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.

Claims

1. An in vitro method for identifying inhibitors of breast cancer metastasis, comprising the steps of: a) providing a CXCL5 protein: b) contacting in vitro said CXCL5 protein with at least one putative ligand of said CXCL5 protein in the presence of a peptide having the sequence of ALWPPNLHAWVP (SEQ ID NO: I) and/or a peptide having the sequence of AHSVSNSDVLGI (SEQ ID NO: 2), and wherein the at least one putative ligand competes for binding to the CXCL5 protein with one or both of the peptides of SEQ ID NO: 1 and SEQ ID NO: 2, and c) detecting in vitro a binding between said at least one putative ligand and said CXCL5 protein.

2. The method according to claim 1, further comprising the steps of: d) in case of a binding of said putative ligand to said CXCL5 protein, detecting whether said binding between said putative ligand to said CXCL5 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.

3. The method according to claim 1, wherein said putative ligand is an inhibitor of the expression, stability and/or biological function of said CXCL5 protein.

4. The method, according to claim 2, wherein said breast cancer metastasis is a bone and/or lung metastasis.

5. The method, according to claim 3, wherein said putative ligand binds to the pocket of the CXCL5 protein.

Description

FIGURES

(1) FIGS. 1A-1B: Analysis of the secretome of chondrocytes for (1A) cytokines and (1B) chemokines (see also tables and 1 and 2).

(2) 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.

(3) 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.

(4) 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.

(5) FIG. 5: Chondrocyte-conditioned medium promotes mitogentic signaling through PI3K/AKT pathway in MCF7AWestern blot showing activation of AKT and ERK1/2 pathway in MCF7A upon treatment with chondrocyte-conditioned medium (AC CM).

(6) FIG. 6: Inhibition of AC CM induced proliferation in MCF7A using PI3K inhibitors (e.g. LY294002).

(7) 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.

(8) 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.

(9) 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.

(10) 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.

EXAMPLES

(11) 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.

(12) 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.

(13) 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 m filter.

(14) 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 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.

(15) 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 L MTT (10 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.

(16) Cell shape: Cell shape was analyzed using the program imageJ.

(17) 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.

(18) Phage display: 12 amino acid binding peptides towards CXCL5 were identified using a commercial phage display system from New England Biosystems.

(19) 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.

(20) Results

(21) 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

(22) TABLE-US-00001 Chondrocytes Osteo- Osteo- Name Norm BMC blasts clasts 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 0.062287 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

(23) 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.

(24) TABLE-US-00002 Chondrocytes Osteo- Osteo- Name Norm BMC blasts clasts 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 0.02659 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

(25) 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.

(26) 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.

(27) Furthermore, using phage display, two peptides that recognize recombinant human CXCL5/ENA-78 were identified:

(28) TABLE-US-00003 Peptide1(001418C,preferredpeptide): H-Pro-Val-Trp-Ala-his-leu-Asn-Pro-Pro-Trp-Leu- Ala-NH2(ALWPPNLHAWVP,SEQIDNO:1); Peptide2(001417C,morepreferredpeptide): H-Ile-Gly-Lys-Val-Asp-Ser-Asn-Ser-Val-Ser- His-Ala-NH2(AHSVSNSDVLGI,SEQIDNO:2).

(29) 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 LY294002 (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.