Pharmaceutical composition for preventing or treating cancer, comprising LRP6 o-glcnacylation inhibitor as active ingredient

Abstract

The present invention relates to a pharmaceutical composition comprising an LRP6 O-GlcNAcylation inhibitor as an active ingredient for preventing or treating cancer, and to a method for determining sensitivity to an LRP6 O-GlcNAcylation inhibitor and a screening method. Based on the finding of the mechanism by which DKK1 inhibits the Hippo signaling pathway, the LRP6 O-GlcNAcylation inhibitor interferes with the O-GlcNAcylation of LRP6 to activate the Hippo signaling pathway, thereby inhibiting the proliferation and metastasis of cancer cells. The LRP6 O-GlcNAcylation inhibitor specifically inhibits only the O-GlcNAcylation of LRP6 without inhibiting the activity of OGT itself, and this action occurs specifically in DKK1-overexpressing cells, and thus is expected to function as an anticancer drug with very few side effects.

Description

A pharmaceutical composition for the prevention or treatment of cancer comprising an LRP6 O-GLCNACYLATION inhibitor as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, comprising an LRP6 O-GlcNAcylation inhibitor as an active ingredient.

[0002]

[0003] The Hippo pathway (or Salvador-Warts-Hippo pathway) is an evolutionarily conserved mechanism whose importance has been proven across various species, including mammals, since its initial discovery in fruit flies. The Hippo pathway plays a crucial role in diverse biological processes, such as organ size regulation, tissue regeneration, and the maintenance of stem cell function; it is receiving significant attention in the field of biomedical research, particularly due to its close association with cancer development.

[0004] At the center of the Hippo pathway are two major kinases, MST1 / 2 and LATS1 / 2. These form a complex with the adapter proteins SAV1 and MOB1 to phosphorylate the downstream proteins YAP and TAZ. YAP / TAZ are transcriptional co-activators; in the unphosphorylated state, they translocate into the nucleus, bind to transcription factors such as TEAD, and promote cell proliferation by inducing the expression of target genes. However, when the Hippo pathway is activated, phosphorylation of YAP / TAZ by LATS1 / 2 occurs, inducing cytoplasmic sequestration of YAP / TAZ and preventing its translocation to the nucleus. Consequently, target gene expression is suppressed, thereby regulating cell proliferation and apoptosis.

[0005] The core function of the Hippo pathway is to maintain the appropriate size of organs by regulating the balance between cell proliferation and apoptosis. Furthermore, it plays a crucial role in stem cell function and tissue regeneration processes, determining cell fate in response to various external stimuli such as mechanical signals, intercellular contact, and energy states. In particular, the Hippo pathway performs an important function as a tumor suppressor pathway. By inhibiting the activity of YAP / TAZ, it prevents excessive cell proliferation and suppresses cancer development by inducing apoptosis. However, dysfunction of the Hippo pathway—specifically, the overactivation of YAP / TAZ—leads to uncontrolled cell proliferation and resistance to apoptosis, becoming a significant cause of cancer development. Indeed, overexpression of YAP / TAZ has been observed in various cancer types, including liver, colorectal, lung, and breast cancer, and it is known to play a significant role in the development and progression of cancer.

[0006] However, Zhengjin He et al. (2021) reported that inhibition of the Hippo signaling pathway in cancer is generally caused by factors other than genetic mutations in the pathway itself. Mutations in key components such as MST1 / 2, LATS1 / 2, and SAV1 are relatively rare in human cancers. In other words, inhibition of the Hippo pathway in cancer occurs more frequently due to dysregulation or disruption of upstream signaling pathways than due to genetic mutations. For example, G-protein coupled receptor (GPCR) signaling and disruption of cellular polarity proteins are presumed to act as major factors influencing inhibition of the Hippo pathway.

[0007] Although inhibition of the Hippo signaling pathway is clearly an important factor in the development of cancer, since mutations found in cancer are often associated with surrounding factors rather than the pathway itself, therapies that simply target mutations may have limitations (Fig. 1).

[0008] Meanwhile, DKK1 is a member of the Dickkopf protein family and is known as a representative inhibitor of the Wnt / β-catenin signaling pathway (Fig. 2). DKK1 blocks signal transduction by binding to the Wnt co-receptors LRP5 and LRP6 and interfering with their interactions with Wnt ligands. The Wnt signaling pathway plays a crucial role in various cellular processes, such as cell proliferation, differentiation, and stem cell maintenance, and unregulated pathways can lead to pathological conditions such as cancer development. By inhibiting Wnt signaling, DKK1 can prevent excessive cell proliferation and function as a tumor suppressor.

[0009] DKK1 is traditionally known as a tumor suppressor protein. By inhibiting Wnt signaling, it reduces the expression of β-catenin-mediated genes related to cell proliferation and survival (Fig. 3). For example, if abnormal activation of the Wnt pathway is a major factor in tumor growth, DKK1 plays a role in inhibiting cancer progression by blocking this process. Therefore, the tumor suppressor effect of DKK1 suggests potential as a therapeutic target in a state where the Wnt pathway is abnormally activated.

[0010] Paradoxically, recent studies demonstrate that DKK1 can act as a tumor-inducing protein under specific conditions. In particular, increased expression of DKK1 has been observed in various cancer types, contributing to the promotion of tumor growth and metastasis. For instance, elevated DKK1 expression in hepatocellular carcinoma (HCC), prostate cancer, and esophageal cancer has been reported to enhance the motility and invasiveness of cancer cells.

[0011]

[0012] It was observed that inhibiting DKK1 expression reduced cancer growth and suppressed metastasis. This suggests that DKK1 may play a role in promoting cancer cell proliferation by activating other pathways, going beyond the simple function of blocking Wnt signaling. In particular, activation of the DKK1-CKAP4 axis induces cancer cell survival and proliferation through PI3K-AKT signaling, thereby accelerating tumor progression and metastasis. These findings demonstrate that DKK1 can perform opposing roles as both a tumor suppressor and a promoter depending on environmental and molecular contexts. Furthermore, the external stimulus EGF plays a role in increasing cancer progression by raising DKK1 expression. This occurs through the MEK-ERK and PI3K-AKT pathways, activating DKK1 transcription through epigenetic changes, including histone phosphorylation and acetylation. These mechanisms suggest that DKK1 does not function as a mere suppressor protein in cancer cells, but rather may contribute to cancer survival and metastasis.

[0013]

[0014] The inventor of the present invention identified the mechanism by which DKK1 induces the overexpression of YAP / TAZ through the inhibition of Hippo signaling, thereby promoting the proliferation and metastasis of cancer (Fig. 4), and completed the invention of an anticancer composition targeting a specific step of this mechanism.

[0015] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising an LRP6 O-GlcNAcylation inhibitor as an active ingredient.

[0016] Another objective of the present invention is the step of measuring the expression level of DKK1 from a biological sample derived from a subject; and

[0017] The present invention provides a method for determining sensitivity to an LRP6 O-GlcNAcylation inhibitor, comprising the step of determining that sensitivity to the LRP6 O-GlcNAcylation inhibitor is high when the expression level of DKK1 measured above is higher than that of a normal control sample.

[0018] Another objective of the present invention is the step of treating DKK1 overexpressing cells with a candidate substance;

[0019] A step of measuring the degree of O-GlcNAcylation of LRP6 in cancer cell lines treated with the above candidate substance; and

[0020] The present invention provides a screening method for an LRP6 O-GlcNAcylation inhibitor, comprising the step of selecting the candidate substance as an LRP6 O-GlcNAcylation inhibitor when the measured degree of O-GlcNAcylation of LRP6 is reduced compared to before treatment with the candidate substance.

[0021]

[0022] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer comprising an LRP6 O-GlcNAcylation inhibitor as an active ingredient. The present invention is based on elucidating the mechanism of inhibition of the Hippo signaling pathway by DKK1, wherein the LRP6 O-GlcNAcylation inhibitor inhibits the membrane localization of OGT and the O-GlcNAcylation of LRP6 induced by DKK1 by interfering with the interaction between OGT and PIP3. This leads to a weakening of the binding affinity between LRP6 and Merlin and an interaction between LATS1 and Merlin, thereby inducing activation of the Hippo signaling pathway by LATS1, which inhibits the proliferation and metastasis of cancer cells.

[0023] Since this method of inhibiting LRP6 O-GlcNAcylation does not inhibit the activity of OGT itself, it does not inhibit the O-GlcNAcylation of other proteins, so it is expected to have few side effects.

[0024] In addition, it was confirmed that the LRP6 O-GlcNAcylation inhibitor of the present invention acts specifically on cells. Specifically, it was confirmed that inhibition of LRP6 O-GlcNAcylation and activation of the Hippo signaling pathway were prominent only when DKK1-overexpressing cells were treated with the LRP6 O-GlcNAcylation inhibitor. This suggests that side effects that may occur during anticancer treatment can be significantly reduced.

[0025]

[0026] Figure 1 is a schematic diagram showing the technical challenges in the development of cancer treatments related to the Hippo signaling pathway.

[0027] Figure 2 is a schematic diagram showing the regulation of the Wnt / β-catenin signaling pathway by DKK1.

[0028] Figure 3 is a schematic diagram showing the tumor suppression mechanism by DKK1.

[0029] Figure 4 is a schematic diagram regarding the hypothesis of inhibition of the Hippo signaling pathway by DKK1.

[0030] Figure 5 shows the change in mRNA expression levels of CTGF and CYR61 upon treatment with the TEAD inhibitor (MGH-CP1).

[0031] Figure 6 compares the expression levels of DKK1 between hepatocellular carcinoma-derived cell lines.

[0032] Figure 7 shows the changes in the expression levels of YAP target genes (CTGF, CYR61, ANKRD1) when Hep3B cells are treated with siDKK1.

[0033] Figure 8 shows the changes in the expression levels of YAP target genes (CTGF, CYR61, ANKRD1) when Huh7 cells are treated with DKK1 CM.

[0034] Figures 9 and 10 show changes in metastasis and invasion upon treatment of hepatocellular carcinoma with DKK1 CM and / or MGH-CP1.

[0035] Figure 11 shows the change in mRNA expression levels of YAP and TAZ upon siYAP / TAZ treatment.

[0036] Figures 12 and 13 show changes in metastasis and invasion upon treatment of hepatocellular carcinoma with DKK1 CM and / or siYAP / TAZ.

[0037] Figure 14 shows the change in YAP expression levels when Hep3B cells are treated with siDKK1.

[0038] Figure 15 shows the increase in p-YAP levels upon treatment of Hep3B cells with siDKK1.

[0039] Figure 16 is a schematic diagram of the Hippo signaling pathway.

[0040] Figure 17 shows the decrease in p-YAP levels when Huh7 cells are treated with DKK1 CM.

[0041] Figure 18 is the result of an immunofluorescence analysis showing changes in YAP expression when DKK1 expression is upregulated or downregulated in Hep3B cells.

[0042] Figure 19 shows the decrease in LATS1 phosphorylation upon DKK1 CM treatment of Huh7 cells.

[0043] Figure 20 is the result of a proximity ligation analysis showing an increase in the interaction between LATS1 and Merlin when Hep3B cells are treated with a DKK1 inhibitor (WAY-262611).

[0044] Figure 21 is the result of a proximity ligation analysis showing increased binding of LRP6 and OGT when rhDKK1 is treated to Hep3B cells with DKK1 knocked down.

[0045] Figure 22 shows the results of changes in O-GlcNAcylation of LRP6 when Huh7 cells are treated with DKK1-EGFP and / or DKK1 inhibitors.

[0046] [Correction pursuant to Rule 91 20.01.2026] Figures 23a, 23b, and 23c are liquid chromatography-mass spectrometry (LC-MS) results for identifying the O-GlcNAcylation site of LRP6.

[0047] Figure 24 shows the results of comparing the changes in O-GlcNAcylation of LRP6 when DKK1 was applied to three LRP6 mutants (T1466A, S1533A, S1575A), respectively.

[0048] Figure 25 shows the results of comparing the phosphorylation levels of LATS1 and YAP in three LRP6 mutants (T1466A, S1533A, S1575A).

[0049] Figure 26 shows the results of comparing 8XGTIIC-Luc activity in three LRP6 mutants (T1466A, S1533A, S1575A).

[0050] Figure 27 shows the results of a co-immunoprecipitation analysis comparing the interaction between three LRP6 mutants (T1466A, S1533A, S1575A) and Merlin.

[0051] Figure 28 shows the results of observing the change in OGT film distribution upon DKK1 CM or rhDKK1 treatment using a confocal microscope.

[0052] Figure 29 shows the results of comparing the amount of OGT in the membrane and the total amount within the cell, respectively, upon DKK1 CM treatment.

[0053] Figure 30 shows the structure of the designed Tm-EGFP-OGT.

[0054] Figure 31 shows the results of comparing the total amount of OGT in the membrane and within the cell following EGFP-OGT and Tm-EGFP-OGT treatments, respectively.

[0055] Figure 32 shows the results of comparing the amount of O-GlcNAcylation of LRP6 according to EGFP-OGT and Tm-EGFP-OGT treatments.

[0056] Figure 33 shows the results of comparing the phosphorylation levels of LATS1 and YAP according to EGFP-OGT and Tm-EGFP-OGT treatments.

[0057] Figure 34 is a schematic diagram of the mechanism of PIP3-dependent LRP6 O-GlcNAcylation induction by DKK1.

[0058] Figure 35 is the result of a proximity ligation analysis showing the interaction between OGT and PIP3.

[0059] Figure 36 shows the increase in O-GlcNAcylation of LRP6 by DKK1 treatment and the inhibition of O-GlcNAcylation of LRP6 by PI3K inhibitor treatment.

[0060] Figure 37 shows the increase in O-GlcNAcylation of LRP6 upon PI3KCB overexpression or siPTEN treatment.

[0061] Figure 38 shows the increased interaction between Merlin and LRP6 upon siPTEN treatment.

[0062] Figure 39 is a schematic diagram showing the mechanism of Hippo signaling pathway activation through inhibition of the interaction between PIP3 and OGT.

[0063] Figures 40 and 41 show the structures of the designed LRP6 O-GlcNAcylation inhibitors, respectively.

[0064] Figure 42 is the result of a proximity ligation analysis showing the interaction between the LRP6 O-GlcNAcylation inhibitor and PIP3.

[0065] Figure 43 shows the results of changes in O-GlcNAcylation of LRP6 following treatment with DKK1 CM and / or LRP6 O-GlcNAcylation inhibitors.

[0066] Figure 44 is a schematic diagram showing the cell-specific mechanism of the LRP6 O-GlcNAcylation inhibitor.

[0067] Figure 45 shows the results of comparing the change in YAP expression levels when HEK293 cells and Hep3B cells were treated with an LRP6 O-GlcNAcylation inhibitor, respectively.

[0068] Figure 46 shows the structure of an LRP6 O-GlcNAcylation inhibitor with a PPO domain bound to the Tet-ON system and the results of expressing it.

[0069] Figure 47 shows the results of comparing the levels of the cell proliferation marker (Ki-67) when HEK293 cells and Hep3B cells were treated with the LRP6 O-GlcNAcylation inhibitor, respectively.

[0070] Figure 48 shows the results of confirming DKK1, YAP, and CTGF expression through IHC analysis of a hepatocellular carcinoma (HCC) tissue microarray.

[0071] Figure 49 shows the results of identifying mutations that mainly occur in HCC patients and confirming DKK1 expression in the corresponding mutations using the TCGA database.

[0072] Figure 50 shows the results of predicting transcription factors by analyzing the increased genes in DKK1 high-expression HCC when comparing DKK1 high-expression HCC and DKK1 non-expression HCC patient samples.

[0073] Figure 51 shows the results of comparing DKK1 expression in normal cell lines HEK293T and HCC cell lines HepG2, Huh7, and Hep3B.

[0074] Figure 52 shows the results of oral administration of the DKK1 inhibitor WAY-262611 after the creation of a mouse xenograft model using Hep3B.

[0075] Figure 53 shows the results of tumor volume measurements over days in a Hep3B xenograft model.

[0076] Figure 54 shows the results of IHC analysis of tissue from a Hep3B xenograft model.

[0077] Figure 55 shows the results of qPCR analysis performed on tissue from a Hep3B xenograft model.

[0078] Figure 56 is a schematic diagram showing TmOGT (adding a domain for membrane transport), OGTⅿPPO (removing a PIP3 interaction domain), and TmOGTⅿPPO (adding a domain for membrane transport and removing a PIP3 interaction domain).

[0079] Figure 57 shows the results that OGT membrane localization can be induced by attaching SP and Tm domains even without a PPO domain.

[0080] Figure 58 shows the results of observing that when the PPO domain is absent, LRP6 O-GlcNAcylation induction does not occur, but when the SP and Tm domains are attached, the degree of LRP6 O-GlcNAcylation increases again.

[0081] Figure 59 is a schematic diagram of the development of a PPO competitive inhibitor by cloning only the PPO domain of OGT.

[0082] Figures 60 and 61 are the results of a colony forming assay showing that the proliferation of Hep3B cell lines and Huh7 cell lines is inhibited upon overexpression of the PPO structure.

[0083] Figure 62 shows the results indicating that metastatic properties, such as migration and invasion of Hep3B cell lines, are inhibited when the PPO structure is overexpressed.

[0084] Figure 63 shows the results indicating that migration and invasion by treatment with DKK1-conditioned media (DKK1CM) were inhibited in Huh7 cells overexpressing the PPO structure.

[0085] Figure 64 shows the results indicating that PPO overexpression has a negligible effect on normal cell lines such as HEK293.

[0086] Figures 65 and 66 show the results of observing that PPO overexpression effectively inhibits YAP in HCC with high DKK1 expression or cancer cell lines with high PIP3 levels due to PTEN mutations.

[0087]

[0088] The "LRP6 (Low-Density Lipoprotein Receptor-Related Protein 6)" of the present invention acts as a co-receptor of the Wnt signaling pathway and is a protein that regulates cell growth and differentiation. It interacts with the Hippo signaling pathway to regulate the activation of YAP, and when O-GlcNAcylation occurs, it binds to Merlin to inhibit the activation of LATS1 and activate YAP, thereby inhibiting the Hippo signaling pathway.

[0089] The "O-GlcNAcylation" of the present invention is a type of post-translational modification in which a sugar molecule called N-acetylglucosamine (O-GlcNAc) is attached to specific amino acid residues of a protein. O-GlcNAcylation attaches to serine (Ser) or threonine (Thr) residues of a protein, is attached by OGT (O-GlcNAc transferase), and is removed by OGA (O-GlcNAcase). In vivo, O-GlcNAcylation is known to be involved in cell signaling, stress response, metabolic regulation, and gene expression.

[0090] The “DKK1 (Dickkopf-1)” of the present invention acts as a Wnt signaling inhibitor and is a protein that promotes cell proliferation by binding to the CKAP4 receptor and LRP6 receptor and activating the PI3K-AKT pathway. In particular, DKK1 is known to be overexpressed in various cancers, such as hepatocellular carcinoma (HCC), which increases the aggressiveness of tumors.

[0091] The "Hippo signaling pathway" of the present invention is a major pathway regulating cell growth and apoptosis, and includes MST1 / 2, LATS1 / 2, and YAP / TAZ as key components. When this pathway is activated, YAP / TAZ is isolated in the cytoplasm or degraded, thereby inhibiting gene expression and cell proliferation.

[0092] "LATS1 (Large Tumor Suppressor Kinase 1)" of the present invention is a major kinase of the Hippo pathway that regulates cell growth and proliferation by inhibiting YAP / TAZ. It is activated through interaction with Merlin and plays a tumor suppressor role.

[0093] The "YAP (Yes-Associated Protein)" of the present invention is a terminator of the Hippo pathway; when activated, it moves to the nucleus to promote gene expression and increase cell proliferation. Overactivation of YAP is associated with cancer and is inhibited by LATS1 / 2.

[0094] The "PI3K-AKT signaling pathway" of the present invention is an intracellular signaling pathway that regulates cell survival, growth, metabolism, migration, and proliferation, and is activated by stimulation such as growth factors or cytokines. When this pathway is activated, PI3K is activated, converting PIP2 (Phosphatidylinositol-4,5-bisphosphate) in the cell membrane into PIP3 (Phosphatidylinositol-3,4,5-trisphosphate), and PIP3 activates AKT to act on various target proteins. The pathway can be inhibited by dephosphorylating PIP3 back to PIP2 by PTEN (Phosphatase and Tensin Homolog).

[0095]

[0096] The present invention will be described in more detail below.

[0097] One aspect of the present invention is a pharmaceutical composition for the prevention or treatment of cancer, comprising an LRP6 O-GlcNAcylation inhibitor as an active ingredient.

[0098] The above LRP6 O-GlcNAcylation inhibitor may include a PPO domain, but is not limited thereto. The above PPO domain refers to a region among the domains constituting the OGT enzyme that binds to PIP3. Therefore, if the above LRP6 O-GlcNAcylation inhibitor includes a PPO domain, the above LRP6 O-GlcNAcylation inhibitor can bind to PIP3 and competitively inhibit the action of OGT. Specifically, if the above LRP6 O-GlcNAcylation inhibitor includes a PPO domain, the above LRP6 O-GlcNAcylation inhibitor can inhibit the action of OGT binding to PIP3 present on the cell membrane, causing membrane localization, and the attachment of O-GlcNAc to LRP6. The inventors of the present invention confirmed that upon PPO treatment, O-GlcNAcylation of LRP6 is inhibited, YAP expression is reduced, and cell proliferation is inhibited.

[0099] The above PPO domain may include the amino acid sequence of SEQ ID NO. 1, for example, may include an amino acid including the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence having substantial identity with respect to the amino acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0100] The term "substantial identity" as used in the present invention means that by aligning each amino acid sequence with any other amino acid sequence to correspond as much as possible and analyzing the sequences, the any other amino acid sequence has at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology with each amino acid sequence.

[0101] The above LRP6 O-GlcNAcylation inhibitor may further include a transmembrane region. The transmembrane region may be utilized to enhance the interaction between PIP3 attached to the cell membrane and the LRP6 O-GlcNAcylation inhibitor.

[0102] The above LRP6 O-GlcNAcylation inhibitor may further include a signal peptide. The signal peptide is an important domain for transmembrane proteins to move to specific cellular organelles, and can be attached to move them to the endoplasmic reticulum, nucleus, mitochondria, peroxysomes, lysosomes, cell membranes, etc., and different signal peptides are attached depending on the target location. Therefore, the above signal peptide may be a signal peptide for moving the above LRP6 O-GlcNAcylation inhibitor to the cell membrane.

[0103] The above LRP6 O-GlcNAcylation inhibitor may inhibit the O-GlcNAcylation of LRP6 by interfering with the interaction between OGT (O-linked N-acetylglucosamine transferase) and PIP3. The inventors of the present invention have elucidated the mechanism of inhibition of the Hippo signaling pathway by DKK1. Specifically, they revealed that when DKK1 binds to LRP6 and CKAP4, PI3K is activated, converting PIP2 attached to the cell membrane into PIP3. When OGT binds to PIP3, membrane localization occurs, and O-GlcNAcylation of LRP6 is induced. Consequently, the binding affinity between LRP6 and Merlin is strengthened, thereby interfering with the interaction between Merlin and LATS1, which inhibits the activation of the Hippo signaling pathway by LATS1. In other words, the LRP6 O-GlcNAcylation inhibitor of the present invention prevents cell proliferation and metastasis by activating the Hippo signaling pathway through the inhibition of O-GlcNAcylation of LRP6 within the mechanism of inhibition of the Hippo signaling pathway by DKK1.

[0104] The LRP6 O-GlcNAcylation inhibitor according to the present invention can be used to induce inhibition of cell proliferation in cancers in which the PI3K-PIP3-OGT pathway is activated in a DKK1-dependent manner. Accordingly, the present invention relates to a pharmaceutical use of the inhibitor of the present invention for the prevention or treatment of cancers in which DKK1 is overexpressed, such as hepatocellular carcinoma (HCC), lung cancer, or multiple myeloma, including but not limited to these.

[0105] The above LRP6 O-GlcNAcylation inhibitor may act specifically on DKK1 overexpressing cells. Since the LRP6 O-GlcNAcylation inhibitor of the present invention is designed based on the mechanism of inhibition of the Hippo signaling pathway by DKK1, it primarily acts on cells in which DKK1 is overexpressed. In particular, the inventors of the present invention confirmed that inhibition of LRP6 O-GlcNAcylation and inhibition of YAP expression are significantly more pronounced in DKK1 overexpressing cell lines than in cell lines with low DKK1 expression. Overexpression of DKK1 may occur in specific cells in cases such as cancer, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, myocardial infarction, osteoporosis, osteoarthritis, obesity, type 2 diabetes, nutritional deficiency, and mechanical stress.

[0106] The above cancer may be one or more selected from the group consisting of brain tumors, benign astrocytomas, malignant astrocytomas, pituitary adenomas, meningiomas, brain lymphomas, oligodendrogliomas, intracranial tumors, ependymomas, brainstem tumors, head and neck tumors, laryngeal cancer, oropharyngeal cancer, nasal cavity cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, thoracic tumors, lung cancer, thymic cancer, mediastinal tumors, esophageal cancer, breast cancer, abdominal tumors, stomach cancer, liver cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, small intestine cancer, colorectal cancer, anal cancer, bladder cancer, urethral cancer, kidney cancer, cervical cancer, endometrial cancer, uterine sarcoma, ovarian cancer, vaginal cancer, prostate cancer, testicular cancer, penile cancer, multiple myeloma, blood cancer, and skin cancer, but is not limited thereto. Preferably, the cancer may be a cancer in which DKK1 overexpression occurs, and it has been reported that DKK1 overexpression occurs in, for example, hepatocellular carcinoma (HCC), pancreatic cancer, esophageal cancer, lung cancer, breast cancer, multiple myeloma, gastric cancer, prostate cancer, colorectal cancer, biliary tract cancer, etc.

[0107] In addition, the LRP6 O-GlcNAcylation inhibitor according to the present invention can also be used for the prevention or treatment of cancer in which the downregulation of the Hippo signaling pathway or the functional activation of YAP contributes to tumor growth. For example, in cancers having NF2 mutations, reduced LATS1 expression, or other Hippo pathway inhibitory characteristics, cell growth and metastasis can be inhibited by administering the inhibitor of the present invention to inhibit YAP activation.

[0108] Furthermore, the inhibitor of the present invention can also be applied for medicinal purposes to induce inhibition of cell proliferation by inhibiting O-GlcNAcylation of LRP6 in cancers accompanied by hyperactivation of the PI3K-PIP3 axis, such as cancers in which PIK3CA mutations or PTEN deletions are observed. In such cancer types, the inhibitor of the present invention can be used alone or in combination with existing anticancer agents.

[0109] The term "prevention" as used in this invention refers to any act of suppressing a viral infectious disease or delaying its onset by administering a pharmaceutical composition according to this invention.

[0110] The term "treatment" as used in this invention refers to any act in which symptoms caused by a viral infectious disease are improved or beneficially altered by the administration of a pharmaceutical composition according to this invention.

[0111]

[0112] The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient.

[0113] The above-mentioned pharmaceutically acceptable carrier may be used as a mixture of saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Additionally, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets. Furthermore, it may be preferably formulated according to each disease or component using appropriate methods in the art or by using the methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

[0114] The above excipients and diluents may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the product is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0115] The composition of the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) according to the desired method, and the pharmaceutical administration form of the composition according to the present invention may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable combination. The pharmaceutical composition according to the present invention may be formulated and used in the form of oral formulations such as capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions, each according to conventional methods.

[0116] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above-mentioned LRP6 O-GlcNAcylation inhibitor. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0117] Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included.

[0118] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used.

[0119] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. A composition according to one embodiment of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above-mentioned factors into account, and this can be easily determined by a person skilled in the art.

[0120] Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the patient's age, gender, and weight, and generally, 0.1 mg to 1,000 mg, 1 mg to 100 mg, or 3 mg to 10 mg per kg of body weight may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dosage may be increased or decreased depending on the route of administration, severity of the disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0121]

[0122] Another aspect of the present invention is the step of measuring the degree of expression of DKK1 from a biological sample derived from a subject; and

[0123] A method for determining sensitivity to an LRP6 O-GlcNAcylation inhibitor, comprising the step of determining that sensitivity to an LRP6 O-GlcNAcylation inhibitor is high when the expression level of DKK1 measured above is higher than that of a normal control sample.

[0124] The description of the above LRP6 O-GlcNAcylation inhibitor is the same as above.

[0125] Since the above LRP6 O-GlcNAcylation inhibitor can act specifically on DKK1 overexpressing cells, it may be necessary to determine sensitivity before treating with the LRP6 O-GlcNAcylation inhibitor.

[0126] The biological sample derived from the above subject may be cancer cells taken from a cancer patient, synovial cells from an inflamed area taken from a rheumatoid arthritis patient, intestinal tissue cells taken from an inflammatory bowel disease patient, vascular endothelial cells or smooth muscle cells taken from an atherosclerosis patient, cardiomyocytes taken from a myocardial infarction patient, osteoblasts taken from an osteoporosis patient, chondrocytes or osteoblasts taken from an osteoarthritis patient, adipose tissue cells or liver cells taken from an obese or type 2 diabetes patient, but is not limited thereto, and any cell reported to show DKK1 overexpression may be adopted as a biological sample by a person skilled in the art as needed.

[0127] The expression level of the above DKK1 may be measured by at least one method selected from the group consisting of reverse transcription-polymerase chain reaction (RT-PCR), competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern Blotting, DNA microarray, enzyme immunoassay (ELISA), immunohistochemistry, Western Blotting, flow cytometry (FACS), radioimmunoassay, radioimmunodiffusion, tissue immunostaining, immunoprecipitation assay, and protein chip, but is not limited thereto. The above methods may be selected by a person skilled in the art as needed and may be performed in a manner known in the art.

[0128]

[0129] Another aspect of the present invention comprises the step of treating DKK1 overexpressing cells with a candidate substance;

[0130] A step of measuring the degree of O-GlcNAcylation of LRP6 in cancer cell lines treated with the above candidate substance; and

[0131] A screening method for an LRP6 O-GlcNAcylation inhibitor comprising the step of selecting a candidate substance as an LRP6 O-GlcNAcylation inhibitor when the measured degree of O-GlcNAcylation of LRP6 is reduced compared to before treatment with the candidate substance.

[0132] The description of the above LRP6 O-GlcNAcylation inhibitor is the same as above.

[0133] The above cells may be cancer cells, but are not limited thereto.

[0134] The step of measuring the degree of O-GlcNAcylation of LRP6 may further include measuring the degree of YAP expression, the degree of LATS1 phosphorylation, or the degree of interaction between Merlin and LATS1. According to the mechanism of inhibition of the Hippo signaling pathway by DKK1, the effect of the LRP6 O-GlcNAcylation inhibitor may manifest not only as a decrease in LRP6 O-GlcNAcylation, but also as a decrease in YAP expression, an increase in the phosphorylation level of YAP, an increase in the phosphorylation level of LATS1, a decrease in the interaction between LRP6 and Merlin, and an increase in the interaction between Merlin and LATS1.

[0135]

[0136] One or more specific embodiments are described in more detail below through examples. However, these examples are intended to illustrate one or more specific embodiments and the scope of the present invention is not limited to these examples.

[0137]

[0138] Experimental Example: Western blot

[0139] Protein samples were separated by size using standard Western blot with 8% or 10% polyacrylamide gels by electrophoresis at 120V. The electrophoresed gels were transferred to PVDF membranes via a transfer kit at 230mA for 3 hours. To measure protein signals on the membranes, the samples were treated with the primary antibody overnight, washed five times for 10 minutes each, treated with the secondary antibody for 1 hour, and washed five times for 15 minutes each. Afterward, ECL was applied using a Western blot detection kit, and Western blot data was measured using Microchemi 2.0.

[0140]

[0141] Experimental Example: Immunofluorescence analysis

[0142] After placing an ECM-coated cover glass in a 6-well glass, cells from each experimental group were seeded. Transfection or reagent treatment was performed 24 hours after seeding. Cells treated with the experimental conditions were transferred to a 96-well plate while attached to the cover glass and fixed by treatment with 4% paraformaldehyde for 20 minutes. Subsequently, the cells were washed three times with PBS, treated with 0.1% Triton X-100 for 15 minutes for permeabilization, and then washed three times with PBS. Afterward, blocking was performed with 5% BSA (in PBS) for 1 hour, followed by treatment with the primary antibody for 16 hours, washing with 1% BSA, treatment with the secondary antibody for 1 hour, washing with 1% BSA, and DAPI staining. The prepared samples were mounted onto a slide glass using mounting solution, and the edges of the slide were coated with nail polish.

[0143]

[0144] Experimental Example: Proximity Ligation Assay (PLA)

[0145] In the same manner as the immunofluorescence analysis method, an ECM-coated cover glass was placed in a 6-well glass, and cells from each experimental group were seeded. After moving the cover glass to a 96-well, the cells were immobilized and permeabilized. Subsequently, sampling was performed using the Navinci PLA kit, followed by mounting onto a slide glass using a mounting solution and coating with nail polish. Finally, the PLA signal was measured using a confocal microscope.

[0146]

[0147] Experimental Example: Liquid Chromatography-Mass Spectrometry (LC-MS)

[0148] To identify the LRP6 O-GlcNAcylation site, HEK293T cells were seeded at 80% density in 10 100 mm dishes and transfected with LRP6-EGFP and Flag-OGT. Additionally, to prevent the inhibition of O-GlcNAcylation by OGA (O-GlcNAse), the cells were cultured for 24 hours with the OGA inhibitor (Thiamet-G) added to the medium. During the lysis process, another OGA inhibitor (PUGNAc) was added to the lysis buffer to suppress O-GlcNAcylation inhibition during sampling. To obtain only LRP6-EGFP protein, sampling was performed by treating with the primary EGFP antibody, followed by treatment with agarose beads rotated for 16 hours and pull-down. The prepared samples were subjected to electrophoresis on an 8% polyacrylamide gel. The LRP6-EGFP band was identified on the electrophoresed gel using Coomassie blue staining, and only that portion was cut out and submitted for LC / MS analysis.

[0149]

[0150] Experimental Example: Co-Immunoprecipitation Analysis

[0151] Twenty-four hours after cell seeding, transfection was performed using Lipofectamine 2000. Six hours after transfection, the cell media was changed, and harvesting was carried out after 24–48 hours. The harvested cells were treated with Triton X-100 lysis buffer and quantified using the Bradford assay. Subsequently, for immunoprecipitation, 1 μg / μl of the primary antibody against the target protein was added, followed by rotation for 16 hours. Afterward, agarose beads were applied for one hour, followed by five washes of 15 minutes each with lysis buffer, sampling, and Western blotting.

[0152] For the sWGA assay to capture O-GlcNACylation, sWGA beads that specifically interact with O-GlcNACylation proteins were treated for 3 hours without primary antibody treatment on the lysed protein, followed by 5 washes of 15 minutes each and sampling, after which Western blotting was performed.

[0153]

[0154] Example 1: Mechanism of Hippo Signaling Pathway Inhibition by DKK1

[0155] Example 1-1: YAP / TAZ-dependent induction of metastasis / invasion of hepatocellular carcinoma (HCC) by DKK1

[0156] CTGF and CYR61 are genes directly activated by the YAP / TEAD complex in the Hippo signaling pathway, respectively; CTGF is involved in cell growth, differentiation, and angiogenesis, while CYR61 is involved in cell migration, angiogenesis, and ECM regulation. When the Hippo signaling pathway is inhibited, YAP moves to the cell nucleus, binds to TEAD, and induces the expression of CTGF and CYR61; therefore, CTGF and CYR61 can be used as biomarkers reflecting the activation of YAP. In fact, when treated with the TEAD inhibitor MGH-CP1, it was confirmed that the mRNA expression levels of CTGF and CYR61 were significantly reduced compared to the control group treated with DMSO (Fig. 5).

[0157] Meanwhile, anticipating that cancer proliferation and metastasis induced by DKK1 would be related to the Hippo signaling pathway, the expression levels of DKK1 were compared among hepatocellular carcinoma-derived cell lines (Fig. 6). Subsequently, experiments were conducted in which DKK1 was knocked down in Hep3B cells, a hepatocellular carcinoma cell line with high DKK1 expression, and treated with DKK1 conditioned medium (CM) in Huh7 cells, a hepatocellular carcinoma cell line with low DKK1 expression. As a result, it was confirmed that as DKK1 expression decreased in Hep3B cells, the expression of CTGF, CYR61, and ANKRD1 also decreased (Fig. 7), while the expression of CTGF, CYR61, and ANKRD1 increased upon treatment of Huh7 cells with DKK1 (Fig. 8). This demonstrates that DKK1 expression is related to the Hippo signaling pathway.

[0158] Next, we aimed to confirm that DKK1 induces cancer cell metastasis and invasion in a YAP / TAZ-dependent manner within the Hippo signaling pathway. When liver cancer cells were treated with DKK1 conditioned culture medium (DKK1 CM) and standard culture medium (CON CM), respectively, it was confirmed that metastasis and invasion significantly increased when treated with DKK1 CM (Figs. 9 and 10). In contrast, when treated with the TEAD inhibitor MGH-CP1, no metastasis or invasion was observed regardless of the type of culture medium. This implies that DKK1 promotes cell metastasis or invasion through the YAP / TEAD complex, demonstrating that DKK1 is involved in the Hippo signaling pathway.

[0159] To directly demonstrate that the DKK1-induced metastasis and invasion of cancer cells is dependent on YAP / TAZ, YAP and TAZ were knocked down. Knockdown was achieved through treatment with siYAP / TAZ, and it was confirmed that mRNA expression levels were significantly reduced, as shown in Figure 11. While metastasis and invasion of cancer cells (HCC) significantly increased upon treatment with DKK1 CM, it was confirmed that when YAP / TAZ was knocked down via siYAP / TAZ treatment, cell metastasis or invasion did not significantly increase despite DKK1 CM treatment (Figures 12, 13). Through this, it was found that the DKK1-induced metastasis and invasion of cancer cells is dependent on YAP / TAZ.

[0160]

[0161] Example 1-2: Inhibition of Hippo signaling pathway through reduced LATS1 phosphorylation by DKK1

[0162] The effects of DKK1 on the Hippo signaling pathway were investigated using Hep3B liver cancer cell lines that express high levels of DKK1. When DKK1 was knocked down by treating Hep3B with siRNA against DKK1 (siDKK1), the expression level of YAP significantly decreased (Fig. 14). Simultaneously, the level of phosphorylated YAP (phospho-YAP; p-YAP) increased (Fig. 15), suggesting that the Hippo signaling pathway was activated (Fig. 16). Conversely, when treated with DKK1 CM, the level of p-YAP decreased (Fig. 17).

[0163] Immunofluorescence analysis was performed to compare the expression levels of YAP following the upregulation or downregulation of DKK1 expression in Hep3B cells. As a result, DKK1 knockdown reduced the accumulation of nuclear YAP, whereas DKK1 CM treatment increased the concentration of nuclear YAP (Fig. 18).

[0164] Additionally, it was confirmed that treatment with DKK1 CM reduced the phosphorylation level of LATS1, an upstream regulator of the Hippo pathway (Fig. 19). Since LATS1 requires phosphorylation for activation, this suggests that DKK1 plays a role in inhibiting the Hippo pathway by inhibiting the phosphorylation of LATS1.

[0165]

[0166] Examples 1-3: Inhibition of Hippo signaling pathway through increased O-GlcNAcylation of LRP6 by DKK1

[0167] Previous studies have shown that O-GlcNAcylation of LRP6 induces the binding of Merlin (NF2) and LRP6, whereas in the absence of O-GlcNAcylation, Merlin binds to LATS1 and activates the Hippo signaling pathway. Accordingly, in this example, changes in O-GlcNAcylation of LRP6 induced by DKK1 were analyzed to elucidate the mechanism by which DKK1 reduces the phosphorylation of LATS1 and increases the level of YAP.

[0168] We investigated whether Merlin binds to LATS1 and activates Hippo signaling when treated with WAY-262611, a drug that inhibits DKK1 binding to LRP6, to induce an effect similar to the knockdown of DKK1. As a result of performing proximal ligation analysis (PLA), it was confirmed that the interaction between LATS1 and Merlin significantly increased upon treatment with WAY-262611 (Fig. 20). This suggests that when DKK1 binds to LRP6, the interaction between Merlin and LATS1 is inhibited, thereby reducing the phosphorylation of LATS1 and increasing the level of YAP.

[0169] In addition, when DKK1 was knocked out using the CRISPR-Cas9 system in Hep3B cell lines and then treated with recombinant DKK1 (rhDKK1), it was confirmed that the binding of LRP6 and OGT increased (Fig. 21). This indicates that DKK1 directly mediates the binding of LRP6 and OGT.

[0170] Next, to confirm O-GlcNAcylation of OGT-mediated LRP6, sWGA (succinylated Wheat Germ Agglutinin), a reagent that specifically detects or isolates O-GlcNAcylated proteins, was used. When DKK1-EGFP was overexpressed, no significant change was observed in the bands in Whole Cell Lysate (WCL), but the intensity of the LRP6 band pulled down by sWGA beads was confirmed to increase significantly (Fig. 22). This indicates that the overexpression of DKK1 increases O-GlcNAcylation of LRP6. Furthermore, it was confirmed that this increase in O-GlcNAcylation was reduced by WAY-262611 treatment.

[0171] In summary, this study demonstrates that as the binding of LRP6 and OGT and the O-GlcNAcylation of LRP6 are increased by DKK1, the interaction between LRP6 and Merlin increases, while the interaction between Merlin and LATS1 decreases, thereby inhibiting Hippo signaling. In other words, DKK1 plays an important role in the Hippo signaling pathway by regulating the O-GlcNAcylation of LRP6.

[0172]

[0173] Example 2: Identification of the O-GlcNAcylation site of LRP6 by DKK1

[0174] To identify the O-GlcNAcylation site of LRP6 induced by DKK1, liquid chromatography-mass spectrometry (LC-MS) was used to identify the estimated O-GlcNAcylation site of LRP6 (Fig. 23). The estimated sites were T1466, S1533, and S1575, respectively, and functional analysis was performed by constructing mutants in which these sites were substituted with alanine.

[0175] When comparing the degree of O-GlcNAcylation in each mutant after DKK1 treatment, it was confirmed that the intensity of the O-GlcNAcylation band decreased slightly in the T1466A and S1533A mutants (Fig. 24).

[0176] Next, we analyzed the phosphorylation levels of LATS1 and YAP to select mutants that could not inhibit the phosphorylation of LATS1. As a result, we observed increased phosphorylation of LATS1 and YAP in the T1466A mutant (Fig. 25).

[0177] Next, 8XGTIIC was used to compare YAP-mediated reporter activity among mutants. As a result, the activity of the T1466A mutant was closest to that of the wild type, confirming that the T1466A mutant did not effectively increase YAP-mediated reporter activity (Fig. 26).

[0178] The results were similar when investigating mutants affecting the interaction with Merlin through co-immunoprecipitation analysis with Merlin (Fig. 27). It was confirmed that the interaction with Merlin was significantly weakened in the T1466A mutant.

[0179] Synthesizing the above results, it can be seen that T1466 is the most important site for the O-GlcNAcylation of LRP6.

[0180]

[0181] Example 3: LRP6 O-GlcNAcylation induced by DKK1-mediated OGT membrane localization

[0182] To investigate the effect of DKK1 on the membrane localization of OGT, changes in OGT membrane localization following treatment with DKK1 CM or rhDKK1 were analyzed using confocal microscopy with EGFP-tagged OGT. As a result, it was confirmed that OGT membrane localization significantly increased upon treatment with DKK1 CM or rhDKK1 (Fig. 28). This was further verified through Western blot analysis. While there was no significant change in the total expression level of OGT, a significant increase in OGT expression in the membrane fraction was observed (Fig. 29). These results demonstrate that DKK1 treatment promotes the migration of OGT to the membrane.

[0183] To investigate the direct effect of OGT membrane localization on LRP6 O-GlcNAcylation, Tm-EGFP-OGT was designed by fusing a trans-membrane domain (TM) to OGT (Fig. 30). Tm-EGFP-OGT exhibited a significantly higher level of membrane localization compared to wild-type OGT (Fig. 31) and was found to further increase LRP6 O-GlcNAcylation (Fig. 32). Additionally, a decrease in LATS1 phosphorylation was observed in Tm-EGFP-OGT-expressing cells (Fig. 33).

[0184] Synthesizing the above results, it can be seen that the membrane translocation of OGT induced by DKK1 treatment is an important mechanism mediating the increase in O-GlcNAcylation of LRP6.

[0185]

[0186] Example 4: Induction of PIP3-dependent LRP6 O-GlcNAcylation in DKK1

[0187] We aimed to elucidate the mechanism by which DKK1 induces membrane localization of OGT in a PIP3-dependent manner and mediates the increase in O-GlcNAcylation of LRP6 (Fig. 34). According to the study by Akira Kikuchi et al. (2022), DKK1 is reported to bind to LRP6 and CAKP4 to activate the PI3K signaling pathway. PI3K is an enzyme that phosphorylates PIP2 (Phosphatidylinositol 4,5-bisphosphate) present in the cell membrane to produce PIP3 (phosphatidylinositol-3,4,5-trisphosphate), and activation of the PI3K signaling pathway leads to the accumulation of PIP3 within the cell membrane.

[0188] To confirm the interaction between OGT and PIP3, a proximity ligation assay (PLA) was performed, and as a result, binding between OGT and PIP3 was confirmed, and it was also observed that this interaction was reduced upon treatment with the PI3K inhibitor Wortmannin (Fig. 35).

[0189] Next, it was confirmed that the increase in O-GlcNAcylation of LRP6 by DKK1 treatment was inhibited by wortmannin treatment (Fig. 36). This indicates that the induction of O-GlcNAcylation of LRP6 by DKK1 is mediated by PI3K activity.

[0190] In addition, it was confirmed that O-GlcNAcylation of LRP6 increased when PI3KCB, an active form of PI3K that increases PIP3 levels, was expressed, or when PTEN, a PIP3-degrading enzyme, was knocked down (Fig. 37). These results support the fact that PIP3 plays a direct role in regulating O-GlcNAcylation of LRP6. Furthermore, it was confirmed that the interaction between Merlin and LRP6 increased under conditions where O-GlcNAcylation of LRP6 was increased through siPTEN treatment (Fig. 38).

[0191] Synthesizing the above results, DKK1 binds to LRP6 and CKAP4 to activate the PI3K signaling pathway. This induces the production of PIP3 within the cell membrane, which in turn interacts with OGT, leading to membrane localization of OGT and an increase in O-GlcNAcylation of LRP6. O-GlcNAcylation of LRP6 increases the interaction between LRP6 and Merlin, thereby hindering LATS1 activation by Merlin and leading to the inhibition of the Hippo signaling pathway.

[0192]

[0193] Example 5: PPO competitor-mediated activation of the Hippo signaling pathway in hepatocellular carcinoma

[0194] It is known that increased PIP3 levels and activation of OGT are observed in many cancers. Based on the mechanism of inhibition of the Hippo signaling pathway by DKK1 newly identified in the above example, the inventors of the present invention predicted that activating the Hippo signaling pathway by inhibiting the interaction between PIP3 and OGT could suppress the proliferation and metastasis of cancer cells (Fig. 39). Accordingly, an LRP6 O-GlcNAcylation inhibitor was developed by isolating only the PPO domain that binds to PIP3 from the OGT domain (Figs. 40, 41).

[0195] Proximate ligation analysis was performed to determine whether an LRP6 O-GlcNAcylation inhibitor could inhibit the O-GlcNAcylation of LRP6 by binding to PIP3. In the experiment, EGFP-PPO, in which EGFP is conjugated to the PPO domain of the LRP6 O-GlcNAcylation inhibitor, was used, and the analysis confirmed that EGFP-PPO binds to PIP3 (Fig. 42).

[0196] It was confirmed that O-GlcNAcylation of LRP6 was increased by DKK1 CM treatment, but decreased again when EGFP-PPO was expressed (Fig. 43). Since LRP6 O-GlcNAcylation inhibitors using this PPO domain do not inhibit the expression or activity of OGT itself, they do not affect O-GlcNAcylation of other proteins, which is expected to reduce the side effects of anticancer drugs.

[0197] Furthermore, LRP6 O-GlcNAcylation inhibitors act specifically on cells (Fig. 44). Specifically, LRP6 O-GlcNAcylation inhibitors act specifically on cancer cells, inhibiting only the O-GlcNAcylation of LRP6, thereby preventing the side effects associated with existing OGT inhibitors. To verify this, the results were compared by treating normal human embryonic kidney cell line HEK293 and liver cancer cell lines Huh7 and Hep3B with OSMI-1 (an OGT inhibitor) or overexpressing EGFP-PPO. As a result of treatment with the OGT inhibitor OSMI-1, it was confirmed that overall O-GlcNAcylation decreased and YAP expression also decreased in all three cells (Fig. 45a). Since O-GlcNAcylation plays various roles in cells, treatment with OSMI-1 implies a high likelihood of in vivo side effects. On the other hand, when EGFP-PPO was overexpressed, overall O-GlcNAcylation was not significantly reduced in all cell lines, and YAP expression was specifically reduced only in the liver cancer cell lines Huh7 and Hep3B (Fig. 45b). This demonstrates that LRP6 O-GlcNAcylation inhibitors containing PPO can act specifically on cancer cells.

[0198] Next, DNA conjugated with the PPO domain gene to the Tet-ON system, whose expression is regulated by doxycycline (Dox), was transfected into HEK293 cells and Hep3B (Fig. 46a). As a result, it was confirmed that YAP expression was specifically reduced only in cancer cells even when the PPO domain was expressed by treatment with various concentrations of Dox (Fig. 46b).

[0199] Immunofluorescence analysis also confirmed the cancer cell-specific action of EGFP-PPO. In HEK293 cells, YAP was expressed regardless of EGFP-PPO expression; however, in Hep3B cells, a liver cancer cell line, YAP expression (red) was significantly reduced only in cells expressing EGFP-PPO (Fig. 47a). Additionally, changes in the expression of Ki-67, a marker for cell proliferation, and Cleaved caspase3, a marker for apoptosis, were examined (Figs. 47b, 47c). As a result, Ki-67 expression decreased significantly with EGFP-PPO expression, while no particular change was observed in Cleaved caspase3. This demonstrates that EGFP-PPO inhibits cell proliferation but does not induce apoptosis.

[0200]

[0201] Example 6: Analysis of DKK1 expression in the Hippo signaling pathway of hepatocellular carcinoma

[0202] Example 6-1: Tissue microarray sample and IHC

[0203] 4 μm thick TMA sections containing 67 HCCs and 10 normal livers were purchased (TissueArray.Com), paraffin was removed, and the sections were rehydrated. The antigens were recovered in Tris buffer, and the sections were cultured with primary antibodies YAP XP (cat. 14074S, CST), DKK1 (cat. AB61034, Abcam), and CTGF (cat. AB6992, Abcam), followed by further culture with HRP-labeled secondary antibodies. The conjugated antibodies were visualized using 3,3' diaminobenzidine chromogen and counterstained with hematoxylin. IHC scores were determined based on the percentage of cells stained by the pathologist and staining intensity. The immunoreactivity of DKK, YAP, and CTGF was evaluated on four staining intensity levels: negative, weak (1+), moderate (2+), and strong (3+). Cytoplasmic staining showing a 2+ or 3+ positive reaction in more than 30% of HCC cells was considered to indicate overexpression of the corresponding protein. The correlations between DKK and YAP or CTGF, and between YAP and CTGF, were evaluated using the chi-square test. A p-value of less than 0.05 was considered statistically significant (Fig. 48). The analysis revealed significant correlations between YAP, CTGF, and DKK1 expression in HCC tissue.

[0204]

[0205] Example 6-2: Confirmation of DKK1 gene expression in patients with hepatocellular carcinoma (LIHC)

[0206] Using the TCGA database, the frequency of mutations primarily found in samples of patients with hepatocellular carcinoma (LIHC) was identified (left), and subsequently, the degree of DKK1 gene expression for each mutation sample was compared with that of general solid tumor samples (right). As a result, it was found that DKK1 expression was increased in HCC patients regardless of mutation (Fig. 49).

[0207]

[0208] Example 6-3: Prediction of increased signal in DKK1 high-expression HCC

[0209] Using the TCGA database, gene expression patterns in liver hepatocellular carcinoma (LIHC) patient samples with high DKK1 expression and no DKK1 expression were examined using a volcano plot (left), and subsequently, genes upregulated in the high-DKK1 expression group in the volcano plot were collected and transcription prediction was performed (right). As a result of the analysis, TEAD2 and TEAD4 of the TEAD family were predicted to be activated transcription factors in high-DKK1 expressing HCC (Fig. 50).

[0210]

[0211] Example 7: Inhibition of hepatocellular carcinoma growth and YAP target gene expression upon DKK1 inhibition

[0212] Example 7-1: Comparison of DKK1 Expression in Normal and HCC Cell Lines

[0213] Quantitative PCR analysis was performed on DKK1 mRNA expression in HEK293T, HepG2, Huh7, and Hep3B cells. Relative expression levels were normalized to the expression levels of HEK293T cells. The analysis results showed that DKK1 expression was high in all HCC cell lines, with the highest expression found in Hep3B (Fig. 51).

[0214]

[0215] Example 7-2: Comparison of tumor size by DKK1 inhibitor treatment in a Hep3B xenograft mouse model

[0216] A Hep3B xenograft model was established by injecting nude mice with Hep3B cell lines. Subsequently, once the tumor size reached 50–100 mm³, WAY-262611 was orally administered at a dose of 16 mg / kg once daily for 10 days, after which the mice were sacrificed (Fig. 52). Tumor volume was measured every two days (Fig. 53), and the calculation formula is as follows:

[0217]

[0218] Analysis of tumor size showed that WAY-262611-treated Hep3B tumor tissues exhibited a decrease in tumor size and weight compared to the control group (Fig. 52), and it was found that tumor growth in Hep3B tumor tissues was inhibited by WAY-262611 treatment (Fig. 53).

[0219]

[0220] Example 7-3: Comparison of YAP and CTGF levels induced by DKK1 inhibitor treatment in a Hep3B xenograft mouse model

[0221] Hep3B cell lines were cultured and subcutaneously injected into female BALB / c nude mice, resulting in a tumor size of approximately 100 mm 3 Half of the tissue obtained when [the condition was met] was fixed in 10% neutral formalin and embedded in paraffin to prepare blocks for IHC analysis. IHC analysis was performed using YAP XP (cat. 14074S, CST) and CTGF (cat. AB6992, Abcam). As a result, it was confirmed that the levels of YAP and CTGF were reduced in the tumor tissue of WAY-262611-treated mice (Fig. 54).

[0222]

[0223] Example 7-4: Comparison of CTGF and CYR61 gene expression induced by DKK1 inhibitor treatment in a Hep3B xenograft mouse model

[0224] Tumor tissue derived from a Hep3B xenograft model was treated with a carrier or WAY-262611, and the expression of representative YAP target genes (CTGF and CYR61) was evaluated by qPCR. As a result, it was confirmed that the expression of YAP target genes was reduced by WAY-262611 treatment (Fig. 55).

[0225]

[0226] Example 8: Membrane-localized OGT via PPO domain required for LRP6 O-GlcNAcylation

[0227] Example 8-1: Immunoblot and Immunoprecipitation Analysis

[0228] HEK293T cells were cultured until the cell density (confluency) reached approximately 80%, at which point the cells were scraped with a scraper to obtain the cells, and then centrifuged in phosphate-buffered saline (PBS) at 12,500 lb for 5 minutes at 4°C. The obtained pellet was resuspended in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 μg / mL leupeptin. For the experiment to detect protein O-GlcNAcylation, 10 μM PUGNAc (OGT inhibitor) was additionally added to the lysis buffer. After incubating on ice for 30 minutes, the lysate was centrifuged at 13,000 µg for 10 minutes at 4°C. The supernatant was collected and used for immunoblotting and immunoprecipitation analysis. For immunoblotting, the protein concentration of each lysate was determined using the Bradford assay. Equal amounts of protein were mixed with 4 µS SDS loading buffer (200 mM Tris, pH 6.8; 8% SDS; 0.05% bromophenol blue; 40% glycerol; 200 mM β-mercaptoethanol) and boiled for 10 minutes. Samples were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated overnight at 4°C with the primary antibody diluted in 5% skim milk or bovine serum albumin (BSA). The next day, the membrane was washed five times with 1 ¼ TBST buffer for 10 minutes per wash. After washing, the membrane was incubated with a secondary antibody diluted in 5% skim milk at room temperature for 1 hour, followed by three washes in 1 ¼ TBST buffer for 15 minutes each. ECL (ELPIS or Millipore) was applied to the membrane, and signal detection was performed using X-ray film or MicroChemi 4.2 (DNR Bio-ImagingSystems).For immunoprecipitation, cell lysates were incubated with the primary antibody in a rotary rig at 4°C overnight. Then, Protein A / G and agarose beads (Fast Flow, 50% slurry, Millipore) were added, and the mixture was incubated in a rotary rig at 4°C for 1 hour. The beads were washed five times with lysis buffer for 10 minutes each at 4°C. Finally, the beads were boiled in 4X SDS loading buffer for 10 minutes before analysis by SDS-PAGE (Figs. 32, 58). Immunoprecipitation analysis revealed that membrane-localized OGT induced LRP6 O-GlcNAcylation better than WT-OGT (Fig. 32), demonstrating that PIP3 interactions via the PPO domain are important for LRP6 O-GlcNAcylation, and that membrane localization of OGT via OGT-PIP3 interactions plays a crucial role in this process (Fig. 58).

[0229]

[0230] Example 8-2: Intracellular Fraction Analysis

[0231] Intracellular fractionation was performed as previously described. HEK293A cells seeded in culture dishes were harvested using a scraper when the confluency reached approximately 80%, and the cell pellet was collected by centrifugation at 12,500 rpm for 5 minutes at 4°C. A portion of the pellet was used to prepare the whole cell lysate. The remaining cells were resuspended in S100 / P100 buffer supplemented with 1 mM phenylmethylsulfonylfluoride and 1 μg / ml leupeptin, and incubated on ice for 20 minutes. The cell suspension was then passed through a 26-gauge needle 20 times. The homogenate was centrifuged at 1,000 µg for 2 minutes at 4°C to remove the nuclei (pellet), and the resulting supernatant was further centrifuged at 20,000 µg for 60 minutes at 4°C. The pellet (membrane fraction) and the supernatant (cytoplasmic fraction) were collected separately. Immunoblot analysis was performed on the membrane, cytoplasm, and total lysate. As a result, it was found that OGT membrane localization could be induced by attaching SP and TM domains even without the PPO domain (Fig. 57).

[0232]

[0233] Example 9: PPO domain acting as a competitive inhibitor of LRP6 O-GlcNAcylation and an inhibitor of HCC cell proliferation and progression

[0234] Example 9-1: Colony-forming assay

[0235] For Huh7 and Hep3B cell lines, transfected cells were treated with trypsin and counted using a Countess™ II automated cell counter (Thermo Fisher Scientific, USA). A total of 5,000 cells were seeded into each well of a 12-well plate. Cells were cultured for 5–7 days in DMEM or RPMI medium supplemented with 10% FBS at 37°C in a humidified incubator containing 5% CO₂. For cell fixation, cells were treated with 4% paraformaldehyde for 20 minutes and then washed once with PBS. For colony staining, a staining solution (1% crystal violet in PBS) was added to each well and incubated for 10 minutes. The wells were then gently rinsed with tap water. Three wells were photographed using a digital camera, and colony area and colony number were quantified using ImageJ software (Figs. 60, 61).

[0236]

[0237] Example 9-2: Transwell migration and invasion assay

[0238] After trypsin treatment, transfected cells were counted using a Countess™ II automated cell counter (Thermo Fisher Scientific, USA). Huh7 cells (1 x 10⁴) and Hep3B cells (1 x 10⁴) 5The cells were resuspended in 200 μL of serum-free medium or serum-free condition medium (CM) and inoculated into the upper chamber of a polycarbonate (PC) membrane hanging insert (SPLInsert™ Hanging, SPL) with 8 μm pores. The lower chamber was filled with 700 μL of the corresponding medium supplemented with 10% FBS. Cells were cultured in a humidified incubator with 5% CO₂ at 37°C for 16–24 hours. After culture, the chambers were gently washed with PBS and fixed in 4% paraformaldehyde for 10 minutes. Cells were stained with 0.5% crystal violet for 5 minutes and washed with PBS to remove excess dye. Non-invasive cells remaining on the upper surface of the membrane were carefully removed using a cotton swab. Three random field images per upper chamber were taken at 200x magnification using a Nikon Eclipse Ti microscope. The area of ​​invasive or migrated cells on the underside was quantified using ImageJ software. For the invasion analysis, the top chamber was pre-coated with Matrigel (BD Biosciences) diluted in serum-free medium, and the analysis was performed using the same Transwell protocol. The results showed that DKK1 inhibits LRP6 O-GlcNAcylation and YAP activity, and inhibits HCC tumor proliferation and progression (Figs. 62, 63).

[0239]

[0240] Example 10: PPO construct inhibiting YAP levels in HCC and PIP3-upregulated cancer cells

[0241] Immunoblotting was performed as described in Example 8-1. Cells were scraped with a cell scraper to obtain the cells, which were then centrifuged in phosphate-buffered saline (PBS) at 12,500 lb for 5 minutes at 4°C. The obtained pellet was resuspended in a lysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM sodium β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 μg / mL leupeptin. For the experiment to detect protein O-GlcNAcylation, 10 μM PUGNAc (OGT inhibitor) was additionally added to the lysis buffer. After incubating on ice for 30 minutes, the lysates were centrifuged at 13,000 µg for 10 minutes at 4°C, and the supernatant was collected for immunoblotting. The protein concentration of each lysate was determined using the Bradford assay. Equal amounts of protein were mixed with 4 µS SDS loading buffer (200 mM Tris, pH 6.8; 8% SDS; 0.05% bromophenol blue; 40% glycerol; 200 mM β-mercaptoethanol) and boiled for 10 minutes. Samples were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were incubated overnight at 4°C with the primary antibody diluted in 5% skim milk or bovine serum albumin (BSA). The following day, the membranes were washed five times with 1 µS TBST buffer for 10 minutes per wash. After washing, the membrane was incubated with a secondary antibody diluted in 5% skim milk at room temperature for 1 hour, and then washed three times for 15 minutes each in 1 lb-TBST buffer. ECL (ELPIS or Millipore) was applied to the membrane, and signal detection was performed using X-ray film or MicroChemi 4.2 (DNR Bio-ImagingSystems).Experimental results showed that existing O-GlcNAcylation inhibitors exhibit broad-spectrum inhibition of protein O-GlcNAcylation, which carries a high risk of various side effects, and that PPO overexpression enables tumor-specific YAP inhibition while minimizing effects on normal cells (Figs. 45a, 64-66).

[0242]

[0243] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0244]

[0245] The present invention identified a novel link between DKK1 and the Hippo signaling pathway and, based on this, developed an LRP6 O-GlcNAcylation inhibitor that specifically inhibits cancer progression in cancer cells. Unlike existing OGT inhibitors, EGFP-PPO does not affect overall O-GlcNAcylation and exerts effects only in specific cells, thus presenting the possibility of minimizing side effects. Therefore, the LRP6 O-GlcNAcylation inhibitor of the present invention suggests the possibility of developing a targeted therapy for liver cancer patients with high DKK1 expression.

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