Method for treating cancer exhibiting high expression of SFRP4

Abstract

The present invention relates to a method for treating cancer exhibiting high expression of SFRP4. In particular, the present invention was developed through intensive research to identify a highly reliable treatment method for stem cell-type gastric cancer that has the worst prognosis and for which effective therapeutic agents are difficult to develop. As a result, it was confirmed that the cancer stem cell properties decreased when Wnt protein was injected to the MKN1 gastric cancer cell line, which exhibits high expression of SFRP4. In addition, it was confirmed that secreted frizzled-related protein 4 (SFRP4), phosphorylated and secreted by protein kinase A (PKA) can form a complex with β-catenin and TCF4 to increase transcription of Wnt target genes, and by inhibiting PKA to prevent phosphorylation of SFRP4, a decrease in cancer stem cell properties was observed, which leads to the completion of the present invention. The invention can be advantageously used as a novel therapeutic agent for stem cell-type gastric cancer.

Description

Treatment of cancers with high SFRP4 expression

[0001] The present invention relates to a method for treating cancer showing high expression of SFRP4.

[0002] Cancer has a high mortality rate worldwide, and in Western societies, it is the second most common cause of death after cardiovascular disease. In particular, the incidence of stomach, colon, breast, and prostate cancers is steadily increasing due to factors such as an aging population, the widespread consumption of high-fat diets resulting from Westernized diets, a rapid increase in environmental pollutants, and increased alcohol consumption. Among these, stomach cancer is the fourth leading cause of cancer death worldwide, with a particularly high incidence in Asia, making it the leading cause of cancer-related deaths. In Korea, it is estimated that 16.2% of cancer patients (20.3% of male cancer patients and 11.2% of female cancer patients) are stomach cancer patients.

[0003] Gastric cancer can be classified into three tumor types based on gene expression patterns: immune subtype (IM), stem-like subtype (ST), and epithelial subtype (EP). Among the three subtypes, stem-like gastric cancer has the worst prognosis and is unresponsive to chemotherapy. Therefore, identifying therapeutic targets for stem-like gastric cancer is essential to improving gastric cancer mortality, and ongoing research is becoming increasingly necessary.

[0004] Therefore, the present invention was conducted to treat stem cell-type gastric cancer, which has a very poor prognosis and lacks effective treatments. As a result, it was confirmed that cancer stem cell potential was reduced when Wnt protein was injected into MKN1, a gastric cancer cell line with high SFRP4 expression. Furthermore, it was confirmed that secreted frizzled-related protein 4 (SFRP4), phosphorylated by protein kinase and secreted, can increase the transcription of Wnt target genes. In addition, it was confirmed that inhibition of protein kinase inhibited the phosphorylation of SFRP4, thereby reducing cancer stem cell potential. Therefore, the present invention is expected to be widely utilized in the medical field as a novel treatment for stem cell-type gastric cancer with high SFRP4 expression.

[0005] The present inventors have diligently researched to discover a reliable treatment method for gastric cancer, especially stem cell-type gastric cancer, which has the worst prognosis and for which the development of effective treatments is difficult. As a result, we confirmed that when Wnt protein was injected into the MKN1 gastric cancer cell line with high expression of SFRP4, cancer stem cell potential was reduced. Furthermore, we confirmed that secreted frizzled-related protein 4 (SFRP4), which is phosphorylated by protein kinase A (PKA) and secreted, forms a complex with β-catenin and TCF4 to increase transcription of Wnt target genes. Furthermore, we discovered that inhibiting the phosphorylation of SFRP4 by inhibiting PKA reduced cancer stem cell potential, thereby completing the present invention.

[0006] Therefore, the purpose of the present invention is to provide a treatment method for refractory gastric cancer (SEM (Stem-like / EMT / Mesenchymal) positive gastric cancer) using the expression pattern of SFRP4.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0008] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0009] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0010] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0011] According to one aspect of the present invention, the present invention provides a method for treating cancer comprising a Wnt (Wingless-related integration site) protein or a nucleic acid encoding the same as an active ingredient.

[0012] In particular, we have made extensive research efforts to discover a highly reliable treatment method for stem cell-type gastric cancer, which has the worst prognosis and is difficult to discover effective treatments. As a result, we confirmed that cancer stem cell capacity was reduced when Wnt protein was injected into MKN1, a gastric cancer cell line with high expression of SFRP4. In addition, we confirmed that frizzled-related protein 4 (SFRP4), which is phosphorylated by protein kinase A (PKA) and secreted, can form a complex with β-catenin and TCF4 to increase transcription of Wnt target genes. In addition, we discovered that inhibiting the phosphorylation of SFRP4 by inhibiting PKA decreased cancer stem cell capacity, thereby completing the present invention.

[0013] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of immune subtype (IM), stem-like subtype (ST), and epithelial subtype (EP) gastric cancer.

[0014] According to the present invention, the effectiveness of chemotherapy can be predicted in advance through genetic analysis of gastric cancer. Gastric cancer can be broadly classified into three types based on gene expression: immune type, epithelial type, and stem cell type. However, it has been reported that immune type, epithelial type, and stem cell type each respond differently to chemotherapy. First, it has been confirmed that gastric cancer patients belonging to the immune type are hardly helped by chemotherapy, while epithelial type patients respond best to chemotherapy. Third, patients classified as stem cell type have the worst overall prognosis, and it has been reported that stem cell type patients are minimally responsive to chemotherapy.

[0015] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16.

[0016] According to a specific embodiment of the present invention, the composition is wherein the cancer is primary cancer.

[0017] According to a specific embodiment of the present invention, the composition is a composition in which secreted frizzled-related protein 4 (SFRP4) is overexpressed. In the present invention, overexpression or increased expression of SFRP4 means an increase in expression of 30% or more, 50% or more, and preferably 100% or more compared to the average of a normal sample or all gastric cancer samples.

[0018] According to the present invention, the composition may include Wnt protein at a concentration of 20 ng / ml to 60 ng / ml, but is not limited thereto.

[0019] According to the present invention, "Wnt-3a" refers to a protein encoded by the WNT3A gene in humans. The WNT gene family consists of structurally related genes encoding secreted signaling proteins, which play a crucial role in tissue homeostasis, embryonic development, and disease.

[0020] According to the present invention, “critical value” refers to the state of the boundary where the state changes when the state of a substance changes due to a certain phenomenon. At this time, the critical value is the value representing the physical quantity of the substance. The boundary where the state changes usually includes the solid-liquid boundary and the liquid-gas boundary, and the factor that changes this state is the temperature according to the internal energy of the substance. Therefore, the melting point / freezing point, which is the temperature where the solid-liquid changes, and the vaporization point / liquefaction point, which is the temperature where the liquid-gas changes, can be referred to as the critical value. The boundary point where the state changes by reaching this critical value is called the critical point. Therefore, the critical value represents a physical quantity, and the critical point represents a moment in time or a location in space.

[0021] According to a specific embodiment of the present invention, the composition is one in which the Wnt protein is increased in transcription by SFRP4.

[0022] The term "Wnt signaling pathways" used herein refers to signaling pathways that begin with proteins that transmit signals to cells via cell-surface receptors. The name Wnt is a portmanteau of Wingless and Int-1, and the Wnt signaling pathway utilizes either paracrine communication between neighboring cells (paracrine) or autocrine communication within the same cell (autocrine). Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt / calcium pathway. All three pathways are activated by the binding of Wnt-protein ligands to Frizzled family receptors, which then transmit biological signals to Dishevelled proteins within the cell.

[0023] According to a specific embodiment of the present invention, the composition is one in which the SFRP4 is phosphorylated by a protein kinase.

[0024] The term “Secreted frizzled-related protein 4 (SFRP4)” herein refers to the protein encoded by the SFRP4 gene in humans. As a member of the SFRP family containing a cysteine-rich domain homologous to the putative Wnt binding site of frizzled proteins, SFRPs act as soluble modulators of Wnt signaling. SFRP4 is a hub gene of a Type 2 diabetes-associated gene co-expression module in human islets and reduces glucose-induced insulin secretion through reduced β-cell exocytosis. The expression and release of SFRP4 in islets is enhanced by interleukin-1β, and its level is elevated in serum for years before the clinical diagnosis of Type 2 diabetes.

[0025] In this specification, the term "phosphorylation" refers to the chemical process of attaching a phosphate group to a molecule. Phosphorylation and its reverse reaction, dephosphorylation, are crucial to many biological processes. Protein phosphorylation is particularly important for function; for example, phosphorylation regulates the function of nearly half of the enzymes in Saccharomyces cerevisiae by activating (or inactivating) them.

[0026] According to a specific embodiment of the present invention, the protein kinase is a composition selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

[0027] The term "protein kinase" in this specification refers to an enzyme that catalyzes a phosphorylation reaction, which transfers a phosphate group from a high-energy donor molecule, such as ATP, to a specific substrate. Proteins, lipids, or carbohydrates change in activity, reactivity, and ability to bind to other molecules depending on their phosphorylation state. Therefore, kinases play a crucial role in metabolism, cell signaling, protein regulation, cell transport, secretion, and numerous other cellular response pathways.

[0028] As used herein, the term “protein kinase A (PKA)” refers to an enzyme whose activity depends on cellular levels of cyclic AMP (cAMP). PKA, also known as cAMP-dependent protein kinase, has multiple functions in cells, including the regulation of glycogen, sugar, and lipid metabolism.

[0029] As used herein, the term “protein kinase C (PKC)” refers to a family of protein kinase enzymes involved in regulating the function of other proteins through phosphorylation of the hydroxyl groups of serine and threonine amino acid residues. These proteins, or PKC enzymes that are members of this family, interact with diacylglycerol (DAG) or calcium ions (Ca 2+ ) are sequentially activated by signals such as an increase in the concentration of PKC. Therefore, PKC enzymes play an important role in several signal transduction cascades.

[0030] As used herein, the term “mitogen-activated protein kinases (MAPKs)” refers to a type of protein kinase that is specific for the amino acids serine and threonine (i.e., serine / threonine-specific protein kinases). MAPKs are involved in directing cellular responses to various stimuli, such as mitogens, osmotic stress, heat shock, and proinflammatory cytokines, and regulate cellular functions including proliferation, gene expression, differentiation, mitosis, cell survival, and apoptosis.

[0031] The term “AKT (Protein kinase B)” in this specification refers to a set of three serine / threonine-specific protein kinases that play a central role in various cellular processes, such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Akt kinases regulate cell survival and metabolism by binding and regulating numerous downstream effectors, such as nuclear factor-κB and Bcl-2 family proteins.

[0032] According to a specific embodiment of the present invention, the composition is one in which the SFRP4 is overexpressed in a primary tumor.

[0033] As used herein, the term "primary tumor" refers to a tumor that grows at the initial anatomical site where tumor progression begins and forms a cancerous mass. Most cancers originate at the primary site, but they can metastasize to other parts of the body. These additional tumors are called secondary tumors.

[0034] According to a specific embodiment of the present invention, the composition is one in which the SFRP4 forms a complex with β-catenin and TCF4.

[0035] As used herein, the term "β-catenin" refers to a part of a protein complex that forms adherens junctions. This cell-cell adhesion complex is necessary for the formation and maintenance of epithelial cell layers and barriers, and as a component of the complex, β-catenin can regulate cell growth and adhesion between cells.

[0036] The term “transcription factor 4 (TCF-4)” as used herein refers to the protein encoded by the TCF4 gene, also known as immunoglobulin transcription factor 2 (ITF-2), located on chromosome 18q21.2 in humans. The TCF4 protein acts as a transcription factor that binds to the immunoglobulin enhancer mu-E5 / kappa-E2 motif, and TCF4 activates transcription by binding to the E-box (5'-CANNTG-3') typically found in the SSTR2-INR or somatostatin receptor 2 initiation element. TCF4 is involved in fetal neurological development during pregnancy, primarily by binding to DNA and initiating neural differentiation. During early development, it is found in the central nervous system, somites, and gonadal protuberances, and later in development, it is found in the thyroid, thymus, and kidney. In adulthood, TCF4 is found in lymphocytes, muscle, mature neurons, and the gastrointestinal system.

[0037] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating cancer, comprising Wnt protein and SFRP4 as active ingredients.

[0038] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of immune subtype (IM), stem-like subtype (ST), and epithelial subtype (EP) cancer.

[0039] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16.

[0040] According to a specific embodiment of the present invention, the composition is wherein the cancer is primary cancer.

[0041] According to a specific embodiment of the present invention, the composition is a composition in which SFRP4 is overexpressed.

[0042] According to the present invention, the composition may include Wnt protein at a concentration of 20 ng / ml to 60 ng / ml, but is not limited thereto.

[0043] According to another aspect of the present invention, the present invention provides a composition for treating cancer comprising Wnt protein, SFRP4, and a protein kinase A inhibitor as active ingredients.

[0044] As used herein, the term "protein kinase inhibitor" refers to a type of enzyme inhibitor that blocks the action of one or more protein kinases (phosphorylation enzymes). Protein kinases are enzymes that can add a phosphate (PO4) group to proteins and regulate their function. The phosphate group is typically added to the amino acids serine, threonine, or tyrosine in proteins. Most kinases act on both serine and threonine, tyrosine kinases act on tyrosine, and many (dual-specificity kinases) act on all three.

[0045] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of immune subtype (IM), stem-like subtype (ST), and epithelial subtype (EP) cancer.

[0046] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16.

[0047] According to a specific embodiment of the present invention, the composition is wherein the cancer is primary cancer.

[0048] According to a specific embodiment of the present invention, the composition is a composition in which SFRP4 is overexpressed.

[0049] According to the present invention, the composition may include Wnt protein at a concentration of 20 ng / ml to 60 ng / ml, but is not limited thereto.

[0050] According to a specific embodiment of the present invention, the protein kinase A inhibitor is a composition selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof.

[0051] The term “GSK690693” herein refers to a pan-Akt inhibitor targeting Akt1 / 2 / 3 with an IC50 of 2 nM / 13 nM / 9 nM in cell-free assays. It also acts on the AGC kinase family members PKA, PrkX, and PKC isoenzymes. GSK690693 also potently inhibits AMPK and DAPK3 of the CAMK family with an IC50 of 50 nM and 81 nM, respectively, affects Unc-51-like autophagy-activating kinase 1 (ULK1) activity, and potently inhibits STING-dependent IRF3 activation.

[0052] The term “H-89” as used herein refers to a protein kinase inhibitor that has the greatest effect on protein kinase A (PKA). Derived from H-8 (N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide), H-89 initially acts specifically as an inhibitor of PKA and is more potent than H-8 in inhibiting PKA. It works by competitive inhibition of the adenosine triphosphate (ATP) site in the PKA catalytic subunit. According to a specific embodiment of the present invention, the C may be a compound of Cas No. 127243-85-0.

[0053] The term “AT13148” herein refers to an oral ATP-competitive substance and a potent ROCK-AKT inhibitor. Like GSK690693, it also acts on the AGC kinase family of enzymes, including PKA, PrkX, and PKC. In a specific embodiment of the present invention, AT13148 may be a compound with Cas No. S7563.

[0054] According to another aspect of the present invention, the present invention provides a composition for killing cancer cells, comprising a Wnt protein or a nucleic acid encoding the same as an active ingredient.

[0055] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of immune subtype (IM), stem-like subtype (ST), and epithelial subtype (EP) cancer.

[0056] According to a specific embodiment of the present invention, the composition is one selected from the group consisting of Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16.

[0057] According to a specific embodiment of the present invention, the composition is wherein the cancer is primary cancer.

[0058] According to a specific embodiment of the present invention, the composition is a composition in which secreted frizzled-related protein 4 (SFRP4) is overexpressed.

[0059] According to a specific embodiment of the present invention, the composition is one in which the Wnt protein is increased in transcription by SFRP4.

[0060] According to a specific embodiment of the present invention, the composition is one in which the SFRP4 is phosphorylated by a protein kinase.

[0061] According to a specific embodiment of the present invention, the protein kinase is a composition selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

[0062] According to a specific embodiment of the present invention, the composition is one in which the SFRP4 is overexpressed in a primary tumor.

[0063] According to a specific embodiment of the present invention, the composition is one in which the SFRP4 forms a complex with β-catenin and TCF4.

[0064] According to another aspect of the present invention, the present invention provides a method for screening a candidate substance for cancer treatment, comprising the steps of: (a) confirming overexpression of SFRP4 (compared to a control group) in a cancer tissue or cancer cell sample isolated from an individual; (b) confirming Wnt protein expression in the sample; (c) administering a candidate substance for cancer treatment to the sample; (d) re-confirming Wnt protein expression in the sample; and, (e) determining that the candidate substance has a cancer treatment effect when Wnt expression decreases and cancer tissue or cancer cells die.

[0065] According to the present invention, the control group refers to a sample with low expression of SFRP4 in a cancer tissue or cancer cell sample isolated from a healthy person or individual.

[0066] According to a specific embodiment of the present invention, the screening method is one selected from the group consisting of immune subtype (IM), stem-like subtype (ST), and epithelial subtype (EP) cancer.

[0067] According to a specific embodiment of the present invention, the screening method is one in which the cancer tissue or cell has overexpressed SFRP4.

[0068] According to another aspect of the present invention, the present invention provides a method for selecting a patient group amenable to cancer treatment by administration of a protein kinase A (PKA) inhibitor, comprising the steps of: (a) confirming SEM (Stem-like / EMT / Mesenchymal) positivity in a gastric cancer sample isolated from a subject; (b) measuring the level of phosphorylated SFRP4 (Secreted frizzled-related protein 4) expression in the sample; and (c) predicting that the subject is amenable to cancer treatment by administration of a PKA (protein kinase A) inhibitor when the level of phosphorylated SFRP4 expression is high.

[0069] According to a specific embodiment of the present invention, the SEM (Stem-like / EMT / Mesenchymal) benign gastric cancer belongs to Classification 3 of Table 1 below.

[0070] Category 1 Category 2 Category 3 PrognosisFavorableModerateUnfavorableYCCImmuneEpithelialStem-likeTCGAMSI, EBVCINGSACRGMSIMSS / TP53-MSS / EMTSingapore-DukeProliferativeMetabolicMesenchymal

[0071] According to a specific embodiment of the present invention, the phosphorylation of the SFRP4 is phosphorylated by a protein kinase, and the protein kinase may be any one selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

[0072] In addition, according to a specific embodiment of the present invention, the PKA inhibitor may be H-89 (Cas No. 127243-85-0) or AT13148 (Cas No. S7563).

[0073] According to another aspect of the present invention, the present invention provides a method for screening a candidate substance for PKA inhibition, comprising the steps of: (a) confirming SEM (Stem-like / EMT / Mesenchymal) positivity in a gastric cancer sample isolated from an individual; (b) measuring the level of phosphorylated SFRP4 expression in the sample; (c) administering a candidate substance for PKA inhibition to the sample; (d) re-measuring the level of phosphorylated SFRP4 expression in the sample; and, (e) determining that the candidate substance has a PKA inhibitory effect when the level of phosphorylated SFRP4 expression decreases.

[0074] According to a specific embodiment of the present invention, the SEM (Stem-like / EMT / Mesenchymal) benign gastric cancer belongs to Classification 3 of Table 1.

[0075] According to a specific embodiment of the present invention, the phosphorylation of the SFRP4 is phosphorylated by a protein kinase, and the protein kinase may be any one selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

[0076] In addition, according to a specific embodiment of the present invention, the candidate substance for PKA inhibition may be H-89 (Cas No. 127243-85-0) or AT13148 (Cas No. S7563).

[0077] According to another aspect of the present invention, the present invention provides a method for screening a candidate substance for cancer treatment, comprising the steps of: (a) confirming SEM (Stem-like / EMT / Mesenchymal) positivity in a gastric cancer sample isolated from a subject; (b) measuring the level of phosphorylated SFRP4 expression in the sample; (c) administering a candidate substance for cancer treatment to the sample; (d) re-measuring the level of phosphorylated SFRP4 expression in the sample; and, (e) determining that the candidate substance has a cancer treatment effect when the level of phosphorylated SFRP4 expression decreases.

[0078] According to a specific embodiment of the present invention, the SEM (Stem-like / EMT / Mesenchymal) benign gastric cancer belongs to Classification 3 of Table 1.

[0079] According to a specific embodiment of the present invention, the phosphorylation of the SFRP4 is phosphorylated by a protein kinase, and the protein kinase may be any one selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

[0080] In addition, according to a specific embodiment of the present invention, the candidate substance for cancer treatment may be H-89 (Cas No. 127243-85-0) or AT13148 (Cas No. S7563).

[0081] According to another aspect of the present invention, there is provided a method for treating cancer, comprising the steps of: (a) confirming SEM (Stem-like / EMT / Mesenchymal) positivity in a gastric cancer sample isolated from a patient; (b) measuring the expression level of phosphorylated SFRP4 (Secreted frizzled-related protein 4) in the sample; and, (c) administering a PKA (protein kinase A) inhibitor to the patient when the expression level of phosphorylated SFRP4 is high. In the method for treating cancer, the PKA inhibitor may be any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof, but is not limited thereto.

[0082] According to another aspect of the present invention, the present invention provides a use of a PKA inhibitor for the treatment of patients with SEM (Stem-like / EMT / Mesenchymal) positive and high levels of phosphorylated SFRP4 (Secreted frizzled-related protein 4) expression. In the use of the PKA inhibitor, the PKA inhibitor may be any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof, but is not limited thereto.

[0083] Hereinafter, the present invention will be described in detail based on examples.

[0084] The features and advantages of the present invention are summarized as follows:

[0085] (a) The present invention provides a method for treating cancer showing high expression of SFRP4.

[0086] (b) The present invention has been made with great effort to discover a highly reliable treatment method for stem cell-type gastric cancer, which has the worst prognosis and is difficult to discover effective treatments. As a result, it was confirmed that cancer stem cell capacity was reduced when Wnt protein was injected into MKN1, a gastric cancer cell line with high expression of SFRP4. In addition, it was confirmed that secreted frizzled-related protein 4 (SFRP4), which is phosphorylated by protein kinase A (PKA) and secreted, can form a complex with β-catenin and TCF4 to increase transcription of Wnt target genes. In addition, it was discovered that inhibiting the phosphorylation of SFRP4 by inhibiting PKA reduced cancer stem cell capacity, thereby completing the present invention. The present invention can be usefully utilized as a new treatment for stem cell-type gastric cancer.

[0087] Figures 1 to 3 illustrate clinical analyses of SFRP4 in gastric cancer patients according to one embodiment of the present invention. Figure 1 shows SFRP4 expression in normal and primary tumor samples from TCGA analyzed in UALCAN, and Figure 2 shows the top 25 genes overexpressed in gastric adenocarcinomas in the UALCAN database. Figure 3 shows the results of a Kaplan-Meier plot analysis using overall survival, first progression, and post-progression survival classified by SFRP4 expression level (gene symbol: 204051_s_at).

[0088] Figures 4 to 9 illustrate the effect of SFRP4 on stemness-related properties dependent on Wnt signaling in gastric cancer cells according to an experimental example of the present invention. Figure 4 shows the difference in SFRP4 expression in non-refractory or refractory gastric cancer cells. Figure 5 shows the results of a cell viability test performed after 72 hours of treatment with 5-FU, and the IC50 value calculated after 72 hours of treatment with 5-FU and oxaliplatin. Figure 6 shows the value of Wnt luciferase activity measured after transfection of pCVM-SFRP4 in SK4. RLA (relative luciferase activity) is an assay used to determine whether a protein can activate or inhibit the expression of a target gene. Figure 7 evaluates the sphere-forming ability after transfection of pCMV-SFRP4 in SK4 and MKN1. Figures 8 and 9 show the results of evaluating the wound healing capacity of SK4 and MKN1 cells after transfection with pCMV-SFRP4, respectively. Cell motility was captured 24 hours after scratching and measured using ImageJ. The data in Figures 4 to 9 were obtained from three independent experiments, and error bars represent standard deviations. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

[0089] Figures 10 and 11 illustrate, according to one experimental example of the present invention, that the putative kinase PKA interacts molecularly with SFRP4. Figure 10 lists site-specific kinases capable of phosphorylating SFRP4 in the Quokka database. Figure 11 shows that proteins solubilized in non-reduced buffer were immunoprecipitated with an anti-SFRP4 antibody. Equal amounts of protein were loaded onto SDS-PAGE and immunoblotted with antibodies against the putative kinase.

[0090] Figures 12 to 15 illustrate the evaluation of PKA inhibition on Wnt signal-dependent stem cells in gastric cancer according to an experimental example of the present invention. In Figure 12, Wnt luciferase activity was measured after 24 hours of treatment with H-89 or AT13148, and relative luciferase activity was calculated. RLA represents relative luciferase activity, and FC represents fold change. Figure 13 shows the activity of Wnt3A according to the critical concentration that can act on SFRP4 in the presence or absence of H-89 or AT13148 as a PKA inhibitor. Figure 14 shows that cells were fractionated, and each fraction was immunoblotted with an antibody. T represents total, C represents cytoplasm, and N represents nucleus. Figure 15 confirms that SFRP4 is activated by the influence of H-89 (a PKA inhibitor). The data in Figures 12 to 15 were obtained from three independent experiments, and the error bars represent the standard deviation. *p<0.05, **p<0.01, ***p<0.005, ****p<0.001.

[0091] Because mutant SFRP4 did not induce Wnt signaling-dependent stem cells, SFRP4 was dephosphorylated using the PKA inhibitors H-89 or AT13148. Treatment with either H-89 or AT13148 decreased Wnt activity. Interestingly, treatment with Wnt3a also decreased Wnt activity compared to the control. Since SFRP4 is secreted and is generally known as a Wnt antagonist, these data suggest that secreted SFRP4 inhibits Wnt3 activity. Furthermore, treatment with either H-89 or AT13148 not only decreased the amount of β-catenin binding to SFRP4, but also reduced nuclear SFRP4. This suggests that PKA induces a higher affinity for SFRP4 and β-catenin than for the dephosphorylated form, and that PKA-mediated phosphorylation of SFRP4 contributes to its function in cancer cells. Therefore, we confirmed that inhibition of PKA can reduce the phosphorylation of SFRP4, and nuclear SFRP4 can act as an agonist of Wnt signaling.

[0092] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0093] Example

[0094] cell culture

[0095] Human gastric cancer cell lines MKN1 and SK4 were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum, 100 μg / ml penicillin / streptomycin, and 1 mM sodium pyruvate in a humidified incubator at 37°C in a 5% CO2 atmosphere.

[0096] To establish cell lines in which SFRP4 overexpression or knockdown is stably maintained, DH5-alpha competent cells were transfected with retroviral SFRP4 ORF vectors and lentiviral shSFRP4 vectors purchased from Origene, and selected on agar plates containing appropriate antibiotics such as 100 μg / ml ampicillin or 15 μg / ml chloramphenicol. Transfected cells were selected on RPMI-1640 containing appropriate antibiotics such as 1 mg / ml kanamycin or 1 μg / ml puromycin, and verified by Western blot data.

[0097] Western blot

[0098] Cells were lysed in RIPA lysis buffer (Pierce) supplemented with a protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF), and total protein lysates were quantified using the BSA assay. Equal amounts of protein were separated by SDS-PAGE and then transferred to PVDF membranes for 1 h. After blocking with 5% skim milk for 1 h, the membranes were incubated with primary antibodies (Table 2).

[0099] AntibodyProduct codeSourcesSFRP4ab154167AbcamGAPDHG9545Sigmaβ-cateninsc-7199Santa cruzTCF42569Cell signaling technologiesLamin A / C2032Cell signaling technologiesSFRP4LS-C314315LifeSpan BiosciencesPhospho serine / threonineab17464Cell signaling technologiesPKAab187515Abcamp-PKAab75991AbcamNormal mouse IgGsc-2025Santa cruz

[0100] Wnt luciferase activity assay

[0101] TopFlash / FopFlash plasmids were purchased from Addgene and transfected into cells with a Renilla luciferase plasmid as a control. Four days after transfection, firefly and Renilla luciferase activities were measured according to the Dual-Luciferase Reporter Assay protocol (Promega).

[0102] Co-Immunoprecipitation

[0103] Cells were lysed in IP lysis buffer (Pierce) supplemented with a protease inhibitor cocktail and PMSF. Equal amounts of protein lysates were spun down with specific antibodies for incubation, followed by the addition of appropriate magnetic beads and incubation overnight at 4°C. The incubated samples were washed five times with PBS-T (PBS containing 0.1% Tween), boiled at 98°C for elution, and the magnetized samples were loaded onto SDS-PAGE.

[0104] Kinase assay

[0105] In vitro kinase activity was measured using the ADP-Glo ​​kinase assay (Promega, USA). ATP-to-ADP conversion curves were generated to generate standard curves according to the manufacturer's protocol. Kinase, substrate, and ATP were prepared for the kinase reaction, and after the kinase reaction, the remaining ATP was depleted. The ADP generated by the kinase reaction was converted to newly synthesized ATP, which was detected by luminescence, and kinase activity was measured as the amount of newly synthesized ATP.

[0106] Nuclear / cytoplasmic fractionation

[0107] Cells were dissociated with trypsin, treated with 20 μM H-89 or 10 μM AT13148 for 24 h, and then washed with PBS (Cellec Chemical, Houston, USA). Cell lysates were fractionated using a nuclear / cytoplasmic fractionation kit (Biovision, K266). Cytoplasmic proteins were identified with GAPDH, and nuclear proteins were identified with Lamin A / C.

[0108] Sphere-forming assay

[0109] 1X10 5 Cells / ml were seeded in 6-well ultra-low attachment plates (Corning, USA) in DMEM / F12 (Gibco) with 5% FBS supplemented with 10 ng / ml insulin and 1 ug / ml hydrocortisone, and 500 μl of medium per well was added every 2 days. Photographs were taken to assess the size of spheroids relative to cell aggregates, and representative pictures of spheroids were taken under a microscope at 40X magnification.

[0110] Wound-healing assay

[0111] Cells were seeded in 6-well plates and incubated until >80% confluent. The cells were scraped with a sterile 1000 μl pipette tip to create a straight wound line, and the cells were then continuously cultured in medium for 24 h. The wound area was captured at 0 h and 24 h using a digital camera system. Representative images of migrating cells were taken with a microscope at 40X magnification, and the migration distance was measured using the software program Celleste 5 (Thermo Fisher Scientific).

[0112] Chemoresistance test

[0113] Cells were seeded in 96-well plates at a confluency of more than 90% 72 hours after chemotherapy treatment. 5-FU was administered the following day, and 72 hours later, cell viability was measured using the MTS test (Promega, USA) according to the manufacturer's instructions. Twenty microliters of MTS solution was added to each well, and the cells were incubated for 2 hours at 37°C in a 5% CO2 incubator. The absorbance of each well was measured at a wavelength of 490 nm using an ELISA plate reader.

[0114] Phosphoproteomics database analysis

[0115] We used the bioinformatics tool Quokka (http: / quokka.erc.monash.edu / ) to identify site-specific kinases that phosphorylate SFRP4 at T186 and T189. Putative kinases were selected with scores higher than 0.5.

[0116] UALKAN and Kaplan-Meier plot analysis

[0117] SFRP4 expression in gastric cancer patients was analyzed using the UALKAN (http: / ualcan.path.uab.edu / ) and KM-plot databases (https: / kmplot.com / ). TCGA data were used for the UALKAN analysis, and overall survival, first-progression, and post-progression survival were analyzed using the KM-plot database.

[0118] Statistical analysis

[0119] All statistical analyses were performed using Prism5 (GraphPad Software, USA). Differences between two-sample groups and groups with more than two samples were assessed using Student's t-test and two-way ANOVA, respectively, for multiple comparisons, with p<0.05 as the cutoff for statistical significance.

[0120] Overexpressed SFRP4 is positively correlated with poor prognosis in gastric cancer.

[0121] A 2018 study in Lancet Oncology suggested that SFRP4 could be a biomarker indicating the level of SFRP4 in patients with stem-like subtype gastric cancer, a type that is chemotherapy-unresponsive and has a poor prognosis. Using the Ualkan and KM-plot databases, we analyzed the clinical relevance of SFRP4 in gastric cancer. SFRP4 was found to be highly overexpressed in primary tumors compared to normal tissues in the Ualkan analysis (Figure 1). It was the 15th most overexpressed gene in the entire human genome (Figure 2). Consistent with previous reports, the KM-plot showed that high SFRP4 expression correlated with a poor prognosis (Figure 3). Therefore, these results indicate a significant positive correlation between SFRP4 and poor prognosis in gastric cancer.

[0122] SFRP4 induces stemness-related properties dependent on Wnt signaling in gastric cancer

[0123] According to the Human Protein Atlas database, SFRP4 is overexpressed in both the extracellular and cytoplasmic domains. To determine the cellular localization of SFRP4 in gastric cancer, we profiled the steady-state level of endogenous SFRP4 expression in a panel of 27 gastric cancer cell lines. In addition to its secretory nature, SFRP4 exhibited intracellular expression in all gastric cancer cell lines. Although SFRP4 was not exclusively overexpressed in EMT cell lines or downregulated in non-EMT cell lines, it was highly expressed in most suspension cell lines. This may suggest that SFRP4 is highly expressed in circulating tumor cells with high metastatic potential. Seven cell lines with high and low SFRP4 expression were selected from each panel and subjected to chemosensitivity testing to select the most stem-like cell lines (Figures 4 and 5). Interestingly, when SFRP4 was overexpressed in SFRP4-low expressing cell lines, Wnt activity was increased (Fig. 6), as well as sphere formation and wound healing abilities (Figs. 7-9). These results suggest that intracellular SFRP4 may be an agonist of Wnt signaling.

[0124] Furthermore, when cell lines with high SFRP4 expression were knocked down, Wnt activity, spheroidization, wound healing, and chemical resistance were reduced. These results confirmed that SFRP4 induces stemness-related characteristics through Wnt signaling.

[0125] PKA phosphorylates SFRP4 at T186 and T189 in gastric cancer.

[0126] Previous studies have shown that phosphorylated SFRP4 can bind to β-catenin and form a complex with TCF4 to regulate the transcription of Wnt target genes. Phosphoproteomics databases were analyzed to identify putative kinases capable of phosphorylating SFRP4 at T186 and T189 (Fig. 10). PKA was selected due to its high score in Quokka and its availability of phosphorylation sites (Fig. 11).

[0127] Inhibition of PKA reduces Wnt signaling-dependent stemness in gastric cancer.

[0128] Because mutant SFRP4 did not induce Wnt signaling-dependent stem cells, SFRP4 was dephosphorylated by treatment with the PKA inhibitors H-89 or AT13148. Treatment with either H-89 or AT13148 resulted in a decrease in Wnt activity (Fig. 12). Interestingly, treatment with Wnt3a also decreased Wnt activity compared to the control (Fig. 13). Since SFRP4 is secreted and is generally known as a Wnt antagonist, these data suggest that secreted SFRP4 inhibits Wnt3 action. Furthermore, treatment with either H-89 or AT13148 not only reduced the amount of β-catenin bound to SFRP4, but also reduced nuclear SFRP4 (Figs. 14 and 15). This suggests that PKA induces a higher affinity for SFRP4 and β-catenin than for the dephosphorylated form, and that PKA-mediated phosphorylation of SFRP4 may mediate its function in cancer cells. Therefore, PKA inhibition can reduce SFRP4 phosphorylation, and nuclear SFRP4 can act as an agonist of Wnt signaling.

[0129] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0130] The present invention is expected to be widely used in the medical field as a new treatment for stem cell-type gastric cancer with high expression of SFRP4.

Claims

1. (a) A step for confirming SEM (Stem-like / EMT / Mesenchymal) positivity in a gastric cancer sample isolated from an individual; (b) a step of measuring the expression level of phosphorylated SFRP4 (Secreted frizzled-related protein 4) in the sample; and, (c) a step of predicting that cancer treatment is possible by administering a PKA (protein kinase A) inhibitor to the individual if the expression level of phosphorylated SFRP4 is high; a method for selecting a patient group eligible for cancer treatment by administering a PKA inhibitor.

2. In paragraph 1, A method wherein the phosphorylation of the above SFRP4 is phosphorylated by protein kinase.

3. In paragraph 2, A method wherein the protein kinase is any one selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

4. In paragraph 1, A method according to claim 1, wherein the PKA inhibitor is any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof. 5.(a) Step of confirming SEM (Stem-like / EMT / Mesenchymal) positivity in gastric cancer samples isolated from the subject; (b) a step of measuring the expression level of phosphorylated SFRP4 in the sample; (c) a step of administering a candidate substance for PKA inhibition to the sample; (d) a step of re-measuring the level of phosphorylated SFRP4 expression in the sample; and, (e) a step of determining that the candidate substance has a PKA inhibitory effect when the level of phosphorylated SFRP4 expression is reduced; a screening method for a candidate substance for PKA inhibition, comprising:

6. In paragraph 5, A method wherein the phosphorylation of the above SFRP4 is phosphorylated by protein kinase.

7. In paragraph 6, A method wherein the protein kinase is any one selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

8. In paragraph 5, A method according to claim 1, wherein the candidate substance for PKA inhibition is any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof. 9.(a) Step of confirming SEM (Stem-like / EMT / Mesenchymal) positivity in a gastric cancer sample isolated from the subject; (b) a step of measuring the expression level of phosphorylated SFRP4 in the sample; (c) a step of administering a candidate substance for cancer treatment to the sample; (d) a step of re-measuring the level of phosphorylated SFRP4 expression in the sample; and, (e) a method for screening a candidate substance for cancer treatment, comprising a step of determining that the candidate substance has a cancer treatment effect if the level of phosphorylated SFRP4 expression is reduced; 10. In paragraph 9, A method wherein the phosphorylation of the above SFRP4 is phosphorylated by protein kinase.

11. In paragraph 10, A method wherein the protein kinase is any one selected from the group consisting of protein kinase A, protein kinase C, Mos / Raf kinases, mitogen-activated protein kinases (MAPKs), phosphorylase kinase, and protein kinase B (AKT).

12. In paragraph 9, A method according to claim 1, wherein the cancer treatment candidate is any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof. 13.(a) Step of confirming SEM (Stem-like / EMT / Mesenchymal) positivity in gastric cancer samples isolated from patients; (b) a step of measuring the expression level of phosphorylated SFRP4 (Secreted frizzled-related protein 4) in the sample; and, (c) a method for treating cancer, comprising administering a PKA (protein kinase A) inhibitor to the patient when the level of phosphorylated SFRP4 expression is high.

14. In paragraph 13, A method according to claim 1, wherein the PKA inhibitor is any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof.

15. Treatment of patients with SEM (Stem-like / EMT / Mesenchymal) positivity and high levels of phosphorylated SFRP4 (Secreted frizzled-related protein 4) expression using PKA inhibitors.

16. In paragraph 15, The use of the above PKA inhibitor is any one selected from the group consisting of GSK690693, Fasudil (HA-1077), A-674563, H-89, AT13148, Staurosporine (AM-2282), Daphnetin, H-1152, and HA-100, or a pharmaceutically acceptable salt thereof.

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