Na+ / K+ATPase inhibitors for use in the prevention or treatment of metastasis

Abstract

The present invention provides a method for the prevention or treatment of metastasis in cancer patients as defined by the presence of CTC clusters in the bloodstream. + / K + In certain embodiments, the present invention relates to an ATPase inhibitor. + / K + The ATPase is a cardiac glycoside and is selected from digitoxin, ouabain, convallatoxin, proscillaridin, lanatoside C, gitoformate, pervoside, strophanthidin, methyldigoxin, deslanoside, bufalin, digoxin, and digoxigenin. The present invention further relates to the use of nucleic acid agents that inhibit the expression of genes involved in the formation and maintenance of CTC clusters.

Description

[Technical field]

[0001] The present invention relates to a method for the prevention or treatment of metastasis comprising administering to a subject a naphthalene derivative thereof. + / K + It concerns ATPase inhibitors.

[0002] This application claims the benefit of priority from European Patent Application No. 18214978.1, filed December 20, 2018, the contents of which are incorporated herein in their entirety. [Background technology]

[0003] The metastatic spread of cancer, usually to bone, lung, liver, and brain, accounts for the majority of cancer-related deaths. The metastasis of epithelial cancers is thought to involve a series of sequential steps: epithelial-mesenchymal transition (EMT) of individual cells within the primary tumor leading to intravasation into the bloodstream, survival of such circulating tumor cells (CTCs) within the bloodstream, and finally, their extravasation at distant sites leading to mesenchymal-epithelial transition (MET) leading to proliferation as epithelial metastatic deposits.

[0004] Circulating tumor cells are cells that leave the cancerous tumor and enter the bloodstream on their way to disseminated metastasis (Alix-Panabiereset et al., Clin Chem 59, 110-118, 2013). The analysis of CTCs is expected to allow elucidating fundamental features of the metastatic process and identifying the vulnerabilities of targeted cancers. Once in the bloodstream, CTCs must overcome the loss of adhesion signals from the primary tumor, as well as the appropriate high shear forces of the circulatory system, to survive. In breast cancer, the ability of CTCs to form clusters has been linked to an increased propensity for metastasis when compared to single CTCs (Aceto et al., Cell 158, 1110-1122, 2014).

[0005] CTCs are found in the blood of cancer patients as single CTCs and CTC clusters (Fidler European Journal of Cancer 9, 223-227 1973; Liotta et al., Cancer Research 36, 889-894 1976), the latter clusters being characterized by a higher capacity to seed metastases (Aceto et al., Cell 158, 1110-1122, 2014). However, it is unknown what increases the metastatic potential and what is the vulnerability of clustered CTCs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Alix-Panabiereset et al., Clin Chem 59, 110-118, 2013 [Non-Patent Document 2] Aceto et al.; Cell 158, 1110-1122, 2014 [Non-Patent Document 3] Fidler European Journal of Cancer 9, 223-227 1973 [Non-Patent Document 4] Liotta et al., Cancer Research 36, 889-894 1976 Summary of the Invention [Problem to be solved by the invention]

[0007] Based on the above state of the art, the object of the present invention is to provide means and methods for preventing and treating metastasis in cancer patients. [Means for solving the problem]

[0008] This object is achieved by the claims herein. [Brief description of the drawings]

[0009] [Figure 1-1] Figure 1 shows DNA methylation analysis of human single CTCs and CTC clusters. A) Live CTCs were stained (Alexa488 or FITC conjugated) for cell surface expression of EpCAM, HER2, and EGFR and counterstained with an antibody against CD45 to identify contaminating leukocytes. B) Principal component analysis, separating CTCs based on the original patient, primarily with CTC clusters (circles) being more heterogeneous compared to single CTCs (triangles). [Figure 1-2] Figure 1 shows DNA methylation analysis of human single CTCs and CTC clusters. C) NES score representing enrichment of transcription factor binding sites (TFBS) in hypomethylated regions of CTC clusters (n=1305) and single CTCs (n=2042) identified using i-cisTarget. D) Gene Ontology (GO) enrichment analysis was performed for 166 genes located in hypomethylated regions of CTC clusters (p=<0.05). [Diagram 2] Figure 2 shows DNA methylation analysis of single CTCs and CTC clusters in mouse xenografts. A) NES score, representing enrichment of transcription factor binding sites (TFBS) in hypomethylated regions of CTC clusters (n=909) and single CTCs (n=521) identified using i-cisTarget. B) Only a very small subset of TFBSs is preferentially hypomethylated in either single CTCs (n=13) or CTC clusters (n=9). [Figure 3-1] Figure 3 shows RNA sequencing analysis of single CTCs and CTC clusters isolated from breast cancer patients. A) Network analysis of transcripts identified in CTC cluster-associated modules, B) Gene regulatory network analysis showing transcription factor dependence on TFs SIN3A, OCT4, and CBFB that also display hypomethylated binding sites. [Figure 3-2]Figure 3 shows the results of RNA-seq analysis of single CTCs and CTC clusters isolated from breast cancer patients. C) RNA-seq analysis of xenograft-derived CTC clusters in addition to genes found to be enriched in TFs in patient CTC clusters with significantly hypomethylated binding sites, such as SIN3A, NANOG, SOX2, RORA, FOXO1, and BHLHE40. D) Gene Ontology (GO) enrichment analysis of genes located in hypomethylated regions of CTC clusters. [Figure 3-3] Figure 3 shows the results of RNA sequencing analysis of single CTCs and CTC clusters isolated from breast cancer patients. E) Transcription factor target gene analysis of single CTCs further confirmed the activity of c-MYC and E2F4. [Figure 4-1] Figure 4 shows a screen of FDA-approved compounds to dissociate CTC clusters. A) Left panel: Representative images of steady-state "unfiltered" and 40 μM filtered BR16 cells stained with Hoechst and TMRM. Images were taken on a high content screening microscope. Right panel: Representative images of outlines of single and clustered CTCs based on nuclear proximity (derived from each left panel image) as determined using the Colombus imaging data analysis system. Bar graphs show the mean cluster size (area in μm2) and viability (%) of unfiltered and filtered BR16 cells (n=3; NS: not significant; ***p<0.001). [Figure 4-2]Figure 4 shows a screen of FDA-approved compounds that dissociate CTC clusters. B) Top panel: Plots show the average cluster size of BR16 cells treated with each of 39 cluster-targeting compounds at four different concentrations: 5 μM, 1 μM, 0.5 μM, 0.1 μM. Cluster-targeting compounds include Na+ / K+ ATPase (n=6), HDAC (n=2), nucleotide biosynthesis (n=5), kinase (n=4), GPCR (n=2), cholesterol biosynthesis (n=1) and nuclear export (n=1), as well as tubulin (n=9) and DNA binding (n=8) compounds and antibiotics (n=1). Untreated or untreated and 40 μM filtration-treated BR16 cells are shown as controls for comparison. The average of two independent measurements is shown. Bottom panel: Heatmap showing nuclei number, average TMRM intensity, and % viability of BR16 cells treated with cluster-targeting compounds at the indicated concentrations. [Diagram 5] FIG. 5 shows the effect of in vitro treatment of BR16 and BRx50 cell lines with digitoxin, ouabain octahydrate, and rigosertib at concentrations of 50 nM, 20 nM, 10 nM, 5 nM, and 1 nM for 17 days on the reduction in cluster size, number of nuclei, TMRM intensity, and % viability compared to untreated or untreated and further 40 μM filtration-treated cells. [Figure 6] Figure 6 shows the effect of digitoxin and ouabain treatment of CTC-derived cell lines. (A) Western blot of CLDN3, CLDN4 and GAPDH on BR16 cells with CLDN3 and CLDN4 double knockout (KO). KO = knockout. (B) Plot showing reduction in average cluster size (area in μm2) of CLDN3 / 4 double KO BR16 cells compared to control BR16 cells. *P<0.05; **P<0.01 by Student's t-test. Error bars represent standard error of the mean (SEM). [Figure 7]FIG. 7 shows that treatment with Na+ / K+ ATPase inhibitors suppresses the formation of spontaneous metastases; (A) Schematic of the experiment; (B) Plots show total bioluminescence flux at day 0 (left) and day 1 (right) upon tail vein injection of BR16 cells pretreated with 20 nM digitoxin or ouabain. n=5; *P<0.05 by Student's t-test. NS=not significant. Error bars represent standard error of the mean (SEM). (C) Metastatic growth curves over 72 days upon tail vein injection of BR16 cells pretreated with 20 nM digitoxin or ouabain. n=5; *P<0.05 by Student's t-test; **P<0.01; NS=not significant. Error bars represent standard error of the mean (SEM). (D) Schematic of the experiment. (E) Plots show the percentage of spontaneous single CTCs and CTC clusters detected in the blood of ouabain-treated BR16 xenografts. n=11 (control), n=5 (ouabain); ***P<0.001 by Student's t-test, error bars represent SEM. (F) Plot shows metastatic index of BR16 xenografts treated with ouabain. n=11 (control), n=5 (ouabain); **P<0.01 by Student's t-test, NS=not significant. Error bars represent standard error of the mean (SEM). (G) Representative images of bioluminescence signal measured in brain and liver of control and ouabain-treated NSG mice. [Figure 8]FIG. 8 shows that treatment with digitoxin and ouabain reduces metastasis formation. (A) Plots show the percentage of Ki67-positive cancer cells detected in the lungs of NSG mice at day 0 (left) or day 1 (right) after injection with BR16 CTC-derived cells treated in vitro with digitoxin or ouabain. Cancer cells are identified by pancytokeratin staining; n=4 mice for each condition. Error bars represent SEM. NS=not significant. (B) Plots show the percentage of caspase-3-positive cancer cells detected in the lungs of NSG mice at day 0 (left) or day 1 (right) after injection with BR16 CTC-derived cells treated in vitro with digitoxin or ouabain. Cancer cells are identified by pancytokeratin staining; n=4 mice for each condition. *P<0.05 by Student's t-test. Error bars represent SEM. NS=not significant. (C) Plots show total bioluminescence flux emitted from primary tumors of BR16 xenografts treated with vehicle (control) or ouabain. Error bars represent SEM. NS=not significant. (D) Plots show total number of CTCs, including both single CTCs and CTC clusters, detected per mL of blood of BR16 xenografts treated with vehicle (control) or ouabain. n=5 for control, n=5 for ouabain. Error bars represent SEM. NS=not significant. (E) Plots show percent (%) of spontaneously generated single CTCs and CTC clusters detected in blood of LM2 xenografts treated with vehicle (control) or ouabain. n=11 for control, n=8 for ouabain. **P<0.01. (F) Plots show total bioluminescence flux emitted from primary tumors of LM2 xenografts treated with vehicle (control) or ouabain. Error bars represent SEM. NS = not significant. (G) Plots show the total number of CTCs, including both single CTCs and CTC clusters, detected per mL of blood in LM2 xenografts treated with vehicle (control) or ouabain. n = 11 for control, n = 8 for ouabain. Error bars represent SEM. NS = not significant.(H) Plot shows metastatic index of LM2 xenografts treated with vehicle (control) or ouabain. n=19 for control, n=8 for ouabain. **P<0.01 by Student's t-test. Error bars represent SEM. [Figure 9] FIG. 9 shows data obtained in the same manner as the data in FIG. 4b. [Figure 10] The plot shows the tumor growth rate over time for BR16 xenografts treated with vehicle (control) or digoxin (2 mg / kg), with no significant differences observed (all P>0.05). [Figure 11] The plots show the numbers of single CTCs, CTC clusters, and CTC-neutrophil clusters (expressed as single CTC-WBC and CTC cluster-WBC) in BR16 xenografts treated with vehicle (control) or digoxin (2 mg / kg). Digoxin treatment clearly reduces the numbers of CTC clusters and CTC-neutrophil clusters. [Figure 12] The plot shows the metastatic index of BR16 xenografts treated with vehicle (control) or digoxin (2 mg / kg). Treatment with digoxin inhibits metastasis. [Figure 13] The plot shows the tumor growth rate over time for LM2 xenografts treated with vehicle (control) or digoxin (2 mg / kg), with no significant differences observed (all P>0.05). [Figure 14] Figure 14 shows Kaplan-Meier curves showing overall survival of LM2 xenografts treated with vehicle (control) or digoxin (2 mg / kg). Digoxin treatment prolongs overall survival. [Figure 15] The plot shows the CTC fold change in LM2 xenografts treated with vehicle (control) or digoxin (2 mg / kg). Treatment with digoxin reduces the formation of CTC clusters and CTC-neutrophil clusters. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Summary of the Invention The inventors profiled the DNA methylation landscape of single CTCs and CTC clusters on a genome-wide scale, concordant within individual cancer patients and human CTC-derived xenografts. They surprisingly found that stemness-related transcription factors orchestrate an OCT4-centric network that is only active in CTC clusters, and that CTC clusters simultaneously exhibit an activated SIN3A-dependent cell cycle progression program. This finding indicates that the ability of CTCs to form clusters directly influences their DNA methylation patterns, resulting in enhanced stemness and cell cycle progression signals that favor metastatic dissemination.

[0011] We identified drugs that specifically disrupt CTC clusters without altering their cell viability. Disruption of clusters to single cells restores DNA methylation at key sites and arrests cluster-associated stemness and cell cycle programs, resulting in a significant reduction in metastatic dissemination capacity.

[0012] A first aspect of the present invention relates to a method for the prevention or treatment of metastasis in a cancer patient comprising administering to said patient a nascent naphthalene derivative. + / K + Concerning ATPase inhibitors.

[0013] A second aspect of the invention relates to a protein selected from: - CLDN3 - CLDN4, and - Na + / K + ATPase or any of its constituent subunit isoforms The present invention relates to the expression of nucleic acid-mediated therapeutic downregulation or inhibition of a target nucleic acid sequence encoding a

[0014] A third aspect of the invention relates to the use of a NaCl solution according to the invention in the prevention and treatment of venous thromboembolism in cancer patients. + / K + The present invention relates to the use of an ATPase inhibitor or a nucleic acid molecule.

[0015] Terms and Definitions For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall include the plural and vice versa. In the event that a definition set forth below conflicts with any document incorporated herein by reference, the set forth definition shall control.

[0016] As used herein, the words "comprising," "having," "containing," "including," and other similar forms and their grammatical equivalents are intended to be equivalent in meaning and open ended, and that an item following any one of these words is not meant to be an exhaustive list of such items, nor is it meant to be limited to only the items in the list. For example, "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can include not only components A, B, and C, but also one or more other components. Thus, "comprising" and its similar forms and their grammatical equivalents are intended and understood to include disclosure of embodiments that "consist essentially of" or "consist of."

[0017] Where a range of values ​​is provided, unless the context clearly dictates otherwise, every intermediate value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, and any other stated or intermediate value within the stated range, is encompassed within the disclosure, excluding any specific limit in the stated range. Where the stated range includes one or both limits, ranges excluding either or both of the included limits are also included in the disclosure.

[0018] With respect to "about," a value or parameter herein includes (and describes) a variation directed to the value or parameter itself. For example, a statement indicating "about X" includes the statement "X."

[0019] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (generally, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition (2012) Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Edition, John Wiley & Sons, Inc.) and chemical methods.

[0021] In the present context, the term "capable of forming hybrid or hybridizing sequences" refers to sequences that are capable of selectively binding to their target sequence under conditions present in the cytosol of a mammalian cell. Such hybridizing sequences can be continuously reverse-complementary to the target sequence or can contain gaps, mismatches or additional non-matching nucleotides. The minimum length of a sequence capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the binding energy than A or T / U nucleotides, and on the backbone chemistry.

[0022] The term "nucleotide" in the present context relates to a nucleic acid or nucleic acid analog building block, an oligomer capable of forming selective hybrids with an RNA or DNA oligomer based on base pairing. The term "nucleotide" in this context includes the classical ribonucleotide building blocks adenosine, guanosine, uridine (and ribosylthymine), cytidine, and the classical deoxyribonucleotides deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. It further includes analogs of nucleic acids such as phosphothioates, 2'O-methylphosphothioates, peptide nucleic acids (PNA; N-(2-aminoethyl)-glycine units linked by peptide bonds, with the nucleobase attached to the alpha-carbon of glycine), or locked nucleic acids (LNA; 2'O,4'C methylene bridged RNA building blocks). Whenever a "hybridizing sequence" is mentioned herein, such a hybridizing sequence may be composed of any of the above nucleotides, or a mixture thereof.

[0023] The term "gene" refers to a polynucleotide that contains at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. Polynucleotide sequences can be used to identify larger fragments or full-length coding sequences of the genes to which they are linked. Methods for isolating sequences of larger fragments are known to those of skill in the art.

[0024] The term "gene expression" or alternatively "gene product" refers to the process and its product, a nucleic acid (RNA) or amino acid (e.g., a peptide or polypeptide) produced when a gene is transcribed and translated.

[0025] As used herein, "expression" refers to the process by which DNA is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0026] The term "antisense oligonucleotide" in the context of this specification relates to an oligonucleotide having a sequence that is essentially complementary to and capable of hybridizing to an RNA. Such antisense action on an RNA results in the modulation, specific inhibition or suppression of the biological action of the RNA. If the RNA is an mRNA, the expression of the resulting gene product is inhibited or suppressed. Antisense oligonucleotides can consist of DNA, RNA, nucleotide analogues and / or mixtures thereof. The skilled artisan knows various commercial and non-commercial sources for calculating the theoretically optimal antisense sequence for a given target. Optimization can be performed both in terms of the sequence of the nucleobases and in terms of the composition of the backbone (ribo, deoxyribo, analogue). There are many sources for the delivery of the actual physical oligonucleotides, which are generally synthesized by solid phase synthesis.

[0027] The term "siRNA" (small / short interfering RNA) in the context of this specification relates to an RNA molecule capable of interfering with (in other words inhibiting or preventing) the expression of a gene that contains a nucleic acid sequence that is complementary to or hybridizes to the sequence of the siRNA in a process called RNA interference. The term siRNA is meant to encompass both single-stranded and double-stranded siRNAs. siRNAs are usually characterized by a length of 17-24 nucleotides. Double-stranded siRNAs can be derived from longer double-stranded RNA molecules (dsRNA). According to a common theory, the longer dsRNA is cleaved by an endoribonuclease (called Dicer) to form double-stranded siRNAs. In a nucleoprotein complex (called RISC), the double-stranded siRNAs are unwound to form single-stranded siRNAs. RNA interference often works through the binding of siRNA molecules to mRNA molecules with complementary sequences, resulting in the degradation of the mRNA. RNA interference can also occur when siRNA molecules bind to intronic sequences of pre-mRNA (immature, unspliced ​​mRNA) in the cell nucleus, causing the degradation of the pre-mRNA.

[0028] The term "shRNA" (small hairpin RNA) in the present context relates to an artificial RNA molecule with a tight hairpin turn that can be used to silence the expression of a target gene via RNA interference (RNAi).

[0029] The term "sgRNA" (single guide RNA) in the present context relates to an RNA molecule capable of sequence-specific inhibition of gene expression via the CRISPR (clustered regularly interspaced short palindromic repeats) mechanism.

[0030] The term "miRNA" (microRNA) in the present context relates to small non-coding RNA molecules (comprising about 22 nucleotides) that function in RNA silencing and post-transcriptional regulation of gene expression.

[0031] The term "inhibitor" in the context of this specification relates to a compound capable of significantly reducing or completely abolishing the physiological function, activity or synthesis of a target molecule. At an abstract level, inhibition encompasses interference with the biosynthesis of a target, prevention of enzyme-substrate binding (the target being a substrate or an enzyme), prevention of ligand-receptor interaction, etc.

[0032] As used herein, the term "treating" or "treatment" of any disease or disorder (e.g., cancer) refers, in one embodiment, to ameliorating the disease or disorder (e.g., slowing, preventing, or reducing at least one of the disease or its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or improving at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Methods for assessing the treatment and / or prevention of a disease are generally known in the art, unless otherwise described below.

[0033] In the context of this specification, the term "prevention or treatment of metastasis" refers to the process of inhibiting the formation of new metastases that did not exist before treatment, including, but not limited to, reducing the survival rate of circulating cancer cells, inhibiting the extravasation of cancer cells from the bloodstream, and inhibiting the dissemination process at the site of extravasation.

[0034] Detailed Description of the Invention A first aspect of the present invention relates to a method for the prevention or treatment of metastasis in a cancer patient comprising administering to said patient a nascent naphthalene derivative. + / K +ATPase Inhibitors Any cancer patient, particularly one at a stage where there is a high risk of metastasis, may be considered at risk for developing metastatic disease that is mediated by or associated with the presence of CTCs.

[0035] In certain embodiments, Na + / K + ATPase inhibitors are provided for the treatment of cancers characterized by the presence of CTC clusters in the bloodstream. In such embodiments, the presence of CTCs is a criterion for treatment according to the present invention.

[0036] Na + / K + ATPase is a transmembrane protein complex found in all higher eukaryotes that functions as a key energy-consuming pump that maintains intracellular ionic and osmotic balance. It is the enzyme (EC 3.6.3.9) that pumps sodium out of the cell and potassium into the cell. Both ions are pumped kinetically against their electrochemical gradients, consuming energy in the form of ATP.

[0037] Na + / K + ATPases are composed of subunits that can be targeted by antisense or other nucleic acid mediated interventions (such as CRISPR). The subunits are the alpha isoforms: ATP1A1 (α1), ATP1A2 (α2), ATP1A3 (α3), and ATP1A4 (α4), and the beta isoforms: ATP1B1 (β1), ATP1B2 (β2), ATP1B3 (β3), and ATP1B4 (β4). Interventions can be targeted specifically to any subunit, or to combinations of subunits based on common sequence content, or to all isoforms of the alpha and / or beta subunits based on identical mRNA sequence tracts.

[0038] In the particular context of the present invention, Na + / K +Inhibitors of ATPase significantly reduce or abolish the target enzyme function, i.e., the pumping of sodium and potassium ions.

[0039] Na + / K + Various classes of chemical compounds are known as examples of ATPase inhibitors. One class includes the well-studied cardiac glycosides, which include both naturally occurring and synthetic inhibitors. + / K + Other examples of ATPase inhibitors are androstene and azaheterocyclyl derivatives of androstene, in particular steroidal NaCl, such as istaloxime (CAS 203737-93-3). + / K + It is an ATPase inhibitor.

[0040] In certain embodiments, inhibitors according to the invention reduce or prevent the formation of new metastases. In certain embodiments, inhibitors according to the invention are useful in the treatment of already existing metastases. In certain embodiments, inhibitors according to the invention are active in both the prevention and treatment of metastases.

[0041] Without wishing to be bound by theory, the inventors have demonstrated that NaCl for use in the prevention or treatment of metastasis according to the present invention is + / K + ATPase inhibitors destroy CTC clusters, resulting in single CTCs compared to CTC clusters, which significantly reduces the possibility of forming metastasis.Cancer patients who have CTC clusters in bloodstream and / or are at high risk of CTC clusters are expected to benefit most from the inhibitors of the present invention.Because metastasis is related to the presence of CTC clusters, and this is not related in all situations, CTC clusters can be detected, and the treatment disclosed herein is beneficial for cancer patients who are suspected to be at risk of developing distant metastasis from primary tumor.

[0042] In certain embodiments, the inhibitors (or nucleic acid agents, as further described below) are provided for use in breast cancer or prostate cancer.

[0043] Typically, patients with breast and prostate cancer have the highest incidence of CTC clusters. However, CTC clusters have been detected in all cancer types, and therefore, the present invention + / K + ATPase inhibitors are expected to be beneficial for cancer patients in general.

[0044] The term "presence of CTC clusters" in bloodstream refers to cancer patients who have CTC clusters somewhere in the bloodstream. In particular, large CTC clusters can be difficult to detect in peripheral blood samples, because CTC clusters are immediately retained in the capillary bed of blood vessels. Therefore, the absence of detectable CTC clusters in peripheral blood samples does not necessarily indicate the absence of CTC clusters in all locations in the bloodstream. Therefore, those skilled in the art recognize that the location of blood collection for detecting CTC clusters must be selected according to the location of the primary tumor or metastasis that releases CTC clusters.

[0045] Known methods for detecting and / or isolating CTC clusters in blood samples include physical property-based methods that exploit differences in cell density, size, dielectric properties, or mechanical plasticity. For example, size selection-based methods exploit the large size of CTCs (and CTC clusters) compared to other blood cells. A non-limiting example of a size-based detection / isolation method is the use of Parsortix devices (Xu et al., PLoS One 10, e0138032, 2015). Another method is that published by Shim et al. (Biomicrofluidics 2013, 7(1):11807 doi:10.1063 / 1.4774304). In certain embodiments, the device for detecting CTCs is a microfluidic device as disclosed in International Patent Application No. 2015 / 077603 / US Patent Application No. 2016 / 279637(A1) or International Patent Application No. 2018 / 005647(A1) / US Patent Application No. 2019 / 160464(A1). In certain embodiments, the device is a microfluidic device as disclosed in US Patent Application No. 2014 / 271990(A1). All patent documents cited herein are incorporated by reference in their entirety.

[0046] Other known methods for the detection / isolation of CTC clusters in cancer patients are antibody-based methods. The antibodies used are primarily specific for epithelial cell surface markers that are not present in blood or stromal cells. See also Balasubramanian et al. (PLoS 1 April 12, 2017; https: / / doi.org / 10.1371 / journal.pone.0175414).

[0047] In the present context, the term circulating tumor cells (CTCs) refers to cells that originate from cancerous tumors, enter the bloodstream, and go on to seed metastases. CTCs can originate from primary tumors and established metastases. Thus, the inhibitors of the present invention are useful for treating cancer patients, regardless of whether they already have established metastases.

[0048] In the present context, the term "CTC cluster" refers to an aggregate of circulating tumor cells that typically contains 2-50 CTCs (Aceto et al., Cell 2014 supra).

[0049] The term "cancer" as used herein may refer to cancers including lung cancer, bladder cancer, breast cancer, colon cancer, kidney cancer, rectal cancer, liver cancer, brain cancer, esophageal cancer, uterine cancer, gallbladder cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, prostate cancer, skin cancer, and hematopoietic tumors; tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; tumors of the central and peripheral nervous system including astrocytoma, neuroblastoma, glioma, and schwannoma; and other tumors including melanoma, seminoma, teratocarcinoma, osteosarcoma, pigmented heterogeneous skin cancer, corneal cancer, thyroid follicular carcinoma, and Kaposi's sarcoma, in particular prostate cancer, lung cancer, breast cancer, liver cancer, stomach cancer, renal cancer, or uterine cancer.

[0050] In certain embodiments, Na + / K + ATPase inhibitors are for cancer patients with breast or prostate cancer.

[0051] In certain embodiments, the cancer is a solid cancer. Solid cancers are characterized by tumors that do not contain cysts or liquid areas.

[0052] In certain embodiments, Na + / K + ATPase inhibitors are cardiac glycosides.

[0053] In the context of this specification, the term "cardiac glycoside" refers to an organic compound comprising a steroid moiety, a lactone moiety covalently attached to C-17 of the steroid, and a glycoside moiety covalently attached to C-3 of the steroid via a glycosidic bond. The steroid and lactone moieties form the aglycone steroid nucleus of the cardiac glycoside. Some cardiac glycosides are aglycones without a glycoside moiety. Two classes of cardiac glycosides are known, which are distinguished by the lactone moiety of the aglycone. Cardenolides have an unsaturated butyrolactone ring as the lactone moiety, and bufadienolides have an α-pyrone ring as the lactone moiety.

[0054] Cardiac glycosides are + / K + ATPase inhibitor that binds potassium and transports it into the cell. + / K + Binds to the extracellular portion of the ATPase. + / K + Extracellular potassium, which induces dephosphorylation of the alpha subunit of ATPase, reduces the action of cardiac glycosides. + / K + Inhibiting ATPase reduces Na + increases in cells. + / Ca 2+ The exchanger pumps calcium out of the cell and sodium into the cell down their concentration gradient. When the concentration gradient of sodium into the cell decreases, Na + / Ca 2+ The ability of the exchanger to function is reduced, resulting in elevated intracellular calcium levels. In the heart, this increases myocardial contractility and increases cardiac vagal tone. Cardiac glycosides exert a characteristic positive inotropic effect on the heart (increasing the strength of myocardial contractions).

[0055] In certain embodiments, the cardiac glycoside is selected from a cardenolide and a bufadienolide.

[0056] In certain embodiments, the cardiac glycoside is selected from digitoxin, ouabain, convallatoxin, proscillaridin, lanatoside C, gitformate, pervoside, strophanthidin, methyldigoxin, deslanoside, bufalin, digoxin, and digoxigenin.

[0057] Digitoxin (CAS 71-63-6) is a cardiac glycoside that occurs naturally in the leaves of the foxglove plant (Digitalis purpurea). Digitoxin is commonly used to treat congestive heart failure. [ka]

[0058] Ouabain (g-strophanthin, (CAS 630-60-4)) is a + / K + It is a cardiac glycoside that acts by inhibiting -ATPase and is primarily used to treat hypotension and cardiac arrhythmias. [ka]

[0059] Convallatoxin (CAS 508-75-8) is a cardiac glycoside of the cardenolide group, occurring naturally in lily of the valley (Convallaria majalis). Convallatoxin is about five times more potent than digitoxin and is primarily used to treat cardiac arrhythmias.

[0060] Proscillaridin (CAS 466-06-8) is a cardiac glycoside of the bufanolide class, primarily used in the treatment of congestive heart failure and cardiac arrhythmias.

[0061] Lanatoside (CAS 17575-22-3) C is a cardiac glycoside of the cardenolide class, primarily used in the treatment of congestive heart failure and cardiac arrhythmias.

[0062] Gitformate (CAS 10176-39-3) is a cardiac glycoside of the bufanolide class, primarily used in the treatment of congestive heart failure and cardiac arrhythmias. Gitformate is a derivative of the naturally occurring cardiac glycoside gitoxin.

[0063] Peruvoside (CAS No. 1182-87-2) is a cardiac glycoside of the bufanolide class, primarily used in the treatment of congestive heart failure and cardiac arrhythmias.

[0064] Strophanthidin is a cardiac glycoside of the cardenolide class, primarily used to treat congestive heart failure and cardiac arrhythmias. Strophanthidin is the aglycone of k-strophanthin, an analogue of ouabain. [ka]

[0065] Digoxin is a naturally occurring cardiac glycoside of the cardenolide class and is primarily used to treat congestive heart failure and cardiac arrhythmias. [ka]

[0066] Digoxigenin (CAS 1672-46-4) is a cardiac glycoside of the cardenolide class. Digoxigenin is the aglycone of digoxin.

[0067] Metildigoxin (CAS 30685-43-9), also known as methyldigoxin, is a cardiac glycoside of the cardenolide class, primarily used in the treatment of congestive heart failure and cardiac arrhythmias.

[0068] Deslanoside (CAS 17598-65-1) is a naturally occurring cardiac glycoside of the cardenolide class, primarily used in the treatment of congestive heart failure and cardiac arrhythmias.

[0069] Bufalin (CAS 465-21-4) is a naturally occurring cardiac glycoside of the bufadienolide class.

[0070] In certain embodiments, the cardiac glycoside is selected from digoxin, digitoxin, and ouabain.

[0071] In a particular embodiment, the cardiac glycoside is digoxin.

[0072] In a particular embodiment, the cardiac glycoside is digitoxin.

[0073] In a particular embodiment, the cardiac glycoside is ouabain.

[0074] In certain embodiments, Na + / K + ATPase inhibitors are for use in disrupting CTC clusters.

[0075] A second aspect of the present invention provides a compound comprising the following components for use in the treatment or prevention of metastatic cancer: - CLDN3 - CLDN4, and - Na + / K + ATPase or any of its constituent subunit isoforms The present invention relates to a nucleic acid molecule comprising or consisting of a nucleic acid sequence of an inhibitor capable of downregulating or inhibiting the expression of a target nucleic acid sequence encoding a protein selected from:

[0076] The data provided in the Examples show that inhibition of the functional expression of any of these proteins leads to a significant inhibition of CTC formation, which in turn is associated with improved clinical outcomes for cancer patients.

[0077] Claudin 3 (CLDN3; Entrez code 1365) and claudin 4 (CLDN4; Entrez code 1364) are components of tight junctions and facilitate cell-cell interactions.

[0078] In general, both antisense targeting of gene targets involved in promoting CTC cluster formation and metastasis and CRISPR or similar approaches are considered.

[0079] In certain embodiments, the nucleic acid sequence of the inhibitor is - an exon contained in the target nucleic acid sequence; - an intron contained in the target nucleic acid sequence; - a promoter region regulating the expression of said target nucleic acid sequence, and / or - an auxiliary sequence controlling the expression of said target nucleic acid sequence, The nucleic acid sequence can specifically hybridize to a sequence or subsequence of

[0080] In certain embodiments, the nucleic acid sequence of the inhibitor is an antisense oligonucleotide, siRNA, shRNA, sgRNA, or miRNA.

[0081] In certain embodiments, the nucleic acid sequence of the inhibitor comprises or consists of a nucleoside analogue.

[0082] Hybridization of the nucleic acid sequence of the inhibitor with an exon, intron, promoter or auxiliary sequence of the target nucleic acid sequence as described above results in a reduction or inhibition of transcription or translation of the target nucleic acid sequence. The mechanism employed can be degradation of the mRNA, for example by RNA interference, CRISPR / Cas systems, inhibition of translation, or blocking of the promoter or enhancer region.

[0083] In certain embodiments, the auxiliary sequence is an enhancer sequence. An enhancer sequence is a short (50-1500 bp) region of DNA that, upon binding by an activator, can increase the likelihood that transcription of a target nucleic acid sequence will occur. An inhibitor nucleic acid sequence will reduce the activity of the enhancer sequence.

[0084] In certain embodiments, the auxiliary sequence is a long non-coding RNA sequence. A long non-coding RNA is a transcript longer than 200 nucleotides that is not translated into protein but controls the transcription or translation of a target nucleic acid sequence.

[0085] In certain embodiments, the nucleic acid sequence of the inhibitor is an antisense oligonucleotide. In certain embodiments, the nucleic acid sequence of the inhibitor is an siRNA. In certain embodiments, the nucleic acid sequence of the inhibitor is an shRNA. In certain embodiments, the nucleic acid sequence of the inhibitor is an sgRNA. In certain embodiments, the nucleic acid sequence of the inhibitor is an miRNA.

[0086] In certain embodiments, the nucleic acid sequence of the inhibitor comprises or consists of a nucleoside analogue.

[0087] One of skill in the art can select appropriate antisense sequences based on genetic information contained in public databases for the target sequence.

[0088] CTC clusters have a higher probability of metastatic dissemination compared to single circulating tumor cells. Therefore, the present invention provides a method for destroying CTC clusters to single CTCs. + / K + ATPase inhibitors and their nucleic acid sequences are advantageous in the prevention and treatment of cancer patients.

[0089] The third aspect of the present invention is a method for producing a Na 2 O 3 according to the first aspect of the present invention. + / K +It relates to an ATPase inhibitor or a nucleic acid molecule according to the second aspect of the invention for use in the prevention and treatment of venous thromboembolism in cancer patients.

[0090] The presence of CTCs in cancer patients is associated with an increased risk of venous thromboembolism. Without wishing to be bound by theory, this is likely due to activation of coagulation via circulating coagulation factors or tissue factor and / or CTC cluster interactions with other cell types such as platelets and endothelial cells (Bystricky et al., critical review in: Oncology / Hematology 114:33-42, 2017).

[0091] The Na + / K + ATPase inhibitors and their nucleic acid sequences can also significantly reduce CTC cluster size and therefore the incidence of venous thromboembolism in cancer patients.

[0092] Na according to the first aspect of the present invention + / K + All embodiments relating to ATPase inhibitors also relate to the third aspect of the invention.

[0093] Another aspect of the present invention relates to the use of the above characterized Na in the manufacture of a medicament for the treatment of cancer as outlined above. + / K + The present invention relates to the use of ATPase inhibitors.Alternatively, the present invention relates to a method for treating cancer.In such a method, an effective amount of the compound described herein (including the dosage form or combination as described) is administered to a subject in need thereof, thereby treating cancer or preventing metastatic spread or recurrence.

[0094] Pharmaceutical Compositions and Administration Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of the present invention, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0095] In certain embodiments, Na according to the present invention and any of its aspects and embodiments. + / K + The ATPase inhibitor is formulated as a dosage form for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation form or suppository. Alternatively, parenteral administration, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms, can be used. Optionally, pharmaceutically acceptable carriers and / or excipients may be present.

[0096] In certain embodiments of the present invention, the compounds of the present invention are generally formulated into pharmaceutical dosage forms that allow for easily controllable drug administration and provide the patient with an accurate and easily administered product.

[0097] In embodiments of the invention relating to topical use of the compounds of the invention, the pharmaceutical compositions are formulated in a manner suitable for topical administration, such as aqueous solutions, suspensions, ointments, creams, gels, or sprayable formulations (e.g., formulations for delivery by aerosol, etc.), which contain the active ingredient together with one or more solubilizing agents, stabilizers, tonics, buffering agents, and preservatives known to those of skill in the art.

[0098] The pharmaceutical compositions can be formulated for oral, parenteral, or rectal administration. Furthermore, the pharmaceutical compositions of the present invention can be configured in a solid form (including, but not limited to, capsules, tablets, pills, granules, powders, or suppositories) or in a liquid form (including, but not limited to, solutions, suspensions, or emulsions).

[0099] The dosing regimen for the compounds of the invention will vary depending on known factors such as the pharmacodynamic properties of the particular drug and its method and route of administration, the race, age, sex, health, medical condition, and weight of the recipient, the nature and extent of the condition, type of concurrent treatment, frequency of treatment, route of administration, renal and hepatic function of the patient, and the desired effect. In certain embodiments, the compounds of the invention may be administered once daily, or the total daily dosage may be administered in divided doses, two, three, or four times daily.

[0100] The pharmaceutical compositions of the present invention may be subjected to conventional pharmaceutical operations such as sterilization, and / or may contain conventional inert diluents, lubricants, or buffers, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, and buffers. They may be produced by standard processes, such as conventional mixing, granulation, dissolution, or lyophilization processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al., The Theory and Practice of Industrial Pharmacy, 4th Edition, 2013 (ISBN 8123922892).

[0101] The present invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived, these examples being intended to illustrate the invention without limiting its scope. EXAMPLES

[0102] Aberrant DNA methylation patterns, including both genome-wide hypomethylation and hypermethylation, have been associated with several types of human cancer (Klutstein et al., Cancer research 76, 3446-3450, 2016; Ehrlich Epigenomics 1, 239-259, 2009; Ehrlich, M, Oncogene 21, 5400-5413, 2002; Feinberg et al., Nat Rev Genet 7, 21-33, 2006). In general, these cancer-associated epigenetic modifications affect distinct genomic regions, with hypermethylation being more frequent in CpG islands, whereas hypomethylation appears to favor regulatory and repetitive elements (Ehrlich, M, Oncogene 21, 5400-5413, 2002). Nevertheless, both modifications have the capacity to alter the expression of neighboring genes and contribute to the cancer phenotype (Klutstein et al., Cancer research 76, 3446-3450, 2016; Ehrlich Epigenomics 1, 239-259, 2009). With regard to regulatory elements, loss of DNA methylation at transcription factor binding sites (TFBS) can indicate transcription factor (TF) networks that are active or networks that are primed for activation at later stages, e.g., during the derivation of induced pluripotent stem cells from differentiated cells (Lee et al., Nat Commun 5, 5619, 2014) or during cancer progression. However, it is unknown about the forces that shape the DNA methylome in breast cancer patients and whether distinct DNA methylation patterns determine the metastatic potential of CTCs.

[0103] DNA methylation patterns in circulating tumor cells (CTCs) and CTC clusters from breast cancer patients We sought to identify transcription factor networks active by available TFBS of single and clustered human breast CTCs, matched within individual liquid biopsies through genome-wide single-cell resolved DNA methylation analysis (bisulfite sequencing). To this end, blood samples were collected from four patients with advanced metastatic breast cancer (Table 1) and processed with Parsortix (Xu et al., PLoS One 10, e0138032, 2015), a microfluidic device that allows size-based, antigen-independent enrichment of CTCs from unmanipulated blood samples. Upon capture, live CTCs were stained for cell surface expression of EpCAM, HER2, and EGFR (Alexa488 or FITC conjugated) and counterstained with antibodies against CD45 to identify contaminating leukocytes (Figure 1a). Upon validation of staining, a total of 18 marker-positive single CTCs and 24 marker-positive CTC clusters (average of 5 ± 2.58 single CTCs and 6 ± 4.24 CTC clusters per patient) were then individually micromanipulated (CellCelector) and deposited in lysis buffer for single-cell whole-genome bisulfite sequencing (Farlik et al., Cell Rep 10, 1386-1397, 2015; Farlik et al., Cell Stem Cell 19, 808-822, 2016).

[0104] [Table 1]

[0105] Principal component analysis (PCA) separated the CTC clusters, which were primarily more heterogeneous compared to single CTCs, from the original patient CTCs (Fig. 1b). To identify differentially methylated regions (DMRs) between single CTCs and CTC clusters, we assessed methylation in 5 kb windows common between at least two different samples in each group, and identified 3347 DMRs with ≥80% methylation differences between single CTCs and CTC clusters. Of these, 1305 DMRs were hypomethylated in CTC clusters and 2042 were hypomethylated in single CTCs. DMRs were analyzed using i-cisTarget, an integrative genomics method that predicts cis-regulatory properties of co-regulated sequences (Herrmann et al., Nucleic Acids Res 40, e114, 2012). Within the hypomethylated DMRs of CTC clusters, we found significant enrichment of several TFBSs, including stemness-related TFs such as OCT4 and STAT3 (Fig. 1c). In contrast, hypomethylated DMRs in single CTCs were enriched in the TFBSs of TFs such as MEF2C and SOX18 (Fig. 1c). To identify specific genes associated with hypomethylated regions in CTC clusters, we used the genomic regions enrichment of annotations tool (GREAT) (McLean et al., Nat Biotechnol 28, 495-501, 2010). Using association rules of a maximum stretch of base plus 50 kb, this analysis revealed 166 genes associated with gene ontology (GO) categories related to processes including cell-cell junctions and membrane receptor activity such as adherens junctions, NMDA receptor activity, lipid transport, and immune responses including NK cell activation and leukocyte apoptosis (Fig. 1d and Table 2). As a parallel approach, global DNA methylation differences were assessed in the TFBSs (Farlik et al., Cell Stem Cell 19, 808-822, 2016), and OCT4 binding sites were found to be consistently hypomethylated in CTC clusters (Table 3). In addition, binding sites for other TFs associated with pluripotency, such as SOX2 and ESRRB, as well as binding sites for TFs associated with cell cycle progression, such as SIN3A, were also hypomethylated (Table 3).On the other hand, in single CTCs, this method observed hypomethylation at the TFBS of several TFs, including c-MYC and E2F4 (Table 3). Collectively, these results indicate that CTC clusters have access to a stem cell-related OCT4-centered TF network and a cell cycle progression-related SIN3A-centered TF network, paralleling embryonic stem cell (ESC) biology, thus suggesting that these networks simultaneously control self-renewal and proliferation (Niwa, Development 134, 635-646, 2007; Kim et al., Cell 132, 1049-1061, 2008; van den Berg et al., Cell Stem Cell 6, 369-381, 2010). In contrast, single CTCs appear to feature a c-MYC-centric network that is commonly enriched in various cancers, but is largely independent of the core pluripotency network and is more involved in regulating genes related to metabolism (Kim et al., Cell 132, 1049-1061, 2008; Kim et al., Cell143, 313-324, 2010).

[0106] [Table 2]

[0107] [Table 3]

[0108] DNA methylation patterns of circulating tumor cells (CTCs) and CTC clusters from an established mouse model Spontaneously generated GFP-labeled single CTCs and CTC clusters from three independent mouse xenograft models, including two human breast CTC-derived cell lines (BR16 and BRx50) and the breast cancer cell line MDA-MB231 (lung metastatic variant, called LM2), were isolated to test the robustness of the findings (Yu et al., Science 345, 216-220, 2014; Minn et al., Nature 436, 518-524, 2005). In this setup, 71 single CTCs and 47 CTC clusters (Table 4) were individually micromanipulated and processed for single-cell whole genome bisulfite sequencing (Farlik et al., Cell Rep 10, 1386-1397, 2015; Farlik et al., Cell Stem Cell 19, 808-822, 2016). Similar to patient CTCs, PCA analysis of xenograft CTCs showed separation mainly based on cell line of origin, yet higher overall homogeneity of the samples compared to patient CTCs. We assessed DMRs with >70% methylation difference between single CTCs and CTC clusters and found a total of 1430 DMRs, of which 909 were hypomethylated in CTC clusters and 521 were hypomethylated in single CTCs. Using i-cisTarget analysis, we identified 40 TFBSs hypomethylated in CTC clusters and 74 TFBSs hypomethylated in single CTCs (Fig. 2a). Interestingly, in agreement with patient data, both binding sites of OCT4-centric TF networks, such as those belonging to SOX2, NANOG, STAT3, and REX1, and binding sites of SIN3A were hypomethylated in xenograft CTC clusters. On the other hand, in contrast to patient CTCs, the availability of stem cell-related TF networks seemed to be controlled by local DNA methylation remodeling at DMRs, rather than influencing the global DNA methylation profile of CTCs. This was supported by the finding that only a handful of TFBSs were preferentially hypomethylated in either single CTCs (n=13) or CTC clusters (n=9) (Figure 2b). Thus, the individual DNA methylation profiles of patient and xenograft CTCs seem to reflect their clustering status.We also show that in breast cancer, an interplay between methylation dynamics and phenotypic properties of CTCs occurs, and that CTC clustering is associated with an epigenetic predisposition to undergo stem cell-related processes and cell cycle progression.

[0109] [Table 4]

[0110] Stem cell-like related transcription factor network To identify whether the available stem cell-related TF network is also transcriptionally active, we performed single-cell degraded RNA-seq analysis of 48 single CTCs and 24 CTC clusters isolated from six breast cancer patients with advanced metastatic disease matched within individual liquid biopsies, and 49 single CTCs and 54 CTC clusters isolated from three xenograft mouse models (Table 4). We further investigated a set of 335 genes previously shown to be consistently upregulated in mouse and human embryonic stem cells and embryonic carcinoma cells in contrast to their differentiated counterparts (Wong et al., Cell Stem Cell 2, 333-344, 2008). A subset of 301 of these 335 genes was found to be expressed in CTC samples. Using these genes, we performed weighted gene co-expression network analysis (WGCNA) to identify four expression modules in human breast cancer samples (blue, grey, turquoise, brown) and four expression modules in xenograft CTC clusters (green, yellow, orange, purple), revealing module-trait relationships in CTCs. Notably, 85 transcripts enriched in patient CTC clusters and 153 transcripts enriched in xenograft CTC clusters were identified (Tables 5 and 6), with 90% overlap between patient and xenograft CTC cluster-enriched stem cell-related transcripts. Interestingly, while transcripts enriched in patient CTC clusters and those overlapping between patient and xenograft are primarily involved in cell cycle progression as judged by network analysis (Fig. 3a), TF target gene analysis confirmed the activity of SIN3A, OCT4, and CBFB, among others, TFs with significantly hypomethylated binding sites (Fig. 3b). Similarly, in xenograft-derived CTC clusters, in addition to genes enriched and detected in patient CTC clusters, TF target gene analysis highlighted the activity of OCT4, including TFs with significantly hypomethylated binding sites, such as SIN3A, NANOG, SOX2, RORA, FOXO1, and BHLHE40 (Figure 3c).TF target gene analysis of single CTCs further confirmed the activity of c-MYC, as well as p53 and E2F4, among others (Figure 3e). Collectively, the gene expression data support the model proposed by the DNA methylation analysis and demonstrate that CTC clusters are primed for an OCT4-centered stem cell-associated TF network and exhibit activation of SIN3A-dependent cell cycle progression programs. Activation of these programs plays a role in determining the metastatic proliferation capacity of CTC clusters.

[0111] DNA methylation patterns of CTC clusters shape accessible and active transcription factor networks that confer a growth advantage to CTC clusters over single CTCs in breast cancer patients. The forces shaping the DNA methylome involve both global differences at TFBSs and local events that mediate responses to environmental cues and phenotypic traits. The ability to dynamically shape the DNA methylome in response to environmental stimuli can be exploited therapeutically by repurposing FDA-approved compounds.

[0112] [Table 5] JPEG0007671988000010.jpg211166

[0113] [Table 6] JPEG0007671988000012.jpg220166

[0114] Dissociation of CTC clusters To identify actionable vulnerabilities of CTC clusters and to test whether epigenetic and transcriptional features of clustered CTCs are reversible upon dissociation of clusters into single cells, we performed the following steps. First, we assessed the expression of all known cell-cell junction (CCJ) components in patient samples from normal breast (TGCA REF), breast cancer (TCGA REF), single CTCs, and CTC clusters (Aceto et al., Cell 2014). Breast cancer cells tend to only partially reduce their CCJ repertoire compared to normal breast cells, whereas CTCs express only a small fraction of CCJ components, probably as a result of increased motility. Nevertheless, CTC clusters retain more CCJs compared to single CTCs. This analysis highlights therapeutic opportunities, showing that CTC clusters rely on a limited number of CCJ components for multicellular adhesion and that approaches aimed at dissociating them could rescue normal tissue expressing a larger variety of CCJs. To this end, 2486 FDA-approved compounds were evaluated for their ability to dissociate clusters of human breast CTC-derived cells. Cluster dissociation was assessed using high-content screening microscopy, comparing cells treated with individual compounds with the negative and positive controls, steady-state clustered BR16 cells and 40 μm filtered BR16 single cell suspensions, respectively (Figure 4a). Interestingly, the significant reduction in average cluster size upon filtration did not affect viability, but did reduce mitochondrial membrane potential, as measured by tetramethylrhodamine methyl ester perchlorate (TMRM) intensity (Figure 4a). The majority of the 2486 FDA-approved compounds did not detect any reduction in cell viability (>70% viability) in BR16 CTC-derived cells when used at 5 μM concentration for 2 days under hypoxic conditions, nor did they reduce detectable average cluster size (>450 μm). 2 However, we identified 39 compounds that significantly reduced the average cluster size without compromising viability. These compounds included Na + / K +Inhibitors of ATPases (n=6), HDACs (n=2), nucleotide biosynthesis (n=5), kinases (n=4), GPCRs (n=2), cholesterol biosynthesis (n=1), nuclear export (n=1), tubulin (n=9), and DNA-binding compounds (n=8) and antibiotics (n=1) were included. Reducing compound concentrations to 1 μM, 0.5 μM, and 0.1 μM resulted in a concomitant increase in the average cluster size of BR16 and BRx50 human CTC-derived cells (Figure 4b). Concomitant with the effect on cluster size, an increase in the number of detected nuclei, mitochondrial membrane potential, and overall viability of both cell lines was observed with decreasing compound concentrations, indicating that cluster size correlates with the overall fitness and proliferation capacity of CTCs (Figure 4b). Under these conditions, six compounds, namely Na + / K + The ATPase inhibitors digitoxin and ouabain octahydrate, the tubulin-binding agents podofilox (also known as podophyllotoxin), colchicine and vincristine sulfate, and the tubulin-binding agent and dual kinase inhibitor rigosertib consistently resulted in a significant reduction in the average cluster size of BR16 and Brx50 CTC cell lines, even at the lowest concentration tested (0.1 μM) (Figure 4b).

[0115] Effect of CTC cluster dissociation on DNA methylation To directly assess the effect of clustering on DNA methylation patterns and proliferation signatures, BR16 and BRx50 cell lines were cultured for 17 days to ensure at least four divisions, allowing adequate time for DNA methylation remodeling to occur. Long-term culture in the presence of 20 nM of ATPase and kinase inhibitors was found to be optimal for cell proliferation and reduction in average cluster size (Figure 5a), and an average of n = 20 cells in triplicate were further processed for WGBS and RNA sequencing.

[0116] Under these conditions, for both CTC-derived cell lines, a subset of cluster-associated hypomethylated DMRs from patients and xenografts regains 20% or more methylation. Interestingly, this gain of methylation occurs at DMRs that contain binding sites for stem cell-associated TFs, and ouabain treatment of BR16 cell lines simultaneously affects binding sites for OCT4, SOX2, and NANOG. This indicates that dissociation of CTC clusters in patient-derived CTC lines leads to DNA remodeling that reduces the availability of binding sites for stem cell-associated TFs.

[0117] [Table 7]

[0118] CLDN3 / CLDN4 knockout We assessed whether disruption of cell-cell junctions in CTC-derived cells would result in cluster dissociation as well as DNA methylation remodeling at CTC cluster-associated DMRs. To this end, we used CRISPR technology to simultaneously knock out both claudin 3 (CLDN3) and claudin 4 (CLDN4), two of the most highly expressed strong junction proteins in CTC clusters, in BR16 CTC-derived cells. Using two independent sgRNAs for each gene, we generated three BR16 lines with double CLDN3 / 4 knockouts, which also showed a significant reduction in the average CTC cluster size (Figure 16F, G).

[0119] Whole-genome bisulfite sequencing of CLDN3 / 4 double knockout cells revealed that upon dissociation into single cells and after Na + / K +Similar to the events that occurred upon ATPase inhibition, many CTC cluster-associated hypomethylated regions showed gain in methylation (Figure ​(Figure9E).9E). Interestingly, i-cis Target analysis of regions that gained higher levels of methylation revealed enrichment of binding sites for OCT4, SOX2, NANOG, and SIN3A (Figure​(Figure9F),Further indicating that CTC clustering directly affects DNA methylation dynamics at binding sites for stem cell- and proliferation-related TFs.

[0120] From these results, Na + / K + ATPase inhibition increases intracellular Ca ++ We show that increased concentrations of IL-16 induce dissociation of CTC clusters through the consequent inhibition of cell-cell junction formation, leading to DNA methylation remodeling at key stem cell- and proliferation-associated junction sites.

[0121] Na + / K + Treatment with ATPase inhibitors suppresses the formation of spontaneous metastases To test whether ouabain and digitoxin also allow the destruction of CTC clusters in vivo, we adopted a two-fold approach. First, we tested whether in vitro treatment with ouabain and digitoxin for 17 days leads to a reduced capacity of treated cells to efficiently seed metastases in untreated mice (Figure 7A). To this end, BR16 cells stably expressing GFP-luciferase were injected into the tail vein of NSG mice at the time of treatment and non-invasively monitored by luminescence imaging for their capacity to seed and grow metastatic lesions. We found that treatment with digitoxin or ouabain did not affect the capacity of BR16 cells to persist in lung tissue immediately after injection ("day 0"; see Figure 7B), but led to a reduced capacity to survive during the first day of arrival, as confirmed by a significant increase in the expression of cleaved caspase 3 compared to control cells (see "day 1"; Figures 7B and 8A,B). Overall, this difference in survival potential at the very early stage of metastatic dissemination resulted in a delay in metastatic growth, as measured over a period of 72 days after injection, despite the absence of further treatment in vivo (Figure 7C).

[0122] Second, to more closely mimic the clinical setting and evaluate the effect of the CTC cluster dissociation strategy on the spontaneous formation of CTC clusters and metastasis from primary tumors, we injected BR16 cells into the mammary fat pad of NSG mice. Fourteen weeks after primary tumor formation, we administered ouabain daily for three weeks and assessed CTC composition and the occurrence of spontaneous metastatic lesions (Fig. 7D). Importantly, we observed that ouabain treatment reduced the frequency of spontaneously generated CTC clusters while increasing the frequency of single CTCs (Fig. 7E), without altering either the size of the primary tumor or the overall CTC number (Fig. 8C,D). In addition to reducing the frequency of CTC clusters, ouabain treatment also led to a significant inhibition (80.7-fold) of the total metastatic burden (Fig. 7F,G). Similarly, when ouabain treatment was administered to NSG mice bearing spontaneously metastatic LM2 tumors, we also observed an increase in the proportion of single CTCs and a decrease in CTC clusters (Fig. 8E), without altering either the size of the primary tumor or the overall number of CTCs (Fig. 8F, G), leading to a reduced metastatic burden compared to controls (Fig. 8H).

[0123] Digoxin treatment Digoxin treatment in BR16 xenograft mice did not significantly affect tumor size (Figure 10), but it significantly reduced the number of CTC clusters and CTC-neutrophil clusters (Figure 11) and inhibited metastasis (Figure 12).

[0124] Similarly, treatment of LM2 xenografted mice did not result in significant differences in tumor size (Figure 13). However, digoxin treatment increased overall survival (Figure 14) and reduced the formation of CTC and CTC-neutrophil clusters (Figure 15).

[0125] Taken together, these results suggest that in vivo Na + / K + We show that ATPase inhibition suppresses the ability of cancerous lesions to spontaneously release CTC clusters, resulting in a significant reduction in metastatic seeding capacity.

[0126] Clinical Trials Patients receive a daily maintenance dose of digoxin. The daily dose of digoxin is calculated according to renal function and target serum digoxin concentration and is applied in the morning (before 10 am) with the treatment plan adjusted based on the efficacy of 0.125 mg and 0.25 mg tablets. Blood samples for analysis of mean CTC cluster size are taken at screening, on days 0 (2 hours after the first oral intake), 3 and 7. If the digoxin serum level on days 7 or 14 is less than 0.70 ng / ml, maintenance therapy with digoxin is continued for up to 3 weeks depending on the digoxin serum level. During the third week of maintenance therapy, individual dose adjustments are made as necessary.

[0127] Materials and Methods cell culture CTC-derived cells were maintained under hypoxia (5% oxygen) on ultra-low attachment (ULA) 6-well plates (Corning, Cat. No. 3471-COR). CTC growth medium containing 20ng / ml recombinant human epidermal growth factor (Gibco, Cat. No. PHG0313), 20ng / ml recombinant human fibroblast growth factor (Gibco, Cat. No. 100-18B), 1x B27 supplement (Invitrogen, Cat. No. 17504-044) and 1x antibiotic-antimycotic (Invitrogen, Cat. No. 15240062) in RPMI 1640 medium (Invitrogen, Cat. No. 52400-025) was added every 3 days. For passaging, cells were spun down at 800g for 5 min using a Heraeus Multifuge X3R centrifuge (Invitrogen, Cat. No. 75004515). The supernatant was then aspirated and the cells were resuspended in 2 ml / well CTC medium and seeded in 6-well ULA plates. BR16 CTC-derived cells were generated in the inventor's laboratory. Brx50 CTC-derived cells were obtained from the Haber and Maheswaran labs (MGH Cancer Center, Harvard Medical School, Boston, MA). MDA-MB-231 (LM2) cells were donated by Joan Massague's lab (MSKCC, New York, NY, USA) and passaged in DMEM / F-12 medium (Invitrogen, Cat#11330057) supplemented with 10% FBS (Invitrogen, Cat#10500064) and 1× antibiotic-antimycotic (Invitrogen, Cat#15240062). For passaging, LM2 cells were washed once with D-PBS (Invitrogen, Cat. No. 14190169) and dissociated using 0.25% trypsin (Invitrogen, Cat. No. 25200056).

[0128] CTC capture and identification Blood samples for CTC analysis were obtained from the University Hospital Basel after obtaining patient consent according to protocols EKNZ BASEC2016-00067 and EK321 / 10, which received ethical approval from the Swiss authorities (EKNZ, Ethics Committee for Northwest / Central Switzerland). On average, 7.5 ml of blood was collected per patient in EDTA vacutainers. Blood was processed in Parsortix GEN3D6.5 cell separation cassettes (Angle Europe) within 1 h of collection. For mouse studies, blood was collected by cardiac puncture and 1 ml of blood was processed similarly through the Parsortix device. Captured CTCs were further stained with EpCAM-AF488-binding antibody (CellSignaling, Cat. No. CST5198), HER2-AF488-binding antibody (Cat. No. 324410, BioLegend), EGFR-FITC-binding antibody (GeneTex, Cat. No. GTX11400), and CD45-BV605-binding antibody (Biolegend, Cat. No. 304042 (anti-human); Cat. No. 103140 (anti-mouse)) in Parsortix cassettes. In all other models (xenografts) with cancer cells stably expressing the GFP-luciferase reporter, only anti-CD45 staining was performed, whereas CTCs were identified based on GFP expression. The number of captured CTCs, including single CTCs, CTC clusters, and CTC-WBC clusters, was determined while the cells were still in the cassettes. CTCs were then released from the cassette in DPBS (no. 14190169, Gibco) onto ultra-low attachment plates (no. 3471-COR, Corning). Representative images were taken at 40x magnification on a Leica DMI4000 fluorescent microscope using a Leica LAS and analyzed with ImageJ.

[0129] Differential white blood cell staining in CTC-WBC clusters Live CTCs captured in Parsortix microfluidic cassettes were stained with anti-biotin-CD45 (cat. no. 103104, BioLegend) and detected with streptavidin-BV421 (cat. no. 405226, BioLegend), anti-mouse Ly-6G-AF594 (cat. no. 127636, BioLegend) and anti-CD11b-AF647 (clone M1 / 70, a gift from Dr. Roxane Tussiwand, University of Basel) or anti-F4 / 80-AF594 (cat. no. 123140, BioLegend) and CD11b-AF647. Additionally, MMTV-PyMT-derived CTCs were marked with EpCAM-AF488 (cat. no. 118210, BioLegend). The cells were then gently released from the microfluidic system onto an ultra-low attachment plate and immediately imaged (Leica DMI400). + CD11b med ), monocytes (Ly-6G - CD11b med / high ) and macrophages (F4 / 80 + CD11b + The number of CTC-WBC clusters with chromatin-specific morphology was evaluated. Immediately after imaging, cells were centrifuged (500 rpm, 3 min) on glass slides and fixed with methanol for 1 min. After a short air-drying, slides were stained using a Wright-Giemsa staining kit (no. 9990710, ThermoFisher) to visualize the nuclear morphology of captured cells, according to the manufacturer's instructions.

[0130] Tumorigenesis assay All mouse experiments were performed in accordance with institutional guidelines.

[0131] For tail vein experiments, NOD SCID Gamma (NSG) mice (Jackson's laboratory) were inoculated with 1 × 10 IgG resuspended in 100 μl of D-PBS. 6 BR16-mCherry cells were injected and monitored with an IVIS Lumina II (Perkin Elmer). For isolation of CTC xenograft mouse models, 1 × 10 6LM2-GFP, 1 x 10 6 BRx50-GFP, or 1 x 10 6 BR16-GFP cells were resuspended in 100 μl of 50% Cultrex PathClear low growth factor basement membrane extract (R&D Biosystems, Cat. No. 3533-010-02) in D-PBS and injected orthotopically into NSG mice. Blood samples were taken 4–5 weeks after tumor onset for LM2 cells, 5–6 months after tumor onset for BR16, and 6–7 months after tumor onset for BRx50 cells.

[0132] Micromanipulation of single cells Enriched CTCs were collected from Parsortix cassettes in 1 ml D-PBS solution (Invitrogen, Cat. No. 14190169) in 6-well ultra-low attachment plates (Corning, Cat. No. 3471-COR) and visualized using a CKX41 Olympus inverted fluorescent microscope (part of the AVISO CellCelector Micromanipulator-ALS). Single CTCs and CTC clusters were identified based on intact cell morphology, AF488 / FITC positive staining, and lack of BV605 staining. Target cells were individually micromanipulated using a 30 μM glass capillary on an AVISO CellCelector micromanipulator (ALS) and deposited into individual PCR tubes (Axygen, catalog no. 321-032-501) containing 10 μl of 2× digestion buffer (EZ DNA Methylation Direct Kit- Zymo, catalog no. D5020) for WGBS or 2 μl of RLT lysis buffer (Qiagen, catalog no. 79216) supplemented with 1 U / μl SUPERase In RNAse inhibitor (Invitrogen, catalog no. AM2694) for RNA-seq, and immediately flash frozen in liquid nitrogen.

[0133] Single-cell whole-genome bisulfite sequencing Proteinase K digestion and bisulfite treatment were performed according to the manufacturer's instructions for the EZ DNA Methylation Direct Kit (Zymo, catalog no. D5020). Bisulfite-treated DNA was eluted using 9 μl of elution buffer and used to generate libraries using the TruSeq DNA Methylation Kit (Illumina, catalog no. EGMK91396) according to the manufacturer's instructions. For amplification, 18 cycles were performed using the FailSafe Enzyme (Illumina, catalog no. FSE51100) and indexes were introduced using the Index Primers Kit (Illumina, catalog no. EGIDX81312). Library purification was performed using Agencourt AMPure XP beads in a 1:1 ratio according to the manufacturer's instructions. To avoid DNA loss during pipetting steps, Corning DeckWork low-binding barrier pipette tips were used (Sigma, catalog no. CLS4135-4X960EA). Library concentrations were estimated using the Qubit DS DNA HS assay kit according to the manufacturer's instructions (Invitrogen, catalog no. Q32854).

[0134] Creating an RNA-seq library RNA was captured on beads conjugated with oligo-dT primers according to Macaulay et al. (Nat Protoc 11, 2081-2103, 2016). cDNA was generated according to the Smart-Seq2 protocol by Picelli et al. (Nat Protoc 9, 171-181, 2014). Sequence libraries were generated and indexed from 0.25 ng of cDNA per sample using the Nextera XT DNA Library Preparation Kit (Illumina, catalog no. FC-131-2001) according to the manufacturer's instructions.

[0135] FDA Approved Compound Screening A library containing 2486 FDA-approved compounds was purchased from Nexus Platform-ETH Zurich. Each compound was resuspended using CTC medium at a concentration of 15 μM and 20 μl was aliquoted in duplicate into a total of 64 flat-bottom clear ultra-low attachment 96-well plates (Corning, Cat. No. 3474).

[0136] To reduce the cluster size of the CTC-derived cell lines, a 40 μm cell strainer was used (Corning, Cat. No. 431750). 40 μl containing 5000 CTC-derived cells were seeded per well into a 96-well ultra-low attachment plate containing 20 μl of pre-dispensed FDA-approved compounds at a concentration of 15 μM, resulting in a final compound concentration of 5 μM. Plates were incubated for 2 days in hypoxia (5% oxygen) and then 20 μl was transferred to 96-well black / clear tissue culture treated plates (BD Falcon, Cat. No. 353219) containing 40 μl D-PBS (Invitrogen, Cat. No. 14190169) and stained for 1 h at 37°C with final concentrations of 4 μM Hoechst 34580 (Invitrogen, Cat. No. H21486), 2 μM TMRM (Invitrogen, Cat. No. T668), and 4 μM TOTO-3 (Invitrogen, Cat. No. T3604). Each plate included two positive controls (untreated cells) and two negative controls (untreated and 40 μM filtered cells). Z-factors were calculated for each individual plate using the following formula: Z'=1-3(σ s +σ c ) / |μ s -μ c | 3 (σ: standard deviation, μ: mean, s: positive control, c: negative control) (Martin et al., PLoS One9, e88338, 2014) with a range between 0.62 and 0.937. Plates were scanned using an Operetta high-content imaging system (Perkin Elmer) and cluster analysis was performed using the Harmony high-content imaging system and analysis software (Perkin Elmer).

[0137] Enrichment Score The enrichment score (ES) indicates the over- or under-representation of a particular object in a sample containing many objects. A positive ES indicates that a particular feature is over-represented (=enriched) compared to other features in the set of analyzed functions. A negative enrichment score indicates the opposite, i.e., the presence of the feature is less than would be expected given the values ​​of other features in the sample. In other words, a positive ES for a transcription factor binding site (TFBS) indicates that the TFBS is represented in the sample to a higher extent than other TFBSs (=enriched). The enrichment score can be normalized by dividing the particular ES by the average enrichment score of all objects in the dataset to obtain the normalized enrichment score (NES). The normalization of the enrichment score accounts for differences in the size of the gene sets and differences in correlation between the gene sets and the expression dataset, and thus the normalized enrichment score (NES) can be used to compare the analysis results across gene sets. In the analysis, only TFBSs with an NES score of 3.0 or higher are considered significant.

[0138] CRISPR-CAS9 CLDN3 / 4 double knockout in BR16 We used lentiviral delivery of pLenti-Cas9-EGFP vector (Addgene) to generate BR16 CTC-derived cell lines stably expressing Cas9 protein along with GFP. Then, in the BR16-Cas9-GFP line, we introduced sgRNA sequences targeting either CLDN3 or CLDN4. In detail, the sgRNA sequences were designed using the GPP web portal (https: / / portals.broadinstitute.org / gpp / public / analysis-tools / sgrna-design). Two sgRNAs targeting CLDN3 (sense 5'-CACGTCGCAGAACATCTGGG-3' (SEQ ID NO: 01) and sense 5'-ACGTCGCAGAACATCTGGGA-3' (SEQ ID NO: 02)) were cloned into the vector pLentiGuide-Puro (Addgene), and two sgRNAs targeting CLDN4 (sense 5'-CAAGGCCAAGACCATGATCG-3' (SEQ ID NO: 03) and sense 5'-ATGGGTGCCTCGCTCTACGT-3' (SEQ ID NO: 04)) were cloned into the vector pLentiGuide-Blast. The vector pLentiGuide-Blast was generated by replacing the puromycin resistance gene of the plasmid pLentiGuide-Puro with a blastidine resistance gene using the MluI and BsiWI restriction enzyme sites. Double-positive clones were selected based on puromycin (1 μg / mL) and blasticidin (10 μg / mL) antibiotic selection for 2 weeks, and CLDN3 / CLDN4 knockout was verified by Western blot.

[0139] Survival analysis Survival analysis was performed using the survival R package (v2.41-3). Kaplan-Meier curves were generated and the significance of survival differences between groups was estimated using the log-rank test. For patients, progression-free survival was defined as the time from primary tumor diagnosis to first recurrence. For the analysis of mouse models, death was chosen as the end point of the analysis and was defined as the time when a given animal had to be euthanized according to our mouse protocol guidelines.

Claims

1. A pharmaceutical composition for use in the prevention or treatment of cancer metastasis, comprising: ＋ / K ＋ The present invention relates to a method for treating cancer, the method comprising administering to a patient a therapeutically effective amount of a Na + / K + ATPase inhibitor, the cancer being characterized by the presence of CTC clusters in the bloodstream, the Na + / K + ATPase inhibitor being an organic compound that includes an aglycone steroid nucleus of a cardiac glycoside formed from a steroid moiety and a lactone moiety covalently attached to C-17 of the steroid, ＋ / K ＋ A pharmaceutical composition, wherein the ATPase inhibitor is effective in disrupting CTC clusters.

2. The Na ＋ / K ＋ 2. The pharmaceutical composition for use in the prevention or treatment of metastasis according to claim 1, wherein the ATPase inhibitor is a cardiac glycoside.

3. The Na ＋ / K ＋ 3. A pharmaceutical composition for use in the prevention or treatment of metastasis according to claim 1 or 2, wherein the ATPase inhibitor is selected from cardenolides and bufadienolides.

4. The pharmaceutical composition for use in preventing or treating metastasis according to any one of claims 1 to 3, wherein the lactone moiety is an unsaturated butyrolactone ring or an α-pyrone ring.

5. The Na ＋ / K ＋ 2. The pharmaceutical composition for use in the prevention or treatment of metastasis according to claim 1, wherein the ATPase inhibitor is selected from digitoxin, ouabain, convallatoxin, proscillaridin, lanatoside C, gitformate, pervoside, strophanthidin, methyldigoxin, deslanoside, bufalin, digoxin and digoxigenin.

6. The Na ＋ / K ＋ 6. The pharmaceutical composition for use in the prevention or treatment of metastasis according to claim 5, wherein the ATPase inhibitor is selected from digoxin, digitoxin and ouabain.

7. The Na ＋ / K ＋ The pharmaceutical composition for use in the prevention or treatment of metastasis according to claim 6, wherein the ATPase inhibitor is digoxin and the daily dose of digoxin is 0.125 mg to 0.25 mg.

8. The Na ＋ / K ＋ The pharmaceutical composition for use in the prevention or treatment of metastasis according to claim 6, wherein the ATPase inhibitor is digoxin and the digoxin serum level is adjusted to between 0.70 ng / ml and 1.0 ng / ml.

9. The pharmaceutical composition for use in preventing or treating metastasis according to any one of claims 1 to 8, wherein the cancer is breast cancer or prostate cancer.

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