WNT5A PEPTIDES IN REDUCTION OF CANCER STEM CELLS

Abstract

A peptide derived from WNT5A protein, said peptide being for use in the prevention of recurrence or relapse of a cancer in a patient or for use in the reduction or elimination of cancer stem cells in a patient diagnosed with a cancer.

Claims

1. A peptide derived from WNT5A protein according to SEQ ID NO: 1 for use in the reduction or elimination of cancer stem cells in a patient diagnosed with colon cancer, which peptide has a length of 20 amino acids or less and comprises the amino acids sequence XDGXEL (SEQ ID NO: 2), or a formylated derivative thereof, wherein X is any amino acid.

2. The peptide for use according to claim 1, wherein expression of WNT5A in cancer cells is 35% or less of the expression in surrounding non-cancer cells.

3. The peptide for use according to claim 1, wherein expression of WNT5A in cancer cells is 35% or less of the expression in surrounding non-cancer cells.

4. The peptide for use according to claim 1, wherein X in position 1 is M or norleucine and X in position 4 is C or A.

5. The peptide for use according to claim 1, wherein said peptide is selected from the group consisting of: TABLE-US-00003 (SEQ.ID.NO.3) MDGCEL, (SEQ.ID.NO.4) GMDGCEL, (SEQ.ID.NO.5) EGMDGCEL, (SEQ.ID.NO.6) SEGMDGCEL, (SEQ.ID.NO.7) TSEGMDGCEL, (SEQ.ID.NO.8) KTSEGMDGCEL, (SEQ.ID.NO.9) NKTSEGMDGCEL, (SEQ.ID.NO.10) CNKTSEGMDGCEL, (SEQ.ID.NO.11) LCNKTSEGMDGCEL, (SEQ.ID.NO.12) RLCNKTSEGMDGCEL, (SEQ.ID.NO.13) GRLCNKTSEGMDGCEL, (SEQ.ID.NO.14) QGRLCNKTSEGMDGCEL, (SEQ.ID.NO.15) TQGRLCNKTSEGMDGCEL, (SEQ.ID.NO.16) GTQGRLCNKTSEGMDGCEL, and (SEQ.ID.NO.17) LGTQGRLCNKTSEGMDGCEL, or a formylated derivative thereof.

6. The peptide for use according to claim 1, wherein said peptide is MDGCEL (SEQ ID NO: 3), or a formylated derivative thereof.

7. The peptide for use according to claim 1, wherein reduction or elimination comprises the following step: a) immediately after diagnosis of cancer and/or during surgery and or after removing the tumour by surgery, administering an effective amount of the peptide, optionally wherein step a) is repeated at least 3 times a week for 2 weeks or more.

8. The peptide for use according to claim 1 in combination with at least one tumour suppressing chemotherapeutic drug wherein prevention, reduction or elimination comprises the following steps: a) administering an effective amount of a tumour suppressing chemotherapeutic drug; b) prior to, simultaneously with and/or subsequently after treatment with said at least one tumour suppressing chemotherapeutic drug, administering an effective amount of the peptide, optionally wherein step b) is repeated at least 3 times a week for 2 weeks or more.

9. (canceled)

10. The peptide for use according to claim 1 in combination with at least one tumour suppressing chemotherapeutic drug, wherein said peptide and said tumour suppressing chemotherapeutic drug are either combined or separate and/or are administered either simultaneously or sequentially.

11. The combination for use according to claim 8 wherein said at least one tumour suppressing chemotherapeutic drug is a combination of 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX) or anthracycline or taxane.

12. The combination for use according to claim 11 wherein the anthracycline is epirubicin or doxorubicin, or the taxane is docetaxel or paclitaxel.

13. The combination for use according to claim 11 wherein said tumour suppressing chemotherapeutic drug is a combination of 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX) for use in treatment of a patient diagnosed with colon cancer.

14. The combination according to claim 10 wherein said tumour suppressing chemotherapeutic drug is an anthracycline or a taxane for use in treatment of a patient diagnosed with breast cancer.

15. (canceled)

16. A peptide agonist derived from WNT5A protein according to SEQ ID NO: 1 for use in the reduction or elimination of cancer stem cells in a patient diagnosed with colon cancer, which peptide agonist has a length of 20 amino acids or less and comprises the amino acids sequence XDGXEL (SEQ ID NO: 2), or a formylated derivative thereof, wherein X is any amino acid.

17. A method of treatment comprising: administering a peptide to a patient diagnosed with colon cancer, wherein the peptide is derived from WNT5A protein according to SEQ ID NO: 1, and wherein the peptide has a length of 20 amino acids or less and comprises the amino acids sequence XDGXEL (SEQ ID NO: 2), or a formylated derivative thereof, wherein X is any amino acid, thereby reducing or eliminating cancer stem cells in the patient.

18. The method of claim 17, wherein X in position 1 is M or norleucine and X in position 4 is C or A.

19. The method of claim 17, wherein the peptide is selected from the group consisting of: TABLE-US-00004 (SEQ.ID.NO.3) MDGCEL, (SEQ.ID.NO.4) GMDGCEL, (SEQ.ID.NO.5) EGMDGCEL, (SEQ.ID.NO.6) SEGMDGCEL, (SEQ.ID.NO.7) TSEGMDGCEL, (SEQ.ID.NO.8) KTSEGMDGCEL, (SEQ.ID.NO.9) NKTSEGMDGCEL, (SEQ.ID.NO.10) CNKTSEGMDGCEL, (SEQ.ID.NO.11) LCNKTSEGMDGCEL, (SEQ.ID.NO.12) RLCNKTSEGMDGCEL, (SEQ.ID.NO.13) GRLCNKTSEGMDGCEL, (SEQ.ID.NO.14) QGRLCNKTSEGMDGCEL, (SEQ.ID.NO.15) TQGRLCNKTSEGMDGCEL, (SEQ.ID.NO.16) GTQGRLCNKTSEGMDGCEL, and (SEQ.ID.NO.17) LGTQGRLCNKTSEGMDGCEL, or a formylated derivative thereof.

20. The method of claim 17, wherein the peptide is MDGCEL (SEQ ID NO: 3), or a formylated derivative thereof.

21. The method of claim 17, wherein the peptide is administered to the patient in combination with at least one tumour suppressing chemotherapeutic drug.

22. The method of claim 21, wherein the tumour suppressing chemotherapeutic drug is an anthracycline or a taxane.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0040] The figures described in the following are to support the detailed description. The invention will be described with reference to the figures in which:

[0041] FIG. 1 is a schematic overview of the animal experiments described in examples 1 to 3.

[0042] FIG. 2 shows the in vivo effect of Foxy-5 treatment on ALDH protein expression in colon cancer tissue as visualized by immunohistochemistry (IHC). Representative photos from saline- and Foxy-5-treated animals are presented. From each such slide, 4 boxes were put on top of the stained tissue and for each of them the ALDH staining was scored as a percentage of stained cells multiplied by the staining intensity. The following score was used for percentage stained cells; 0 for positive stained cells <5%, 1 for positive cells 5-25%, 2 for positive cells 26-50%, 3 for positive cells 51-75%, 4 for positive cells >75%.

[0043] For staining intensities, the stained areas were scored as; 1 for weak staining, 2 for medium staining and 3 for strong staining. For each box a final score was obtained by multiplying the score for percentage of positively stained cells with the score for staining intensity. Finally, a mean value was obtained from the 4 boxes for each tumour. The same scoring protocol was then used for all IHC analyses presented in the present study.

[0044] FIG. 3 shows the in vivo effect of Foxy-5 treatment on Dck1 (also named DCAMKL1) protein expression in colon cancer tissue as visualized by IHC. Representative photos from saline- and Foxy-5-treated animals are presented. From each such slide 4 boxes were put on top of the stained tissue and for each of them the Dck1 (DCAMKL1) staining was scored as a percentage of stained cells multiplied by the staining intensity as described in detail for FIG. 2.

[0045] FIG. 4 shows the in vivo effect of Foxy-5 treatment on ALDH and Dck1 (DCAMKL1) mRNA expression in colon cancer xenograph tissue compared with that of tumors from vehicle-treated control mice.

[0046] FIG. 5 shows the in vivo effect of Foxy-5 treatment on Cox-2 and 15-PGDH protein expression in colon cancer tissue as visualized by IHC. The IHC stainings were scored exactly as described in detail for the IHC staining in FIG. 1.

[0047] FIG. 6 shows the in vivo effect of Foxy-5 treatment on active -catenin nuclei expression in HT-29 colon cancer tissue as visualized by IHC.

[0048] The IHC stainings were scored exactly as described in detail for the IHC staining in FIG. 1. In the lower right panel, the size of the tumours for both the saline- and Foxy-5-treated animals are presented.

[0049] FIG. 7 shows the in vivo effect of Foxy-5 treatment on active -catenin nuclei expression in Caco-2 colon cancer tissue as visualized by IHC. The IHC staining were scored exactly as described in detail for the IHC staining in FIG. 2. In the lower right panel, the size of the tumours for both the saline- and Foxy-5-treated animal tumour are presented.

[0050] FIGS. 6 and 7 further shows the in vivo effect of Foxy-5 treatment on tumour volume in HT-29 and Caco-2 colon cancer xenograph tissue, respectively. The knowledge that colon cancers have elevated -catenin signalling makes it possible to validate the effects outlined in FIGS. 6 and 7, respectively, by determining the its relation to changes in tumour volume. The figures outlines the effects of Foxy-5 treatment on tumour volume of HT29 and Caco-2 derived tumours compared to that from control (vehicle-treated) mice.

[0051] FIG. 2B is based on a repeated and extended analysis as in FIG. 2 and shows the in vivo effect of Foxy-5 treatment on ALDH protein expression in colon cancer xenograph tissue as visualized by immunohistochemistry (IHC). Representative images from vehicle (saline) and Foxy-5-treated animals are presented. From each such slide, 6 boxes were randomly put on top of the stained tissue and for each of them the ALDH staining was scored as a percentage of stained cells multiplied by the staining intensity. The following score was used for percentage stained cells; 0 for positive stained cells <5%, 1 for positive cells 5-25%, 2 for positive cells 26-50%, 3 for positive cells 51-75%, 4 for positive cells >75%.

[0052] For staining intensities, the stained areas were scored as; 1 for weak staining, 2 for medium staining and 3 for strong staining. For each box a final score was obtained by multiplying the score for percentage of positively stained cells with the score for staining intensity. Finally, a mean value was obtained from the 6 boxes for each tumour sample. The same scoring protocol was then used for all IHC analyses presented in the present study.

[0053] FIG. 3B is based on a repeated and extended analysis as in FIG. 3 and shows the in vivo effect of Foxy-5 treatment on Dck1 (also named DCAMKL1) protein expression in colon cancer xenograph tissue as visualized by IHC. Representative images from vehicle- and Foxy-5-treated animals are presented. From each such slide 6 boxes were put on top of the stained tissue and for each of them the Dck1 staining was scored for staining intensity, as described in detail for FIG. 2.

[0054] FIG. 5B is based on a repeated and extended analysis as in FIG. 5 and shows the in vivo effect of Foxy-5 treatment on Cox-2 protein expression in colon cancer xenograph tissue as visualized by IHC. Representative images from vehicle- and Foxy-5-treated animals are presented. From each such slide 6 boxes were put on top of the stained tissue and for each of them the Cox-2 staining was scored as a percentage of stained cells multiplied by the staining intensity as described in detail for FIG. 2.

[0055] FIG. 5C is based on a repeated and extended analysis as FIG. 5 shows the in vivo effect of Foxy-5 treatment on 15-PGDH protein expression in colon cancer xenograph tissue as visualized by IHC. Representative images from vehicle- and Foxy-5-treated animals are presented. From each such slide 6 boxes were put on top of the stained tissue and for each of them the 15-PGDH staining was scored as a percentage of stained cells multiplied by the staining intensity as described in detail for FIG. 2.

[0056] FIG. 8 is based on a repeated and extended analysis as FIGS. 6 and 7, respectively, and shows the in vivo effect of Foxy-5 treatment on active -catenin nuclei expression in HT-29 and Caco-2 colon cancer xenograph tissue as visualized by IHC. Representative images from vehicle- and Foxy-5-treated animals are presented. From each such slide 6 boxes were put on top of the stained tissue and for each of them the active -catenin nuclei staining was scored as a percentage of stained cells multiplied by the staining intensity as described in detail for FIG. 2.

[0057] FIG. 9 shows the in vivo effect of Foxy-5 treatment on Ascl2 protein expression in HT-29 and Caco-2 colon cancer xenograph tissue as visualized by IHC. The Ascl2 protein is a transcription factor activated by -catenin signaling that has been show to promote the CSC niche. Representative images from vehicle- and Foxy-5-treated animals are presented. From each such slide 6 boxes were put on top of the stained tissue and for each of them the Ascl2 staining was scored as a percentage of stained cells multiplied by the staining intensity as described in detail for FIG. 2.

[0058] FIG. 10 shows the absence of a Foxy-5 effect on FOLFOX-induced cytotoxicity. Colon cancer HT29 cells were treated with either FOLFOX alone (green triangles), 5-FU alone (orange triangles), oxaliplatin alone (black squares), Foxy-5 alone (red squares) or FOLFOX+Foxy-5 (blue circles). The cytotoxic effects of these different treatments were evaluated by CellTiter-Blue fluorescence cell viability assay. The dose-response curves of all these different treatments in HT29 are outlined in the figure. The data are normalized and shown as meansSEM.

DETAILED DESCRIPTION

[0059] The Wnt (Wingless-related integration site) protein family contains highly conserved proteins that play a role in embryonic development such as body axis patterning, cell proliferation and migration. The Wnt signalling pathways are either canonical or non-canonical and they primarily trigger the regulation of gene transcription and increased proliferation via canonical signalling or regulation of several non-proliferative functions via activation of different non-canonical signalling pathways in the cells. The Wnt proteins are further involved in tissue regeneration in adult bone marrow, skin and intestine. Genetic mutation in the Wnt signalling pathway may cause breast cancer, prostate cancer glioblastoma, type II diabetes and other diseases.

[0060] The canonical Wnt pathway activates -catenin and is integral in regulating self-renewal of normal stem cells and the subversion of the canonical Wnt signalling has been implicated in tumourigenesis. In contrast, non-canonical Wnt signalling is characterized by an absence of an increase in -catenin signalling and has been studied for its role in embryonic patterning, gastrulation, and organogenesis. Moreover, non-canonical Wnt is proposed to antagonize canonical signalling. WNT5A is an example of a non-canonical Wnt ligand. WNT5A is tumour-suppressive in acute myelogenous leukemia (AML), colon cancer, breast and prostate cancer, and ovarian carcinoma. Over-expression of WNT5A in a WNT5A homozygous mouse model was shown to correlate with a reduced number of breast CSCs in a study by Borcherding et al., Paracrine WNT5A signalling inhibits expansion of tumour-initiating cells, Cancer Research 75:1972-1982, 2015 suggesting that heterozygous loss of WNT5A correlates with shorter survival of breast cancer patients. Interestingly, WNT5A has the opposite effect in malignant melanoma, gastric cancer as well as a few other cancer types, as exemplified by the fact that high expression of WNT5A in primary malignant melanoma is correlated with a shortened survival time.

[0061] WNT5A is a protein expressed by many normal cells in the body. WNT5A is secreted from the cells and exerts its action on the same or neighbouring cells by binding to and activating a receptor complex primarily involving a Frizzled receptor. The WNT5A protein is known to activate a receptor called Frizzled 5. Upon activation of the Frizzled 5 receptor a series of signalling events inside of the cells are activated, where one of the first events, is generation of short-lived increase in calcium inside of the cell, a so called calcium-signal. The calcium-signal in turn triggers a series of forthcoming signalling events leading to a change in the functions of the cells, such as adhesion and migration. Thus, activating such a Frizzled receptor leads to signalling events inside the cell, resulting in increased adherence of the cell to its neighbouring cells and its adhesion to the surrounding connective tissue resulting in decreased ability of the tumour cell to migrate to structures in the vicinity, such as lymph nodes and blood vessels. In healthy breast epithelial cells for example, WNT5A is highly expressed and secures a firm adherence between cells and to the surrounding basement membrane and thereby restricts migration of the cells.

[0062] In order to reconstitute WNT5A signalling in cancer tissue that lack an endogenous expression of WNT5A, a small peptide, i.e. equal to or less than 20 amino acids derived from the amino acid sequence of the WNT5A molecule has been developed and then additionally modified. An example of such a peptide is Foxy-5, which is a true WNT5A agonist in that it triggers the same signaling events and functional responses as WNT5A and in comparison, with WNT5A it is much simpler molecule and it can be administered systemically and still reach the tumor tissue. Thus, the term signalling properties, as used herein, means binding of the WNT5A or the Foxy-5 peptide to primarily a Frizzled receptor protein (Fz) followed by an intracellular signalling cascade in the cell eventually leading to reduction or elimination of CSCs.

[0063] The term surrounding non-cancer cells, as used herein, means morphologically normal cells, of the same type from which the tumour has originated, enclosing or encircling the tumour tissue.

EXAMPLES

Example 1

[0064] Tumours from two different human colon cancer cell types HT29 and Caco-2 cells were examined by immunohistochemistry (IHC) and mRNA (see FIG. 1).

[0065] On day 0: Subcutaneous injections of HT29 or Caco-2 colon cancer cells in nude mice were done.

[0066] On day 7 after tumours have been established in the mice, intraperitoneal (I.P) injections of vehicle (saline) alone (gr 1) or Foxy-5 (2 g/g; gr 2) were done.

[0067] On days 9-23 tumour growth and animal weight was monitoredthen +8 I.P. injections every 2nd day of either vehicle alone (gr 1) or Foxy-5 (2 g/g; gr 2).

[0068] On day 24 the two types of tumours (Caco-2 and HT29 derived) (divided in two parts of which one was fixed and the other frozen at 80 C.) were analyzed by IHC for their protein expression of COX-2, 15PGDH, -catenin, Ascl2, ALDH and Dckl1 and for their mRNA content of ALDH and Dckl1 (ALDH is a general stem cell marker and Dck1 is a specific marker for colon CSCs).

[0069] Foxy-5 was shown to significantly reduce the expression of the two stem cell biomarkers in both Caco-2- and HT29-derived colon cancers (see FIGS. 2 to 4).

Example 2

[0070] To further validate the findings outlined in FIGS. 2-4 we analyzed possible mechanisms responsible for the decreased number of CSCs in the present experiments. The enzyme cyclooxygenase-2 (COX-2) is often upregulated in colon cancer. COX-2 activity leads to generation and release of prostaglandin E2 (PGE2). PGE2 promotes cancer progression and was shown to favor expansion of colon CSCs. (Wang et al., Prostaglandin E2 promotes colorectal cancer stem cell expansion and metastasis in mice. Gastroenterology 149:1884-1895, 2015). While perturbation of canonical WNT-signaling pathway is believed to account for the initiation of colorectal tumors, the increased expression of cyclooxygenase-2 (COX-2) that occurs in the majority of colorectal tumors is thought to play a crucial role during colorectal cancer development. Increased COX-2 expression leads to an increased abundance of its principal metabolic product, prostaglandin E2 (PGE2). Treatment of the animals in the present study with Foxy-5 resulted in a decrease not only in the expression of the PGE2 generating enzyme COX-2 (see FIGS. 5 and 5B and but also in an increase in the PGE2 degrading enzyme 15-PGDH (see FIGS. 5 and 5C). These data demonstrate that treatment with the Foxy-5 peptide can by a unique dual action cause a reduction in the intra-tumor level of PGE2 and thus explain the observed reduction of CSCs (FIGS. 2-4 and 2B-3B).

Example 3

[0071] In vivo effect of Foxy-5 treatment on -catenin signalling in HT-29 or Caco-2 colon cancer tissue could be another possible explanation for the observed reduced number of CSCs. Decreased amounts of active -catenin nuclei expression were observed in both Caco-2 and HT29 derived colon cancer tumors (see FIGS. 7 and 8) as a result of Foxy-5 treatment. The observations of Foxy-5-reduced -catenin signalling were validated by reductions in tumour volumes (FIGS. 6 and 7). Essential for the present study is the fact that it was also observed that Foxy-5-induced reductions of the -catenin downstream target Ascl2, a transcription factor promoting the cancer stem cell niche, indicate that the ability of Foxy-5 treatment to reduce the number of CSCs is also dependent on reduced -catenin/Ascl2 signaling (see FIG. 6-9). Consequently, the Foxy-5 peptide exhibit a unique property to reduce the number of CSCs by its ability to target three different elements (COX-2, 15-PGDH and -catenin) that leads to reduction in two separate signaling pathways promoting CSCs.

Example 4

[0072] The aim of this study was to test the possibility of simultaneous treatment of Foxy-5 and FOLFOX, by evaluating the in vitro effect of the Foxy-5 peptide on the cytotoxic effect of FOLFOX. FOLFOX is a made up from a combination of the two chemotherapeutic agents 5-FU and oxaliplatin and folinic acid, the latter is also called leucovorin not a chemotherapeutic agent but is used to potentiate the effect of 5-FU on human HT29 colon cancer cells. FOLFOX is the most commonly used chemotherapy in the treatment of colon cancer patients.

[0073] The initial experiment was to evaluate the cytotoxic effect of the Foxy-5 peptide, oxaliplatin, and 5-FU as monotherapy (FIG. 10). The results confirmed that as folinic acid there was no cytotoxic effect of the Foxy-5 peptide on cell viability and consequently no IC.sub.50 value could be determined. Whereas the IC.sub.50 values of oxaliplatin and 5-FU were calculated from the dose-response curves to be 3.4 and 6.8 M, respectively.

[0074] The combination results indicate that the Foxy-5 peptide do not enhance or reduce the inhibitory effect of FOLFOX on cell viability. The IC.sub.50 value of FOLFOX treatment alone was 1.5 M and a combined treatment with the Foxy-5 peptide and FOLFOX exhibited the same IC.sub.50 value, as is also clear from their overlapping dose-response curves in FIG. 10.

[0075] In conclusion, addition of Foxy-5 did not alter the effect of FOLFOX treatment on the viability of human H T2 9 colon cancer cells, thus no interaction can be anticipated. There is therefore no indication that Foxy-5 will diminish the cytostatic effect of FOLFOX treatment.