells towards osteoblast conditioned media supplemented
CXCL5 binding peptides InhPEP-1 or InhPEP2, n=3. 10C) Cell shape
analysis of MDA-MB-231 cells that migrated towards chondrocyte
conditioned media supplemented with CXCL5 binding peptides InhPEP-1
or InhPEP2. 10D) Filters were coated with Matrigel and MDA-MB-231
cells were allowed to invade through the matrix. Chondrocyte
conditioned media was used as chemoattractant supplemented with
CXCL5 binding peptides InhPEP-1 or InhPEP2.
DETAILED DESCRIPTION
[0027] According to a first aspect thereof, the object of the
present invention is solved by providing a method for identifying
inhibitors of breast cancer metastasis, comprising the steps of: a)
providing at least one protein that is specific for the secretome
of a chondrocyte; b) contacting said at least one protein with at
least one putative ligand of said at least one said protein of the
secretome, and c) detecting a binding between said at least one
putative ligand and said at least one said protein of the
secretome.
[0028] The term "ligand" in context of the present invention shall
be understood in the broadest sense, and specifically shall include
compounds that target the cellular/biological/molecular function of
the proteins. Of course the term "ligand" also includes any kind of
molecule binding to the proteins of interest, and thereby
modulating the proteins stability and/or function, such as, for
example inhibiting a signaling pathway of at least one protein that
is specific for the secretome of a chondrocyte, such as, for
example, the PI3K/AKT pathway in case of CXCL5 signaling.
[0029] ENA-78 also referred to as CXCL5 is usually associated with
bacterial induced inflammation. In the context of the present
invention, it was found that this molecule is also secreted by
chondrocytes that can produce hyaline cartilage, and that the
inhibition of ENA-78 using, for example, both purified monoclonal
antibody and peptides obtained through phage display screening can
inhibit the migration of cancer cells induced by the cartilage
secretome. Thus, the mature chondrocyte secretome provides a
preferable environment for breast cancer metastasis formation,
through secretion of chemotatic and mitogenic signals. Migration of
MDA-MB-231 cells could be inhibited with monoclonal antibodies
(mAb) and proliferation of MCF7A cell could be abrogated by PI3K
inhibitor (see examples).
[0030] Hsu et al (in: Hsu Y L, Hon M F, Kuo P L, Huang Y F, Tsai E
M. Breast tumor-associated osteoblast-derived CXCL5 increases
cancer progression by ERK/MSK1/Elk-1/Snail signaling pathway.
Oncogene. 2012 Oct. 8 (online)) describe a study that analyzes the
soluble factors secreted by breast tumor-associated osteoblasts
(TAOBs), which are described to be responsible for promoting cancer
progression. They showed that the addition of CXCL5 did not
increase cell proliferation in either MCF-7 or MDA-MB-231 cancer
cells, but did enhance the migration and invasiveness of MCF-7 and
MDA-MB-231 as well as 4T1 cancer cell lines. Treatment of mice by
CXCL5 (0.5 mg/kg) increased the metastasis of 4T1 cells. Tumor
nodules in various organs (livers, lungs and intestines) of the
CXCL5-treated mice were both more numerous and larger than those
found in the control group of mice. Finally, treatment of mice by
anti-CXCL5 monoclonal antibody inhibited the metastasis of 4T1
cells in 62.5% (5 of 8) of the mice, in comparison with 100% (8 of
8) of the control mice. The tumor nodules in various organs
(livers, lungs and intestines) of the CXCL5 antibody-treated mice
were far fewer than those found in the control group mice. The
authors conclude that CXCL5 may be a major factor enhancing the
metastatic ability of breast cancer cells, and that anti-CXCL5
strategies may be used to target metastasis in breast cancer.
[0031] Hsu et al. showed that tumor nodules in livers, lungs and
intestines of the CXCL5 antibody-treated mice were far fewer than
those found in the control group mice, nevertheless, no effect
could be seen in ribs (i.e. bones). Furthermore, regarding the
cancer cell line 4T1, Pulaski and Ostrand-Rosenberg (in: Mouse 4T1
breast tumor model. Curr Protoc Immunol. 2001 May; Chapter 20: Unit
20.2) describe the 4T1 mammary carcinoma as a transplantable tumor
cell line that is highly tumorigenic and invasive and, unlike most
tumor models, can spontaneously metastasize from the primary tumor
in the mammary gland to multiple distant sites including lymph
nodes, blood, liver, lung, brain, and bone. Thus, the progressive
spread of 4T1 metastases to the draining lymph nodes and other
organs is very similar to that of human mammary cancer.
Nevertheless, Hsu et al. did not show bone metastases in the mouse
model.
[0032] In contrast to Hsu et al., the present invention is based on
the surprising finding that the chondrocyte secretome possesses
molecules that induce breast cancer cell migration and metastasis.
Chondrocytes are markedly different from osteoblasts, and are a
critical component of the cartilage and bone environment and play a
role in the renewal and repair of fractures. Thus, the present
approach represents a hitherto unknown niche in bone for tumor
metastasis.
[0033] Preferred is a method according to the present invention,
wherein said at least one protein that is specific for the
secretome of a chondrocyte is selected from the group consisting of
cytokines and chemokines, and more preferably selected from the
group consisting of CXCL5, human granulocyte chemotactic protein-2
(GCP-2 or CXCL6), macrophage inflammatory protein-3 alpha (MIP-3a
or CCL20), neutrophil-activating protein-2 (NAP-2), interleukin-6
(IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10
(IL-10), and chemokine (C-X-C motif) ligand 1 (CXCL1, GRO-alpha)
(hereinafter also designated as "marker", "markers", "gene" or
"genes"). Preferred are GCP-2, GRO-a, MIP3a, CXCL5, NAP-2, IL-7,
and IL-10.
[0034] Particularly preferred is a method according to the present
invention, wherein said at least one protein that is specific for
the secretome of a chondrocyte is CXCL5. It was surprisingly found
in the context of the present invention that a particular region of
the protein CXCL5 is suitable for a screening according to the
present invention ("pocket", see FIGS. 7 and 8). Therefore, another
aspect of the present invention relates to a method according to
the present invention, wherein the protein used for screening is
CXCL5 and screening is performed in order to identify identifying
potential inhibitors of breast cancer metastasis that are ligands
of the pocket of CXCL5. Preferably, said screening using CXCL5 is
performed with CXCL5 (see SEQ ID No: 3) as a dimer. In the context
of said screening and as an example, two peptides comprising (or
consisting of) the amino acid sequence selected from ALWPPNLHAWVP
(SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID NO: 2) were identified that
bind to said region. Another aspect of the present invention
therefore relates to the CXCL5-related method as described that is
used in order to identify potential binding compounds as described
herein (e.g. small molecular drugs) that bind to CXCL5 in a similar
or the same manner as the peptides comprising (or consisting of)
the amino acid sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1)
and AHSVSNSDVLGI (SEQ ID NO: 2). Encompassed is also a competitive
screening, where the potentially binding compounds competes with
one (or both) of the peptides for a binding to CXCL5, and in
particular for the region as described.
[0035] The term "contacting" in the context of the present
invention means any interaction between the potentially binding
substance(s) with said at least one protein that is specific for
the secretome of a chondrocyte, whereby any of the two components
can be independently of each other in a liquid phase, for example
in solution, or in suspension or can be bound to a solid phase, for
example, in the form of an essentially planar surface or in the
form of particles, pearls or the like.
[0036] In a preferred embodiment, a multitude of different
potentially binding substances are immobilized on a solid surface
like, for example, on a compound library chip and said at least one
protein that is specific for the secretome of a chondrocyte (or a
functional part thereof) is subsequently contacted with such a
chip. Then, a binding between said at least one putative ligand and
said at least one protein that is specific for the secretome of a
chondrocyte is detected.
[0037] The above method can be performed simultaneously (i.e. in
one screening reaction) or in parallel (in separate screenings)
with one or more of said at least one protein that is specific for
the secretome of a chondrocyte, and thus several genes can be
arranged to be screened as a "panel". Preferred is a panel with all
markers, other preferred panels include 2, 3 or more markers. For
reasons of handling, the markers can be pre-screened in pooled
fractions of compounds (ligands), and then analyzed further in
individual reactions. Based on pre-screenings, also panels of the
above genes can be combined that show similar binding
characteristics. In this way, different panels can be designed for
different ligands (or mixtures of ligands). Particularly preferred
is a screening with CXCL5 (e.g. as described above) and/or
GRO-alpha.
[0038] Preferred is a method according to the present invention,
wherein said identifying is a screening and takes place in vitro or
in vivo.
[0039] The gene product employed in a method of the present
invention can be a full length protein (marker) or a fragment with
N/C-terminal and/or internal deletions. Preferably the fragment is
either an N-terminal fragment or a C-terminal fragment comprising
the cytoplasmatic region, depending on whether potentially
interacting compounds are sought that specifically interact with
the N- or C-terminal fragment.
[0040] The potentially binding substance, whose binding to the
markers is to be measured, can be any chemical substance or any
mixture thereof. For example, it can be a substance selected from
the group of a peptide library molecule, an aptamer, a combinatory
library molecule, a cell extract derived molecule, a small
molecular drug, a bacterial metabolite, a phage display molecule,
an antibody or fragment thereof, a protein, a protein fragment, and
combinations thereof, and preferably a peptide comprising the amino
acid sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and
AHSVSNSDVLGI (SEQ ID NO: 2), an aptamer, and/or an antibody or
fragment thereof. In the context of the present invention, a small
molecular drug is a low molecular weight (with a maximum weight of
approx. of 800 Daltons) organic compound that serves as a ligand.
The upper molecular weight limit for a small molecule is
approximately 800 Daltons which allows for the possibility to
rapidly diffuse across cell membranes so that they can reach
intracellular sites of action. In addition, this molecular weight
cutoff is a necessary but insufficient condition for oral
bioavailability. Preferred is a lower maximum molecular weight of
500 Daltons based on the observation that clinical attrition rates
are significantly reduced if the molecular weight is kept below
this 500 Dalton limit.
[0041] Measuring of binding of the compound to the at least one
marker can be carried out either by measuring a marker or label
that can be attached either to the protein or to the potentially
interacting compound. Suitable markers or labels are known to
someone of skill in the art and comprise, for example, fluorescence
or radioactive markers. The binding of the two components can,
however, also be measured by the change of an electrochemical
parameter of the binding compound or of the protein, e.g. a change
of the redox properties of either the marker or the binding
compound, upon binding. Suitable methods of detecting such changes
comprise, for example, potentiometric methods. Further methods for
detecting and/or measuring the binding of the two components to
each other are known in the art and can without limitation also be
used to measure the binding of the potential interacting compound
to the at least one marker or fragments thereof.
[0042] Then, preferred is a method according to the present
invention, further comprising the steps of: d) in case of a binding
of said ligand to said at least one protein, detecting, if said
binding between said ligand to said at least one protein leads to a
decrease of the migration and/or a re-differentiation of a breast
cancer cell and/or a reduction of the number and/or size of breast
cancer metastases. Methods to detect these effects of the ligand(s)
are known to the person of skill, and preferred methods are
described in the examples below, and in the literature as cited
herein. If a decrease of the migration and/or a re-differentiation
of a breast cancer cell and/or a reduction of the number and/or
size of breast cancer metastases is detected, the ligand is
selected for further analysis and improvement, if required.
[0043] Preferred is a method according to the present invention,
wherein said identifying is performed with at least two of said
proteins simultaneously, or in parallel. Most preferred is an
identification based on the markers CXCL5 and/or GRO-alpha.
[0044] Further preferred is a method according to the present
invention, wherein breast cancer metastases are bone and/or lung
metastases.
[0045] The method according to the present invention identifies,
for example, ligands selected from an inhibitor of the expression,
stability and/or biological function of said at least one protein
that is specific for the secretome of a chondrocyte. In the context
of the present invention, the term "protein that is specific for
the secretome of a chondrocyte" relates to secreted molecules that
are either uniquely and/or predominantly (i.e. in higher amounts
than in other cells) secreted by chondrocytes, when compared with
osteoblasts (OBs), osteoclasts (OCs), and/or bone marrow derived
stromal cells (BMCs). As examples, for chondrocytes, uniquely
secreted are at least GCP-2, GRO-a, MIP3a, CXCL5, NAP-2, IL-7, and
IL-10, and predominantly secreted are at least IL-8 and IL-6.
[0046] The method according to the present invention also
encompasses several rounds of screening in order to detect, if said
ligand is active on several breast cancer cell lines, and/or is
binding to several markers as described. Also combinations of
ligands can be screened jointly or separately, in order to identify
suitable combinations of preferably synergistically active
combinations of ligands that inhibit the markers.
[0047] The thus selected binding compound(s) (ligands) is then in a
preferred embodiment modified in a further step. Modification can
be effected by a variety of methods known in the art, which include
without limitation the introduction of novel side chains or the
exchange of functional groups like, for example, introduction of
halogens, in particular F, Cl or Br, the introduction of lower
alkyl groups, preferably having one to five carbon atoms like, for
example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
tert-butyl, n-pentyl or iso-pentyl groups, lower alkenyl groups,
preferably having two to five carbon atoms, lower alkynyl groups,
preferably having two to five carbon atoms or through the
introduction of, for example, a group selected from the group
consisting of NH2, NO2, OH, SH, NH, CN, aryl, heteroaryl, COH or
COOH group.
[0048] The modified binding substances are than individually tested
with a method of the present invention, i.e. they are contacted
with the markers again, and subsequently binding of the modified
compounds to the marker polypeptide is measured. In this step, both
the binding per se can be measured and/or the effect on the
migration and/or a re-differentiation of a breast cancer cell
and/or a reduction of the number and/or size of breast cancer
metastases. If needed, the steps of selecting the binding compound,
modifying the binding compound, contacting the binding compound
with a marker polypeptide and measuring the binding of the modified
compounds to the protein can be repeated a third or any given
number of times as required. The above described method is also
termed "directed evolution", since it involves a multitude of steps
including modification and selection, whereby binding compounds are
selected in an "evolutionary" process optimizing its capabilities
with respect to a particular property, e.g. its binding activity,
its ability to activate, inhibit or modulate the activity of the
protein.
[0049] As mentioned above, all assays as described herein can be
performed either in vitro and/or in vivo: Preferred are in vitro
assays.
[0050] The method according to the present invention can
furthermore encompass a step of detecting and/or measuring the
sensitivity to an anti-breast cancer treatment using a protein that
is specific for the secretome of a chondrocyte in the absence and
presence of the ligand as identified according to the present
invention (see also below in the context of diagnosis). The results
of such a detection and/or measurement can be used either in order
to adjust an already begun anti-cancer treatment using a ligand,
e.g. by increasing or decreasing the dosage, and/or to determine,
whether a resistance against a ligand has already developed or will
develop.
[0051] Yet another aspect of the present invention then relates to
a ligand for at least one protein that is specific for the
secretome of a chondrocyte according to the present invention,
wherein said ligand is selected from the group of an inhibitor,
activator, competitor or modulator of the expression and/or
biological function of said at least one protein, and is preferably
a substance selected from the group of a peptide library molecule,
an aptamer, a combinatory library molecule, a cell extract derived
molecule, a small molecular drug, a bacterial metabolite, a phage
display molecule, an antibody or fragment thereof, a protein, a
protein fragment, and combinations thereof, and preferably a
peptide comprising the amino acid sequence selected from
ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID NO: 2), an
aptamer, and/or an antibody or fragment thereof. Preferably, said
antibody is a human, humanized, mouse or chimeric antibody. Further
preferred is a ligand that binds to CXCL5 in a similar or the same
manner (e.g. in the same pocket as described herein) as the
peptides comprising (or consisting of) the amino acid sequence
selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID
NO: 2).
[0052] As mentioned, preferred is a ligand that is an inhibitor of
the expression and/or biological function of said at least one
protein. More preferred is a ligand that is an inhibitor of the
expression and/or biological function of at least two, three, four
or five proteins, even more preferred is a ligand that is an
inhibitor of the expression and/or biological function of all
proteins as described herein. Of course, the activities and/or
affinities of a chosen ligand with respect to the different
proteins can vary between markers and ligands.
[0053] The term "inhibitor" includes any molecule interfering with
the expression and/or function of the above proteins. Preferred
are, for example RNAi, based molecules to target protein
expression, or molecules binding to the proteins and thereby
inhibiting their biological function(s). The latter can be
antibodies targeting the proteins, peptides or small molecules,
preferably as described and/or screened herein. Specifically
preferred are all kinds of cytokine and/or chemokine
inhibitors.
[0054] Preferred is a ligand according to the present invention,
wherein said at least one protein is selected from the group
consisting of chemokines, cytokines, CXCL5, GCP-2, MIP-3a, NAP-2,
IL-6, IL-7, IL-8, IL-10, GRO, and GRO-a, and preferably CXCL5 and
GRO-alpha.
[0055] Preferred is the ligand according to the present invention,
wherein said ligand is part of a fusion protein, is part of a
carrier molecule that optionally comprises at least one anticancer
agent, such as, for example, a chemotherapeutic, peptide, small
molecule drug, and/or radionucleotide that is conjugated to said
ligand, is covalently bound to bisphosphonate or other bone-tissue
homing moieties, preferably via a linker that is enzymatically
cleaved in the bone-tissue environment, and/or is part of a
diagnostic agent that optionally comprises at least one detectable
moiety. Preferred is a peptide comprising the amino acid sequence
selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID
NO: 2) that is coupled or conjugated to a chemotherapeutic
substance, such as, for example, at least one anticancer agent,
such as, for example, a cytotoxic moiety. The linker can be one
that is cleaved by cathepsins and specifically cathepsin-K, or one
that is cleaved by MMP-9 and/or MMP-2.
[0056] Yet another aspect of the present invention then relates to
method for producing a pharmaceutical composition, comprising the
steps of: a) optionally, performing a method according to the
present invention as above, and b) formulating said at least one
ligand as identified, or a ligand according to the present
invention as described above, with at least one pharmaceutically
acceptable excipient.
[0057] Thus, in a further embodiment of the methods of the present
invention, the interacting compound (ligand) identified as outlined
above, which may or may not have gone through additional rounds of
modification and selection, is admixed with suitable auxiliary
substances and/or additives. Such substances comprise
pharmacological acceptable substances, which increase the
stability, solubility, biocompatibility, or biological half-life of
the interacting compound or comprise substances or materials, which
have to be included for certain routs of application like, for
example, intravenous solution, tablets, injectables, sprays,
band-aids or pills.
[0058] Carriers, excipients and strategies to formulate a
pharmaceutical composition, for example to be administered
systemically or topically, by any conventional route, in particular
enterally, e.g. orally, e.g. in the form of tablets or capsules,
parenterally, e.g. in the form of injectable solutions or
suspensions, topically, e.g. in the form of lotions, gels,
ointments or creams, or in nasal or a suppository form are well
known to the person of skill and described in the respective
literature.
[0059] Administration of an agent, e.g., a compound can be
accomplished by any method which allows the agent to reach the
target cells, such as metastatic breast cancer cells. These methods
include, e.g., injection, deposition, implantation, suppositories,
oral ingestion, inhalation, topical administration, or any other
method of administration where access to the target cells by the
agent is obtained. Injections can be, e.g., intravenous,
intradermal, subcutaneous, intramuscular or intraperitoneal.
Implantation includes inserting implantable drug delivery systems,
e.g., microspheres, hydrogels, polymeric reservoirs, cholesterol
matrices, polymeric systems, e.g., matrix erosion and/or diffusion
systems and non-polymeric systems, e.g., compressed, fused or
partially fused pellets. Suppositories include glycerin
suppositories. Oral ingestion doses can be enterically coated.
Inhalation includes administering the agent with an aerosol in an
inhalator, either alone or attached to a carrier that can be
absorbed. The agent can be suspended in liquid, e.g., in dissolved
or colloidal form. The liquid can be a solvent, partial solvent or
non-solvent. In many cases, water or an organic liquid can be
used.
[0060] Yet another aspect of the present invention is directed at a
pharmaceutical composition for treating or preventing breast cancer
metastasis, obtainable by a method according to the invention as
above. Another aspect of the present invention then relates to the
pharmaceutical composition as above, wherein said pharmaceutical
composition further comprises additional pharmaceutically active
ingredients, for example, anti-cancer chemotherapeutics. Thus, the
ligand according to the invention is for administration in
combination with other ligands, and/or chemotherapeutically active
substances, such as anti-cancer chemotherapeutics.
[0061] Preferred is a pharmaceutical composition according to the
present invention, wherein said ligand of the at least one marker
is selected from an inhibitor, activator, competitor or modulator
of the expression and/or biological function of said at least one
marker, and is preferably a substance selected from the group of a
peptide library molecule, an aptamer, a combinatory library
molecule, a cell extract derived molecule, a small molecular drug,
a bacterial metabolite, a phage display molecule, an antibody or
fragment thereof, a protein, a protein fragment, and combinations
thereof, and preferably a peptide comprising the amino acid
sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI
(SEQ ID NO: 2), an aptamer, and/or an antibody or fragment thereof.
Preferably, said antibody is a human, humanized, mouse or chimeric
antibody. Further preferred is a ligand that binds to CXCL5 in a
similar or the same manner (e.g. in the same pocket as described
herein) as the peptides comprising (or consisting of) the amino
acid sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and
AHSVSNSDVLGI (SEQ ID NO: 2).
[0062] Further preferred is a pharmaceutical composition according
to the present invention, wherein said ligand of the at least one
marker is part of a fusion protein, is part of a carrier molecule
that optionally comprises at least one anticancer agent, such as,
for example, a chemotherapeutic, peptide, small molecule drug,
and/or radionucleotide that is conjugated to said ligand, and/or is
part of a diagnostic agent that optionally comprises at least one
detectable moiety. Preferred is a peptide comprising the amino acid
sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI
(SEQ ID NO: 2) that is coupled or conjugated to a chemotherapeutic
substance, such as, for example, at least one anticancer agent,
such as, for example, a cytotoxic moiety.
[0063] Yet another aspect of the present invention then relates to
a ligand for at least one protein that is specific for the
secretome of a chondrocyte according to the present invention or
the pharmaceutical composition according to the present invention
for use in the diagnosis of diseases and/or for use in the
prevention and/or treatment of diseases. Preferred is the ligand or
the pharmaceutical composition for use according to the present
invention, wherein said disease to be prevented and/or treated is
selected from breast cancer, breast cancer metastases, in
particular breast cancer-derived bone metastases, and breast
cancer-derived lung metastases.
[0064] Preferred is the ligand for use according to the present
invention, wherein said ligand of the at least one marker is
selected from an inhibitor, activator, competitor or modulator of
the expression and/or biological function of said at least one
marker, and is preferably a substance selected from the group of a
peptide library molecule, an aptamer, a combinatory library
molecule, a cell extract derived molecule, a small molecular drug,
a bacterial metabolite, a phage display molecule, an antibody or
fragment thereof, a protein, a protein fragment, and combinations
thereof, and preferably a peptide comprising the amino acid
sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI
(SEQ ID NO: 2), an aptamer, and/or an antibody or fragment thereof.
Preferably, said antibody is a human, humanized, mouse or chimeric
antibody. Further preferred is a ligand that binds to CXCL5 in a
similar or the same manner (e.g. in the same pocket as described
herein) as the peptides comprising (or consisting of) the amino
acid sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and
AHSVSNSDVLGI (SEQ ID NO: 2).
[0065] Further preferred is a ligand for use according to the
present invention, wherein said ligand of the at least one marker
is part of a fusion protein, is part of a carrier molecule that
optionally comprises at least one anticancer agent, such as, for
example, a chemotherapeutic, peptide, small molecule drug, and/or
radionucleotide that is conjugated to said ligand, and/or is part
of a diagnostic agent that optionally comprises at least one
detectable moiety. Preferred is a peptide comprising the amino acid
sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI
(SEQ ID NO: 2) that is coupled or conjugated to a chemotherapeutic
substance, such as, for example, at least one anticancer agent,
such as, for example, a cytotoxic moiety.
[0066] Further preferred is the ligand for use according to the
present invention, wherein said ligand is administered systemically
and/or administered locally. Even further preferred is the ligand
for use according to the present invention, wherein said ligand is
for administration in combination with other chemotherapeutically
active substances, such as anti-cancer chemotherapeutics as
described above.
[0067] Yet another aspect of the present invention then relates to
a method for treating or preventing breast cancer metastasis in a
patient in need thereof, comprising the step of administering an
effective amount of at least one ligand for one protein that is
specific for the secretome of a chondrocyte or of a pharmaceutical
preparation according to the present invention to said patient in
need thereof.
[0068] Preferably, an active agent (ligand) is administered in the
form of a pharmaceutical composition, such as an antibody, peptide,
or a binding compound. Preferably, said patient is a human being or
a domesticated animal. Treating is meant to include, e.g.,
preventing, treating, reducing the symptoms of, or curing the
disease or condition. The invention also includes a combination
comprising (a) a ligand according to the present invention for a
first marker and (b) at least one ligand according to the present
invention for a second marker, for use in the treatment/prevention
of breast cancer metastasis. Preferred agents (a) and (b) are
described in the present application. The combinatorial use of
ligands inhibiting the above mentioned proteins yields surprising
synergistic results that are translated into new therapeutic uses
by the herein disclosed invention.
[0069] An "effective amount" is an amount of the ligand(s) as
mentioned above that acts on the at least one protein and decreases
the migration and/or a re-differentiation of a breast cancer cell
and/or a reduction of the number and/or size of breast cancer
metastases, and thus alleviates symptoms as found for the disease.
Alleviating is meant to include, e.g., preventing, treating,
reducing the symptoms of, or curing the disease or condition. Thus,
preferred is a method according to the present invention, wherein
said administering of said ligand to said at least one protein
decreases the migration and/or a re-differentiation of a breast
cancer cell and/or reduces the number and/or size of breast cancer
metastases. Preferably, said breast cancer metastases to be treated
are bone and/or lung metastases.
[0070] Preferred is a method according to the present invention,
wherein said at least one protein that is specific for the
secretome of a chondrocyte is selected from the group consisting of
cytokines, chemokines, CXCL5, GCP-2, MIP-3a, NAP-2, IL-6, IL-7,
IL-8, IL-10, GRO, and GRO-a.
[0071] Preferred is a method according to the present invention,
wherein, wherein said ligand is selected from an inhibitor of the
expression, stability and/or biological function of said at least
one protein, and is preferably selected from a peptide library
molecule, an aptamer, a combinatory library molecule, a cell
extract derived molecule, a small molecular drug, a bacterial
metabolite, a phage display molecule, an antibody or fragment
thereof, a protein, a protein fragment, and combinations thereof,
and preferably a peptide comprising an amino acid sequence selected
from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID NO: 2),
an aptamer, and/or an antibody or fragment thereof. Further
preferred is a ligand that binds to CXCL5 in a similar or the same
manner (e.g. in the same pocket as described herein) as the
peptides comprising (or consisting of) the amino acid sequence
selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI (SEQ ID
NO: 2).
[0072] Further preferred is a method according to the present
invention, wherein said ligand of the at least one marker is part
of a fusion protein, is part of a carrier molecule that optionally
comprises at least one anticancer agent, such as, for example, a
chemotherapeutic, peptide, small molecule drug, and/or
radionucleotide that is conjugated to said ligand, and/or is part
of a diagnostic agent that optionally comprises at least one
detectable moiety. Preferred is a peptide comprising the amino acid
sequence selected from ALWPPNLHAWVP (SEQ ID NO: 1) and AHSVSNSDVLGI
(SEQ ID NO: 2) that is coupled or conjugated to a chemotherapeutic
substance, such as, for example, at least one anticancer agent,
such as, for example, a cytotoxic moiety.
[0073] Even further preferred is a method according to the present
invention, wherein said ligand is for administration in combination
with other chemotherapeutically active substances, such as
anti-cancer chemotherapeutics, as described above.
[0074] Another aspect of the present invention is based on the fact
that bone matrix remodeling occurs throughout the lifetime of an
individual. The remodeling of long bone occurs via a process
involving endochondral ossification, wherein the formation of a
cartilage callus is a necessary first step. Such cartilaginous
environment is also associated with healing of micro-fractures.
Furthermore, long bones constitute the primary targets of breast
cancer metastasis. Chondrocytes and their activities during the
formation of cartilage play an essential role in this process.
[0075] Interestingly, Ulici et al. (in: Ulici V, Hoenselaar K D,
Gillespie J R, Beier F. The PI3K pathway regulates endochondral
bone growth through control of hypertrophic chondrocyte
differentiation. BMC Dev Biol. 2008 Apr. 11; 8:40) describe an
essential role of PI3K signaling in chondrocyte differentiation and
as a consequence of this, in the endochondral bone growth
process.
[0076] Yet another aspect of the present invention then relates to
methods of screening for ligands for preventing and/or treating
tumor metastasis, in particular in breast cancer, by inhibiting
and/or limiting the formation of cartilaginous entities within long
bones of a patient, for example, by inhibiting and/or limiting the
processes involved in endochondral ossification. The screening can
be done in analogy to the methods as described herein, and
preferably involves the use of chondrocytes and/or targets
chondrocyte-specific functions in the formation of cartilaginous
entities in vivo and/or in vitro.
[0077] Another aspect of this invention then relates to a method
for the prevention and/or treatment of tumor metastasis, in
particular in breast cancer, by limiting or inhibiting the
formation of cartilaginous entities within long bones in analogy to
the methods as described herein. Another aspect of this invention
then relates to the development of targeted therapeutics to be
directed at and for detecting the cartilaginous moieties within
long bones of a patient, for example, by inhibiting and/or limiting
or detecting the processes involved in endochondral ossification,
again in analogy to the methods, to the ligands and to the
pharmaceutical preparations as described herein.
[0078] Yet another aspect of the present invention then relates to
a method for detecting breast cancer metastasis, comprising the
step of detecting at least one protein that is specific for the
secretome of a chondrocyte selected from the group consisting of
cytokines, chemokines, CXCL5, GCP-2, MIP-3a, NAP-2, IL-6, IL-7,
IL-8, IL-10, GRO, and GRO-a in a biological sample obtained from a
subject having primary breast cancer; wherein the presence of said
at least one protein that is specific for the secretome of a
chondrocyte is indicative for breast cancer metastasis and/or an
increased risk for breast cancer metastasis.
[0079] Preferred is a method according to the present invention,
wherein said detecting comprises detecting of a binding of a ligand
according to the present invention, preferably coupled to a
detectable moiety as described above. Suitable markers or labels
that can be attached either to the protein or to the ligand are
known to someone of skill in the art and comprise, for example,
fluorescence or radioactive markers. The binding of the two
components can, however, also be measured by the change of an
electrochemical parameter of the binding compound or of the
protein, e.g. a change of the redox properties of either the marker
or the binding compound, upon binding. Suitable methods of
detecting such changes comprise, for example, potentiometric
methods. Further methods for detecting and/or measuring the binding
of the two components to each other are known in the art and can
without limitation also be used to measure the binding of the
potential interacting compound to the at least one marker or
fragments thereof.
[0080] The results of such a detection and/or measurement can also
be used either in order to adjust an already begun anti-cancer
treatment using a ligand or a pharmaceutical composition according
to the present invention, e.g. by increasing or decreasing the
dosage, and/or to determine, whether a resistance against a ligand
according to the present invention has already developed or will
develop.
[0081] Preferred is a method according to the present invention,
wherein said metastases are bone and/or lung metastases.
[0082] Methods to detect binding are well known to the person of
skill and may comprise methods involving cell sorting, marker
antibody-based assays, gel analyses, protein or nucleic acid based
blots, rtPCR, and/or chip analyses.
[0083] Preferred is a method according to the present invention,
wherein said sample is selected from a blood, plasma, urine, a
sample comprising chondrocytes, and a tissue sample, such as, for
example, a biopsy comprising breast, bone, cartilage, lung, liver,
brain or tumor tissue.
[0084] Another aspect of the present invention then relates to a
screening tool for a ligand for at least one protein that is
specific for the secretome of a chondrocyte, wherein said tool is a
cell which recombinantly expresses at least one product of a gene
selected from the group consisting of selected from the group
consisting of cytokines, chemokines, CXCL5, GCP-2, MIP-3a, NAP-2,
IL-6, IL-7, IL-8, IL-10, GRO, and GRO-a. The expression constructs
can be present extrachromosomally or integrated into the
chromosome. The marker polypeptide (or part thereof) can be
expressed in the form of a fusion protein, for example together
with an enzymatically active moiety as reporter-construct, in order
to be able to detect the expression product.
[0085] The screening tool can be part of a kit, optionally
comprising additional compounds and instructions for performing the
methods as described herein.
[0086] Preferred is the screening tool according to the present
invention, wherein said cell is a chondrocyte in a non-human
transgenic mammal. Preferred is a transgenic mouse, rat, pig,
monkey, goat, cow or sheep. Methods to produce these non-human
transgenic mammals are well known to the person of skill in the
art.
[0087] Similar to the kit including a screening tool according to
the present invention, diagnostic kits can be designed that include
diagnostic tools, which are cells and/or animals as above. The
diagnostic tool can be part of a kit, optionally comprising
additional compounds and instructions for performing the methods as
described herein.
EXAMPLES
[0088] The present methods have been performed with CXCL5 as an
example, but can be used for other proteins of the secretome of
chondrocytes as well, such as, for example GCP-2, MIP-3a, NAP-2,
IL-6, IL-7, IL-8, IL-10, GRO, and/or GRO-a.
[0089] Cell differentiation: Primary human monocytes, articular
chondrocytes, and adipose tissue derived stromal cells were
isolated from healthy donors and differentiated into osteoclasts
(OC), articular chondrocytes (AC) and osteoblasts (OB)
respectively. Cell lines: MDA-MB-231 (MDA), a metastatic, and
MCF7A, an epithelial like breast cancer cell line, were used in
this study.
[0090] Serum free conditioned media: After differentiation, cells
were washed 2 times with PBS and incubated with DMEM for 24 hours.
After 24 hours, conditioned media was harvested and passed through
a 0.45 .mu.m filter.
[0091] Migration: MDA-MB-231 cells were serum-starved for 24 hours
before testing of the migration. Migration media was plated in the
bottom of the well and a transwell insert (8 .mu.m pores) was
placed on top. 25.000 cells were seed on top of the filter. After
15 hours, the filters were washed and non-migrated cells were
removed. Migrated cells were stained with H&E and counted under
a microscope. Numbers were obtained as the average number of 5
random fields per filter.
[0092] MTT: Proliferation was measured using MTT assay. Cells were
seeded in a 96 well plate. After one day, the medium was changed to
the appropriate medium. At the time for analysis, 5 .mu.L MTT (10
.mu.g/mL) was added to each well and incubated for 4 hours.
Afterwards, the solution was removed, and DMSO was added. The
absorbance was measured at 550 nm.
[0093] Cell shape: Cell shape was analyzed using the program
imageJ.
[0094] Antibody array: The detection of proteins in the secretome
was done using commercially available membrane antibody arrays from
RayBiotech. CM was incubated with antibody arrays against
chemokines and cytokines and detected using chemiluminescence.
[0095] Phage display: 12 amino acid binding peptides towards CXCL5
were identified using a commercial phage display system from New
England Biosystems.
[0096] Intracellular Ca2+ imaging was done using Fura-2. Cells were
incubated with Fura-2 and the ratio between fluorescence
intensities at 340 nm and 380 nm was measured and plotted.
Results
[0097] Using the above methods, first, a cytokine pattern was
identified for cytokines that are specific (unique) (in bold) for
the secretome of chondocytes according to the following table 1
(see also FIGS. 1A-1B). Predominant cytokines as identified are
indicated in italics. POS=positive control; NEG=negative control,
BMC=Bone marrow derived stromal cells
TABLE-US-00001 TABLE 1 Chondrocytes Name Norm BMC Osteoblasts
Osteoclasts POS 1 1 1 1 NEG 0.066679 0.017539 0.027469 0.020553 IL2
0.06822 0.029074 0.027572 0.022438 MCP-1 1.217602 0.3431 0.339299
0.963559 TNF-a 0.083163 0.046156 0.036696 0.034415 IL3 0.109001
0.057641 0.053618 0.059465 MCP-2 0.114293 0.040159 0.044784
0.077262 TNF-b 0.080495 0.055858 0.039132 0.036499 IL4 0.065596
0.026211 0.027045 0.020462 MCP-3 0.069777 0.041931 0.036496 0.02842
EGF 0.173708 0.145263 0.124084 0.10602 IL5 0.070889 0.028318
0.027409 0.020449 M-CSF 0.094147 0.054951 0.043839 0.053754 IGF-1
0.069955 0.046404 0.039877 0.030477 ENA-78 0.580272 0.039544
0.033606 0.026904 IL6 4.458285 0.312157 0.210344 0.028285 MDC
0.06951 0.044773 0.032661 0.077925 Angiogenin 0.130348 0.049213
0.128664 0.053362 G-CSF 0.073201 0.030328 0.026645 0.020192 IL7
0.674108 0.054183 0.044566 0.021315 MIG 0.066486 0.038226 0.031662
0.025064 Oncastatin M 0.122209 0.078958 0.094876 0.068533 GM-CSF
0.122743 0.085959 0.067787 0.063404 IL8 1.971182 0.056809 0.067922
0.778721 MIP-1d 0.066842 0.031235 0.027645 0.024996 Thrombopoietin
0.075069 0.040592 0.036405 0.027825 GRO 3.603709 0.069439 0.333483
0.847619 RANTES 0.11883 0.08383 0.064541 0.066543 VEGF 0.067953
0.045875 0.035369 0.025091 GRO-a 2.552966 0.037847 0.0522 0.114478
IL12 p40p70 0.067642 0.042385 0.032334 0.033116 SCF 0.073957
0.045659 0.038223 0.0334 PDGF BB 0.065285 0.055458 0.045311 0.06404
I-309 0.093258 0.046966 0.043567 0.038475 IL13 0.065819 0.026038
0.026463 0.020016 SDF-1 0.068709 0.049419 0.046384 0.040627 Leptin
0.092324 0.052001 0.040222 0.037338 IL1a 0.064796 0.031397 0.031443
0.024549 IL15 0.075558 0.036248 0.031916 0.021626 TARC 0.070533
0.059218 0.04433 0.037474 IL-1b 0.062973 0.034325 0.034879 0.028596
INF-g 0.067375 0.039403 0.035133 0.027364 TGF-b1 0.072089 0.054443
0.04144 0.034063
Second, a chemokine pattern was identified for cytokines that are
specific (unique) (in bold) for the secretome of chondocytes
according to the following table 2. Predominant cytokines as
identified are indicated in italics. (see also FIGS. 1A-1B). Abbr.
see Table 1.
TABLE-US-00002 TABLE 2 Chondrocytes Name Norm BMC Osteoblasts
Osteoclasts POS 1 1 1 1 NEG 0.02855 0.01466 0.02252 0.02853
Eotaxin-3 0.03941 0.03665 0.05101 0.05389 MCP2 0.034 0.02502
0.02885 0.04779 PARC 0.02727 0.02446 0.02918 0.03463 Fractalkine
0.03062 0.02841 0.03547 0.03988 MCP-3 0.032 0.02357 0.02709 0.03633
Rantes 0.05469 0.04292 0.05864 0.06533 GCP-2 0.376229 0.032181
0.046655 0.044334 MCP-4 0.04013 0.0235 0.07659 0.0353 SDF-1 a
0.04002 0.02583 0.04274 0.0375 GRO 2.08272 0.03558 1.16742 0.45779
MDC 0.04859 0.03593 0.04364 0.0737 SDF-1 b 0.03548 0.03057 0.04493
0.04088 BLC 0.02893 0.0263 0.0345 0.03796 GRO a 1.657005 0.026569
0.118857 0.047771 MIG 0.03617 0.02547 0.0331 0.0362 TARC 0.03788
0.02988 0.03977 0.04457 CCL28 0.02845 0.02467 0.02448 0.03463 HCC-4
0.03687 0.02371 0.03106 0.03831 MIP-1 a 0.03908 0.03419 0.04794
0.08943 TECK 0.02784 0.02461 0.03232 0.03341 Ckb8-1 0.04352 0.03516
0.04026 0.04801 I-309 0.06397 0.04242 0.04853 0.0584 MIP-1b 0.09124
0.0734 0.10668 0.67802 CTSCK 0.05321 0.04033 0.05319 0.06158 I-TAC
0.03436 0.03131 0.03593 0.04165 MIP-1g 0.03116 0.02555 0.02649
0.04729 CXCL16 0.03417 0.02506 0.0304 0.09064 MIP-3a 0.452969
0.022053 0.024229 0.029753 ENA-78 0.511243 0.023615 0.029447
0.03794 IP-10 0.08671 0.04606 0.05728 0.06331 MIP-3b 0.04593
0.02595 0.02763 0.03415 Eotaxin 0.02944 0.02283 0.03761 0.03288
Lymphatactin 0.0355 0.03193 0.04076 0.03973 MPIF-1 0.02872 0.0238
0.02756 0.03984 Eotaxin-2 0.03329 0.02705 0.03645 0.05566 MCP-1
0.94123 0.72813 1.17109 1.49859 NAP-2 0.300804 0.038889 0.065753
0.080037
[0098] As can be seen examples, for chondrocytes, uniquely secreted
are at least GCP-2, GRO-a, MIP3a, CXCL5, NAP-2, IL-7, and IL-10,
and predominantly secreted are at least IL-8 and IL-6.
[0099] Then, also using the above methods, the migration of
MDA-MB231 breast cancer cells in the presence of the chondrocyte
secretome has been severely inhibited by blocking the signaling of
ENA-78 (CXCL5) using monoclonal antibody to ENA-78 and peptides
identified through phage display that have high binding affinity to
ENA-78.
[0100] Furthermore, using phage display, two peptides that
recognize recombinant human CXCL5/ENA-78 were identified:
TABLE-US-00003 Peptide 1 (001418C, preferred peptide):
H-Pro-Val-Trp-Ala-his-leu-Asn-Pro-Pro-Trp-Leu- Ala-NH2
(ALWPPNLHAWVP, SEQ ID NO: 1); Peptide 2 (001417C, more preferred
peptide): H-Ile-Gly-Lys-Val-Asp-Ser-Asn-Ser-Val-Ser-His- Ala-NH2
(AHSVSNSDVLGI, SEQ ID NO: 2).
[0101] Finally, it was found that chondrocyte conditioned medium
promotes mitogentic signaling through PI3K/AKT pathway in MCF7A
cells. As shown in FIGS. 5 and 6, chondrocyte conditioned medium
exclusively induces proliferation in MCF7A and can be inhibited by
LY-294002 (PI3K inhibitor). A Western blot showed the activation of
AKT and the ERK1/2 pathway in MCF7A upon treatment with AC CM.
Finally, the inhibition of AC CM induced proliferation in MCF7A
through PI3K.
Sequence CWU 1
1
3112PRTartificial sequencepeptide identified using phage display
1Ala Leu Trp Pro Pro Asn Leu His Ala Trp Val Pro1 5
10212PRTartificial sequencepeptide identified using phage display
2Ala His Ser Val Ser Asn Ser Asp Val Leu Gly Ile1 5 103114PRThomo
sapiens 3Met Ser Leu Leu Ser Ser Arg Ala Ala Arg Val Pro Gly Pro
Ser Ser1 5 10 15Ser Leu Cys Ala Leu Leu Val Leu Leu Leu Leu Leu Thr
Gln Pro Gly 20 25 30Pro Ile Ala Ser Ala Gly Pro Ala Ala Ala Val Leu
Arg Glu Leu Arg 35 40 45Cys Val Cys Leu Gln Thr Thr Gln Gly Val His
Pro Lys Met Ile Ser 50 55 60Asn Leu Gln Val Phe Ala Ile Gly Pro Gln
Cys Ser Lys Val Glu Val65 70 75 80Val Ala Ser Leu Lys Asn Gly Lys
Glu Ile Cys Leu Asp Pro Glu Ala 85 90 95Pro Phe Leu Lys Lys Val Ile
Gln Lys Ile Leu Asp Gly Gly Asn Lys 100 105 110Glu Asn
* * * * *